GB1582068A - Powder composition comprising viable bifidobacteria cells containing powder and lactulose containing powder - Google Patents

Description

(54) POWDER COMPOSITION COMPRISING VIABLE BIFIDOBACTERIA CELLS CONTAINING POWDER AND LACTULOSE CONTAINING POWDER (71) We, MORINAGA MILK INDUSTRY CO., LTD., a Japanese body corporate of 33-1, Shiba 5-chome, Minato-ku, Tokyo, Japan, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed to be particularly described in and by the following statement: This invention generally relates to a powdery composition comprising dried viable cell mass of the genus Bifidobacterium (hereinafter described as Bifidobacteria cells) to be orally administered for initiation or conservation of favorable state in intestinal flora.

More particuarly this invention relates to a powdery composition comprising viable Bifidobacteria cells containing powder and lactulose containing powder which composition may keep high survival rate for a long period of storage.

It has been well known that Bifidobacterium is normally found in the human intestine, and is one of useful micro-organisms.

Also it has been known that oral administration of viable Bifidobacteria cells is effective for preventing and treating bowel complaints. particularly diarrhea, intestinal catarrh, dyspepsia, constipation and intestinal microbial substitution (microbisme substitute') after treatment of antibiotics. For such purposes, a variety of powdery composition products containing dried viable Bifidobacteria cells have been sold in the market.

In the conventional powdery compositions, however, the survival rate of Bifidobacteria cells was unsatisfactorily low, and it has been a basic problem to improve the cell survival rate and to preserve a viable count of Bifidobacteria cells at a certain level for a desired long period of storage.

For instance, numbers of viable Bifidobacteria cells in 1 gram of the powdery products supplied by 12 companies in Japan and of the powdered milk containing Bifidobacteria cells and small portion of lactulose supplied by a certain German company is generally as low as 5 or 6x107 or so. Moreover, the viable count of the cells in these products rapidly decreases during storage.

Under such circumstances. many attempts for improving the shelf life of products containing Bifidobacteria cells have been made.

Ohta reported his research note entitled "Research for freezing and freeze drying Lactobacillus bifidus" in Japanese journal "Ochanomizu Igaku Zasshi (Ochanomizu Medical Journal)" Vol. 7. No. 11 (195C)). pp. 2967-2975, concerning viability of freeze dried Bifidobacteria cells which were prepared with varying drying methods and suspending agents.

In this research report, he reported that sodium glutamate as a suspending medium for freeze drying of Bifidobacteria cells may give a better cell survival rate when freeze dried Bifidobacteria cells were stored at 37"C. and skim milk. soluble starch and sucrose followed thereafter. And the best results were observed when sodium glutamate in 3% (by weight) concentration was used. but the survival rate in this best case decreased to 1% after only 2 months storage Smiha reported in the "Journal of Food Science'' Vol. 39 (1974) pp. 641-642 that 55C/c of survival rate after 2 months storage at 3() C was obtained when Lactobacillus bifidus (old scientific nomenclature of genus Bifidobacterium) cells were subjected to freeze drying using suspending agent of skim milli added ascorbic acid, thiourea and ammonium chloride.

This value is somewhat higher but the product prepared by this method is not preferable for the usual distributing systems since the viable count of Lactobacillus bifidus in this product is considered to rapidly decrease during prolonged storage period of more than 2 months.

As mentioned in the above, decrease of survival rate during storage largely impairs commercial value of such powdery products, since cell density of Bifidobacteria cells decreased during storage even if the products initially have a high cell density.

Consequently, in the conventional products containing viable Bifidobacteria cells, lactose, and starch have been added as suspending medium for protection of the cells.

Nevertheless, the conventional powdery compositions containing known suspending mediums do not achieve sufficient viability. Such suspending mediums, therefore, have not actually function of protection of the cells, and rather function only for diluting freeze dried Bifidobacteria cells. In fact, cell density of viable Bifidobacteria cells in the powdery products presently distributed in the market is very low.

There has not been known a powdery product containing dried Bifidobacteria cells which is capable of keeping a high cell density of viable cells during prolonged storage period.

Therefore, it is an object of the present invention to provide a powdery composition containing dried viable Bifidobacteria cells in a high cell density which is capable of a higher cell survival rate during a long period of storage.

It is another object of the invention to provide a powdery composition containing dried viable Bifidobacteria cells which is capable of establishing Bifidobacterium flora in intestinal tract when it is orally administered.

