CA1169798A - Process for the production of polysaccharide - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Process of producing a polysaccharide having a molecular weight higher than 200,000 and which is effective in lowering the level of cholesterol in serum and liver and the atherogenic index, which comprises culturing Bacillus polymyxa No. 271 in an aqueous culture medium containing assimilable saccharides until said polysaccharide accumulates in said medium and recovering said accumulated polysaccharide from said medium. Cationic salt can also be produced.

Description

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This invention relates to a process for the production of polysaccharide.
A method for the preparation o~ polysaccharide using Bacillus polymyxa No. 271, is known according to Japanese Kokoku 67-7600. Bacillus polymyxa No. 271 has been deposited with the Fermentation Research Institute, Agency of Industria Science and Technology, Ministry of International Trade and Industry and the deposit number was designated as FERM-P No.
1824.
Japanese Kokoku 67-7600 discloses a method for the preparation of a highly viscous polysaccharide by culturing Bacillus polymyxa No. 271 in a culture medium containing an assimilable carbon source such as glucose, sucrose, and lactose.
The polysaccharide obtained from the viscous fermentation broth is composed of glucose, mannose, galactose and glucuronic acid.
It is also known that a mixture of the above described acidic polysaccharide with a neutral polysaccharide can be obtained from a medium containing sucrose as the carbon source.
The molecular weight of the polysaccharide prepared by using Bacillus polymyxa No. 271 in a glucose containing medium was determined by the Staudinger formula and it was found to be approximately 1,300,000 as disclosed in the Journal of the Agricultural Chemical Society of. Japan, vol. 42, No. 7 page 431-434, (1968).
An object of the invention is to provide a process of producing a polysaccharide having a molecular weight higher than 200,000 and which is effective in lowering the level of cholesterol in serum and liver and,the atherogenic index.
Another object of the invention is to provide a process of producing a cationic salt of a polysaccharide having a .~

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cation degree of substitution of more than 0.2 and is effective in lowering the level of cholesterol in serum and liver and the atherogenic index~
These objects of the invention can be attained by providing a process for producing a polysaccharide wherein Bacillus polymyxa No. 271 is cultured in a culture medium containing an assimilable carbon source until the poly-saccharide, preferably in highly viscous form, accumulates in the medium and said polysaccharide is recovered from the medium. Bacillus p~lymyxa No. 271 is preferably cultured in the medium for more than 20 hours. The accumulated polysaccharide can be purified by ultrafiltration and/or precipitation with alcohol. Preferably, the culture medium is an aqueous culture medium containing assimilable saccharides~
Another object of the invention is to provide a process for producing a cationic salt of a polysaccharide wherein Bacillus polymyxa No. 271 is cultured in a culture medium con-taining àn assimilable carbon source and a sufficient amount of ~ ~i9~

a cation to produce a cationic salt of a polysaccharide having a substitution degree of more than 0.2 until a cationic salt of the polysaccharide, preferably in highly viscous form, accumulates in the medium and said cationic salt of polysaccharide is recovered from the medium.
The culture medium preferably contains a tctal cation equivalent of at least 0.005 per 1 of the medium and is maintained at a pH of more than 4.5. If the culture condition is not suitable for the production of the desired salt of polysaccharide or if the accumulated salt of polysaccharide is subject to decationa-tion, the desired salt of polysaccharide can be produced,by adding the desired cationic salt to the fermented broth or to the free polysaccharide followed by stirring for a period long enough to produce a cationic saIt of polysaccharide having a degree of substitution of more than 0.2.
The accumulated salt of polysaccharide in the medium can be purified directly or after the treatment with the cationic salt by ultrafiltration and/or precipitation with alcohol.
These polysaccharides, having a molecular weight higher than 200,000 and the cationic salt of polysaccharide having a substitution degree of more than 0.2 have a remarkable effect in lowering the cholesterol level in serum and liver and the atherogenic index and are useful in the therapeutical or pro-phylactic treatment of arteriosclerosis or hypercholesterolemia.
This invention relates to a process for the production of polysaccharides and the salts thereof by culturing Bacillus polymyxa No. 271 in a culture medium. These polysaccharides and the salts thereof are remark~bly effective in lowering the cholesterol level in serum and liver and the atherogenic index.
It is well known that the cholesterol level in the blood is affected greatly by the amount and kinds of foods.
Cholesteremia is deemed to be one of the dangerous factors ..

