GB2077101A - Antiallergic composition containing streptococci - Google Patents

Abstract

An antiallergic composition consists of a lactobacterium belonging to the Streptoccoccus genus, such as Streptoccoccus lactis, Streptococcus bovis and Streptococcus faecalis or an extract thereof.

Description

SPECIFICATION Antiallergic composition and process of preparing same The present invention relates to antiallergic substances and to the process for the preparation thereof. More particularly this invention relates to a new antiallergic substance making the use of lactobacteria belonging to the Streptococcus genus.

Many kinds of medicines are being produced from either microorganisms or parts thereof.

Medicines based on microorganisms, such as drugs for controlling intestinal functions prepared from strains belonging to the Streptococcus genus, more particularly active interococ'ci as well as polyvalent antigen preparations prepared from various pathogenic microbes or microbes in the respiratory tract are widely used. The main component of these microorganisms is a kind of protein which is foreign to man and animals. All pharmaceutical enzyms are derived from substances or bodies other than man except urokinase, and they are heterogenous to man.

When a certain kind of protein foreign to the body is fed into a mammal, antibodies are produced automatically. The tendency of producing antibodies is generally prominent when such proteins are injected into a muscle, though the problem is that this may produce antibody IgE capable of inducing allergy. When allergens are produced in a living body, a serious anaphlatic shock may be caused therein owing to immunological reaction to the toxic effect of antigens introduced into the body.

Pathological symptoms resulting from various immunological reactions caused by the entry of foreign heteroantigens in the body are called "heteroimmune sickness".

Allergic diseases have been classified by Gell and Coombs (Gell PGH, Coombs RRA, Lachmann PJ (editors): Clinical Aspects of Immunology, 3rd ed. Blackwell, 1975) into four types on the nature of the immunological reactions: Type I: IgE antibodies fixed to waste cells react with antigen, triggering release of histamine and activation of slow-reacting substance (SRS-A) and eosiuophil chemotactic factor (ECF-A).

This is the mechanism responsible for Asthma atopy, Hay fever, urticaria, Atopic dermatitis, anaphylaxis, and angioedema.

Type II: IgE or IgM antibodies react with antigen on target cells and activate complement, causing cell lysis. This occurs in certain types of drug reactions.

Type Ill: IgG or IgH antibodies from complexes with antigen and complement neutrophil chemotactic factors with resultant local tissue inflamation.

Serum sickness involves this mechanism.

Type IV: Sensitized Tlymphocytes react with antigen, producing inflamation through the action of lymphokines.

The principal example is allergic contact dermatitis.

These allergic reactions are found in various mammals as well as man.

The inventors have conducted a series of researches on the production of antibodies in guinea pigs using foreign proteins applied thereto. Interesting findings were obtained in the application of Streptococci derived from cow milk to guinea pigs suffering from a rise of antibody value and which the bacteria inhibited an over-producing of IgE, regardless it being alive or dead.

From the capability of inhibiting the over-production of IgE by the introduction of heteroprotein in vivo owing to the application of the Streptococci, the inventors speculated upon the possibility of using Streptococci for remedy and prevention of various heteroimmune sicknesses.

They investigated further the effect of inhibition of an allergic reaction, using a typical heteroimmune sickness for which an experimental system could readily be established and which yielded very valuable and successful results.

The present invention was carried out in accordance with the above observations.

There exist many numbers of lactobacteria belonging to the Streptococcus genus, such as Streptococcus p yo genes, Streptococcus equismilis, Streptococcus zooepidemicus, Streptococcus equi, Streptococcus dysgalatiae, Streptococcus sanguis, Streptococcus pneumoniae, Streptococcus anginosus, Streptococcus agalactiae, Streptococcus acidominimum, Streptococcus salivarius, Streptococcus mitis, Streptococcus bovis, Streptococcus equinus, Streptococcus thermophilus, Streptococcus faecalis, Streptococcus faecium, Streptococcus avium, Streptococcus uberis, Streptococcus lactis and Streptococcus cremoris.

Among these lactobacteria, Streptococcus Jactis is a typical non-pathogenic microorganism found in most animal milk and which is widely used for making dairy products, butter, cheese, yoghurt and the like.

Streptococcus bovis is another kind of lactobacterium belonging to the Streptococcus genus found in most animal milk.

Streptococcus facilas is derived from plants and has been used for preparing drugs to control intestinal functions.