In accordance with this invention, these objects mentioned in the above are accomplished by homogeneously admixing viable Bifidobacteria cells containing powder with lactulose containing powder.

In other words. this invention is predicated upon the discovery that admixing specific quantity of lactulose containing powder. having major part of lactulose, as a suspending agent with viable Bifidobacteria cells containing powder had a surprising effect for improving the cell survival rate.

The powder containing Bifidobacteria cells used in this invention can comprise dried Bifidobacteria cells and suspending agent such as powdered skim milk, sodium glutamate, gelatine or lactose and includes at least 20x1010 of viable Bifidobacteria cells in 1 gram thereof. Such a powder may- be prepared bv conventional methods.

The lactulose containing powder used in this invention can include at least 55% by weight of lactulose in total solid content thereof and slight moisture content. The remaining solid content consists essentially of lactose and lactose which are residues in the reaction mixture from lactulose preparation.

It is necessary that the final powdery composition product in accordance with this invention which may be prepared by homogeneously admixing 40 - 70% by weight of the viable Bifidobacteria cells containing powder with 60 - 30% by weight of the lactulose containing powder contains at least 8x1010 of viable Bifidobacteria cells in I gram of the final product. 28 - 57'Sf bv weight of lactose and less than 2.5c by weight of moisture content in the final product.

The powdery composition in accordance with this invention has a largely improved cell survival rate of the Bifidobacteria cells after relatively long period of storage and prevents decrease in the cell survival rate if the storage period is prolonged.

The microbial strain of Bifidobacteria cells used in this invention may be anv known strains of species belonging to the genus Bifidobacteria. but it is preferable to use one or more of Bifidobacterium adoleseentis (ATCC 15703 and others). Bifidobacterium longum (ATCC 15707 and others). Bifidobacterium bifidum (ATCC 11146 and others) all of which can be normally found in the human intestinal tracts irrespective of age.

In accordance with the present discovery. the powdery composition of this invention includes viable Bifidobacteria cells and lactulose both in high densities.

The powdery composition of this invention is obtainable by homogeneously admixing viable Bifidobacteria cells containing powder with lactulose containing powder.

The lactulose containing powder used for preparing the powdery composition of this invention is obtained by an'. known methods. for example, by drying lactulose syrup.

In the Japanese Patent No. 874.954 (Japanese Patent Publication No. 29X4/l977).

Nagasawa et a hive disclosed tillt a lictulose syrup containing lactulose above 8()C/c by weight in total solid content can be produced by admixing -in aqueous solution of lactose with 3 to 10% by weight of aqueous solution of sodium hydroxide containing from ().27 to ().4 C? of sodium hydroxide based on the weight of lactose. heating said mixed solution to a temperature of from 7 C to I 3t)C to isomerize the lactose so as to minimize formation of products such as galactose and the other substances. and thereafter concentrating the reaction liquid to separate lactose from the reaction liquid (see U.S. Pat. No. 3 16174.

Belg. Pat. No. 783690. French Pat. No. 72 18213. Dutch Pat. No. 150513 and German Pat.

No. 2224680).

A process for preparin. a lactulose powder from lactulose syrup is disclosed in the Japanese Patent No. 778,564 (Japanese Patent Publication No. 49-44331(See U.S. Pat. No.

3716408, Belg. Pat. No. 774451, French Pat. No. 71/38472, German Pat. No. 2148159, Dutch Pat. No. 147784 and U.K. Pat. No. 1318494). In accordance with the process, a lactulose powder containing more than 55% by weight of lactulose in solid content can be obtained by mixing an aqueous solution have a pH of less than 7.0 and containing above 60% by weight of lactulose in total solid content with an aqueous solution having dissolved, more than 0.3%, based on the weight of said lactulose, of KONNYAKU powder, and drying the resulting mixture by conventional hot air spray drying apparatus. (The KONNYAKU powder is derived from the corm of a perennial herb, "Amorphophalus konjac" belonging to the Araceae family and is mostly carbohydrate composed of mannan.) In the Japanese Patent Publication No. 52-21063, Nagasawa et al have also disclosed that a highly pure lactulose powder can be obtained by cooling lactulose syrup between -20 C and -45 C in which range the syrup is not freezed, and subjecting it to freeze drying to produce condensed syrup, and then heat-drying the condensed syrup untill a dried porous mass is obtained, and finally pulverizing the porous mass into powder under dehumidified atmosphere.

