DE3118148A1 - Antiallergic product and process for its production - Google Patents

Abstract

An antiallergic product which contains as main ingredient Streptococcus lactis, Streptococcus bovis or Streptococcus faecalis from the Lactobacteriaceae family is described. This product can be produced by cultivating these strains in a culture broth, isolation and freeze-drying.

Description

"Antiallergic preparation and method for his

Manufacturing II Priorities Claimed: May 12, 1980, Japan, No. 55-61755 and March 19, 1981, Japan, No. 56-38790 The invention relates to an antiallergic Preparation, the main active ingredient of a streptococcus strain from the Lactobacteriaceaen family contains, as well as process for its preparation.

Many types of medicine are made from either microorganisms or parts made by microorganisms. Medicinal products derived from microorganisms, are widely used, such as preparations for controlling intestinal functions, those made from streptococcal strains, in particular from active enterococci as well as polyvalent antigen preparations, those from different pathogenic microbes or microbes of the respiratory tract have been produced.

The main active ingredient of these microorganisms is a type of protein that Is alien to humans and animals. Except for urokinase, all pharmacological derive Enzymes from substances or bodies that do not exist in humans; aside from that these enzymes are heterogeneous with respect to humans.

If a certain type of protein, which is foreign to the body, is administered to a mammal, so it automatically forms antibodies. The tendency to form antibodies is especially pronounced when these proteins are injected into the muscle, though The problem then arises that the antibody responsible for the allergy is IgE is also produced. If allergens are formed in a living body, then the consequence could be a severe anaphylactic shock caused by the immunological Response to the toxic effects caused by the antigens introduced into the body will.

The pathological symptoms arising from the various immunological Reactions arise due to the entry of foreign heteroantigens into the body are called "heteroimmune disease".

Allergic diseases are from P.G.H. Gell, R.R.A. Coombs and P.J. Lachmann in "Clinical Aspects of Immunology" 3rd Edition, Blackwell, 1975, in four Types have been classified according to the type of immunological reaction: Type I: The IgE antibodies fixed on the cells react with the antigen, causing the release of histamine and the activation of a slowly reacting substance (SRS-A) and the eosinophil chemotactic factor (ECF-A).

This is the mechanism that is responsible for asthma atopy, hay fever, urticaria, atopic dermatitis, anaphylaxis, and angioedema is responsible.

Type II: The IgE or IgM antibodies react with the antigen Target cells and activate the complement, which leads to cell lysis. This mechanism occurs with certain types of drug reactions.

Type III: The IgG or IgH antibodies form complexes with the antigen and the complementary neutrophil chemotacic factors, causing local tissue inflammation develop.

This mechanism occurs in serum sickness.

Type IV: Sensitized lymphocytes react with the antigen and cause inflammation through the action of lymphokinesis.

The main example of this is allergic contact dermatitis.

All of these allergic reactions are in humans as well found in various mammals.

The inventors have made various attempts on the formation of antibodies carried out in guinea pigs to which foreign protein has been administered. at the administration of streptococci from cow's milk to guinea pigs that are too high Interestingly, it was found that the streptococci Inhibit the overproduction of IgE, regardless of whether the microorganism is alive or is dead.

Using this ability of the streptococci to overproduce To inhibit IgE upon introduction of heteroprotein in vivo, Applicants have the Possibility of studying streptococci also as prevention and therapy in different-heteroimmune To apply diseases. The inhibitory effect of an allergic reaction was compared with a typical heteroimmune disease studied for which an experimental system can be set up easily. There were valuable and promising results can be obtained.

The Lactobacteriaceaen family includes many species of streptococci, such as Streptococcus pyogenes, Streptococcus equismilis, Streptococcus zooepidemicus, Streptococcus equi, Streptococcus dysgalatiae, Streptococcus sanguis, Streptococcus pneumoniae, Streptococcus anginosus, Streptococcus agalactiae, Streptococcus acidominimus, 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 strains, Streptococcus lactis is a typical non-pathogenic one Microorganism found in the milk of most animals and used for its manufacture used by dairy products, such as butter, cheese, or yogurt.

Streptococcus bovis belongs to the Lactobacteriaceaen and is in the Milk found in most animals.

Streptococcus faecalis comes from plants and was used for production of medicinal preparations suitable for the control of intestinal functions are used.

New species of Streptococcus lactis have now been found that have a have effective anti-allergy components and for the production of antiallergic Preparations can be used, namely Streptococcus lactis (Lister) Lohnis BK-042 or BK-045. Both strains have been found in the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan, and the American Type Culture Collection, Maryland, V.St.A., deposited under the following numbers: Streptococcus lactis (Lister) Lohnis BK-042: FERM-P 4590 and ATCC 31861 Streptococcus lactis (Lister) Lohnis BK-045: FERM-P 4591 and ATCC 31862.

