GB2080818A

GB2080818A - A microbiological process for the production of a polysaccharide and its cationic salt and their use in depressing serum and liver cholesterol levels and the atherogenic index

Info

Publication number: GB2080818A
Application number: GB8116459A
Authority: GB
Original assignee: Showa Sangyo Co Ltd
Current assignee: Showa Sangyo Co Ltd
Priority date: 1980-07-28
Filing date: 1981-05-29
Publication date: 1982-02-10
Also published as: IT1195275B; DE3121548A1; CH659085A5; FR2487376B1; NL8102628A; GB2080818B; FR2487376A1; IT8122039A0

Abstract

A microbiological process for producing a polysaccharide with a molecular weight above 200,000 or a cationic salt thereof with a degree of substitution by the cation above 0.2 involves culturing Bacillus polymyxa No. 271 (FERM-P No. 1824) in a medium containing assimilable saccharides and, optionally, a desired cation, and recovering from said medium said polysaccharide or salt thereof. The polysaccharide and its salt are of value in depressing serum and liver cholesterol and the atherogenic index in mammals including man.

Description

SPECIFICATION A microbiological process for the production of a polysaccharide and its cationic salt and their use in depressing serum and liver cholesterol levels and the atherogenic index BACKGROUND OF THE INVENTION Field of the Invention This invention relates to a microbiological process for the production of a polysaccharide and its cationic salt and their use in depressing serum and liver cholesterol levels and the atherogenic index.

Description of the Prior Art A method for the preparation of a polysaccharide using Bacillus polymyxa No. 271, has been disclosed in Japanese Kokoku 67-7600. Bacillus polymyxa No. 271 was deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry and the deposit number was designated as FERM-P No. 1 824.

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 or lactose. The polysaccharide obtained from the viscous fermentation broth is composed of glucose, mannose, galactose and glucuronic acid.

It was also known that a mixture of the above-described acidic polysaccharide with the neutral polysaccharide was obtained from a medium containing sucrose as the carbon source.

The molecular weight of the polysaccharide elaborated by Bacillus polymyxa No. 271 in the glucose-containing medium was determined by the formula of Staudinger, and it was found to be approximately 1,300,000 as disclosed in the Journal of Agricultural Chemical Society of Japan, vol 42, No. 7 page 431-434, (1968).

SUMMARY OF THE INVENTION One object of the invention is to provide a process for producing a polysaccharide having a molecular weight of more than 200,000 and an activity in depressing any increase in serum and liver cholesterol and the atherogenic index.

Another object of the invention is to provide a process for producing a salt of a polysaccharide with a cation having a degree of substitution with the cation of more than 0.2 and an activity in depressing rises in serum and liver cholesterol 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 highly viscous polysaccharide accumulates in the medium and said polysaccharide is recovered from the medium. Bacillus polymyxa No. 271 is preferably cultured in the medium for more than 20 hours. The accumulated polysaccharide can ',e purified by ultrafiltration and/or precipitation with alcohol.

Another object of the invention can be attained by providing a process for producing a salt of a polysaccharide with a cation wherein Bacillus polymyxa No. 271 is cultured in a culture medium containing an assimilable carbon source and a sufficient amount of cation to produce a salt of a polysaccharide having a substitution degree of more than 0.2 until a highly viscous salt of the polysaccharide accumulates in the medium, and said salt of a polysaccharide is recovered from the medium.

The culture medium preferably contains a total equivalent of cation of at least 0.005 per 1 of the medium, and maintains a pH value of more than 4.5. If the culture condition is not suitable for the production of the desired polysaccharide salt, or the accumulated salt of the polysaccharide is subject to decationation to produce the desired polysaccharide salt, the desired salt of the cation can be added to the fermented broth or the free polysaccharide and stirred for a sufficient period to produce a salt of a polysaccharide with a cation having a degree of substitution of more than 0.2.

The accumulated salt of the polysaccharide in the medium can be purified directly or after treatment with a salt of a cation by means of ultrafiltration and/or precipitation with alcohol.

These polysaccharides having a molecular weight of more than 200,000 and the salt of the polysaccharide with a cation having a substitution degree of more than 0.2 have a remarkable effect in depressing serum and liver cholesterol level and the atherogenic index, and are useful in the therapeutic or prophylactic treatment of arteriosclerosis and hypercholesterolemia.

DESCRIPTION OF THE PREFERRED EMBODIMENT 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 have an activity in remarkably depressing rises in serum and liver cholesterol level and in the atherogenic index.

It is well known that the cholesterol level in the blood is greatly affected by both amount and kind of foods ingested. Cholesteremia is deemed one of the dangerous factors in promoting an outbreak of arteriosclerocis such as myocardial infarction.