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which are responsible for an outbreak of arteriosclerosis such as myocardial infarction.
With the exception of digestible and absorbable poly-saccharides such as starch, polysaccharides, one of the components of foodstuffs, have been neglected from the point of view of dietetics.
The inventors have carried out animal tests with rats to develop an agent for depressing the level of cholesterol in serum and liver frc~n the polysaccharide produced by Bacillus 10 polymyxa No. 271 . (The polys~ccharide produced by Bacillus polymyxa No. 271 is abbreviated hereinafter as B.p.~
It has been found that B.p. with a mean molecular weight higher than 200,000 is remarkably effective in lowering the level of cholesterol in serum and liver and in surprisingly increasing the cholesterol level of high density lipoprotein (hereinafter abbreviated as EIDL) which is known as a factor to prevent arteriosclerosis.
Ihe property of increasing the cholesterol level of HDL
indicates the effectiveness in lowering the atherogenic index ~0 which is formulated as follows:

serum cholesterol - HDL cholesterol HDL cholesterol ~his effect of B.p is unforeseeable.
The mean molecular weight (hereinafter abbreviated rnerely as molecular weight) of B.p. was measured by the method of Ninomiya et al in J. Agr. Chem. Soc. Japan 42 (7), P431(1968) .
The polysaccharide of l~he present invention can be prepared by culturing Bacillus polymyxa No. 271 (FERM-P No. 1824) under aerobic conditions in an aqueous culture medium containing 30 3-5% of a carbon source such as glucose, sucrose, lactose, molasses and other saccharides, to which has been added a nitrogen source such as a peptone, corn steep liquor, yeast extracts and i~

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urea, and salts such as magnesium sulfate. The accumulated viscous polysaccharide is purified by precipitation with alcohol and ultrafiltration. It is essential that the molecular weight of s.p. be higher than 200,000 in order to be active in - depressing the level of cholesterol in serum and liver.
The condition for the cultivation of the microorganisms and for the purification of B.p was fully studied since the fermented broth was very viscous and the treatment such as filtration, concentration and drying in the purification process was very difficult. The culturing period is usually more than 20 hours to obtain a polysaccharide having a mean molec,ular weight higher than 200,000. A polysaccharide having a molecular weight of 190,000 is obtained by culturing microorganisms for 16 hours.
The polysaccharide having a mean molecular weight higher than 200,000 can also be obtained if a polysaccharide having a molecular weight lower than 200,000 is treated with alcohols of lower concentration. A polysaccharide of higher molecular weight precipitates with alcohols of lower concentration, and a lower molecular weight polysaccharide precipitates with alcohols of higher concentration. Salts such as sodium chloride or potassium chloride can facilitate the precipitation of polysaccharide with alcohol of lower concentration. A polysaccharide of lower molecular weight precipitates more easily by the addition of salts than without such addition when an alcohol of the same concentration is used.
A polysaccharide of high molecular weight can be hydrolyzed with an acid or an alkali in order to obtain a polysaccharide of lower molecular weight. It is preferable to hydrolyze the poly-saccharide before the removal of the cells from the broth byfiltration, since the filtration resistance is diminished.

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The polysaccharide produced by the method of the invention is active in depressing the cholesterol level in serum and liver, as established by the examination of rats bred with a diet containing 5% of B.p.
The B.p of the invention can be taken in powder form or as an aqueous solution or in suspension in water. It is used alone or is incorporated in the diet or in various kinds of health foo~s.
It has further been found that the salts of poly-saccharide prepared from Bacillus polymyxa No. 271 are alsoachieved in depressing the cholesterol level in serum and liver.
The study was carried out by combining acidic polysaccharide with various cations in order to find a relationship between the lowering of the cholesterol level, the kind of cations to be substituted in B.p.and the degree of substitution of these cations.
The cationic salts of polysaccharide derived from cations comprising at least one member selected from the group consisting of alkali metal, alkali earth metal, transition metal, manganese, aluminium, zinc, copper and ammonium ions an~ having a substitu~ion degree of more than 0.2, have a remarkable activity in lowering the cholesterol level in serum and liver.
The cationic salts of B.~ can be produced by aerobically culturing Bacillus polymyxa No. 271 in a culture medium containing 3-5% of a carbon source such as glucose, galactose, lactose, molasses, and other saccharides,to which has been added a nitrogen source such as peptOne corn steep liquor, yeast extracts and urea, and cationic salts such as phosphate, magnesium sulfate.
The polysaccharide accumulated in the medium is highly viscous and is composed of D-glucose, D-mannose, D-galactose and D-glucuronic acid, the ratio of these components being 3:3:1:2.