These methods are suitable for obtaining lactulose powder including lactulose more than 55% by weight. Nowadays it has come to be possible to industrially produce highly pure lactulose powder reaching to 96%, and in this invention it is desirable to use highly pure lactulose containing powder to the utmost so as to keep a higher cell survival rate of the Bifidobacteria cells in the final product.

As will be described later, the moisture content of the final product is desirable to be less than 2.5% by weight, and for this reason the moisture content in the lactulose powder is desirable to be kept as low as possible, and preferably be less than 0.7%.

The Bifidobacterium used in this invention may be any known strains of species belonging to the genus Bifidobacterium (see Bergey's Manual of Determinative Bacteriology, Eighth Edition, edited by R.E. Buchanan & N.E. Gibbones, pp. 669- 676, 1974, The Williams & Wilkins Co., Baltimore, U.S.A.:), but it is preferable to use one or more of B. adolescentis (ATCC 15703 and others). B. longum (ATCC 15707 and others) and B. bifidum (ATCC 11146 and others) all of which can be normally found in the human intestinal tracts irrespective of age.

These strains are listed in the catalogue of Strains, Eleventh Edition (1974), The American Type Culture Collection, and is believed to be obtainable therefrom.

Any known fermentation methods and culture media for preparation of Bifidobacteria cells may be utilized, but the culture medium disclosed by Nagasawa et al in the Japanese Early Opened Patent Application Publication No. 50-89587 is suitable, since microbial strains of genus Bifidobacterium may grow well in the culture medium and it is cheap in cost. The culture medium contains corn steep liquor and fish liver as nitrogen sources; and lactose, inorganic salts and cystine are added to form a desirable basic composition thereof; and extracts from rice bran or germs of various kinds of cereals are further added thereto.

The H-culture medium bv Yuka Hara et al which contains meat extract, yeast extract, KH2PO4, K2HPO4, CH3COONa. lactose and cystine and having a pH of 6.8 is also preferable. since this is easily prepared (see Journal of Japanese Society of Food and Nutrition, Vol. 18, No. 1 (1965). p. 1)).

After proliferation. Bifidobacteria cells may be collected bv centrifuging the culture medium. The collected cells are then suspended into sterilized isotonic sodium chloride solution of the same quantity as the culture medium. and are washed away the culture medium and metabolites and the like which are adhered to the surfaces of the cells. and are collected again by centrifuging the solution.

Normally about 6 - 10 grams of wet cells are obtained from 1 liter of the culture medium. and these wet cells contain 6(1 - S()xl()' of Bifidobacteria cells per 1 gram thereof. The wet cells are then suspended into the sterilized aqueous solution. in the ratio of hO grams of wet cells per 1 liter of the solution, containing 104 grams of suspending agent which consists of about 38% by weight of powdered skim milk, about 9% by weight of sodium glutamate, about 4% by weight of gelatine, about 49% by weight of sucrose as the suspending agent, thereafter the solution is freeze dried and pulverized into powder according to conventional methods. The thus obtained dried Bifidobacteria cells containing powder has composition of about 74% by weight of the suspending agent, about 22% of dried cells, 4% or less of moisture content, and at least 20x1010 of viable Bifidobacteria cells in 1 gram of the powder.

At least 2()xl()l" of the viable counts in 1 gram of the dried Bifidobacteria cells containing powder is necessarv for obtaining desirable high cell density of Bifi-dobacteri- cells in the final product of the powdery composition. The moisture content in the dried Bifidobacteria cells containing powder is normally about 2.0% - 4.0% by weight, but is desirable to be as low as possible for increasing cell survival rate in the composition.

The thus obtained dried Bifidobacteria cells containing powder and lactulose containing powder are admixed to a ratio of 40 - 70% and 60 - 30% by weight respectively by means of a V-type mixer under low humidity atmosphere. The thus obtained powder composition contains about 28 - 57% by weight of lactulose and 2.5% by weight or less of moisture content, and at least 8x101 of viable Bifidobacteria cells are contained in 1 gram of the powdery composition. It is necessary to keep the powdery composition at cool and dark place, since the composition has a high hygroscopicity. The powdery composition may be aritightly packed or filled in capsules and then airtightly packed for distribution. Proper dose of the powdery composition of this invention is 1 - 1.5 grams for infants, 1.5 - 3 grams for children and 3 - 6 grams for adults in a day.