Polysaccharides, one of the components of foodstuffs, have been neglected from the point of view of dietetics except for the digestible and absorbable polysaccharides such as starch.

We have carried out animal tests with rats to develop an agent for depressing the level of cholesterol in serum and liver from the polysaccharide which is elaborated by Bacillus polymyxa No. 271 (the polysaccharide produced by Bacillus polymyxa No. 271 is abbreviated hereinafter as "B.p.").

It has been found that B.p. having a mean molecular weight of more than 200,000 has the activity of remarkably depressing rises in the cholesterol level in serum and liver, and the activity of surprisingly increasing the cholesterol level of high density lipoprotein (hereinafter abbreviated as HDL) which is known as a factor in preventing arteriosclerosis.

The activity in increasing the cholesterol level of HDL indicates the activity of depressing the atherogenic index which may be formulated as follows: serum cholesterol -- HDL cholesterol HDL cholestrol This is an unforeseenable effect of B.p.

The mean molecular weight (hereinafter abbreviated as merely 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 the present invention can be elaborated by culturing Bacillus polymyxa No. 271 (FERM-P No. 1824) aerobically in an aqueous culture medium containing 3-5% of a carbon source such as glucose, sucrose, lactose, molasses or other saccharides, supplemented with a nitrogen source such as peptone, corn steep liquor, yeast extract or urea, and a salt such as magnesium sulfate. The accumulated viscous polysaccharide is purified by precipitation with alcohol and ultra-filtration. It is essential that the molecular weight of B.p. be more than 200,000 in order to display the activity of depressing the elevation of the cholesterol level in serum and the liver.

The conditions for the cultivation of the micro-organism and for the purification of B.p. were fully studied as the fermented broth was very viscous and treatment such as filtration, concentration and drying in the purification process was very difficult. The culture period is usually more than 20 hours in order to obtain the polysaccharide having a mean molecular weight of more than 200,000. Polysaccharide with a molecular weight of 190,000 is obtained by culturing the micro-organism for 16 hours. The polysaccharide having a mean molecular weight of more than 200,000 can also be obtained if the polysaccharide having a molecular weight of less than 200,000 is treated with alcohols of lower concentration. Polysaccharide of higher molecular weight precipitates with alcohols of lower concentration, and that of lower molecular weight precipitates with alcohols of higher concentration.Salts such as sodium chloride or potassium chloride can facilitate the precipitation of polysaccharide with alcohols of lower concentration. Polysaccharide of lower molecular weight precipitates upon the addition of salts more easily than without the addition of salts when alcohol of the same concentration is used.

Polysaccharide of high molecular weight can be hydrolysed with acid or alkali in order to obtain the polysaccharide of lower molecular weight. It is preferable to hydrolyse the polysaccharide before the removal of the cells from the broth by filtration, since the filtration resistance is diminished.

The polysaccharide produced by the method of the invention exhibits the activity of depressing the cholesterol level in serum and the liver in an examination of rats bred with a diet containing 5% of B.p.

The B.p. of the invention can be taken as a powder or as an aqueous solution or suspension in water. It may be used alone or incorporated in the diet or in various kinds of food (foods for health).

It has further been found that the salts of polysaccharide elaborated by Bacillus polymyxa No.

271 have also an activity in depressing the cholesterol level in serum and the liver. The study was carried out by combining acidic polysaccharide with various cations in order to find the relationship between the activity in depressing the cholesterol level, the kind of cations to be substituted in B.p. and the degree of substitution of these cations.

The salts of polysaccharide with cations at least one member of which is selected from the group consisting of alkali metal, alkaline earth metal, transition metal, manganese, aluminium, zinc, copper and ammonium ions and having a substitution degree of more than 0.2 have an activity in remarkably depressing the cholesterol level in serum and liver.

The salts of B.p. with cations can be produced by culturing aerobically Bacillus polymyxa No.

271 in a culture medium containing 3-5% of a carbon source such as glucose, galactose, lactose, molasses, and other saccharides, supplemented with a nitrogen source such as peptone, corn steep liquid, yeast extracts and urea, and salts combined with cations such as phosphate or 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 ratios of these components to one another being: 3:3:1:2. From the ratio of the component sugars, and the combination of 2 cations with the molecular weight of 1500, the substitution degree with cations is calculated as follows:

Mi: content of cation g/Kg Ni: gram equivalent of cation Alkali metal includes sodium and potassium and alkaline 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 as the value of the total equivalent, and the pH value of the medium needs to be adjusted at more than 4.5, preferably between 6 and 7. If the culture medium is not suitable for the production of the desired salt of B.p., the accumulated salt of B.p. may not have the predetermined substitution degree. The B.p. thus obtained, or the polysaccharide subjected to the 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. having a substitution degree of more than 0.2 before the 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 cation from the salt of B.p. by means of ionexchange or electrolysis to obtain exclusively the salt of B.p. with the special cation.