The mycological properties of these two microorganisms are as follows: I) Streptococcus lactis (Lister) Lohnis BK-042 a) Morphological properties (1) Shape: chains of egg-shaped cells with a diameter of 0.1 to 1.0 µm (2) Agility: immobile (3) Gram stain: positive.

b) Fermentation properties (1) Growing conditions: optional anaerobic (2) growth at 450C: not detectable (3) liquefaction of gelatine: negative (4) reduction of litmus milk: positive (5) fermentation of saccharides: Mannose + mannitol + Maltose + salicin + sucrose # starch -glucose + Inulin - where + fermentation, - mean no fermentation and + not detectable.

c) Physiological properties (1) Oxidase test: negative (2) Catalase test: negative (3) oxidation fermentation test: fermentation (4) growth in a culture broth, which contains 6.5% sodium chloride: no growth noticeable (5) growth at pH 9.6: no growth noticeable (6) Effect of heat on growth (30-minute Heating at 600C): no growth noticeable (7) Lactic acid production from glucose: 96% (8) optical rotation of lactic acid produced by the strain: D + II) Streptococcus lactis (Lister) Lohnis BK-45 a) Morphological properties (1) Shape: chains of ovoid cells 0.6 to 1.0 am in diameter (2) Mobility: immobile (3) Gram stain: positive b) Fermentation properties (1) Growth conditions: facultative anaerobic (2) growth at 450C: positive (3) liquefaction of gelatine: negative (4) reduction of litmus milk: positive (5) fermentation of saccharides: Mannose + mannitol + maltose + salicin + sucrose -starch -glucose + inulin -wherein + Fermentation, - no fermentation and - mean not detectable.

c) Physiological properties (1) Oxidase test: negative (2) Catalase test: negative (3) oxidation fermentation test: fermentation (4) growth in a culture broth, which contains 6.5% sodium chloride: no growth noticeable (5) Growth at pH 9.6: no growth noticeable (6) Effect of heat on growth (30 minutes heating at 600C): no growth noticeable (7) lactic acid production from glucose: 92% (8) optical rotation of the lactic acid produced by the strain: d) Hemolysis (1) horse blood agar: reaction (2) sheep blood agar: reaction.

Since it has been found that other streptococcal strains valuable components for the production of antiallergic preparations the following strains deposited: Streptococcus lactis BK-051 at the Institute For Fermentation of Osaka, Japan, under the number IFO 12007, Streptococcus bovis BK-052 and Streptococcus faecalis BK-026 at the Institute For Fermentation of Osaka, Japan under numbers IFO 12057 or IFO 12964 and at the American Type Culture Collection under the Numbers ATCC 15351 and ATCC 10100 respectively.

The subject of the invention is thus an antiallergic preparation, the main active ingredient is Streptococcus lactis, Streptococcus bovis or Streptococcus faecalis from the family also in the form of cell fragments, Lactobacteriaceaen, / optionally in combination with usual Carriers and / or diluents, contains.

In the preparation according to the invention, however, living microorganisms can also dead trunks that have been treated by heat or by freeze-drying are, or fragments of these strains are used as the active ingredient. The inventive antiallergic preparation can contain conventional additives or stabilizers and as a suspension, grains, granules or powder. Also the method of administration is not critical, - the preparation can be administered orally, intraperitoneally or intravenously will.

The invention also relates to a method for producing the antiallergic Preparation which is characterized in that Streptococcus lactis, Streptococcus bovis or Streptococcus faecalis from the Lactobacteriaceaen family, isolated from the culture broth and freeze-dried to a powder.

Experimental animals (guinea pigs) due to overproduction Immunoglobulin E (IgE) suffered from allergic diseases, became that of the present invention antiallergic preparation administered. The resulting antiallergic reaction confirms the successful administration of this preparation.

The typical advantages of the antiallergic preparation according to the invention are the following: (1) The antiallergic preparation of the present invention is effective in various types of heterogeneous immune diseases such as allergic Bronchial asthma, infantile asthma, allergic rhinitis (hay fever, pollinosis), atopic dermatitis, nutritional allergy, drug allergy, allergic liver toxicosis or allergic gastroenteritis.

(2) The preparation according to the invention consists essentially of the microorganism itself. Neither toxicity nor side effects were found in the tests so that the preparation can be regarded as pharmacologically very safe.

(3) In contrast to conventional antiallergic preparations that are produced from polyvalent antigens, the preparation according to the invention consists from a single bacterium.