As previously mentioned, the cell survival rate of Bifidobacteria cells in the powdery composition of the present invention is highly increased by admixing lactulose containing powder with the powdery composition may contain viable Bifidobacteria cells in such a high cell density as about 10 times of that in the conventional products which are diluted with dispersing medium.

When the ratio of lactulose content to Bifidobacteria cells content is reduced in the powdery composition, the cell survival rate is decreased, and it has been noticed that lactulose content is desirable to be above 28% by weight of final product. It has been also found that when the ratio is increased too much, the cell survival rate is slightly decreased and moreover hygroscopicity of the powdery composition is increased by the lactulose powder which in turn results a tendency of agglomeration of the powdery composition. As will be seen from the examples hereinafter described, it has been found that lactulose content in the powdery composition is desirable to be less than 57% by weight. When the moisture content in the powdery composition is increased above 2.8% by weight, the cell survival rate is rapidly decreased, and therefore it is practically important to keep the moisture content 2.5% by weight or less.

Now it will be understood that it is necessary, for preparing the powdery composition of this invention, to prepare high cell density Bifidobacteria cells containing powder and to prepare high concentration lactulose containing powder.

It will come to be clear from the examples and the results of the comparative tests that the inventor of the present application has provided a powdery composition which contains viable Bifidobacteria cells in a high cell density, and show excellent cell survival rate due to the admixing of lactulose containing powder, and is effective for establishment of favorable state in intestinal flora as compared to the comparative samples wherein lactulose containing powder is substituted with the conventional dispersing medium (such as starch and lactose).

Sample 1 Lactulose containing powder was prepared by the same manner as shown in the example 1 of said Japanese Patent No. 778564. That is, 3 Kg. of lactulose aqueous solution of 6.5 in pH comprising 53.8% of lactulose. 8.26; ofgalactose. 6.3% of lactose, 31.1C/c of water and 0.6% of others (all by weight) was prepared. To 1.5 liter of water. 7.8 g (0.5G/c based on the lactulose content of the aqueous solution) of commercially available Konnyaku powder (milled powder from Fukushima Prefecture, Japan) were added under stirring to be uniformly swollen and filtered using a 100 mesh filter cloth to remove insolubles. The filtrate was added to said 3 Kg. of lactulose aqueous solution and then it was mixed well.

This mixed solution was 140 cp in viscosity at 200C (measured by means of a B-type viscometer manufactured by Tokyo Keiko Co., Ltd. in accordance with the conventional method). The mixed solution was heated at 45 C and then dried under the conditions of 1700C in hot air inlet temperature. 9() C in outlet temperature and 9,001) r.p.m. in rotation velocity of atomizer by means of a spray dryer (manufactured by Anhydro Co.). One and three-quarter Kg. of white powder was obtained. The powder contained 78.2% by weight of lactulose content, 0.51% by weight of moisture content and was free flowable and easily soluble in water.

Bifidobacteria cells containing powder was prepared by the method (hereinafter referred to method A) as described hereunder.

Culture medium was prepared in such a manner that 3.0'" of fish liver, 2.OC/r of corn steep liquor. 2.0% of lactose. 0.1% of KH.PO4 .1C/ of K2HP4, 0.5% of CH3COONa and 0.04cue of 1-cvstine (all bv weight) was dissolved in 10 liter of water, and then pH of the aqueous solution was adjusted to 6.8 by adding IN of aqueous solution of sodium hydroxide, and thereafter the solution was sterilized bv means of an autoclave at 121"C for 15 minutes and then cooled. Bulk starter culture of Bifidobacterium longum (ATCC 15707) was added to the culture medium to a concentration of 4'/c and mixed uniformly by stirring.

Then fermentation was carried out anaerobically in Jar Fermentor by gaseous carbon dioxide at 37 C for 12 hours. During this fermentation, about 20% by weight of aqueous solution of CaC()) was added to the culture medium for adjusting its pH to about 7.

The culture medium was then cooled to 5"C and centrifuged (10,000 r.p.m.) to collect the Bifidobacteria cells suspended therein by means of a cooling high speed continuous centrifuge, thereafter the thus collected cells were resuspended into 1000 ml of sterilized and cooled (5"C) isotonic sodium chloride solution to wash away impurities, and then said resuspended solution was centrifuged (10.000 r.p.m.) to collect the washed bacterial cells.

