DE2106154B2 - Process for the production of a lactic acid bacteria preparation - Google Patents

Description

In recent times, according to the development of antibiotics, chemotherapy is also on treatment of epidemic indigestion has become applicable and shows remarkable therapeutic effects. However, improper use of antibiotics also causes the occurrence of resistant dysentery bacteria, an increase in superinfection and a decrease or Destroying beneficial bacteria in the intestinal tract.

In recent years, therefore, the administration proposed by Metchinikow of lactic acid bacteria preparations again for the treatment and prevention of epidemic digestive disorders such as dysentery, salmonellosis, and the like, as well as the treatment and prevention of superinfection, and the knowledge of them achievable effects is spreading more and more.

However, because the lactic acid bacteria used to manufacture the conventional preparations are sensitive to antibiotics, there is the disadvantage that they are therapeutically ineffective if the drug is used at the same time as chemotherapy using antibiotics is used.

The object of the invention is therefore to develop lactic acid bacteria preparations from lactic acid bacteria, which do not lose their activity when using different types of antibiotics at the same time and sulfonamides, and which have a wide range of resistance to various species of antibiotics and sulfonamides.

Japanese Patent 567,370 has already disclosed a method for producing a preparation described from a strain of Streptococcus faecalis B 10-SMR, a lactic acid bacterium, that is resistant to streptomycin. However, there are many options in the newer chemotherapy are given in which more ah two types of antibiotics and sulfonamides are used simultaneously has such a lactic acid bacteria preparation, which is only against a certain antibiotic Has resistance, has little practical value.

It has been found that lactic acid bacteria preparations, in order to be of medicinal interest, use the must meet the following conditions:

First, it is necessary that the lactic acid bacteria in the preparation in question at the same time

ίο Presence of different types of antibiotics and sulfonamides remain resistant, d. that is, the lactic acid bacteria are required to have multiple drug resistance exhibit. So you have to select those lactic acid bacteria that are at the same time become resistant to two or more different types of antibiotics. It will thereby avoided that the lactic acid bacterium against streptomycin, but not or only with difficulty to other antibiotics, becomes resistant or that

ao the lactic acid bacterium against other antibiotics when streptomycin becomes resistant, however, its already

existing resistance to streptomycin loses more or less.

Second, it is important that the lactic acid bacteria

»5 rium its resistance to other microorganisms transmits, because otherwise viruses and CoIi germs when administering the lactic acid bacteria preparations, which are present in the intestinal canal, also become resistant and thereby the therapy of the

3 "patient is dangerously affected.

Thirdly, the lactic acid bacterium must have a constant resistance and not from certain ones Be dependent on drugs. The reason is that the lactic acid bacteria even if it does Resistance to different types of antibiotics and sulfonamides has gained its value as a preparation loses if this resistance weakens slightly. Lactic acid bacteria with resistance to a particular Medicines like streptomycin can show dependence on the latter that go as far may that they need this as an indispensable substance for their growth. One is then forced add or remove antibiotics that are dependent on breeding to the medium

to be administered at the same time as the lactic acid bacteria.

Fourth and last, it is necessary that that Lactic acid bacteria preparation can be converted into a stable powder preparation after the Lactic acid bacteria have been grown. The rate of growth of lactic acid bacteria in the culture medium must therefore be high. In general, lactic acid bacteria show resistance to drugs have become resistant, a significant decrease in their growth rate in the Culture medium, and therefore one must choose the lactic acid bacterium, its growth rate does not decrease because of drug resistance.

Based on work on the selection and breeding of malchic acid bacteria, it was found that a lactic acid bacterium which satisfies the aforementioned four conditions and against the following Antibiotics as well as various sulfonamide resi-

6s shows stenz obtained by the following procedure can be:

One chooses. against the lactic acid bacterial strains belonging to Streptococcus faecalis

^ 3 4

Sulfonamide-resistant strain from and breeds. In the case of CM up to 30 to 40, im this strain on media containing streptomycin fail from TC 40 to 50, in the case of EM 16 to 17 (hereinafter abbreviated as SM), chlorampheni- and in the case of ABPC 20 to 30 consecutive col (hereinafter abbreviated as CM), Tetra transplant cultivations carried out to obtain the Bakcyclin (hereinafter abbreviated as TC), 5 terium high resistance to any drug Erythromycin (hereafter abbreviated as EM) ye borrow. The degree of resistance of the milk or milk obtained in this way. Aminobenzoylpenicillin (hereinafter referred to as the acid bacteria strain BIO-4R, i.e. a milk ABPC abbreviated) in such a way that the acid bacterium with multiple drug resistance said Strain initially using the substance is below for each of the above-mentioned anti-distance gradient agar dish method and then io biotikum indicated, based on the degree of resistance through repeated transfer in media with sub- of the mother line (BIO):