From the ratio of these sugars, and the combination of 2 cations .;

with a molecular weight of 1500 the substitution degree of the cations is calculated as follows: /
substitution degree = ~ (Ni ~ 17050 where:
Mi : content of cation g/I~g Ni : gram equivalent of cation Alkali metal includes sodium and potassium and alkali earth metal includes calcium and magnesium.
The amount of cation contained in the culture medium is preferably at least 0.005 per 1 of the medium since the value of the total equivalent and the pH value of the medium must be adjusted to more than 4.5 preferably between 6 and 7. If the culture medium is not suitable for the production of desired salt of s.p the accumulated salt of B.p may not have the pre-determined substitution degree. The B.p thus obtained or the polysaccharide sub~ected to a treatment of decationation such as ion-exchange and electrolysis can be treated with the desired salts and stirred for a sufficient period to produce the desired salt of B.p with a substitution degree of more than 0~2, before i 20 refining by ultrafiltration or precipitation with alcohol.
^ Since all cations are not always necessary for culturing microorganisms, it is preferable to treat B.p with the desired salts after removing the cation from the salt of B.p by means of ion-exchange or electrolysis to obtain exclusively the salt of B.p with the special cation.
The salt of polysaccharide produced by the method of the invention having a substitution degree more than 0.2 has been found active in lowering the ch~lesterol level in serum and liver as a result of en examination of rats bred with a diet containing 5% of salt of B.p.
The salt of B.p of the invention can be taken in powder form or an aqueous solution or suspension in water and it may be 7 9 ~

used as an ingredient in various foodg which are called health foods.
The methods for preparing ~.p and the salt thereof and their activities in depressing the cholesterol level are illustrated in the following examples and experiments.
Example l 600 ml of a broth precultured during 24 hours with Bacillus polymyxa No. 271 (FERM-P No. 1824) was added to 12 1 (pH 7.0) of a culture medium containing 4% glucose, 0.1% peptone,

2% corn steep liquor, 0.2% magnesium sulfate and 6 ppm manganese sulfate. The cultivation was carried out at a ternperature of 28C with aeration of 0.8 W M for 72 hours. The viscosity of the cultured broth was as high as 9,500 cP. The broth was diluted with five times its amount of hot water and heated up to 70-80C.
The cells of the microorganisms were filtered and removed from the broth with a filter paper precoated with diat earth. 40 l of the filtered broth was treated with an ultrafilter (UF-SWM-85V-l, membrane module Abcor SWM-85M made by Bioengineering Co., Ltd.), purified by constant volume filtration up to W~Wo = 5 (ratio of the amount of added water to that of original broth~, concentrated and freeze dried to give 50 g of powdered poly-saccharide (sample l).
Examples 2 - 4 Control 1 4 Samples of 41 l of the filtered broth obtained in Example l were adjusted to a p~ value of 1.5 with hydrochloric acid and heated at 80C for 5 minutes (Exp. 2), 15 minutes (Exp.

3) 45 minutes (Exp. 4) and 2 hours (Exp. 5) respectively and then neutralized with sodium hydroxide. Thereafter each sample was ultrafiltered as in Example l, dried to give 48 g of powdered polysaccharide (sample 2), 46 g (sample 3), 45 g (sample 4) and 40 g (sample 5) respectively as Examples 2 - 4 and control l.
The molecular weight and the analytical data of examples 1 - 4 and control 1 are shown in Table 1.

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Table 1 Sample No. molecular weight crude protein(%) ash(%) 1 93 x 104 1.16 7.6 2 54 x 104 1.02 7.5 3 36 x 104 0.87 7.2