(4) The microorganisms used are not pathogenic, so they do not cause infection and offer the possibility of living microorganisms to use.

(5) In contrast to conventional microbial preparations, which by The preparation according to the invention can be administered inoculation or injection administered orally so that there are patients who are associated with the inoculation There is no fear and psychological resistance.

(6) In the treatment of asthma, the use of the inventive Preparation leads to a marked drop in IsE levels, confirming that the preparation of the present invention is satisfactory with conventional preparations and Procedure can be compared.

(7) Extracts of the microorganisms can be used in the preparation according to the invention use as an antiallergic agent. The effect occurs at a very low level Dose, e.g.

1 mg / kg body weight / day up to 50 mg / kg / day, with the preparation either injected under the skin, intramuscularly or intravenously, or administered orally can be.

The examples illustrate the invention.

Example 1 Streptococcus lactis BK-042 and BK-045 are cultivated and isolated using a centrifuge. The microorganisms are detailed with distilled water and washed in a conventional manner to a dry powder freeze dried.

4-week-old female guinea pigs are used for the experiments a body weight of 340 to 460 g observed for a week, then become healthy Animals selected. During this time and during the experiment, the guinea pigs will in a controlled cage at a temperature of 22 + 2 C and humidity held by 50 + 5%, whereby they have solid food and water without Restriction received For the sensitization of the test guinea pigs one is given Rabbits weighing approximately 3 kg receive a suspension in a weight ratio 1: 1 of 1% i-amylase solution (crystalline s-amylase from hay bacillus) and Freund's complete adjuvant at a dose of 1 ml / animal twice a week during Injected 4 weeks. The rabbit is used for passive sensitization obtained anti-s-amylase rabbit serum (antibody value: 32-fold) in one dose of 2 ml / animal 21 guinea pigs injected intraperitoneally.

Each group of three guinea pigs becomes one, two or three Weeks before passive sensitization with the anti-amylase rabbit serum Streptococcus lactis BK-042 or BK-045 administered every day using a metal probe in a dose of 100 mg / animal as a suspension in physiological sodium chloride solution.

The formation of antibodies is based on the IgE values using of a Rhadebas IgE test preparation. The results are shown in Table I with compared to the control group, all values being the mean of three animals each Group are.

Table 1 IgE values in β mol / ml duration Trunk not administered 1 week 2 weeks weeks S. lactis 31.7 17.3 17.0 BK-042 68 7 S. lactis 33.0 16.3 l 9.3 BK-045 As can be seen from Table I, the ability to form antibodies is markedly inhibited in groups treated with the microorganism in comparison with control groups. The slightly lower value after two weeks is attributed to the fact that the examined guinea pigs tended to vomit when the microorganism was forcibly administered by gastric tube and consequently suffered from bronchitis.

Inhibition of the allergic reaction by those given in Table I. Microorganisms are examined as follows: Seven guinea pigs (body weight 340 to 460 g) in a group are 48 hours after the end of passive sensitization with the anti-oC amylase rabbit serum described above in a chamber with a 1% oc-amylase solution for 30 minutes at a rate of 1.46 ml / minute sprayed by means of an ultrasonic atomizer. The one that occurs in the animals allergic shock is observed.

The animals are forcibly given Streptococcus lactis every day in a dose of 400 mg / kg. The inhibition of allergic shock is by comparison of the animals treated with the microorganism determined with control animals. The results are given in Table II, the severity of the seizure is according to the following levels determined: Grade 0: no signs Grade 1: tremors, sneezing Grade 2: dyspnoea, ataxia, Urinary and stool evacuation grade 3: dyspnoea, ataxia, urinary and stool evacuation, cramps, Paralysis, grade 4 cyanosis: death.

T able II S. lactis strain BK-045 S. lactis BK-042 Control control Dose (mg / kg / day) - 400 400 400 - 400 400 400 Duration (days) - 1 2 3 - 1 2 3 Severity grade 4 2 and grade 3 4 1 2 3 2 1 Frequency grade 2 3 6 6 2 2 5 4 3 the degree 1 1 2 1 3 Grade 0 1 1 1 seizures Total number of sea piggy 7 7 7 7 7 7 7 7 middle 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-test - 1.740 2.872 1.845 - 1.977 0.613 1.690 In the untreated control groups, all guinea pigs had to gasp about 3 minutes after being sprayed with the 1% amylase solution, while about 5 minutes later some had to vomit and convulsions and urination were observed. Two animals suffered from cyanosis, hind paw paralysis and had died of general anaphylactic shock at the end of the spraying.

On the other hand, the animals treated with the bacteria show in comparison to the untreated groups an excellent inhibition of general anaphylaxis.