inhibiting substance concentration several times in _SM ; _more ^ ₂₀ 00 times

Presence of the above antibiotics in py innnfnph

the order SM, CM, TC, EM and ABPC kulti- ^ 100 times

is fourth until the desired resistance is 15 ^ M 500 times

of the tribe against these substances. jq ^ 33-fold

After this procedure the ^r jtamm becomes Resi- ρ 100-fold

stenz also against Kanamycin, Fradiomy-ABPC 30x

^C -ü- ° ^ ^nd ^ ^{: i} ! · ^L j ^com y ^ci ° 'Leucomycin, Peni- Oleandomycine '. '.'. '.'. '.'. '.'. '.'. '.'. '. '.'. 15x

cilhn GK, cephaloridin, gentamycin, spiramycin, lincomycin 10-fold

Bicillin Syncillin, Oradllin Staphcillin V, Leucomycin. ". '.'."'.'.'.'.'.'. more than 60 times

Methocillm S, Staphcillm A, and Hetacillin. Peaicäfo GK 20-fold

The invention is thus based on that in the patent cephaloridin. 20x

addressed the specified procedure. Gentamycin ... '. 40x

The mother line of the invention for 25 spiramycin 250-fold

Derivation of a lactic acid bacterium with resistance bicillin V2 3 ^ aCh

against various kinds of antibiotics and SuI- Svncillin * ~ 3 ^ fold

fonamiden is a sulfonamide resistant oracillin 160-fold

⁰ J ⁸⁶ ^ tS ⁸ S ¹ "'° ^Π Λ ^Stre P ^t0 ^ ^0CC _D ^{us faec} f?; ^U " ^d , ™% Staphcillin.'. '.'. '.'. '. ".'. '. '.'. ' more than 5 times

as BIO strain Oaparusche Patent 567 370) 30 _Sta £ _{hcil] in v more than 32-fold}

designated. Methocillin S more than 5 times

To this BIO strain against the aforementioned staphcillin A more than 5-fold

different types of antibiotics become resistant _XT "5" · ιγ _linf ".

. _A > c _A · ι 1 r 1 ι -r-% ι ι INaulClllin .7ZUIaL-Il

it is subjected to the following treatments: 35 In addition, the resistance was more than 20Ofold

a) First, the strip method is used to cultivate against SA as in the Staphylococcus 209 P strain, this strain is found in lactose-precipitated calcium car- ^ge ! l ,,,,. . _{/ Λ} , _T. ,,,. _λ bonat agar medium (hereinafter referred to as substance gradient agar (Ca-LA medium), abbreviated to Ca-LA medium), which according to the invention holds the SM according to the invention to make it resistant, 72 hours at a temperature of 37 ° C ⁴ ° Lactic acid bacterial strain BIO against various in a substance gradient agar dish; 'Kinds of medicines used let down

b) then choose different colonies, ^{prepare them as follows:}

the meat extract 10g spread over the entire shell

th microorganisms on the spot with the peptone 10 g

the highest SM concentration and ⁴⁵ salt (NaCl) 2 g

inoculate each of these colonies in a Ca-LA-lactose 20 g

Medium one that has a higher SM concentration yeast extract 20 ml

has calcium carbonate precipitated by the strip method 10 g

breed; Agar-agar 14 g

c) the culture obtained in this way is also selected, ⁵ ° _. ^ j ,, · 1 by 16 to 17 times in succession in a ^ genffen ^The ingredients are mixed medium is transferred, and which m 1000 m of distilled water solved the anti-. 10 ml of the biotikum (SM) in subinhibiting concentra-

55 shell cast. The liquid is in an un-

According to the aforementioned method, a BTO ferry can be solidified at an angle of inclination of 10 ° and after Strain, which has a high resistance to SM, get the solidification back in the horizontal position will. Then the same are brought, whereupon an amount of 10 ml of the pre-procedural steps for the other antibiotics mentioned liquid with the required concentration repeated, in the order CM, TC, EM 60 antibiotic added to the solidified medium and ABPC, whereby a lactic acid bacterium is poured on and allowed to solidify in layer form is won, which becomes multiple drug inventory. The substance gradient agar prepared in this way can and can be used according to the invention with advantage as a culture medium for obtaining the residences can. This lactic acid bacterium is ver-4R strain as a BIOstenz against different types of antibiotics and referred to under No. 184 of Agency 65. The lactic acid bacteria strain BIO-of Industrial Science and Technology, Fermentation 4R, in such a medium according to the pre-Research Institute, which the named method in the order SM, CM, TC, is an authorized bacterial depository in Japan. EM and ABPC has been made resistant will