The inventors have discovered new varieties of Streptococcus lactis possessing an effective component inhibiting allergy and which can be used for preparing antiallergic agents, namely Streptococcus lactis (Lister) Lohnis BK-042 and BK-045. Both bacteria have been deposited in the Fermentation Research Institute, Agency of Industrial Science and Technology, of Ibaraki, Japan and in the American Type Culture Collection, of Maryland, U.S.A.The deposit numbers of these cultures are: Streptococcus lactis (Lister) Lohnis BK-042: FERM-P 5490 ATCC 31861 dated 1 st April 1981 Streptococcus lactis (Lister) Lohnis BK-045: FERM-P 4591 ATCC 31862 date 1 st April 1981 The mycological characteristics of these microorganisms are as follows: (1) Streptococcus lactis (Lister) Lohnis BK-042 (a) Morphologic characteristics (1) Shape: Chained ovoids Diameter: 0.6 to 1.0 m (2) Mobility: Non-motile (3) Gramstaining: Positive (b) Fermextation characteristics (1) Growth Condition: Facultatively anaerobic (2) Growth at 54 C: Not test designated (3) Liquefaction of Gelatin: Negative (4) Reduction of Litmus Milk: Positive (5) Fermentation on saccharides: Mannose + (Wherein Mannitol + +:Fermentation Maltose + designated Salicin + Sucrose + -: No fermentation Starch - designated Glucose + Inulin - +: Not test designated) (c) Physiological characteristics (1) Oxydase Test: Negative (2) Catalase Test: Negative (2) o-F tested: F (4) Growth in the 6.5% NaCI brath: No growth designated (5) Growth at pH 9.6: No growth designated (6) Effect of Temperature on Growth (Heated for 30 minutes at 60 C): No growth designated (7) Lactic-acid Production Rate of Glucose: 96% (8) Optical Rotation of the Lactic-acid produced by the strain: D + (II) Streptococcus lactis (Lister) Lohnis BK-045 (a) Morphologic characteristics (1) Shape: Chained ovoids Diameter: 0.6 to 1.0 pm (2) Mobility: Non-motile (3) Gramstaining: Positive (b) Fermentation characteristics (1) Growth Condition: Facultatively anaerobic (2) Growth at 45 C: Positive (3) Liquefaction of Gelatin: Negative (4) Reduction of Litmus Milk: Positive (5) Fermentation on saccharidesi Mannose + (Wherein Mannitol + +: Fermentation Maltose + designated Salicin + Sucrose + - : No fermentation Starch - designated Glucose + Inulin - f : Not test designated) (c) Physiological characteristics (1) Oxydase Test: Negative (2) Catalase Test: Negative (3) O-F tested: F (4) Growth in the 6.5% NaCI brath: No growth designated (5) Growth at pH 9.6:No growth designated (6) Effect of Temperature on Growth (Heated for 30 minutes at 60"C): No growth designated (7) Lactic-acid Production Rate of Glucose: 92% (8) Optical Rotation of the Lactic-acid produced by the strain: D + (d) Hemolysis (1) Horse-blood Agar: reaction (2) Sheep-blood Agar: reaction Streptococcus lactis BK-051 has been deposited with the Institute For Fermentation of Osaka, Japan (hereinafter referred to as "IFO") under the deposit number, "IFO 12007").

Streptococcus bovis BK-052 and Streptococcus faecalis BK-026 are respectively deposited with IFO and the American Type Culture Collection. The Catalogue of Strains show the deposit number of these strains as follows: Streptococcus bovis BK-052: IFO 1 2057 ATCC 15351 Streptococcus faecalis BK-026: IFO 12964 ATCC 10100 The inventors have also found that these strains possess useful components for preparing antiallergic agents as well as the new varieties BK-042 and BK-045.

In the course of researches conducted by the inventors to find out a new antiallergic agent yielding a high efficiency in the inhibition of production of immuno globlin E (IgE) for the prevention of allergic sickness as a result of over-production of IgE in the body, allergic symptoms were caused to occur in experimental animals (guinea pigs), to which anti-allergic constituents discovered according to the present invention were subsequently applied produced an appreciable antiallergic reaction confirming the successful application thereof.

It is a principal object of the present invention to provide antiallergic agent and method for preparing the same, the effective constituents of which consist of whole or an extract of strain.

Streptococcus lactis, Streptococcus bovis or Streptococcus faecalis selected from the lactobacteria belonging to the Streptococcus genus.

According to the present invention, living strains of the mentioned microorganisms may be used as the active principle for the antiallergic agent. However, dead strains treated by heat or powdered strains treated by freeze-drying and fragments of the strain may also be used as the active principle for the agent. Common additives or stabilizers may be applied to the substance obtained according to this invention. No particular restriction is imposed upon the form of the antiallergic agent which may accordingly be either a suspension, a grain, a granule or a powder.

The method of administering the agent produced according to the invention is not restricted and may be either oral, intraperithoneal or intravenous.

The specific advantages of the antiallergic agents produced according to the invention are as follows: (1) It is effective for various types of heterogenic immune diseases, such as allergic bronchinal asthma, infantile asthma, allergic rihinits (hay fever, pollinosis etc.), atopic dermatitis, alimentary allergy, drug rash, allergic liver toxicosis, allergic gasteroenteritis, and effective for other types of immune diseases.

(2) The preparation produced in accordance with the present invention mainly consists of microorganism itself. Neither toxicity nor side-effect was found to occur in the experiments so that the preparation may be said as a high pharmacological safety.

(3) Unlike conventional antiallergic preparations made from polyvalent antigens, the present invention uses only a single bacterium.

(4) The strains to be used are non-pathogenic, so that those do not give rise to infection and they offer the possibility of using living microorganism.

(5) Unlike conventional microbic preparations which are to be adminstrated by innoculation or injection, the preparation relevant to the present invention can be administrated orally so that the patient is offered the expectation of freedom from the fear and psychological resistance inherent to inoculation.

(6) The use of the preparation from the invention for the treatment of asthma, yielded a significant drop in IgE level which confirmed that the present preparation can compare satisfactorily be used with any conventional method.

(7) The present invention makes possible to use strain extracts as the antiallergic agent.

Efficacy is ensured by a dosage that may be as small as 1 mg/kg. body weight/day up to 50 mg/kg/day, which means an unusual usefulness for the drug, the application of which may be either by hypodermic, intramuscular or intravenous injection as well or oral administration.

The invention will be understood more readily by inferring to the following examples; however, these examples are only meant as illustrations not to be construed to limit the scope of the invention.

Example 1 Streptococcus lactis BK-042 and BK-045 were respectively fermented and then isolated by means of a centrifuge. Then respective strains were washed thoroughly with distilled water and subsequently freeze-dried in a conventional manner to obtain a dried powder thereof.