Example 2 Streptococcus lactis BK-042 or BK-045 are cultivated and washed thoroughly with water. Each 150 g of microorganism are distilled with Water made up to a volume of 500 ml, the cells are for 60 minutes broken open with ultrasound (20 kHz, 200 W). The smashed microorganism containing liquid is centrifuged at 11,000 rpm for 60 minutes, each supernatant is freeze-dried. About 8 to 9 g of dry product are obtained.

These products are based on their effectiveness in inhibiting the anaphylactic Shocks studied: female guinea pigs with a medium body weight of 240 g are observed for a week, then 30 become healthy animals divided into three groups. During the preliminary examination and the trial will be the animals in a thermostated breeding chamber at a temperature of 22 + 20C and a humidity of 50 + 5%, with no restrictions Commercially available powdered diet food and tap water were given.

10 guinea pigs of each group will be given passive awareness before being given administered a feed with the anti-albumin rabbit serum for 20 days which is mixed with the preparation according to the invention, the amounts calculated as such be that a total dose of 100 mg / animal / day is achieved.

The rabbit anti-albumin serum is obtained by intramuscular injection to a rabbit weighing approximately 4 kg from 1 ml of a suspension in a weight ratio of 1: 1 of a 1% albumin solution and Freund's complete adjuvant three times a week for 5 weeks.

2 ml of this anti-albumin rabbit serum (antibody value: 16-fold) are injected intraperitoneally into each guinea pig. 48 hours later each will Guinea pigs are placed in a 52.5 chamber and using an ultrasonic nebulizer at a rate of 1.5 ml / minute for 30 minutes with 1 90% albumin solution sprayed.

The resulting anaphylactic shock is compared with the from compared to untreated animals. The results are given in Table III, the The degree of severity is determined according to Example 1.

T abe 1 1 e III S. lactis Strain control BK-042 BK-045 Dose (mg / animal / day) - 100 100 Duration (days) 20 20 Grade 4 2 Severity Grade 3 5 2 # 2 and grade 2 2 1 2 frequency the degree 1 1 3 0 Grade 0 4 # 6 seizures Total number of sea 10 10 10 piggy 10 10 10 moderate severity degree 2.8 1.1 1.0 Standard deviation 0.919 1.197 1.333 Mortality (%) 20 0 0 Student's t-test @ 3.564 ** 2.871 * (*: 95% reliability **: 99% reliability) Each guinea pig consumed the total amount of feed mixed with the extract prepared as described above to give 100 mg / animal / day. The guinea pigs gained weight normally.

Two of the untreated animals from the control group suffered 30 Seconds after the start of spraying with 1% animal albumin solution from severe convulsions, one minute after the start of the spray they were dead. Another guinea pig suffered from it severe cyanosis, two animals suffered from cyanosis and paralysis of the hind paws 5 minutes after the start of spraying and five animals showed shortness of breath and paralysis of the hind paws and ataxia.

In contrast, six animals treated with S. lactis showed BK-045 and four guinea pigs treated with S. lactis BK-042 showed no evidence of any Change. Some other animals treated with the preparation according to the invention had however, severe cyanosis and convulsions, but none of the treated guinea pigs died. The reduction in shock symptoms can be clearly observed in these groups.

In other words, all animals in the control group showed severe symptoms, whereas six of the ten animals treated with S. lactis showed no symptoms. In addition, the guinea pigs that were prepared with the according to Example 2 could Product had been treated, no side effects were found.

Five Wistar rats weighing 200 to 240 g were given oral 50 to 70 g / kg body weight / day freeze-dried product (prepared as above) administered every day for a week. At the autopsy, none of the animals were found sign of abnormality can be detected.

Example 3 Streptococcus lactis BK-042, BK-045, BK-051, Streptococcus bovis BK-052 and Streptococcus faecalis BK-026 are processed according to the method of operation of the Example 2 cultivated and the extract obtained.

Each extract is given orally to guinea pigs prior to sensitization administered by mixing with the feed, the dosage is according to Example 2 calculated. The animals receive the extracts in the same way after the sensitization.

An anti-hay bacillus a-amylase rabbit serum is used for sensitization made, which is diluted with physiological saline to 32-fold dilution and injected intraperitoneally into each guinea pig in an amount of 1.5 ml will. The allergic reaction is induced 48 hours after sensitization. The symptoms of the reaction are given according to Example 1.

The allergic reaction is induced by having seven guinea pigs each group in a 52.2 l cage with a 1% 6-amylase solution in physiological Sodium chloride solution for inhalation sprayed at one speed of 1.5 ml / minute for 10 minutes Use of an ultrasonic nebulizer. The results are given in Table IV.