The salt of the polysaccharide produced by the method of the invention, having a substitution degree of more than 0.2, exhibits an activity in depressing the cholesterol level in serum and the liver in the examination with rats bred with the diet containing 5% of the salt of B.p.

The salt of B.p. of the invention can be taken as a powder or as an aqueous solution or a suspension in water, and it is used as an ingredient in various foods known as health foods.

The methods of preparing B.p. and salts thereof, and their activities in depressing the cholesterol level are illustrated in the following examples and records of experiments.

Example 1 600ml of the precultured broth with Bacillus polymyxa No. 271 (FERM-P No. 1824) for 24 hours were added to 1 2 1 (pH 7.0) of a culture medium containing 4% glucose, 0. 1% peptone, 2% corn steep liquor, 0.2% magnesium sulfate and 6ppm manganese sulfate. The cultivation was carried out at the temperature of 28"C with aeration of 0.8VVM for 72 hours. The viscosity of the culture broth was as high as 9,500 cP.The broth was diluted with five times its amount of hot water and heated up to 70-80"C. The cells of the microorganism were filtered and removed from the broth with a filter paper precoated with diatomaceous earth. 40 1 of the filtered broth were treated in an ultrafilter (UF-SWM-85V-1, membrane module Abcor SWM85M 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 yield 50g of powdered polysaccharide (sample 1).

Examples 2-4, Control 1 4 Samples of 40 1 of the filtered broth obtained in example 1 were adjusted with hydrochloric acid to the pH value of 1.5, and heated at 80"C for 5 minutes (Exp. 2), 1 5 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 1, dried to yield 489 of powdered polysaccharied (sample 2), 469 thereof (sample 3), 459 thereof (sample 4) and 409 thereof (sample 5), respectively, as examples 2-4 and control 1, respectively.

The molecular weight and the analytical data of examples 1-4 and control 1 are shown in Table 1, as follows: Table 1 Sample No. the molecular weight crude protein (%) ash (%) 93x104 1.16 7.6 2 54X104 1.02 7.5 3 36 x 104 0.87 7.2 4 25 x 104 0.89 8.1 5 16x104 0.78 8.2 Example 5 The filtered broth was obtained by culturing the microorganism as in example 1 with the culture medium (pH 7.0) containing 4% of hydrol as the dry matter byproduced in the production of crystal glucose, 0. 1% peptone, 2% corn steep liquor, 0.2% magnesium sulfate and 6ppm manganese sulfate. 40 1 of the filtered broth were supplemented with twice the volume of ethanol. The fibrous precipitate thus formed was collected by centrifuging. The precipitate was dehydrated, dried and dissolved again in 1 5 1 of water.After the aqueous solution had been heated up to 70-80"C and filtered, 25 1 of ethanol were added to the filtrate.

The precipitate thus formed was collected again, dried under vacuum at 50"C and crushed into 50g of powdered polysaccharide (sample 6).

Example 6 40 1 of the filtered broth obtained in example 5 were treated with the ultrafilter (UF-SWM85V-1, 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 the original broth), concentrated and freeze dried to yield 50g of powdered polysaccharide (sample 7).

Example 7 600ml of the precultured broth with Bacillus polymyxa No. 271 for 24 hours were added to 12 1 (pH 7.0) of the culture medium containing 5% crystal glucose, 0.3% powdered yeast extract, 0.05% urea, 0.2% magnesium sulfate and 6ppm manganese sulfate. The cultivation was carried out at a temperature of 28"C with aeration of 0.8 WM for 120 hours. 40 1 of the filtered broth were obtained as in example 1, and the purification was carried out also as in example 1 except for using methanol as the precipitant to obtain 409 of powdered polysaccharide (sample 8).

The molecular weight and the analytical data of samples 6-8 in examples 5-7 are shown in Table 2, as follows: Table 2 Sample No. the molecular weight crude protein (%) ash (%) 6 1O4X104 2.0 8.1 7 100 x 104 1.4 6.2 8 136X1O4 1.9 7.5 Example 8 600ml of the precultured broth with Bacillus polymyxa No. 271 for 24 hours were added to 121 (pH 7.0) of the culture medium containing 4% crystal glucose, 0.5% peptone, 0.5% potassium primary phosphate, 0.1% magnesium sulfate (7H2O), 6ppm manganese sulfate (4-6H2O) and 0.1% defoaming silicon. The cultivation was carried out at the temperature of 28"C with aeration of 0.8 VVM for 96 hours. The viscosity of the cultured broth was 14,000 cP. The broth was diluted with five times its volume of hot water and was treated as in example 5 to yield 53g of powdered polysaccharide (sample 9).