In the experiments, four weeks old female guinea pigs having a body weight of 340-460 9 were kept in preliminaries for a period of one week. Healthy animals were then selected accordingly. For the whole period in preliminaries and during the experiment, the guinea pigs were kept in a homeostatic condition in a controlled cage at a temperature of 22 + 2"C and humidity of 50 + 5%, having allowed too take solid food and tap water without any restriction.

For sensitization of the tested guinea pigs, a rabbit with a body weight of about 3 kg was injected with a 1 ml/body does of 1:1 (by weight) suspension of 1%. a-amylase (crystallin aamylase obtained from hay bacillus) solution and Freund's complete adjuvant twice a week for a period of four weeks, whereas 2 ml/body dose of anti-a-amylase rabbit serum (antibody value: 32 times) obtained from the rabbit was intraperitoneally injected to twenty-one guinea pigs for passive sensitization.

Strains of Streptococcus lactis BK-042 and BK-045 were respectively administered to a group consisting of three guinea pigs in such a way that 100 mg/body suspension in physiological salt solution was given forcibly every day with a metal stomach sonde, starting one, two and three weeks prior to conducting the passive sensitization with the anti-a-amylase rabbit serum.

The production of antibodies was investigated by checking the results of IgE tests (using Rhadebas IgE test kit). The results pertaining to the tested and untested control groups are given in Table I, in which the figures indicate the average of three animals from each individual group.

Table I IgE Values Tested (ym/ml) Duration Strain Not administered 1 week 2 weeks 3 weeks S. lactis 31.7 17.3 17.0 BK-042 68.7 S. lactis BK-045 33.0 16.3 19.3 As is evident from Table I, the capability of antibody production of the groups treated with the strain is appreciably inhibited relative to the groups which were kept untreated. The somewhat lower value noted for the two-week treatment group was attributed to the fact that the tested guinea pigs had the tendency of regurgitating when given forcedly the above strain with the stomach snode, the consequence of which caused then to suffer from bronchitis.

The inhibition to the allergic action of the above strains was then tested accordingly.

Tests were performed as per the method of passive sensitization. That is, groups consisting of seven guinea pigs (body weight, 340-460 g) were treated so that 48 hours after completion of passive sensitization with the anti-a-amylase rabbit serum produced as outlined in the foregoing, the animals were put in a chamber in which they were sprayed with a 1 % a-amylase solution at a rate of 1.46 ml/min for a period of 30 minutes by means of an ultrasonic nebulizer to observe the allergic shock suffered by the animals. 400 mg/kg doses of the strain were administrated every day forcibly. The inhibition efficacy of the allergic shock was determined by comparing the average shock of individual test groups having been tested with the stran with that from the groups left untreated Relevant results are as shown in Table II. The degree of severity of attack was accessed as per the following scaling.

Grade 0: No Signs Grade 1: Trembling, Sneezing Grade 2: Dyspenea, Ataxia, Urination, Defaecation Grade 3: Dyspenea, Ataxia, Urination, Defaecation, Convulsions, Paralysis, Cyanosis.

Grade 4: Death Table II Strain Control S. lactis BKL-045 Control S. lactis BK-042 Dosage (mg/kg.day) - 400 400 400 - 400 400 400 Duration (days) - 1 2 3 - 1 2 3 Severity Grade 4 2 Attack and Grade 3 4 1 2 3 2 1 Frequency Grade 2 3 6 6 2 2 5 4 3 Grade 1 1 2 1 3 Grade 0 1 1 1 Total No. of Guinea Pigs 7 7 7 7 7 7 7 7 Average Grade of Severity 2.57 2.14 1.86 1.71 3.00 2.29 1.71 1.29 Standard Deviation 0.535 0.378 0.378 1.113 0.816 0.488 0.951 0.756 Mortality (%) 0 0 0 0 29 0 0 0 Student's t - 1.740 2.872 1.845 - 1.977 0.613 1.690 In the case of untreated control groups, all tested guinea pigs were found to suffer from hyperpnea about three minutes after being sprayed the 1 % solution of a-amylase, whereas occasional regurgitation, convulsion, urination and the like were observed about five minutes later.Two animals were found to suffer from cyanosis, paralysis of hind legs and were killed because of general anaphylatic shock at the end of spraying.

On the other hand, the groups treated with the bacteria were found to be in an excellent inhibition to general anaphylaxis in comparison to the untreated groups.

Example 2 Strains of Streptococcus lactis BK-042 and BK-045 were fermented and then washed thoroughly with water. A portion of distilled water was respectively added to each strain in an amount of 1 50 9 to 500 ml and then broken up ultrasonically (20 kHz, 200W) for 60 minutes.

Each liquid containing clastic S. Lactis strain was centrifuged at 11,000 r.p.m. for 60 minutes, and each resultant supernate was lyophilized to yield about 8 to 9 g of dried products.

Products contained in the respective supernates were studied to investigate the efficacy of inhibiting anaphlatic shock by way of comparison.

For the experiments, female guinea pigs with an average weight of 240 g were selected and kept in preliminaries for a period of one week.

From these guinea pigs, 30 healthy ones were then selected to constitute three individual groups.

Tested animals were kept in preliminaries during the entire period in preliminaries and during the experiment in a thermostatic breeding chamber at a temperature of 22 + 2"C and humidity of 50 + 5%, being allowed to take a commercially available powder diet and tap water without any restriction.