Sixty g of the thus obtained wet Bifidobacteria cells were resuspended into 1 liter of sterilized aqueous solution of suspending agent comprising 40 g of sucrose, 15 g of mucin, 5 g of gelatine, 15 g of glutamic acid and 15 g of aspartic acid (aminosuccinic acid), and then freeze dried in accordance with conventional method to obtain 96 g of Bifidobacteria cells powder containing 23.0% of the cells content, 74.7% of suspending agent content and 2.4% of moisture content (all by weight). The viable count of the Bifidobacteria cells in 1 gram of the powder was 36x10"'.

Finally 90 g of said lactulose containing powder and 90 g of said Bifidobacteria cells containing powder was homogeneously admixed by means of V-type mixer (by Tokuju Seisakusho Co., Ltd.) in a room kept at low humidity, thereafter about 180 g of the powdery composition was filled in a red bottle and after airtightly sealed the bottle was stored at a cool, dark, and low humidity place. Thus obtained powder composition contained 39% by weight of lactulose content and 1.46% by weight of moisture content, and the viable count of the Bifidobacteria cells therein was 18x10i(1/g, Example 2 In accordance with the method described in the example of the Japanese Patent Publication No. 52-21063, lactulose containing powder was prepared as follows: Five hundred g of lactulose syrup composed of 56% of lactulose, 7.0% of galactose, 4.00/cm of lactose, 1.0% of others and 32% of water (all by weight) was poured at a depth of 5 mm in a pan in a shelved lyophilizer, and freezed drying was started at -40 C, under vacuum of 1 mmHg; and about 2 hours later the temperature in the lyophilizer was adjusted to -30"C; and then gradually rised to 80"C spending 4 hours therefor, while the vacuum was gradually decreased to 30 mmHg. During this time, the syrup gradually bubbled up to a depth of 20 cm and became a uniform honeycomb like foamy mass. Spending two hours, the temperature was gradually adjusted to 350C; and then kept the temperature for 16 hours and 340 g of a dried honeycomb like mass was obtained. During this time the vacuum was changed from 6x10-2 mmHg to 'x10- mmHg. The dried foamy mass was pulverized into powder in a dehumidified chamber. The obtained powder was white and sufficiently free flowable and contained 80.8cue by weight of lactulose and ().5C/c by weight of moisture.

Bifidobacteria cells containing powder were separately prepared by the same method as in the example 1 except that Bifidobacterium adolescentis (ATCC 15705) was used (hereinafter referred to method B). Ninety two grams of the Bifidobacteria cells containing powder was obtained. The viable count of the cells was 287x109/g and contained 3.0% by weight of moisture 74.69 by weight of suspending agent and 22,4 C/c by weight of the Bifidobacteria cells.

Forty grams of the previously obtained lactulose containing powder and 60 g of the obtained Bifidobacteria cells containing powder were homogeneously admixed in the same way mentioned in the example 1, and about 100 g of powdery composition was obtained.

The viable count of the Bifidobacteria cells was 172x10''i'g. and contained 32.3% by weight of lactulose and 2.02cur by weight of moisture.

Test I A comparative test was conducted with respect to the relationship between cell survival rate and various storage period.

The samples used in the test were Bifidobacteria cells containing powdery products A, B and C which were distributed in the market by 3 companies A. B and C. a Bifidobacteria cells containing powder D as a comparitive sample in which conventional dispersing medium was used instead of lactulose containing powder in the present invention and the powdery compositions obtained in the examples 1 and 2 in the above.

The products A - C used in the test were the ones which left effective terms about 2.6 months calculated from the indications on their packages. Though it might possibly be that several months have been elapsed from their manufacturing dates when those are purchased, in this test. those were handled on the supposition that those were manufactured on the day on which those were purchased.

Each 50 g of the products A - C was weighed in a dehumidified chamber in a vial bottle and was sealed off for prohibiting it from being moistened.

The comparative sample D was prepared by admixing I part by weight of Bifidobacteria cells containing powder obtained by the method A with ().8 part by weight of starch and 0.2 part by weight of lactose as dispersing agent.

Fifty grams of the comparative sample D and the powdery compositions of examples 1 and 2 were also weighed respectively in the same bottles (capable to hold 100 ml), and then sealed off for eliminating those from being moistened.