Example 9 40 ml of a saturated aqueous solution of NaCI were added as the precipitant to 40 of the filtered broth obtained in example 7, and 80 1 of ethanol were added to the broth and treated as in example 5 to yield 55 g of powdered polysaccharide (sample 10).

Control 2 40 1 of the filtered broth obtained in example 8 were diluted with the same amount of water and 300 ml of ion-exchange resin Amberlite (Registered Trade Mark) 200 (H type), and 600 ml of ion-exchange resin Amberlite (Registered Trade Mark) IRA-411 (OH type) were added to the diluted filtered broth. The resulting mixture was stirred for 3 hours at a temperature of 40"C.

The liquid thus treated was purified with an ultrafilter (UF-SWM-85V-1, 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 yield 509 of powdered polysaccharide (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 were added to the broth. The resulting mixture was left standing for one night and treated as in control 2 to yield 519 of powdered polysaccharide (sample 12).

Example 11 The same treatment for the broth was carried out as in example 10 with 20ml of an aqueous solution containing 5% NaCI instead of the potassium chloride of example 10 to yield 51 9 of powdered polysaccharide (sample 13).

The molecular weight, analytical date, content of cation and the degree of substitution with the cation of samples 9-1 3 are shown in Table 3, as follows: Table 3 content of cation (ppm, equivalent of cation/Kg) Sample molecular crude total degree of No. weight protein ash Na K Mg Ca equivalent substitution x104 % % 9 114 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 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 6,900 130 .270 < 50 0.30 0.003 0.022 0.002 0.33 0.25 Example 12 600 ml of the precultured broth with Bacillus polymyxa No. 271 for 24 hours were added to 1 2 1 (pH 7.0) of the culture medium containing 5% crystal glucose, 0.625% peptone, 0.5% potassium primary phosphate, 0.2% magnesium sulfate (7H20), 0.5% calcium carbonate, 6ppm manganese sulfate (4 - 6H20) and 0.1% soybean oil. The cultivation was carried out at the temperature of 28 C with aeration of 0.8 VVM for 96 hours. The viscosity of the cultured broth was 16,000 cP. 40 l of the filtered broth were treated as in example 5 to yield 58 9 of powdered polysaccharide (sample 14).

Example 13 40 í of the filtered broth obtained in example 1 2 were ultrafiltered under the same conditions as in control 2 to yield 62 9 of powdered polysaccharide (sample 15).

Example 14 40 l of the filtered broth obtained in example 1 2 were diluted with the same amount of water and demineralized by causing the broth to flow down at the flow rate (SV = 3) in a tower (diameter 5 cm) filled with the mixture of 300ml of Amberlite (Registered Trade Mark) 200 (H type) and 600 ml of Amberlite (Registered Trade Mark) IRA-411 (OH type). The mixture of the salts (35 g of potassium primary phosphate and 1 5 9 of magnesium sulfate (7H2O)), the amounts of which were calculated from the salts contained in the original culture medium, was dissolved in a small amount of water, and added to the demineralized broth.The resulting mixture was further neutralized with sodium hydroxide, left standing for one night and then ultrafiltered according to the method described in control 2 to yield 609 of the powdered polysaccharide (sample 16).

Control 3 The broth after ion exchange treatment in example 14 was ultrarefined without the addition of salts to yield 56g 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, as follows: Table 4 content of cation (ppm, equivalent of cation/Kg) Sample molecular crude total degree of No. weight protein ash Na K Mg Ca equivalent substitution x104 % 14 104 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 - 0.29 0.40 0.36 0.21 1.26 0.95 16 89 0.9 5,61d 16,350 6,700 920 0.24 0.42 0.55 0.046 1.26 0.95 17 91 0.9 1.2 670 70 310 3,190 0.029 0.002 0.025 0.16 0.216 0.16 Example 15 600 ml of the precultured broth with Bacillus polymyxa No. 271 for 24 hours were added to 121 (pH 7.0) of the culture medium containing 4% crystal glucose, 0.5% peptone, 0.5% potassium primary phosphate, 0.5% calcium carbonate, 0.1 % magnesium sulfate (7H20), 6ppm manganese sulfate and 0. 1% defoaming silicone. The cultivation was carried out at the temperature of 28"C with aeration of 0.8VVM 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 70-80"C. The cells of the microorganism were filtered and removed from the broth with a filter paper precoated with diatomaceous earth. 40 1 of the filtered broth were treated with the ultrafilter (UF-SWM-85V-1, membrane module Abcor SWM-85V made by Bioengineering Co., LTD), purified to W/Wo = 5 (ratio of the amount of added water to that of the original broth), concentrated and freeze dried to yield 50g of powdered polysaccharide (sample 18).