Ten guinea pigs of each group but control group were tested in such a way that, prior to passive sensitization by applying anti-albumin rabbit serum, these were fed for a period of 20 days food mixed with the agent produced according to the invention, amount of which was carefully calculated so as to yield an addition rate of 100 mg/body.day.

The anti-albumin rabbit serum was taken from a rabbit (body weight: about 4 kg) to which 1 ml of suspenson of 1:1 mixture of 1 % albumin solution and Freud's complete adjuvant had been administrated intramuscularly three times a week and for a continuous period of five weeks.

2 ml of anti-albumin rabbit serum (antibody value 1 6 times) was injected intraperitoneally to each guinea pig for passive sensitization. 48 hours later every guinea pig was placed in a 52.51 chamber in which spraying was applied with 1 % albumin solution by means of an ultrasonic nebulizer at a rate of 1.5 ml/minute for a period of 30 minutes in order to observe the anaphylatic shock occuring on the guinea pig during the spraying and in order to compare with each pig of the control untreated with the product pertaining to the invention.

The observed shock symptoms were evaluated and classified into five grades as described in Example 1, and relevant results are shown in Table Ill.

Table 111 S. lactis Strain Control BK-042 BK-045 Dosage (mg/body.day) - 100 100 Duration (days) - 20 20 Severity Grade 4 2 of Attack Grade 3 5 2 2 and Grade 2 2 1 2 Frequency Grade 1 1 3 0 Grade 0 4 6 Total No. of Guinea Pigs 10 10 10 Average Grade of Severity 2.8 1.1 1.0 Standard Deviation 0.919 1.197 1.333 Mortality (%) 20 0 0 Student's t 3.564** 2.871* (Note: * means 95% reliability means 99% reliability) Each guinea pig took all the food in which the extract prepared according to the method of Example 2 was mixed in order to administer 100 mg/body.day and the weight of the pig increased normally.

Two of the untreated pigs of the control group were found to suffer from serious cramp 30 seconds after being sprayed with 1 % albumin solution and one minute later the beginning of spray they were killed. Another guinea pig was heavily affected by cyanosis, two were found to suffer from cyanosis and paralysis of hind limbs about 5 minutes later the beginning and five animals suffered from tacypnea, paralysis of hind limbs, ataxia etc.

Unlike the animals of the control, six guinea pigs treated with the substance from S. lactis BK-045 and four guinea pigs treated with the substance from S. lactis BK-042 were neither affected whatever nor changed. Some of other guinea pigs treated with the substance prepared according to the present invention showed serious cyanosis, cramp etc., but none of the treated guinea pigs was killed, and the trend of reducing of shock symptoms was significantly observed.

It may also be described in such a manner that all the animals of the control group were found to show an onset of severe symptoms, but as many as six of ten animals treated with the substance from S. lactis showed no symptoms as a result of reduction of shock or effect offered by administration of the substance prepared according to the invention.

It should be noted that no side-effect seemingly based on the above product was not found on the guinea pigs treated with the product prepared according to Example 2.

Five Wistar rats (body weight: 200 to 240 g) were orally administered with 50-70 g/kg (body weight).day of freeze-dried product obtained by the process of Example 2 every day for a continuous period of one week, but autopsy applied to all thereof revealed no sign of anomaly whatever.

Example 3 Streptococcus lactis BK-042, BK-045, BK-051, Streptococcus bovis BK-052 and Streptococcus faecalis BK-026 were respectively fermented in the same manner as disclosed in Example 2 to obtain the extract.

Prior to the sensitization, each extract was orally administered to each guinea pig of testing groups by mixing it in the food and the dosage was calculated in the similar manner to that in Example 2, and also each extract was given to the guinea pig after the sensitization in the same manner.

As sensitizing anti-serum to be applied to the guinea pig was made of anti-hay bacillus aamylase rabbit serum, which was then diluted to 32 times with physiological salt solution, 1.5 ml of the solution was injected intraperitoneally to the guinea pig for sensitization thereof.

Induction of allergic reaction thereof was effected 48 hours after the sensitization. The observed symptoms of the reaction were classified according to the same manner as stated in preceding Example 1.

The induction was performed in such a manner that seven guinea pigs of each group were placed in a 52.21 cage, in which the guinea pigs were then tested by spraying with 1 % aamilase physiological salt solution at a rate of 1.5 ml/sec by means of an ultrasonic nebulizer for a continuous period of ten minutes for inhalation. The results are given in Table IV.

Table IV Intact S. lactis S. bovis S. faecalis Strain Control Control BK-042 BK-K045 BK-051 BK-052 BK-026 Dosage (mg/kg.day) 150 10 150 10 150 10 150 10 150 10 Duration (days) 14 14 14 14 14 14 14 14 14 14 Severity of Grade 4 2 1 2 Attack and Grade 3 4 3 1 2 1 1 2 1 2 2 2 Frequency Grade 2 1 2 3 3 2 1 1 1 1 3 Grade 1 1 2 1 2 2 3 2 3 2 3 Grade 0 5 1 1 1 2 2 1 1 1 2 No. of Animals 5 7 7 7 7 7 7 7 7 7 7 7 Average Grade of Severity 0 3.14 2.00 1.57 1.86 1.29 1.57 1.57 2.14 1.57 2.00 1.29 Standard Deviation 0.690 1.155 0.976 1.069 1.113 1.512 1.134 1.574 1.134 0.816 1.254 Mortality (%) 28.6 0 0 0 14.3 28.6 0 0 0 Student's t 2.336* 3.476** 2.242* 2.607* 2.308* 3.130* 1.540 2.018 2.240* 3.144** (*:95% reliability; **:99% reliability) Example 4 Strain of Streptococcus Jactis BK-045 was fermented in 1001 of culture broth comprising 1 % of glucose, 1% of powdered yeast and 1% of pepton at 32'C for a continuous period of 18 hours and then fermented broth was centrifuged at 10,000 r.p.m. so as to isolate the strain from the broth.