4 25 x 104 0.89 8.1 16 x 104 0.78 8~2 Example 5 The filtered broth was obtained by culturing the microorganisms as in Example 1 with the culture medium (pH 7.0) containing 4% of hydrol as the dry matter byproduct obtained in the production of crystal glucose, 0.1% peptone, 2% corn steep liquor, 0.2% magnesium sulfate and 6 ppm manganese sulfate.
To 40 1 of the filtered broth was added twice its volume of ethanol. The fibrous precipitate thus formed was collected by centrifugation. The precipitate was dehydrated, dried and dissolved again in 15 1 of water. After the aqueous solution was heated up to 70 - 80C and filtered, 25 1 of ethanol was added to the filtrate. The precipitate thus formed was collected again, dried under vacuum at 50C and was crushed into 50 g of powdered polysaccharide (sample 6).
Example ~.
40 1 of the filtered broth obtained in example 5 was treated with an ultrafilter (UF-SWM-85V-l, membrane module Abcor SWM-85V made by Bioengineering Co., LTD), purified with constant volume filtration up to W/Wo 5 (ratio of the amount of added water to that of original broth), concentrated and freeæe dried to give 50 g of powdered polysaccharide (sample 7).
Example 7.
600 ml of the broth precultured for 24 hours with Bacillus polymyxa No. 271 was added to 12 1 (p~ 7.0) of a culture medium containing 5% crystal glucose, 0.3% powdered yeast extract, : ~ g _ 7~a 0.05% urea, 0.2% magnesium sulfate and 6 ppm manganese sulfate.
me cultivation was carried out at a temperature of 28C with aeration of 0.8 VVM for 120 houxs. 40 1 of the filtered broth was obtained as in Example 1, and the purification was carried out also as in Example 1 except that methanol is used as the precipitant to give 40 g of powd~red polysaccharide (sample 8).
The molecular weight and the analytical data of samples 6 - 8 of Example 5 - 7 are given 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 a broth precultured for 24 hours with Bacillus polymyxa No. 271 was added to 12 1 (pH 7.0) of a culture medium containing 4% crystal glucose, 0.5% peptone, 0.5%
po~assium primary phosphate, 0.1% magnesium sulfate (7H20), 6 ppm manganese sulfate (4 - 6H20) and 0.1% defoaming silicon.
The cultivation was carried out at a temperature of 28C with aeration of 0.8 VVM for 96 hours. The viscosity of the cultured broth was 14,000 cP. me broth was diluted with five times its volume of hot water and was treated as in Example 5 to give 53 g of powdered polysaccharide (sample 9).

Example 9 40 ml of a saturated aqueous solution of NaCl was added as the precipitant to 40 1 of the filtered broth obtained in Example 7, 80 1 of ethanol was added further to the broth and the latter was treated as in Example 5 to give 55 g of powdered polysaccharide (sample 10).

1 ~97~8 Control 2.
40 1 of the filtered broth obtained in E~ample 8 was diluted with the same amount of water and 300 ml of the ion-exchange resin Amberlite 200TM (H type) ~nd 600 ml of the ion-exchange resin Amberlite IRA-411TM (OH type) were added to the diluted filtered broth. The resultant mixture was stirred for 3 hours at a temperature of 40C. The liquid thus treated was purified with an ultrafilter (UF-SWM-85V-l), membrane module Abcor SWM-85V made by Bioengineering Co., LTD.), up to W/Wo = 5 (ratio of the amount of added water to that of original broth), concentrated and freeze dried to give 50 g of powdered poly-saccharide (sample 11).
- Example 10.
The filtered broth obtained in Example 8 was treated with the ion-exchange resin as in control 2 and 50 ml of an aqueous solution containing 5% potassium chloride was added to the broth. The resultant mixture was left standing for one night and treated as in control 2 to give 51 g of powdered poly-saccharide (sample 12).
Example 11.
The same treatment for the broth was carried out as in Example 10 with 20 ml of an aqueous solution containing 5% NaCl instead of potassium chloride as in Example 10, to give 51 g of powdered polysaccharide (sample 13).
The molecular weight, analytical data, content of cation and the ~egree of substitution with cation of samples 9 -13 are shown in Table 3.

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~9~9 ~abl e 3.

Sam- molec- crude ~ash ~onte~t of cation(ppm) total degree of ple ula~ pro~eir ~eauivalent of ca ;ion/K~) equiv- substitu-No. wei~ht Na K M~ _ Ca _ alen-t tion x104 % %
9 114 1.46 7.4 5,400 18, ooo 3 ~ 400<5 0.230. ~6 0.28 0.002 o .97 0.73 l O 98 1.23 7.2 5 ' 9 ' 7 2,700 <5 0~650.25 0.22 0.002 1.12 o.84 11 118 - 7~ 3. o 59o 120 270 ~50 0.02500003 0.022 o .002 o . o52 0.039 12 1 i 8 0.73 4o 5 55o 22,300 260 <5 0.02~oo57 0.021 0.002 0062 0.47 13 116 73 4.9 6,90o 130 270 <50 _ _ 0.300~003 0.022 0.002 0:33 0.25 .

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Example 12.
600 ml of a broth preeultured for 24 hours with Baeillus polymyxa No. 271 was added to 12 1 (pH 7.0) of the eulture medium eontaining 5% erystal glucose, 0.625% peptone, 0.5% potassium primary phosphate, 0.2% magnesium sulfate (7H2O), 0.5% calcium carbonate, 6 ppm manganese sulfate (4 - 6H20) and 0.1% soybean oil. The cultivation was carried out at a temperature of 28C with aeration of 0.8 VVM for 96 hours.
The viscosity of the cultured broth was 16,000 cP. 40 1 of the filtered broth was treated as in Example 5 to give 58 g of powdered polysaccharide (sample 14).