Examples 16-20 6 Samples of 40 1 of the filtered broth were divided from the broth obtained in example 1 5.

Each sample was diluted with the same amount of water, and demineralized by flowing the broth down at the flow rate (SV = 3) in a tower (5cm diameter) filled with the mixture of 300 ml of Amberlite (Registered Trade Mark) 200 (H type) and 600 ml of Amberlite (Registered Trade Mark) IRA-411 (OH type). One of the 6 samples is sample 1 9 and each of the other 5 samples was supplemented with 11 of a 10% aqueous solution of NaCI, KCI, MgCl2, CaCI2 and NH4CI, respectively. Each sample was left standing for one night and ultrafiltered according to the method of example 1 to yield 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 ion-exchange resin as in example 1 6. The resulting mixture was left standing for one night with occasional stirring. The precipitate formed was collected with a centrifuge washed twice with demineralized water and dried under vacuum to yield a ferric salt of the polysaccharide (sample 25).

Example 22 1 I of a 10% aqueous solution of aluminium chloride was added to the broth after treatment with an ion-exchange resin as in example 1 6. The pH value was adjusted to 4.5. The resulting mixture was left standing for one night with occasional stirring. The precipitate produced was treated as in example 21 to yield an aluminium salt of the polysaccharide (sample 26).

The molecular weight, content of cation, total equivalent of cation and degree of substitution of samples 18-26 are shown in Table 5, as follows: Table 5 total sample molecular content of cation (ppm equivalent/Kg) equivalent degree of No. treatment weight Na K Mg Ca others of cation substitution 18 ultrafil- x 104 5,860 13,000 3,720 2,850 tration 89 0.25 0.33 0.31 0.14 1.03 0.78 19 deminer- 660 85 250 1,750 alization 59 0.03 0.002 0.02 0.09 0.14 0.10 20 substituted 19.500 210 70 380 with Na 63 0.85 0.005 0.005 0.02 0.88 0.66 21 substituted 1,150 23,400 70 280 with K 54 0.05 0.60 0.005 0.01 0.67 0.50 22 substituted 78 130 13.700 200 with Mg 68 0.003 0.003 1.13 0.01 1.15 0.86 23 substituted .250 110 130 19,500 with Ca - 0.01 0.003 0.01 0.98 1.00 0.75 24 substituted 1.090 75 32 280 NH4 : 18,600 with NH4 70 0.05 0.002 0.02 0.01 1.03 1.09 0.82 25 substituted 93 120 50 75 Fe 24,300 with Fe" - 0.004 0.003 0.004 0.004 1.30 1.32 0.99 26 substituted 25 70 3 25 Al 11,600 with Al - 0.001 0.002 - 0.001 1.29 1.29 0.97 Experiment 1 Seven groups, each group consisting of five 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. upon the activity in depressing the cholesterol level. Seven test groups were established. They are the standard diet group (with no cholesterol), the control diet group (with cholesterol) and the 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 (below).

The rats were freely fed with the diet and water. After the test, the abdomens of the rats were 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 by 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 by HDL-sterozyme made by FUJI ZOKI SEIYAKU CO., LTD.

The results are shown in Table 7 (below).

Table 6. The composition of the diet (% by weight) standard control test composition diet diet diet casein 22 22 22 salt mixture(1) 4 4 4 vitamin mixture2) (water soluble) 0.85 0.85 0.85 soybean oi1(3, (mixed with oil-soluble vitamins) 1 1 1 choline 0.15 0.15 0.15 lard 10 10 10 cholesterol 0 0.5 0.5 sodium salt of colic acid 0 0.25 0.25 B.p. 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 vitamins A(30001U), D(3001U) and E (100mg)/Kg diet.

(4) the bracketed value shows the addition of 3% B.p. to the diet.

Harper's salt mixture: CaCO3, 29.29; CAPO4. 2H2O, 0.43; KH2PO4, 34.31; NaCI, 25.06; MgSO4: 7H2O, 9.98 and other minor ingredients to a total of 100.