The strain was then thoroughly washed with distilled water and 1 50 g thereof was placed in a 500 ml conical beaker and diluted with distilled water to 500 ml in order to make a suspension of the strain. Subsequently the suspension was ultrasonically broken up in ice water bath, at 200 kHz and 200W for a continuous period of one hour. The clastic strain was centrifuged at 11,000 r.p.m. for a period of one hour to precipitate high molecular substances comprising mainly of coarse cell walls of the strain. The supernate was then concentrated at a low temperature and a reduced pressure and then dialized for a continuous period of 48 hours through a cellulose type semipermeable membrane to obtain residue of dialysis. The residue was subsequently treated centrifugally at 12,000 r.p.m. for a continuous period of 20 minutes to take the supernate.The supernate was then lyophilized in order to obtain powder thereof. The powder was white or pale yellow and odorless. The yield of the powder was about 500 mg per 100 g of live strain.

The powder obtained from last supernate in accordance with Example 4 was investigated in order to confirm the efficacy of inhibiting the onset of anaphylatic shock.

Female guinea pigs (average body weight: 210 g) were used for the experiment and kept in preliminaries for ten days, of these, five healthy ones were picked up for each test group.

Maintenance of tested animals was performed, both in preliminaries and during the testing period in a thermostatic chamber, in which the temperature was kept at 23 + 3"C, guinea pigs then being allowed to take a commercially available powdered diet and tap water without any restriction.

The powder was suspended in physiological salt solution, then the suspension was intravenously administered to the fore limbs in such a manner that the dosage was 50 mg (of the powder) per kg (body weight) a day for a continuous period of two days.

In order to sensitize the tested guinea pigs, then they were injected intraperitoneally with 1.5 ml of anti-a-amylase rabbit serum (antibody value 32 times) obtained from a rabbit (body weight: 3 kg) having been injected intramuscularly, three times a week and a continuous period of five weeks, with 1 ml/body of mixtured suspension of 1 % amylase solution and Freund's complete adjuvant for passive sensitization. 48 hours later the sensitization, each guinea pig was placed in a 1.681 cylindrical chamber, in which the guinea pig was then sprayed, at a rate of 0.5 ml/minute, with 1 % amylase solution, by means of an ultrasonic nebulizer, for a continuous period of ten minutes for inhalation, in order to observe the anaphylatic shock on the guinea pig, which was compared with the control guinea pigs.

The observed results were classified according to severity of the shock as described in Example 1.

The classified are shown in Table V.

Table V S. lactis Strain Control BK-045 Dosage (mg/body.day) - 50 Duration (days) - 2 Severity Grade 4 4 of Attack Grade 3 1 and Grade 2 1 Frequency Grade 1 . 2 Grade 0 2 No. of Guinea Pigs 5 5 Average Grade of Severity 3.8 0.8 Standard Deviation 0.447 0.837 Mortalidy (%) 80 0 Student's t 7.076"" (Note: **means 99% reliability) In the experiments, the guinea pigs of the control group were observed to be affected by a serious cramp 30 seconds after being sprayed with the 1 % albumin solution, and one minute after the beginning of the spraying four guinea pigs were killed. The last one was suffered from heavy cyanosis, but was not killed.

On the other hand, one of the guinea pigs treated with the powders prepared according to Example 4 was affected either by dyspnea, cramp or ataxia, other guinea pigs suffered from hyperpenea and convulsion, but none of them were killed. The powders obtained by Example 4 show the great efficacy against allergic reactions.

Example 5 Two 1 935 dried S. Lactis BK-045 strains were respectively broken up, centrifuged and dialized in a manner similar to that used for Example 4. The respective dializates in the membrane were then centrifuged at 12,000 r.p.m. for 20 minutes. One precipitate was discarded and one supernate was freeze-dried, yielding 1 0.05 g of white or pale yellow powdery product. The other precipitate was freeze-dried and the yield was 4.95 g. This product of fraction will be referred to as ''B-1'' hereinafter.

While a portion of 99.5% (by volume) ethyl alcohol was slowly added to the other supernate to yield a solution with an ethyl alcohol concentration of about 25% (by volume) in order to fractionate it. During the addition of ethyl alcohol a white suspension was observed which was thereupon centrifuged at 12,000 r.p.m. for 20 minutes to precipitate the suspension. After decantation the precipitate was freeze-dried, yielding 4.60 g of white or pale yellow powdery product. This product or fraction will be hence referred to as Additional 99.5% ethyl alcohol was poured into the decanted supernate to yield a solution with an ethyl alcohol concentration of about 40% (by volume), and again a white suspension was observed, which was thereupon centrifuged at 12,000 r.p.m. for 20 minutes to separate the suspension.Then the supernate was decanted and the precipitate was freeze-dried, yielding 4.23 9 of white or pale yellow powdery product. This product or fraction will be referred to as "B-3" hereinafter.

Another portion of 99.5% ethyl alcohol was added to the decanted supernate to yield a solution with an ethyl alcohol concentration of about 50% (by volume), and a slightly white suspension was observed which was then centrifuged at 12,000 r.p.m. for 20 minutes. The precipitate was obtained by means of decantation and was freeze-dried, yielding 0.05 9 of white or pale yellow powdery product. This product will be referred to as "B-4" hereinafter.