T able IV Strain intact con- S. lactis S. bovis S. faecalis Control trolleys BK-042 BK-045 BK-051 BK-052 BK-026 Dose (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 grade 4 2 1 2 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 the degree 1 1 2 1 2 2 3 2 3 2 3 Grade 0 5 1 1 1 2 2 1 1 1 2 Total number of sea piggy 5 7 7 7 7 7 7 7 7 7 7 7 middle 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-test 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 Streptococcus lactis BK-045 is cultivated in 100 ml of a culture broth containing 1% glucose, 1% powdered yeast and 1% peptone at 320 ° C. for 18 hours. The culture broth is then centrifuged at 10,000 rpm.

The microorganism is thoroughly washed with distilled water and in an amount of 150 g in a 500 ml conical beaker of distilled Water diluted to 500 ml. This suspension is ultrasound in an ice water bath (200 kHz, 200 W) for one hour, then at 11,000 rpm for one hour centrifuged to remove the high molecular weight substances, mainly from the cell walls insist to remove. The supernatant is at low temperature and reduced Pressure concentrated and passed through a semipermeable cellulose membrane for 48 hours dialyzed.

The residue is centrifuged at 12,000 rpm for 20 minutes.

The supernatant is freeze-dried, a white to pale yellow color is obtained odorless powder in a yield of about 500 mg per 100 g of living organism.

The product from the last supernatant is used for its effectiveness Inhibition of anaphylactic shock studied: female guinea pigs with a mean body weight of 210 g are observed for 10 days, then five become healthy Animals selected for each group. During the preliminary tests and the test will be the animals in a thermostated chamber, in which the temperature 23 + 3 "C, the guinea pigs receive powdered without restriction Diet food and tap water.

The powder described above is in physiological sodium chloride solution suspended, the suspension is administered intravenously in the front paws at a dose of 50 mg / kg body weight / day for two days.

For sensitization, the guinea pigs are given 1.5 intraperitoneally ml of anti-CC amylase rabbit serum (antibody value: 32-fold), which is injected by a Rabbit weighing 3 kg is derived intramuscularly three times a week 1 ml of a suspension of 1% animal d ~ amylase solution and Freund's for 5 weeks Complete adjuvant has been injected. 48 hours after being sensitized each guinea pig in a 1.68 liter cylindrical chamber at one speed of 0.5 ml / minute with a 1 Eigen α-amylase solution using a Ultrasonic atomizer sprayed for 10 minutes. The resulting anaphylactic Shock is compared to that of control animals. The results are in the table V indicated, the severity of the shock is as described in Example 1.

Table V 5. lactis Strain control BK-045 Dose (mg / animal / day) - 50 Duration (days) # 2 Grade 4 4 Severity grade 3 1 and Frequency grade 2 the degree 1 2 Grade 0 seizures 2 Total number at sea- 5 5 Piggy middle. Severity 3.8 0.8 Standard deviation 0.447 0.837 Mortality (%) 80 0 Student's t-test 7.076 ** (**: 99% reliability) The animals in the control group had severe cramps after 30 seconds of spraying with the 1% albumin solution, four guinea pigs were dead one minute after the start of spraying. The fifth suffered from severe cyanosis, but survived.

The animals treated with the preparation according to the invention either suffered shortness of breath, convulsions or ataxia, others showed shortness of breath and convulsions, however, none died. This proves that the invention as described above produced preparation has a good effect against allergic reactions.

Example 5 Two S. lactis BK-045 strains, dried in 1935, are prepared according to the procedure of Example 4 was broken, centrifuged and dialyzed. the Dialysates are centrifuged at 12,000 rpm for 20 minutes. The one precipitate is discarded, a supernatant is freeze-dried, whereby 10.05 g of white or Contains pale yellow powdered product. The other precipitate is freeze-dried, 4.95 g of a product which will hereinafter be referred to as "B-1" are obtained.

The supernatant on this product is made with 99.5 percent by volume ethyl alcohol slowly added, a solution is obtained with an ethyl alcohol concentration of about 25 percent by volume. During the addition of the ethyl alcohol, a white color appears Suspension centrifuged at 12,000 rpm for 20 minutes. After decanting if the precipitate is freeze-dried, 4.60 g of white or pale yellow is obtained powdered product designated B-2 ".

The decanted supernatant is with further 99.5 term ethyl alcohol added, a solution with an ethyl alcohol concentration of about 40 is obtained Volume percent. The resulting white suspension is 20 minutes at 12,000 Centrifuged rpm. The supernatant is decanted, the precipitate is freeze-dried, 4.23 g of a white or pale yellow powdered product are obtained which is given the designation "B-3".