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Example 13.
40 1 of the filtered broth obtained as in Example 12 was ultrafiltered under the condition as in control 2 to give 62 g of powdered polysaccharide (sample 15).
Example 14.
40 1 of the filtered broth obtained as in Example 12 was diluted with the same amount of water and demineralized by flowing down the broth at the flow rate (SV=3) in a tower (diameter 5 cm) filled with a mixture of 300 ml of Amberlite 200TM (H type) and 600 ml of Amberlite IRA-411TM (OH type). A
mixture of salts (35 g of potassium primary phosphate and 15 g of magnesium suLfate(7H2O), the amount of which is calculated from the salts contained in the original culture medium is dissolved in a small amount of water. It was added to the demineralized broth. The resultant mixture was further neutralized with sodium hydroxide and allowed to stand for one night after which it was ultrafiltered according to the method described in control 2 to give 60 g of the powdered poly-saccharide (sample 16).
Control 3~
The broth after treatment with an ion exchange resin a~ in Example 14 was ultrafiltered without the addition of salts to give 56 g of the powdered polysaccharide (sample 17).
The molecular weight, analytical data, content of cation and degree of substitution with cation in Examples 12 - 14 and control 3 are shown in Table 4.

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~ Table L~.
_ Sam- molec- crude ash content of cation (ppm) total degree of ple ~lar prote~ . ( ~ equivalent o~ cation/K~) equiv- substitu-No. weight Na I K Mg I Ca alent tion ~_ x10~ ~ , 1~ 10~ 2~0 8~ 15~7018~800 7~850 ~250 OQ2~OOL~8 o~65 0~21 1 -58 1 o18 100 104 8096 ~ 67015 ~ 600 ~ ~ ~20 ~ ~ 120 0~290~0 0~36 0~21 1 ~26 o~95 16 89 0~9 6~15~61016~350 6~700 920 0~2~0~2 o.55 0~0~6 1 ~26 95 17 91 0~9 102670 7o 310 3~190 _ 000290 ~ 002 0 ~ 025 0 ~ 16 0 ~ 216 0 O 16 .
Example 15.
600 ml of a broth precultured for 24 hours with Bacillus polymyxa No. 271 was added to 12 1 (pH 7.0) of a culture medium containing 4% crystal glucose, 0.5% peptone, 0.5% potassium primary phosphate, 0.5% calcium carbonate, 0.1%
magnesium sulfate (7H20),6 ppm manganese sulfate and 0.1%
defoaming silicone. The cultivation was carried out at a temperature of 28C with aeration of 0.8 W M for 72 hours. The viscosity of the cultured broth was 10,000 cP. The broth was diluted with five times its volume of hot water and heated up to 70 - 80C. The cells of the microbes were filtered and removed from the broth with a filter paper precoated with diatom earth. 40 1 of th^ filtered broth was treated with an ultrafilter (UF-SWM-85V-l, membrane module Abcor SWM-85V made by Bioengineering Co., Ltd.), purified up to W/Wo = 5 (ratio of the amount of added water to that of original broth), concentrated and freeze dried to give 50 g of powdered poly-saccharide (sample 18).

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Example 16 - 20 6 Samples of 40 1 of the filtered broth was divided from the broth obtained in Example 15. Each sample was diluted - with the same amount of water, and demineralized by flowing down the broth at the flow rate (S~=3) in a tower (5cm diameter) filled with the mixture of 300 ml of Amberlite 200 ~ (H Type) and 600 ml of Amb~rlite IRA-411TM (OH type). One of 6 samples is sample 19 and each of the other 5 samples is combined with 1 1 of a I~/o aqueous solution of NaCl, KCl, MgC12, CaC12 and NH4Cl respectively. Each sample was left standing for one night and ultrafiltered according to the method of Example 1 to give 40 -45 g of the special salts of polysaccharide respectively (sample 19 - 24).
Example 21 1 1 of a 10% aqueous solution of ferric chloride was added to the broth after treatment with the ion exchange resin as in Example 16. The resultant mixture was lèft to stand for one night with occasional stirring. The formed precipitate was collected with the centrifuge, it was washed twice with demineralized water and dried under vacuum to give a ferric salt of polysaccharide (sample 25).
Example 22 1 1 of a l~/o aqueous solution of aluminum chloride was added to the broth after treatment with the ion exchange resin as in Example 16. The pH value was adjusted at 4.5. The resultant mixturé was left to stand for one night with occasional stirring. The produced precipitate was treated as in Example 21 to give an aluminum salt of polysaccharide (sample 26).