All of these 6 samples were stored for 6 months at 200C, and further the samples of the examples 1 and 2 were stored for additional 6 months.

The viable counts of the cells in the samples were determined by a method mentioned hereunder at the times immediately after manufacturing (as to the samples A - C, immediately after purchasing), after 2 months storage, after 4 months storage and after 6 months storage for all samples, and after 12 months storage only for the samples of examples 1 and 2. Cell survival rates were calculated by using following equation.

Cell Survival rate (%) = Viable count after storage x 100 Viable count immediately after manufacturing The method for determining viable counts is as follows: Precisely weighed 1 gram of each sa

The results of this test are shown in Table 1.

TABLE 1 Viability of the cells in the samples storage immediately after after after after after samples manufacturing 2 months 4 months 6 months 12 months viable count 76x105 32x105 10x105 12x105 A survival rate (%) 100 42.1 13.2 15.8 viable count 122x105 22x105 13xI05 7xl05 B survival rate (%) 100 18.0 10.7 5.7 viable count 200x104 48xlû4 30x104 28xl04 C survival rate (%) 100 24.0 15.0 14.0 viable count 175x109 102x109 73x109 71x109 D survival rate (%) 1()() 58.3 41.7 40.6 viable count 180x109 152x109 150x1()9 147x1 & 130xl09 Ex.

1 survival rate (Yr) 100 84.4 83.3 81.7 72.2 viable count 172x109 147x109 143x109 141x109 118x109 Ex.

2 survival rate (%) 100 85.5 83.1 82.0 68.6 Note: Viable count shows the viable count of Bifidobacteria cells in 1 gram of each samples.

As will be seen from Table 1. the cell survival rates after 6 months storage in the commercially available Bifidobacteria cells containing powders (samples A - C) are extremely low. The cell survival rate of the sample D is rather good as compared with that in the samples A - C, but it is not to be compared with those in the samples of examples 1 and 2 of this invention.

Test 2 A test was carried out for the influence of the ratio of lactulose containing powder to the bacterial cells containing powder (hereinafter referred to L/B ratio) to cell survival rate.

Lactulose powder containing 95.5% by weight of lactulose in solid content, 0.6% by weight of mositure was prepared in accordance with the example 4 in the Japanese Patent No. 778564 in such a manner that 29.4 g, (0.5 based on the lactulose content) of commercially available konnyaku powder (milled powder, from Fukushima Prefecture, Japan) was added to 4.5 liters of water. while stirring. to cause uniform swelling and it was filtered using a 100 mesh filter cloth to remove insolubles, and the filtrate was added to 3 Kg. of an aqueous lactulose solution of a pH of 6.4 and having a composition of 68.0% of lactulose, 1.2% of galactose, 0.1% of lactose, 30.0% of water and 0.7% of others (all by weight), obtained by epimerization of lactose, and, thereafter, oxidizing the byproduced aldose. This lactulose containing powder was admixed with Bifidobacteria cells containing powder prepared by the method A in different L/B ratio shown in Table 2 to obtain 9 samples of powdery compositions. and a test for viability of the cells was carried out by the same method as in the Test l. Meanwhile quantities of lactulose content and moisture content in the samples were determined by Sweeleys method (Journal of the American Chemical Society, Vol. 85 (1963); p. 1497) and by conventional method respectively. The results of this test are shown in Table 2.

TABLE 2 Cell survival rates in the powdery compositions having different L/B ratios percentage percentage lactulose moisture viable counts cell of of content content content of the B. cells survival Sample lactulose B. cells in the in the in the sample rate containing containing samples samples after Nos. powder powder immediately after 6 months in the in the after 6 months storage composition composition (%) (%) manufacturing storage (%) 1 70 30 66.9 1.14 108x109 77x109 71.3 2 65 35 62.1 1.22 124x109 96x109 77.4 3 62.8 37.2 60.0 1.27 133x109 106x109 79.7 4 60 40 57.3 1.30 1.44x109 119x109 82.6 5 50 50 47.8 1.50 180x109 147x109 81.7 6 40 60 38.2 1.69 215x109 173x109 80.5 7 30 70 28.7 1.86 252x109 202x109 80.2 8 25 75 23.9 1.95 273x109 162x109 59.3 9 20 80 19.1 2.03 287x109 121x109 42.2 Note: Viable count shows the viable count of Bifidobacteria cells in 1 gram of each samples.