The decanted supernatant is mixed with 99.5% ethyl alcohol, man receives a solution with an ethyl alcohol content of about 50 percent by volume. the The pale white suspension is centrifuged at 12,000 rpm for 20 minutes. The precipitation is freeze-dried after decanting, 0.05 g of a white or pale yellow powdered product designated "B-4".

Each of these fractions is used in guinea pigs to determine the Administered anti-anaphylaxis effects. Female guinea pigs are as in example 3 and divided into groups of seven guinea pigs.

The test conditions are as described in Example 3.

Each fraction is mixed into the feed of the guinea pigs Dosage for each guinea pig is 150 mg / kg body weight / day. The dosage is calculated from the feed intake by each guinea pig the day before. The experiment lasts 14 days and the animals are treated as described in Example 3. The results are summarized in Table VI.

T able VI Fraction Control B-1 B-2 B-3 B-4 Dose (mg / kg / day) - 150 150 150 150 Duration (days) - 14 14 14 14 Severity grade 4 5 4 3 1 1 and grade 3 2 3 1 1 3 Frequency grade 2 1 3 1 the degree 1 1 1 1 Grade 0 seizures 1 1 Total number of sea pig 7 7 7 7 7 middle 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-test 0.512 0.971 3.281 ** 2.569 * (*: 95% reliability; **: 95% reliability) After spraying with 1% animal> -amylase solution, all control animals showed dyspnoea and sneezing, five animals being dead about 4 minutes after the start of the spray treatment. Two guinea pigs had severe attacks of cyanosis, convulsions and paralysis. Four guinea pigs given Fraction Bi died, three of whom had severe symptoms. That is, fraction B-1 is not effective against allergic symptoms. Three guinea pigs that received fraction B-2 died, only one survived with severe symptoms. Since the other animals exhibited allergic symptoms to a lesser extent than the animals which had been treated with fraction B-1, fraction B-2 has a certain effect against allergic symptoms.

On the other hand, the fractions B-3 and B-4 are clearly effective against allergic symptoms. One of the animals treated with fraction B-3 died other guinea pigs had severe symptoms, the others only showed mild ones Symptoms.

That is, fraction B-3 is very effective against allergic symptoms. One of the animals that had received fraction B-4 died, three had severity Symptoms, the others only mild symptoms. Fraction B-4 is therefore allergic to Symptoms effective.

The guinea pigs showed no side effects during the study.

Example 6 According to the invention, Streptococcus lactis can be either cultivate in solid form or in a liquid culture medium, the liquid culture medium however, it is preferable to obtain a greater yield.

Although various culture broths are commercially available, For the experiments different culture broths specially prepared to increase the yield of living bacteria per unit of quantity of culture broth.

Culture broth I: 5 to 50 g glucose, 3 to 20 g powdered yeast, 3 to 20 g of peptone and 0 to 20 g of sodium bicarbonate are treated in an ion exchanger Dissolved water and made up to 1 liter. The solution is under increased pressure and a Sterilized at a temperature of 1210C for 20 minutes, then with sodium hydroxide to pH 7.0 set to 8.5.

Culture broth II: 5 to 50 g glucose, 0 to 20 g sodium citrate, 0 to 20 g of sodium bicarbonate and 0 to 20 g of potassium phosphate are in an ion exchanger treated water and made up to 500 ml. Broth A.

Then 5 to 20 g of powdered yeast and 5 to 20 g of peptone are added in ion-exchanged water solved. You get 500 ml of broth B.

Both broths A and B are under high pressure at one temperature sterilized by 1210C for 20 minutes and mixed together. The mixture is with Sodium hydroxide adjusted to pH 7.0 to 8.5.

Culture broth III: 5 to 20 g glucose, 5 to 20 g powdered yeast, 5 up to 20 g peptone, 0 to 20 g sodium citrate and 0 to 20 g potassium phosphate are used in Dissolved water treated with ion exchanger and made up to 1 l. The solution is adjusted to pH 7.5 to 8.5 with sodium hydroxide and passed through an ultrafilter filtered.

Streptococcus lactis BT <-042, BK-045 and BK-051, Streptococcus bovis BK-052 and Streptococcus faecalis BK-026 are cultivated in the respective broths. At the end of the fermentation period, the pE value of the culture broth is 4.3 to 4.5. the Strains are each isolated from their culture broths, the yields are for Streptococcus lactis and Streptococcus faecalis about 6.0 to 6.5 g / l and for Streptococcus bovis about 8 g / l.

In the first stage, the logs obtained in this way are used twice in depth with about five times the volume of physiological sodium chloride solution, then washed once with enough distilled water.