Table 7 rate of cholesterol value (mg/dl) accumulated diet atherogenic cholesterol group serum HDL liver(mg/g) index in the liver standard 91 # 16 59.6 # 16.0 3.7 # 1.2 0.6 + 0.4 diet control diet 291 # 5 31.6 # 7.0 22.2 # 0.6 8.2 # 2.0 50.4 # 4.7 test diet 5% B.p of sample 1 118 # 16 39.5 # 4.8 7.6 # 1.1 1.9 # 0.3 14.2 # 1.7 5% B.p of sample 2 129#40 38.9j8.4 10.2 + 1.9 2.4 + 1.0 16.6 + 6.4 5% B.p of sample 3 165 i 41 34.5 + 4.7 9.6 + 2.5 3.8 + 1.2 15.9 i 4.7 5% B.p of sample 4 190 # 37 40.1 # 9.7 12.9 # 0.9 4.2 # 2.6 20.6 # 6.0 5% B.p of sample 5 243 + 35 30.5 + 4.8 20.6 + 1.7 7.0 # 2.0 35.4 + 5.3 (1) liver cholesterol and liver cholesterol control diet group - standard diet group X 100 cholesterol intake As clearly shown in Table 7, the B.p. having a molecular weight of more than 200,000 (sample 1-4) has the activity of remarkably depressing the cholesterol level in serum and the liver.

The appearance of the liver of rats bred with a standard diet was reddish brown and the liver of rats bred with the control diet was yellowish brown. This was clearly the characteristic colour of fatty liver. On the other hand, the liver of rats bred with B.p. of 1-4 had a healthy reddishbrown color. The liver of rats bred with the control, sample 5, was yellowish brown similarly to that of the rats bred with the control diet. This was clearly the characteristic colour of fatty liver.

Experiment 2 The test was carried out on rats bred with the samples 6-8 shown in Table 2 to prove the activity of depressing the cholesterol level. 8 diet groups were established in this experiment.

The composition of the diet method of the experiment was the same as in experiment 1.

The results are shown in Table 8, as follows: Table 8 rate of cholesterol value(mg/dl) accumulated diet atherogenic cholesterol group serum HDL liver(mg/g) index in the liver standard diet 96 # 13 61.8 # 4.5 2.9 # 0.2 0.6 # 0.2 control diet 323 + 19 36.3 i 1.2 22.4 # 1.0 8.3 # 0.4 52.4 -+ 3.8 test diet 3% B.p of sample 6 213 # 11 32.1 # 1.5 11.9 # 0.6 5.7 # 0.5 19.8 # 2.5 5% B.p of sample 6 150 # 11 39.7 # 2.1 9.5 # 0.5 2.9 # 0.5 14.6 # 1.4 3% B.p of sample 7 138 # 11 34.9 # 1.2 7.9 # 0.5 3.0 # 0.3 9.9 # 1.1 5% B.p of sample 7 90 # 3 45.0 # 2.4 5.4 # 0.6 1.0 # 0.1 3.8 # 1.3 3% B.p of sample 8 201 # 8 35.8 # 2.2 11.3 # 0.8 4.5 # 0.3 18.8 # 2.0 5% B.p of sample 8 155 # 14 37.1 # 1.9 9.6 # 0.3 3.0 # 0.5 14.7 # 1.5 As appears from Table 8, the B.p. of this invention exhibits the activity of remarkably depressing rises in the serum and liver cholesterol level, and of lowering the atherogenic index when 3% or 5% of B.p. is added to the diet. These activities were especially evident in the B.p.

(sample 7) which had been treated by ultrafiltration. Sample 8 having the highest molecular weight of 1,360,000 had nearly the same activities as those of sample 6 having a molecular weight of 1.040.000. It seems to be true that the activities of B.p. will not increase significantly at the level of the molecular weight of about 1,000,000. The liver of the rats bred with the sample of the invention had a reddish brown color in the same way as those of rats on the standard diet.

Experiment 3 Animal tests were carried out with the samples given in table 3 to prove the relationship of the degree of substitution to the activity in depressing the cholesterol level. 5% of the sample was added to the diet. The detail of the method of the test was the same as in experiment 1.