Each fraction was administered to guinea pigs to examine the anti-anaphilaxis effect. In this examination, sample animals, female guinea pigs, were chosen in the same manner as in Example 3, and they were distributed each group which contained seven pigs. The other conditions were the same as those in Example 3. Each fraction was mixed in each guinea pig's food and the dosage to each pig was 1 50 mg/kg.day. The dosage was calculated by accounting the weight of food taken by each pig the day before. The duration was 14 days, and the treatment after the administration was the same as that of Example 3.

The examinated result will be shown in the Table VI.

Table Vl Fraction Control B-l B-2 B-3 B-4 Dosage (mg/kg.day) - 1 50 1 50 1 50 1 50 Duration (days) - 14 14 14 14 Severity of Grade 4 5 4 3 1 1 Attack and Grade 3 2 3 1 1 3 Frequency Grade 2 1 3 1 Grade 1 1 1 1 Grade 1 1 Total No. of Guinea Pigs 7 7 7 7 7 Average Grade of Severity 3.71 3.57 2.57 2.00 2.29 Standard Deviation 1.428 0.535 1.618 1.291 1.033 Mortality (%) 71 57 43 14 14 Student's t 0.512 0.971 3.281"" 2.569" (Note:: "-95% reliabiliW' ""-99% reliability) Upon induction of 1 % a-Amylase solution, all control animals were effected by dispenea and sneezing, whereas five of them were killed about four minutes after the beginning of induction.

Two guinea pigs suffered severe attacks of cyanosis, convulsions and paralysis. Four of the guinea pigs administered with B-l fraction were killed, and three of them were subjected to heavy symtoms, that is to say, B-l fraction was not effective against allergic symptoms. Three of the guinea pigs administered with B-2 fraction were killed and only one of them was brought to heavy symptoms. Since the others were not affected by allergic symptoms in so large an extent as the B-l case, one B-2 fraction was deemed against allergic symptoms.

On the other hand, B-3 and B-4 fractions were significantly effective against allergic symptoms. One of the animals administered with B-3 fraction was killed and another guinea pig was brought to heavy symptoms, but the others were lightly affected by the symptoms. Thus B-3 fraction was found very effective against allergic symptoms. One of the guinea pigs administered with B-4 fraction was killed and three of them were affected by heavy symptoms, but the others were lightly brought to the symptoms. Then B-4 fraction was very effective allergic symptoms.

Consequently no side-effect to the guinea pigs was found during the examination.

Example 6 According to the present invention, the lactobacterium belonging to Streptococcus lactis may be fermented in either solid or liquid culture medium, but it is preferable to choose a liquid culture medium or broth in order to obtain a large amount of strains.

Though different kinds of culture broths were available commercially, but for the experiments some kinds of culture broths were prepared specially in order to increase a yield of live bacterium per unit quantity of the culture broths, according to the practice described below: Culture Broth I: 5 to 50 9 of glucose, 3 to 30 9 of powdered yeast, 3 to 20 9 of pepton and 0 to 20 9 of sodium bicarbonate were dissolved in ion-exchanged water to 11, which was subsequently sterilized under an elevated pressure at a temperature of 121"C for 20 minutes, and then adjusted with caustic soda to pH 7.0 to 8.5.

Culture Broth II: 5 to 50 9 of glucose, 0 to 20 9 of sodium citrate, 0 to 20 9 of sodium bicarbonate, 0 to 20 9 of potassium phosphate were dissolved in ion-exchanged water to obtain 500 ml of broth A.

Subsequently, 5 to 20 9 of powdered yeast and 5 to 20 9 of pepton were dissolved in ionexchanged water to obtain 500 ml of Broth B.

Both broths A and B were sterilized under a high pressure ata temperature of 121"C for 20 minutes and then mixed together. The mixture was adjusted with caustic soda to pH 7.0 to 8.5.

Culture Broth 111: 5 to 20 9 of glucose. 5 to 20 9 of powdered yeast, 5 to 20 9 of pepton, 0 to 20 9 of sodium citrate and 0 to 20 9 of potassium phosphate were dissolved in ion-exchanged water to 11, which was adjusted with caustic soda to pH 7.0 to 8.5, and then treated by ultrafiltration.

Streptococcus lactis BK-042, BK-045 and BK-051; Streptococcus bovis BK-052; and Streptococcus faecalis BK-026 were fermented respectively. Measurement of pH level of the culture broth at the termination of fermentation revealed 4.3 to 4.5.

All fermented strains were respectively isolated from their culture broth, the yields of the strain were as listed below: Streptococcus lactis approx. 6.0-6.5 g/I Streptococcus faecalis approx. 6.0-6.5 g/l Streptococcus bovis approx. 8 g/l.

In the first step, the strains obtained according to the above procedure were thoroughly washed two times with phsiologic salt solution having a volume of about five times as much as that of the strains, and then washed one time with sufficient distilled water.

In the second step, a portion of distilled water was added to 1 50 9 of the washed strains to get about 1 500 ml of suspension of the strain, then the suspension was treated ultrasonically for crushing the strains. The suspension containing clastic strain was centrifuged and the supernate was recovered.

In the third step, the supernate obtained in the second step was lyophilized by means of a freeze drying apparatus to concentrate to one-tenth of the original volume. The concentrate was dialized twice through a cellulose type semipermeable membrane with distilled water of ten times as much as the concentrate for a continuous period of 24 hours in a low temperature room. The residual dializate was centrifuged at 1 2,000 r.p.m. and for a continuous period of 20 minutes, recovering the supernate.