In the second stage, 150 g of washed logs are distilled with as much Water is added so that about 1500 ml of suspension is obtained, which is treated with ultrasound is going to break up the cells. The suspension containing the cell debris is centrifuged, the supernatant is collected.

In the third stage, the supernatant is put up in a freeze dryer narrowed to a tenth of its original volume. The concentrate comes out twice on a semipermeable cellulose membrane against ten times the volume of distilled Dialyzed water for 24 hours at low temperature.

The remaining dialysate is centrifuged at 12,000 rpm for 20 minutes, the supernatant is collected.

99.5% ethyl alcohol is gradually added to this supernatant. During the addition of alcohol, the protein insoluble in the supernatant changes its Properties and is suspended in the supernatant .. The addition of alcohol is at an ethyl alcohol concentration of 30% ended. The supernatant is centrifuged, the precipitate is obtained. In this fourth stage, the amount of added is 99.5% ethyl alcohol 43 ml per 100 ml supernatant.

The precipitate obtained is freeze-dried, one obtains a white or pale gray, colorless and odorless powder in a yield of 0; 23 Weight percent of the washed strain.

Example 7 The procedure of Example 6 is repeated, man receives in the fourth stage 10 g of precipitate, which is mixed with distilled water will. 50 ml of a suspension are obtained, the hot water bath in 1/15 minutes is heated. The thermally denatured protein is at 12,000 rpm for 20 minutes centrifuged. The precipitate is freeze-dried. The powder contains essentially thermally denatured protein, the soluble substances bound to the protein have been removed in the procedure described in Example 6 in /. The yield of powder is about 0.12 percent by weight, based on the washed strain.

The products of Examples 6 and 7 are antiallergic Effect investigated using guinea pigs with in vivo antigens be formed by introducing heteroprotein - and by active sensitization the antigen causes allergic asthma (see e.g. T. Yagura et al. "Experimental Allergic Asthma in Guinea Pigs," Allergology, Excepta Medica P. 148, Amsterdam, 1971). The results are summarized in Table VII.

Table VII Intact Control Control Example 6 Example 7 Dose (mg / kg / day) 1 1 Duration (days) 3 3 Severity grade 4 7 2 and grade 3 2 3 3 Frequency grade 2 1 4 3 the degree 1 2 1 Grade 0 5 1 seizures Total number of guinea pigs 5 10 10 10 medium 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-test 4.422 ** 2.626 * (*: 95% reliability; **: 99% reliability) From the results in Table VII it can be seen that the products of Examples 6 and 7 had amazing effects in inhibiting the formation of antibodies, chemical attack induction substances such as histamine or serotonin Have antagonism between antibodies and chemical products as well as promote the formation of antiallergic antibodies.

In addition, the experimental allergic asthma in the guinea pigs also induced by passive sensitization with anti-rabbit serum. The antiallergic Effect of the products of Examples 6 and 7 is demonstrated by administration to the guinea pigs examined. The results are summarized in Tables VIII and IX.

T able VIII intact control S.latis S.bovis S.faecalis Strain control BK-045 BK-052 BK-026 Dose (mg / kg / day) 3 1 0.5 3 3 Duration (days) 10 10 10 10 10 Severity grade 4 9 1 1 2 3 1 and grade 3 7 2 2 4 1 3 Frequency grade 2 1 2 3 1 2 1 the degree 1 2 3 2 1 1 Grade 0 5 3 1 1 1 Total number of sea- 5 17 10 10 10 7 7 piggy medium 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-test 4.71 ** 4.48 ** 4.57 ** 1.67 2.96 ** (*: 95% reliability **: 99% reliability) T able IX intact control S. lactis S. bovis S. faecalis Strain control BK-045 BK-052 BK-026 Dose (mg / kg / day) 1 1 1 Duration (days) 7 7 7 Severity grade 4 6 1 and grade 3 3 4 4 5 Frequency grade 2 1 1 1 2 the degree 1 2 2 2 Grade 0 5 3 2 1 Total number at sea- 5 10 10 10 10 piggy medium severity 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-test 3,943 ** 3,000 ** 3,385 ** (*: 95% reliability **: 99% reliability) During passive sensitization, the anti-rabbit IgE antibody is formed in the anti-rabbit serum. If this IgE antibody is administered to the guinea pig, it will suffer from allergic reactions which can be inhibited with the preparations according to the invention.

There Streptococcus lactis, Streptococcus bovis and Streptococcus faecalis are very safe, their extracts and thus the antiallergic according to the invention Preparations are of course also very safe. Tables X, XI, XII and XIII in which some of these strains and their extracts are tested for their safety, confirm the.