The results are shown in Table 9, as follows: Table 9 rate of cholesterol value(mg/dl) accumulated diet atherogenic cholesterol group serum HDL liver(mg/g) index in the liver standard diet 88 # 5 50.9 # 4.2 3.2 # 0.3 0.7 # 0.1 - control diet 251 + 26 28.9 + 2.7 20.6 + 0.5 8.2 # 1.8 49.8 + 2.2 test diet 5% B.p of sample 9 106 # 6 49.9 # 6.0 7.7 # 1.1 1.3 # 0.3 8.6 # 2.9 5% B.p of sample 10 116 # 16 41.0 # 6.1 7.4 # 1.4 2.2 # 0.7 7.8 # 4.2 5% B.p of sample 11 291 # 47 31.6 # 3.2 23.4 # 0.5 8.4 # 1.3 51.4 # 1.8 5% B.p of sample 12 105 # 11 32.0+4.2 8.3+1.6 2.3+0.7 7.5+3.1 5% B.p of sample 13 154 # 21 28.1 # 2.7 9.8 # 1.6 4.8 # 1.1 12.9 # 4.0 As clearly shown in table 9, samples 9, 10, 12 and 13, each having a degree of substitution of more than 0.2, i.e., degrees of 0.73, 0.84, 0.47 and 0.25, respectively, demonstrated a remarkable activity in depressing the cholesterol level in serum and the liver.

The B.p. (sample 11) having a degree of substitution of 0.039 had a similar effect to that of the control diet group.

Samples 12 and 13 which are supplemented with salts after demineralization had a fairly good activity similar to that of samples 9 and 10. The existence of the combined cation with B.p. is the essential factor for the exhibition of the activity in depressing the cholesterol level.

The mixing only of cations with B.p. did not display such activity (see table 6 showing 4% of Harper's salt mixture added to the control and test diet groups). These groups do not display any activity.

In observations on the liver of test animals, the liver of rats bred with the standard diet and with the test diet using 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 the B.p. of the invention was far superior to that of the rats bred with the control diet, as described in experiments 1 and 2.

Experiment 4 Animal tests were carried out on rats bred with the samples 14-17 shown in table 4 prepared in examples 12-14 and control 3. 10 diet groups, which were the standard diet, the control diet and 8 test diet groups (with cholesterol and samples 14-17 in Table 4 at the 3% and 5% level, respectively), were established. The composition of the diet and the method of the test were the same as experiment 1.

The results of the test are shown in table 10, as follows: Table 10 rate of cholesterol value(mg/dl) accumulated diet atherogenic cholesterol group serum HDL liver(mg/g) index in the liver standard diet 96 # 13 61.8 # 4.5 2.9 # 0.2 0.6 # 0.2 - control diet 323 # 19 36.3 # 1.2 22.4 # 1.0 8.3 # 0.4 52.4 # 3.8 test diet 3% B.p of sample 14 213 # 11 32.1 # 1.6 11.9 # 0.6 5.7 # 0.5 19.8 # 2.5 5% B.p of sample 14 150 # 11 39.7 # 2.1 9.5 # 0.5 2.9 # 0.5 14.6 # 1.4 3% B.p of sample 15 138 # 11 34.9 # 1.2 7.9 # 0.5 3.0 # 0.3 9.9 # 1.1 5% B.p of sample 15 90 # 3 45.0 # 2.4 5.4 # 0.6 1.0 # 0.1 3.8 # 1.3 3% B.p of sample 16 127 # 16 42.2 # 1.0 6.6 # 0.4 2.0 # 0.4 6.5 # 1.1 5% B.p of sample 16 91.8 45.1 # 4.3 5.3 # 0.4 1.1 # 0.3 2.6 # 1.2 3% B.p of sample 17 251 # 17 33.9 # 1.2 15.6 # 1.6 6.4 # 0.4 31.3 # 3.4 5% B.p of sample 17 227 # 12 32.4 # 1.6 15.5 # 1.3 6.1 # 0.4 29.1 # 3.2 As clearly shown in Table 10, samples 1 4, 1 5 and 16 were superior to the control, and sample 17 having a substitution degree of 0.1 6 had decreased activity. Sample 16, with a substitution degree of 0.95, prepared by the addition of salts to demineralized B.p. and by ultrafiltration, had nearly the same effect as sample 15, with a substitution degree of 0.95 prepared by ultrafiltration without demineralization. The activity of depressing the cholesterol level was fully restored by the addition of salts.