Then a portion of 99.5% ethyl alcohol was gradually added to the supernate. As the result of the addition of ethyl alcohol, the protein insoluble in the supernate was changed its property with the alcohol and suspended in the supernate. Addition of the alcohol was then terminated at an ethyl alcohol concentration of 30%. The supernate was then centrifuged to obtain the precipitate thereof. It is noted that the amount of the addition of 99.5% ethyl alcohol to the supernate was 43 ml to 100 ml of the starting supernate in the fourth step.

The precipitate obtained in the fource step was lyophilized into powder form. The powder was white or light gray, tasteless and odorless. The yield of the powder was 0.23% by weight of the washed strain.

Example 7 The procedure mentioned in Example 6 was repeated and 10 9 of precipitate was prepared as in the fourth step. A portion of distilled water was added to the precipitate to obtain 50 ml of suspension. The suspenson was placed in a vessel and heated in a boiling water both for 1 5 minutes. The thermaly denaturated protein thus realized was centrifuged at 12,000 r.p.m. for a continuous period of 20 minutes to obtain resultant precipitate, which was lyophilized into dried powder. The dried powder contained mostly of thermaly denaturated protein, and soluble substances combined with the protein had been removed by the above mentioned treatment.

The yield of the powder was about 0.12% by weight of the washed strain.

[Resuks of Animal Tests] In general, methods for inducing allergic action to vertebrate animals have been well known.

See, for example, Yagura T., et al. ''Experimental Allergic Asthma in Guinea Pigs", Allergology, Excepta Medica P. 148, 1971 Amsterdam.

The inventors used guinea pigs as the experimental animal to produce antigins in vivo by introducing heteroprotein in the guinea pig, and then allergic asthma was caused to provoke by active sensitization with the antigen and checked the antiallergic reaction of the substances prepared as in Examples 6 and 7. The results of which were reported in Table VII.

Table VII Intact control Control Example 6 Example 7 Dosage (mg/kg.day) 1 1 Duration (days) , 3 3 Severity of Grade 4 7 2 Attack and Grade 3 2 3 3 Frequency Grade 2 1 4 3 Grade 1 2 1 Grade 0 5 1 1 Total No. of Guinea Pigs 5 10 10 10 Average Grade of Severity 0 3.6 1.9 2.4 Standard Deviation 0.699 0.994 1.265 Mortality (%) 70.0 0 20.0 Student's t 4.422"" 2.626" (*-95% reliability, "-99% reliability) From the results shown in Table VII, it will be understood that the substances prepared according to the Examples 6 and 7 have remarkable effects for inhibiting the production of antibodies, chemical attack induction substances, such as histamin, serotonin etc., antagonism between antibodies and chemicals, and for promoting the production of antiallergic antibodies.

Furthermore, the experimental allergic asthma was induced in guinea pigs in accordance with passive sensitizaton by application of anti-rabbit serum. The antiallergic action of the substances prepared according to Examples 6 and 7 was investigated by applying .to the tested guinea pigs.

The results of Examples 6 and 7 are respectively listed in Tables VIII and IX.

Table VIII Intact S. lactis S. bovis S. faecalis Strain Control Control BK-045 BK-052 BK-026 Dosage (mg/kg.day) 3 1 0.5 3 3 Duration (days) 10 10 10 10 10 Severity of Grade 4 9 1 1 2 3 1 Attack and Grade 3 7 2 2 4 1 3 Frequency Grade 2 1 2 3 1 2 1 Grade 1 2 3 2 1 1 Grade 0 5 3 1 1 1 No. of Guinea Pigs 5 17 10 10 10 7 7 Average Grade of Severity 0 3.47 1.60 1.90 2.40 2.85 2.28 Standard Deviation 0.624 1.430 1.197 1.350 1.215 1.380 Mortality (%) 52.9 10.0 10.0 20.0 42.9 14.3 Student's t 4.71** 4.48** 4.57** 1.67 2.96** (*-95% reliability; **-99% reliability) Table IX Intact S. lactis S. bovis S. faecalis Strain Control Control BK-045 BK-052 BK-026 Dosage (mg/kg.day) 1 1 1 Duration (days) 7 7 7 Severity of Grade 4 6 1 Attack and Grade 3 3 4 4 5 Frequency Grade 2 1 1 1 2 Grade 1 2 2 2 Grade 0 5 3 2 1 No. of Guinea Pigs 5 10 10 10 10 Average Grade of Severity 0 3.5 1.6 2.0 2.1 Standard Deviation 0.707 1.350 1.414 1.101 Mortality (%) 60 0 10 0 Student's t 3.943** 3.000** 3.385** (*-95% reliability; **-99% reliability) In the case of the passive sensitization, the anti-rabbit IgE antibody was produced in the antirabbit serum. It will be understood that by applying the above IgE antibody to the tested guinea pig, the guinea pig was affected with allergic reaction which can be inhibited by the substance prepared in accordance with the invention.

As Streptococcus lactis, Streptococcus bovis and Streptococcus faecalis which are highly safe and their extracts were used in this invention, the antiallergic agents of the invention would be naturally very safe. But we chose and examined some of these strains and their extracts in order to confirm their safety. The resulted data are given in Table X. XI, XII and XIII.