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Experiment 1.
Seven groups, each group consisting of ~ive rats raised for four weeks, were bred with a test diet for four days to investigate the influence of the molecular weight of B.p on the depressing of the level of cholesterol. Seven test groups were established. There was a standard diet group (with no cholesterol), a control diet group (with cholesterol) and test diet groups (with cholesterol and 5% of B.p of each of samples 1 - 5 in Table 1). The composition of the diet is shown in Table 6.
The rats were freely fed with the diet and water.
After the test, the abdomen of the rats was opened under anesthesia with ether and -the blood was collected from the abdominal artery. The liver was picked out 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 measured directly with a Determiner TC made by KYOWA HAKKO CO., LTD. The liver was saponified and the unsaponifiable matter was separated. The cholesterol level of unsaponifiable matter was measured by the same Determiner TC.
The level of the HDL-cholesterol was measured with a HDL-sterozyme made by FUJI ZOKI SEIYAKU CO., LTD~
The results are shown in Table 7.

., 7 ~ 8 Table 6. The composition of the Qiet ~% by weight~

composition ¦ diteantdard ¦ control test , casein 22 22 22 salt mixture1) ~ , vitamiremixture2)Oo85 oO85 0.85 (water soluble) soybean oil3) 1 1 1 (mixed with oil so]uble vitamine) choline 0.15 0.15 0.15 lard 10 10 10 cholesterol 0 0.5 005 sodium salt of 0 0.25 9-25) B.p 0 0 5 ~3) sucrose 62 61.25 (5O -25) Total ¦ 100 ¦ 100 100 .

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20 l) Harper's salt mixture.
2) Harper's vitamin mixture.
3) mixed with vitamin A(3000IU), vitamin D(300IU) and vitamin E(lOOmg)/Kg diet.
4) the ~racketed value shcws the addition of 3% B.p to the diet.
Harper's salt mixture CaC03, 29-29; CaHP04-2H20, 0.43, KH2P04, 34.31, NaCl, 25.06, MgS04 7H20, 9.98 and others .

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Ta~le 7, cholesterol value ~mg/dl) atherogenic rate of diet index accurnulated group _ _ _ cholesterol serum HDL liver(mg~:g) in the liver : - .
standard 91+16 5906~1600 307i102 0.6+00~ -control 291+53 31~6+70022.2+0.6 8.2+2.0 50.4+~.7 test diet

5% ~oP of 116~16 39-5~4.87.6+1.1 109iOo3 1~.2+1.7 sample 1 5% B.p of 129+~0 38.9+~.~10.2~1.9 2.~+1.0 1606+6.
sample 2 s5~a ~ieP 3f165i~1 3~.5*4.79.6*2-5 3.8i1.2 15.9+~-7 5% 3-P of 190i37 ~0.1~9.712.9~0.9 ~.2*2.6 20.6*6.0 sample ~
5% ~.p of 2~3i35 3005+~o820.6i1.7 7.0i200 35.~+503 sample 5 1) liver cholesterol and _ liver cholesterol control diet group standard diet group - --- ---- x 100 cholesterol intake .. . .

1 ~9~.3 As clearly shown in Table 7, the s.p having a molecular weight higher than 200,000 (sample 1-4) is remarkably active in depressing the cholesterol level in serum and liver.
The appearance of the liver of rats bred with standard diet was reddish brown and the liver of rats bred with control diet was yellowish brown. This is clearly a characteristic color of fatty liverO On the other hand, the liver of rats bred with B.p of 1-4 had a healthy reddish brown color. The liver of rats bred with the control, sample 5, was yellowish brown like one bred with the control diet.
This is clearly the characteristic color of a fatty liver.
Experiment 2 The test was carried out with rats bred with samples ~-8 shown in Table 2 to prove the activity of lowering the cholesterol level. 8 diet groups were established in this experiment. The composition of the diet and the method of the experiment are the same as in experiment 1.
The results are shown in Table 8.