Tab 1 1 e X: LD50 (mg / kg) administration (mg / kg) Substance animal substance animal sex number ip po BK-026 ddy - Mice M 8 190 480> 3000 "Wistar rats M 8 250> 5000 BK-045 ddy - Mice M 10 250 600> 30000 "Wistar rats M 10> 30,000 BK-045 ICR mice F 10 720-1040 Extract of Example 2 ddy alice M 10 2000> 30000 Table XI At- Substance Animal Sex number Dose (mg / kg) Days Findings BK-045 Wistar- M 4 50000-70000 5 no animal Rats died, Diarrhea 1 ddy mice M 6 100,000 5 3 mice died of Lower approximation T able XII Substance Animal Sex Add Dose Feeding Findings number duration BK-045 Wistar rats F 10 5000 mg / kg 3 months no BK-045 ddy mice F 10 3000 mg / kg 6 months histo- chemical BK-045 ddy mice M 10 3000 mg / kg for 6 months - and pa- BK-045 Wistar-Ratter M 10 10% of the 4 month thological Feed BK-045 Wistar-Ratter F 10 10% of the 4 months Feed change BK-045 gen Fischer M extract 5 5% of 22 weeks Example 2 rat feed *: 86-week-old animals Table XIII Substance Animal Sex Addition Dose Feeding Findings number duration BK-045 Wistar-Ratter M 4 0.05% of the 18 months none Forage histo- 0.5% of the 18 month chemical Feed 5% of the 18 months and pa- Feed tholo- Wistar-Ratter M 5 1500 mg / kg for 18 months BK-026 change ddy mice M 6 250 mg / kg for 24 months

Claims (18)

Claims 1. Antiallergic preparation, as the main active ingredient Streptococcus lactis, Streptococcus bovis or Streptococcus faecalis from the family of the Lactobacteriaceaen, also in the form of cell fragments, optionally in combination with conventional carriers and / or diluents containing. 2. Antiallergic preparation according to claim 1, characterized in that that it contains Streptococcus lactis (Lister) Lohnis BK-042 (ATCC 31861). 3. Antiallergic preparation according to claim 1, characterized in that that it contains Streptococcus lactis (Lister) Lohnis BK-045 (ATCC 31862). 4. Antiallergic preparation according to claim 1, characterized in that that it contains Streptococcus lactis BK-051 (IFO 12007). 5. Antiallergic preparation according to claim 1, characterized marked, that it contains Streptococcus bovis BK-052 (ATCC 15351). 6. Antiallergic preparation according to claim 1, characterized in that that it contains Streptococcus faecalis BK-026 (ATCC 10100). 7. Antiallergic preparation according to claim 1 to 6, characterized in that that it contains the main active ingredient in the form of a freeze-dried powder. 8. Process for the production of an antiallergic preparation according to Claims 1 to 6, characterized in that Streptococcus lactis, Streptococcus bovis or Streptococcus faecalis from the Lactobacteriaceaen family, isolated from the culture broth and freeze-dried to a powder. 9. The method according to claim 8, characterized in that one Streptococcus lactis, Streptococcus bovis or Streptococcus faecalis cultivated, washes, with distilled water is added, smashed, centrifuged and the supernatant is recovered. 10. The method according to claim 8 and 9, characterized in that one Streptococcus lactis, Streptococcus bovis or Streptococcus faecalis cultivated, washes thoroughly, suspended in water, smashed, by precipitation of the impurities a first supernatant is obtained, this first supernatant on a semipermeable membrane dialyzed, centrifuged the dialysis residue and a second supernatant wins. 11. The method according to claim 10, characterized in that this second supernatant in the form of a powder freeze-dried. 12. The method according to claim 8 to 10, characterized in that ethyl alcohol is added to the second supernatant and the precipitate is recovered. 13. The method according to claim 12, characterized in; that one Distilled water was added to the precipitate from the ethyl alcohol solution, the resulting The suspension is heated and the protein is obtained as a precipitate. 14. The method according to claim 8 to 13, characterized in that Streptococcus lactis (Lister) Lohnis BK-042 (ATCC 31861) is used. 15. The method according to claim 8 to 13, characterized in that Streptococcus lactis (Lister) Lohnis BK-045 (ATCC 31862) is used. 16. The method according to claim 8 to 13, characterized in that Streptococcus lactis BK-051 (IFO 12007) is used. 17. The method according to claim 8 to 13, characterized in that Streptococcus bovis BK-052 (ATCC 15351) is used. 18. The method according to claim 8 to 13, characterized in that Streptococcus faecalis BK-026 (ATCC 10100) is used.