The colour and the appearance of the liver of rats bred with the 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 the samples 18-25 (table 5) obtained by the examples 15-22 to prove the activities of the salts of Bp. 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. The results of the test are shown in Table 11, as follows: Table 11 rate of cholesterol value(mg/dl) accumulated diet atherogenic cholesterol group serum HDL liver(mg/g) index in the liver standard diet 92 # 14 43.3 # 7.3 2.6 # 0.3 1.2 # 0.5 control diet 284 # 31 31.8 # 5.4 17.7 # 0.6 8.1 # 1.8 44.1 # 3.1 test diet ultrafiltered B.p of sample 18 114 # 18 39.1 # 13.9 7.3 # 1.1 1.9 # 0.3 12.2 # 2.4 sample 19 Na salt 106 # 13 34.6 # 6.5 7.8 # 1.0 1.8 # 0.6 12.8 # 3.7 sample 20 K salt 92 # 25 40.7 + 12.7 6.5 + 2.2 1.6 + 1.4 8.5 + 5.3 sample 21 Mg salt 88 + 35 50.3 + 13.2 6.2 + 1.8 1.1 + 1.7 7.7 + 5.0 sample 22 Ca salt 85 # 24 47.8 # 11.7 5.8 # 1.6 0.9 # 0.9 6.1 # 3.9 sample 23 NH4salt 72 # 17 46.9 # 4.9 4.6 # 1.8 0.5 # 0.2 3.9 # 5.0 sample 24 Fe salt 102 # 17 37.0 # 0.1 7.7 # 1.3 1.8 # 0.7 12.1 # 3.3 sample 25 Al salt 110 # 17 42.5 # 17.0 6.5 # 1.9 1.9 # 1.1 9.6 # 5.6 As shown in table 11, the diet with the salts of B.p. with combined cations showed the remarkable effect in depressing the serum cholesterol level, liver cholesterol level, atherogenic index and the rate of accumulated cholesterol in the liver as compared with the control diet. This shows that the combined cation in the salt of B.p. has a strong activity in depressing cholesterol levels. The ammonium, calcium and magnesium salts of B.p. had specially high activities in depressing the atherogenic index, which is closely connected with an outbreak of arteriosclerosis. The index showed the same or a lower value 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 colour and the appearance of the liver were far superior to that of the control as described in the foregoing experiments.

Claims

1. A microbiological process for producing a polysaccharide having a molecular weight of more than 200,000 and an activity in depressing rises in serum and liver cholesterol and in the atherogenic index, characterised in that Bacillus polymyxa No. 271 (FERM-P No. 1 824) is cultured in an aqueous culture medium containing assimilable saccharides until said polysaccharide accumulates in said medium, and in that said accumulated polysaccharide is recovered from said medium.

2. A process as set forth in claim 1, characterised in that said Bacillus polymyxa No. 271 is cultured in said medium for more than 20 hours.

3. A process as set forth in claim 1 or claim 2, characterised in that said accumulated polysaccharide in the medium is subject to ultrafiltration before recovering said accumulated polysaccharide.

4. A process of producing a cationic salt of a polysaccharide having a degree of substitution with the cation of more than 0.2 and an activity in depressing rises in the serum and liver cholesterol and in the atherogenic index, which process characterised in that Bacillus polymyxa No. 271 (FERM-P No. 1824) is cultured in an aqueous culture medium containing assimilable saccharides and the cation until said cationic salt of said polysaccharide accumulates in said medium, and in that said cationic salt of said polysaccharide is recovered from said medium.

5. A process as set forth in claim 4, characterised in that said cation is at least one member selected from alkali metals, alkaline earth metals, transition metals, manganese, aluminium, zinc, copper and ammonium ions.

6. A process as set forth in claim 4 or claim 5, characterised in that said culture medium contains a total cation equivalent of at least 0.005 per 1 of the medium, and said Bacillus polymyxa No. 271 is cultured in said culture medium at a pH value of more than 4.5.

7. A process as set forth in any of claims 4 to 6, characterised in that salts are added to the fermented broth before recovery of said salt of said polysaccharide.

8. A process as set forth in any of claims 4 to 7, characterised in that said accumulated salt of po!ysaccharide is subject to ultrafiltration before recovery of said salt of said polysaccharide.

9. A process as set forth in any of claims 4 to 8, characterised in that said accumulated salt of said polysaccharide is recovered from the medium by adding alcohol to the fermented medium as a precipitant.

10. A process as set forth in claim 1 substantially as herein described in any one of the specific examples.

11. A process as set forth in claim 4 substantially as herein described with reference to any one of the specific examples.

1 2. A polysaccharide with a molecular weight above 200,000 when made by a process as claimed in any of claims 1 to 3 and 10.

1 3 A cationic salt of a polysaccharide with a degree of substitution by the cation above 0.2 when made by a process as claimed in any of claims 4 to 9 and 11.

14. A polysaccharide as claimed in claim 1 2 for use in lowering serum and liver cholesterol levels and the atherogenic index.

1 5. A cationic salt of a polysaccharide as claimed in claim 1 3 for use in lowering serum and liver cholesterol levels and the atherogenic index.

16. A pharmaceutical composition comprising a polysaccharide as claimed in claim 1 2 or salt as claimed in claim 13, together with a pharmaceutically acceptable currier or diluent thereof.