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Table 8 rate of diet cholesterol value(mg/dl) a~herogenic accumulated group index cholesterol in serum HDL . liver(mg/g) the liver diet 96*1 3 61 o 8+~ . 5 20 9~0.2 0.6~0.2 dintrl 323i19 36.3i102 22.~ 0 8.3io.~ 52.Lt~3.8 test diet 3% B-p f213+11 32.1+1.5 1109+0.6 5.7+005 19.8+2.5 sample 6 5% ~ P of1 50~11 39 - 7+2 O 1 9 O 5+o O 5 209+005 14.6+1.
sample 6 3% Bop of138+11 3Lt.9+1o2 709+0.5 3.0+0.3 9.9+1.1 sample 7 5% 30 P of 90+ 3 L~500*2. ~ 5. ~+o .6 l o O+O o l 3.8+1.3 sample 7 3% B-p of201~ 8 3508+2.2 1103+0.8 ~05+o.3 18.8~2.0 sample 8 5% Bo P of 1 55*14 37 1 *1 9 906+0 - 3 3 - 0+005 14.7+1 5 As obviously shown in Table 8, the B.p of this ` invention exhibits the activity of remar~ably loweri.ng the level of cholesterol in the serum and liver and of decreasing the atherogenic index when 3% or 5% of B.p was added to the diet. These properties were especially evident in the B.p (sample 7) which was treated by ultrafiltration). Sample 8 having the highest molecular weight ~1,360,000) had nearly the same properties as those of sample 6 having a molecular weight of 1,040,000.

9 ~

It seems that the properties of B.p will not increase at a molecular weight of about 1,000,000. The liver of the rats bred with the sample of the invention had the same reddish brown color as that of the standard diet.
Experiment 3.
The animal test was carried out with samples given in Table 3 to prove the relationship between the degree of substitution with the activity of lowering the cholesterol level. 5% of the sample was added to the diet. The detail of the method of performing the test is the same as in experiment 1. The results are shown in Table 9.

Table 9 cholesterol value atherogenic rate of diet (mg/dl) indexaccumulated group - _ cholesterol - serum ~L liver(mg/-g) in the liver .
stà -d d80-t 5 50.9+4.23-2+0.3 007+0.1 diet 251 i26 28~9i20720.6iO.5 8O2t108~9O8i2.2 test diet 5% 3iP 9E106+ 6 It909+6.07.7+1.1 1.3~0038.6+2.9 s5~ampiPe10f116+16 ~1.0+6.17.~i1.L~ 2.2iO.7708+4.2 5~9 3iP 11f291+~7 31.5i3.223.1tiO.5 ~.4i~.351~4;t1o8 s5%mplPe 12 105~11 32.0~.2 8.3+1.6 2,3+~7 705~3.1 sampie 131 5L~+21 28 o 1+20 7 908+1.64.8+1.1 1 2.9 t40 O

,.
.
'.~

~ ~ B~79 ~
.

As clearly shown in Table 9, samples 9, 10, 12 and 13, each having a substitution degree of more than 0.2, i.e.
of 0.73, 0.84, 0.47 and 0.25 respectively, are remarkably - effective in lowering the cholesterol level in serum and liver.
The B.p ~sample 11) having a substitution degree of 0.039 had a similar effect to that of the control diet group.
Sample 12 and 13 which are combined with salts after demineralization had fairly good activity as in the case of samples 9 and 10. The existence of the cation combined with B.p is the essential factor for the exhibition of the activlty of lowering the cholesterol level. The only mixing of cation with B.p did not display such activity as shown in table 6 where 4%
of Harper's salt mixture was added to the control and test diet group. These groups do not display any activity.
In the observation on the liver of the test animals the liver of rats bred with the standard diet and with the test diet comprising the B.p of the invention was reddish brown, and that of the control diet was yellowish brown. The appearance of the liver of rats bred with B.p of the invention was far superior to that of the control diet as described in experiments 1 and 2.
Experiment 4 The anirnal test was carried out with rats bred with the samples 14 - 17 shown in Table 4 prepared in Examples 12 14 and control 3. 10 diet groups which are the standard diet, the control diet and 8 test diet groups (with cholesterol and samples 14 - 17 in Table 4 at 3% and 5% level respectively) were established. The composition of the diet and the method for carrying out the test were the ~ame as in experiment 1.