As will be apparently noticed from the Table 2, the samples 1-7 which contained above 28.7So of Iactulose showed higher cell survival rates after 6 months storage. When L/B ratio is increased to a certain level as in the samples 1-3, however, the powdery compositions tend to agglomerate due to the hygroscopicity of lactulose powder, though the survival rates are not so much lowered.

Meanwhile, when the L/B ratio is decreased to a certain level as in the samples 8 and 9, survival rate is lowered and the containing powder as a suspending agent is decreased.

Accordingly, it has been found that the powdery composition of this invention preferably be prepared by admixing 40%-70% by weight of freeze dried Bifidobacteria cells containing powder with 60 - 30% by weight of lactulose containing powder, and lactulose content in the powdery composition preferably be resulted within a range of 28 - 57% by weight.

Test 3 A test was carried out for influence of moisture content in the powdery composition of this invention to the survival rate.

6 samples of the powdery compositions were prepared by admixing lactulose containing powder used in the Test 2 (95.5% by weight of lactulose in solid content, 0.6% by weight of moisture content) with Bifidobacteria cells containing powder prepared by the method A but having different moisture contents.

These samples were subjected to viability test of the cell in the same method as in the Test 2. The results of this test are shown in Table 3.

TABLE 3 moisture moisture viable counts cell survival content of content of the B. cells rates after Sample B. cells in the in the sample 6 months containing composition storage Nos. powder immediately after after 6 months ( /c) (%) manufacturing storage 1 5.62 3.13 122x109 9x1 & 7.4 2 5.08 2.83 153x109 68x10" 44.4 3 4.42 2.51 182x10" 145x109 79.7 4 3.75 2.16 182x10 149x109 81.9 5 2.40 1.49 170x10 137x109 80.6 6 2.06 1.33 177x10 148x109 83.6 Note: Viable count shows the viable count of Bifidobacteria cells in I gram of each samples.

As will be clear from the Table 3, the survival rate was considerably lowered when the powdery compositions contained above 2.51cos by weight of moisture, and the survival rate was remarkably lowered when the moisture content was above 3C4 by weight.

It will be seen that the samples which had moisture content less than 2.50% shows above 80% of high survival rate after 6 months storage. Accordingly it is essential that the moisture content in the powdery composition is less than 2.50?? by weight.

Test 4 In order to examine the efficacy of the powdery composition prepared by the invention for establishment of favorable state in intensinal flora. following test was carried out using the powdery composition of the example 1 of this invention. the powdery composition of the sample D in the Test l and the composition of the sample A in the Test 1.

Six subjects who were dosed with antibiotics for treating bowel complaints were taken for the test. Ages and sexualities of the subjects are listed in Table 4. The all of their stools were observed less than 104/g of stool in viable counts of Bifidobacteria cells. As shown in Table 4, these subjects separated into 3 groups, and 2 grams of each sample was dosed to the subjects 3 times a day at fixed times for 7 consecutive days.

After 3 days and after 7 days from the beginning of treatment. stools of 6 subjects were collected and I gram of the each stool was suspended into 9 ml of sterilized isotonic sodium chloride solution. Each of these initial suspension was serially diluted into 102 to 108 times using sterilized isotonic sodium chloride solution. One-tenth ml of the dilution was placed on agar plate of H-culture medium added penicillin and agar in the ratio of 0.1 IU and 0.015 g per 1 ml of the culture medium respectively, and was spreaded evenly with L-shaped glass rod. The colonies of the Bifidobacteria cells were counted after 72 hours anaerobic incubation at 37 C by steel wool method (Azuma et al, Japanese Journal of Bacteriology, Vol. 17 (1962), pp. 802-806, This method is a modification of Parker's method described in Australian Journal of Experimental Biology, Vol. 33 (1955), pp. 33-38).

The reason why penicillin added H-culture medium was used was for restrainig growth of bacteria other than genus Bifidobacterium on the agar plate. The results of this test are shown in Table 4.