Table X LD50 (mg/kg) Substance Animal sex no. iv (mg/kg) ip po BK-026 ddy mice M 8 190 - 480 > 3000 BK-026 Wister rats M 8 250 > 5000 BK-045 ddy mice M 10 250 600 > 30000 BK-045 Wister rats M 10 > 30000 BK-045 ICR mice F 10 720-1040 Extract by Expl.2 ddy mice M 10 2000 > 30000 Table Xl Substance Animal sex no. dose (mg/kg) days Findings BK-045 Wister rats M 4 50000-70000 5 No animal died Diarrhea BK-045 ddy mice M 6 100000 5 3 mice died malnutrition Table XII Duration Substance Animal Sex no. dose of feeding Findings BK-045 Wister rats F 10 5000 mg/kg 3 months BK-045 ddy mice F 10 3000 mg/kg 6 months No histochemical BK-045 ddy mice M 10 3000 mg/kg 6 months and pathological BK-045 Wister rats M 10 10% feed 4 months changes BK-045 Wister rats F 10 10% feed 4 months BK-045 Extract Fischer M 5 5% feed 22 weeks by Expl. 2 rats ": :86 weeks old aged animal were used.

Table XIII Duration Substance Animal Sex no. Dose of feeding Findings BK-045 Wister rats M 4 0.05% in feed 18 months No histochemical and pathological 0.5% in feed 18 months changes 5% in feed 1 8 months BK-026 Wister rats M 5 1500 mg/kg 18 months ddy mice M 6 250 mg/kg 24 months While the invention has been illustrated with reference to various particular embodiments thereof, it is to be appreciated that modification and variations can be made within the scope of the invention.

Claims (18)

1. An antiallergic composition comprising a principal component of lactobacterium belonging to the Streptococcus genus.

2. An antiallergic composition claimed in claim 1 wherein said lactobacterium is Streptococcus lactis (Lister) Lohnis BK-042 (ATCC 31861).

3. An antiallergic composition claimed in claim 1 wherein said lactobacterium is Streptococcus lactis (Lister) Lohnis BK-045 (ATCC 31862).

4. An antiallergic composition claimed in claim 1 wherein said lactobacterium is Streptococcus lactis BK-051 (IFO 12007).

5. An antiallergic composition claimed in claim 1 wherein said lactobacterium is Streptococcus bovis BK-052 (ATCC 15351).

6. An antiallergic composition claimed in claim 1 wherein said lactobacterium is Streptococcus faecalis BK-026 (ATCC 10100).

7. An antiallergic composition claimed in any one of claims 1 to 6 wherein said principal component of lactobacterium is freeze-dried in the form of powder.

8. A process for the production of an antiallergic composition which comprises fermenting a lactobacterium belonging to the Steptococcus genus, isolating said lactobacterium from the resulting fermentation, and freeze-drying said lactobacterium in the form of dried powder.

9. A process for the production of an antiallergic composition which comprises fermenting a lactobacterium belonging to the Streptococcus genus, washing the strain of said lactobacterium, adding distilled water to said strain, crushing said strain, and recovering supernate obtained by centrifuging the liquid containing sai'd crushed strain.

10. A process for the production of an antiallergic composition which comprises fermenting a lactobacterium belonging to the Streptococcus genus, washing throughly the fermented strain, suspending said washed strain in water, crushing said strain in said suspension, obtaining a first supernate from the suspension containing said crushed strain by precipitating impurities, dialyzing said first supernate by means of a semipermeable membrane, and centrifugalizing the residue of dialysis and obtaining a second supernate.

11. A process for the production of an antiallergic composition claimed in claim 10 wherein said second supernate is freeze-dried in the form of powder.

1 2 A process for the production of an antiallergic composition which comprises: (A) washing the fermented strain of a lactobacterium belonging to the Streptococcus genus; (B) suspending said washed strain in water, crushing said strain and isolating said crushed strain from said suspension and recovering a first supernate from said suspension; (C) dialyzing said supernate by means of a semipermeable membrane, and recovering a second supernate after having been centrifugalized; and (D) adding ethyl alcohol to said second supernate for precipitating the protein contained in said second supernate, and recovering the precipitation from said ethyl alcohol solution.

1 3. A process for the production of an antiallergic composition which comprises: (A) washing the fermented strain of a lactobacterium belonging to the Streptococcus genus; (B) suspending said strain in water, crushing said strain in said suspension, and recovering a first supernate of said suspension after removing solid substances; (C) dialyzingsaid first supernate by means of a semipermeable membrane, and recovering the second supernate after isolating residue from said first supernate; (D) adding ethyl alcohol to said second supernate for precipitating the protein contained in said second supernate, and recovering said precipitation from said ethyl alcohol solution; (E) adding distilled water to said precipitation to obtain the suspension of said precipitation;; and (F) heating said suspension for recovering said protein contained in said suspension as the precipitation.

14. A process for the production of an antiallergic composition claimed in any one of Claims 8 to 1 3 wherein said lactdbacterium belonging to the Streptococcus genus is Streptococcus lactis (Lister) Lohnis BK-042 (ATCC 31861).

1 5. A process for the production of an antiallergic composition claimed in any one of claims 8 to 1 3 wherein said lactobacterium belonging to the Streptococcus genus is Streptococcus lactis (Lister) Lohnis BK-045 (ATCC 31862).

1 6. A process for the production of an antiallergic composition claimed in any one of claims 8 to 1 3 wherein said lactobacterium belonging to the Streptococcus genus is Streptococcus lactis BK-051 (IFO 12007).

1 7. A process for the production of an antiallergic composition claimed in any one of claims 8 to 1 3 wherein said lactobacterium belonging to the Streptococcus genus is Streptococcus bovis BK-0052 (ATCC 15351).

18. A process for the production of an antiallergic composition claimed in any one of claims 8 to 13 wherein said lactobacterium belonging to the Streptococcus genus is Streptococcus faecalis BK-026 (ATCC 10100).