9 7 ~ ~

The results of the test are sho~Jn in table 10.
Table 10 cholesterol value~ ~therogenic rate of ~ie~ index accumulated group cholesterol serum(mg/dl) .~L(mg/dl) liver~mg/g) in the liver standard 96+13 61 ~ 8 1-4 o 5 2 ~ 9+0 ~ 2 0 ~ 6+0 ~ 2 diet diontrOl 323~19 36~3*l~2 2204+100 803~tOoLJ- 52~ 3~8 test diet sampie 11t 21 3+1 1 32 ~ 1 +1 ~ 5 1 1 ~ 9;~ 0 ~ 6 5~ 7iO ~ 5 1 9 ~ 8i2 ~ 5 samp;e 14 150+11 39-7+2-1 9~5+0 5 2~9+oO5 14.6+1.LI-3% Bop f 138+11 34~9+102 709+005 3~0~0~3 9~9i101 sample 1 5 5% B-p of 90i 3 ~5 ~ Oi2 o ~ 5 ~ 4iO ~ 6 1 ~ OiO ~ 1 3 ~ 8i1 o 3 sample 15 ` 3% B-p Of 127+16 42~2+1 ~0 6~6+004 2~0+0~4 6~5+1~1 : sample 16 B.p of 91+ 8 45~1+403 503+0~4 101+0~3 2~5+102 sample 16 3% B-p f 251 +1 7 33 o 9 t1 ~ 2 1 50 6*1 ~ 5 6 ~ Lt+0 ~ 4 31 3+3 ~ Lj sample 1 7 5% Bop of 227i12 3204i106 15~5~1~3 6~1+0~4 29~1*3~2 sample 1 7 As clearly shown in Table 10, samples 14, 15 and 16 were superior to the control sample, and sample 17 having a substitution degree of 0.16 had lower activity. Sample 16 with a substitutlon degree of 0.95 prepared by the addition of salts to demineralized iB.p by ultrafiltration had nearly the same effect as sample 15, (substitution degree 0.95 prepared by ultrafiltration without demineralization). The activity of lowering the cholesterol level was fully restored by the addition of salts.

11~979,~

rhe color and the appearance of the liver of rats bred with B.p of the invention were far superior to the control as described in the foregoing experiments 1 to 3.
Experiment 5 The animal test was carried out with samples 18 - 25 (Table 5) obtained in Examples 15 - 22 to prove the activities of the salts of B.p with various cations.
The amount of the salt of B.p added to the diet was 4%. The experiment was carried out as in experiment 1.
lO The results of the test are shown in Table ll.

~able 11 cholesterol value atherogenic rate of diet _ _ index accumulated groupserum HDL liver cholesterol (mg/dl~(mg/dl) (mg/g) in the liver standard92+11t~t3.3+7.3 ~2. 5-~o. 3 1o2+0~ 5 diet28~i313108+5.1t 17.7+0.6 8.1 +1.8 Lt401+3-20 test diet ultrafil-tered Bop of114ti16 39.1i13.97.3~t1.1 1.9*0-3 1202+2.4 sample 18 NamPsalt19 106+13 31t.6+605 7.8t1.0 108+0.6 12.8~t3.7 Kamplt 20 92+25 LtOo7+1 2.7 6.5+2.2 1.6+1.~ 8.5+5-3 30 Mampllt21 88+35 5003+13.2 6.2+1.8 1.1+1.7 7.7+5.0 campllt22 85+24 47.8+1107 5.8+1.6 0.9+0.9 601+3.9 NH~ Psalt3 7~+17 It6.9ilt.9 4.6+1.8 0.5+0.2 3-9+5-Fampllt24t 102i17 37-0+6.1 7-7+1-3 1.8+0.7 12.1+3.3 Almpaelt25 110i17 Lt2.5~17-0 6-5i1-9 1.9+1.1 8.6*5.6 .,, l~
.~,~, .

As shown in Table 11, the diet with the salts of B.p with combined cations showed a remarkable effect in lowering the serum cholesterol level, ~iver cholesterol level, atherogenic index and the rate of accumulated cholesterol in the liver compared with the control diet. This shows that the combined cation in the salt of B.p has a strong activity in lowering the cholesterol level. The ammonium, calcium and magnesium salts of B.p are especially active in lowering the atherogenic index which is closely associated with an outbreak of arteriosclerosis. The index is the same or lower than that obtained from the standard diet.
In the observation on the liver of rats bred with the salts of B.p of the invention, the color and the appearance of the liver are far superior to that of the control as described in the foregoing experiments.

~. . . ~.

Claims (3)

The embodiments of the invention in which an exclu-sive property or privilege is claimed are defined as follows:-

1. A composition for lowering the level of choles-terol in serum and liver and the atherogenic index consist-ing of a mixture of a polysaccharide having a molecular weight higher than 200,000 which is prepared by culturing Bacillus polymyxa No. 271 in an aqueous culture medium con-taining assimilable saccharides until said polysaccharide accumulates in said medium and recovering said accumulated polysaccharide from said medium and a pharmaceutical carrier.

2. A composition according to claim 1 consisting of a mixture of a polysaccharide having a degree of substitution of more than 0.2 which is prepared by culturing Bacillus polymyxa No. 271 in an aqueous culture medium containing assimilable saccharides and a cation until said cationic salt of polysaccharide accumulates in said medium and re-covering said cationic salt of polysaccharide from said medium and a pharmaceutical carrier.

3. A composition according to claim 2, wherein said cation is selected from the group consisting of sodium, potassium, magnesium, calcium, ammonium, Fe and Al.