TABLE 4 Effect of Bifidobacteria cells containing powder on establishnient of intestinal flora subject viable age & sex after after count of subject 3 days 7 days Nos. samples of B. cells/g * * in powdery age sex composition 1 Powdery composition 27 # 38x109 28x1010 of Example 1 180x109/g 2 of this invention 45 # 29x108 10x1010 3 Powdery composition 23 # 10x105 19x109 of sample D 175x109/g 4 in the Test 1 48 # < 105 72x108 5 Powdery composition ** 28 # < 105 < 105 of sample A 1x107/g 6 in the Test 1 23 # < 105 71x106 * Viable count of Bifidobacteria cells Viable count in the powdery composition of sample A is based on the instruction for dosage.

As will be seen from Table 4. viable counts of the Bifidobacteria cells in the stools of the subjects (No. 1 and No. 2) who were treated with the powdery composition of this invention were 38x109/g, 20x108/g only after 3 days from beginning of treatment, on the 7th day after treatment these increased to 28x10/g and 10x1010/g respectively. The powdery composition of this invention, therefore, induced the predominance of Bifidobacteria cells in the intestinal flora of the subjects. since total number of intestinal flora was around the level of 10" to 1011 in l gram of stool.

Meanwhile. in the stools of the subjects (No.5 and No.6) who were treated with the powdery composition of sample A, viable counts of Bifidobacteria cells were not increased, and in one case (No.5) among them viable count of Bifidobacteria cells was not found in the level of 10' per 1 gram of stool after 7 days from the beginning of treatment. In the other case (No.6). viable count of Bifidobacteria cells was observed in the level of 107/g on the 7th day after the beginning of treatment. but such a low viable count cannot be considered to establish favorable state in intestinal flora.

In case of subjects (No 3 and No. 4) who were treated with the powdery composition of sample D, only 19x109/g and 72x108/g of viable counts of Bifidobacteria cells were observed even on the 7th day after the beginning of treatment, though these values were rather close to these of the subjects (Cases 1 and 2) who were treated with the powdery composition of the present invention, but growing and indwelling of Bifidobacteria cells in the intestine was apparently delayed as compared to these of the above cases l and 2.

Now it will be understood that there are remarkable difference from growing and indwelling of Bifidohacteria cells in the intestine between the subjects treated with sample D and the subjects treated with the powdery composition of the present invention, even though they are administered almost same level of Bifidobacteria cells, and therefore it is clear that the powdery composition of Bifidobacteria cells containing powder and lactulose containing powder in accordance with this invention is excellently efficient for establish ment of favorable state in intestinal flora.

As previously mentioned, the powdery composition of the present invention shows excellently high cell survival rate of Bifidobacteria cells during storage and may keep the high cell survival rate for a considerably long period, and moreover it is apparent that it may have effect of establishment of favorable state in intestinal flora.

In the light of foregoing descriptions, the powdery composition of the present invention is effective for establishing favorable state in intestinal flora due to reduce saprogenic bacteria in the human intestine, and for treating contipation due to increase of viable count of Bifidobacteria cells upon administration, and also it is effective for treating portosystemic encephalopathy due to reduction of ammonium concentration in blood by restraining absorption of ammonium gas which was produced by digestion of protein in intestinal tract.

Claims (6)

WHAT WE CLAIM IS

1. A powdery composition which contains 28 - 57 percent by weight of lactulose, less than 2.5 percent by weight of moisture and at least 8x10tu of freeze dried viable cells of genus Bifidobacterium per gram of said composition.

2. A composition according to Claim 1 which comprises (a) 40-70 percent by weight of a powdered mixture of said cells and a suspending agent therefor, containing at least 2X101 of said cells per gram 9f said mixture, and (b) 60 - 30 percent by weight of powdered mixture containing at least 55 percent by weight of lactulose, the remainder consisting essentially of lactose and galactose.

3. A powdery composition according to either of Claims 1 and 2, wherein the microbial strain of said cell mass is selected from Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium bifidum and mixtures thereof.

4. A composition according to either of Claims 2 and 3, wherein said powdered mixture of cells and suspending agent consists of about 74 percent by weight of suspending agent, about 22 percent by weight of freeze dried viable cell mass of genus Bifidobacterium and about 4 percent by weight of moisture.

5. A composition according to Claim 4, wherein said suspending agent consists of about 38 percent by weight of powdered skim milk, about 9 percent by weight of sodium glutamate, about 4 percent by weight of gelatin and about 49 percent by weight of sucrose.

6. A powdery composition containing lactulose and cell mass of genus Bifidobacterium substantially as hereinbefore described in the Examples.