then inoculated on a small amount of milk medium in a bottle, about 10 to 15 ml in total, and cultured ^{for a long time at a temperature of 37 D C.} The culture that solidifies the milk is then selected and inoculated again in fresh milk medium for further cultivation. After successive transfers more than ten times, the strain that has a high viability in milk medium is selected. By means of such a successive transfer culture, the strain becomes only one growth rate when it is first cultivated in milk medium. of 50 to 60 per cent, finally brought it to 100 per cent, and the solidification of the milk regularly takes two to three days.

The lactic acid bacterial strain BIO-4R, which is based on obtained in this way grows easily in a milk medium, the main component of which is dry skimmed milk and that can therefore be easily pulverized by spray drying. The technology for spray-drying lactic acid bacteria cultures for the purpose of producing stable, powdery ones Lactic acid bacteria preparations are described in Japanese Patent Publication 502,315. The essentials is to pulverize the culture obtained by growing the aforementioned by means of a spray dryer Multiple drug resistance lactic acid bacterial strain in the medium high concentration of skimmed milk. Usually, the Preparation still added a carrier substance to the powder obtained in this way.

The following test was carried out to determine the multiple resistance and the effect of the To confirm the lactic acid bacteria preparation obtained in the invention:

The preparation of the invention was administered orally to mice together with SNi every day. Included an abnormal increase in bacteria such as yeast in the feces could not be detected. On the other hand, when only SM was given, the amount of bacteria such as yeast in the excrement increased remarkably at. Furthermore, mice were fed with SM and EM and with the strain Salmonella enteiitidis No. 11 infected, after which the mortality increased to 80 to 100%. As opposed to the lactic acid bacteria preparation from BIO-4R according to the invention at the same time given with or before the administration of SM and EM, the mortality decreased to about 50 ° 0. It can thus be determined that the lactic acid bacterial preparation according to the invention against SM and EM is resistant and at the same time the abnormal increase in yeast (phenomenon of superinfection) controls and reduces the susceptibility to infection by Salmonella enteritidis.

The invention is illustrated below by way of example.

example

The strain Streptococcus faecalis BIO was inoculated onto the substance gradient agar (Ca-LA medium), which had been prepared as described above and added with SM. This tribe became grown by the strip method for 72 hours at a temperature of 37 ° C.

Then the colony that had grown at the site with the highest SM concentration was selected from the colonies grown on the plate and repeated the successive transfer culture using the same type of medium subject to this A lactic acid bacterium with high resistance to SM was obtained.

Subsequently, this SM-resistant lactic acid bacterium became a total of 30 times the successive one Transfer culture subjected in the same manner as above but using of a medium offset with CM. A lactic acid bacterium which was active against SM and CM is resistant. Now, this SM- and CM-resistant lactic acid bacterium became 40 times in the same In the same way, but with a TC-containing medium, subjected to the successive transfer culture until a lactic acid bacterium obtained wv de that was resistant to SM, CM and TC. Then this SM-, CM- and TC-resistant lactic acid bacteria 16 times in the same manner as above but using a medium mixed with EM subjected to the successive transfer culture, from which the lactic acid bacterium with resistance to SM, CM, TC and EM was taken. Eventually this lactic acid bacterium became resistant to SM, CM, TC, and EM 20 times in the same way, but using an ABPC-spiked medium of the subjected to successive transfer culture, after which a lactic acid bacterium BIO-4R was isolated, which not only against SM, CM, TC, EM and ABPC, but also against the others mentioned above Antibiotics as well as sulfonamides was resistant.

It has been confirmed that the degree of resistance to the aforementioned various kinds of drugs reaches a limit when the successive transfer culture is 16 to 17 times in the case of SM, 30 to 40 times in the case of CM. 40 to 50 times in the case of TC, 16 to 17 times in the case of EM and 20 to 30 ^{times in the F 7} ClI of ABPC.

The lactic acid bacterial strain BIO-4R with multiple drug resistance obtained in the manner described was then inoculated into a milk medium (about 15 ml) obtained by mixing in 2000- / ml SM (mg), 15 // ml CM, 10; / ml TC, 100 // ml EM and 20 // ml ABPC have been prepared v / ar, whereupon the strain which had a high ability to solidify milk was selected. This selected strain was subjected to such a treatment about 10 to 15 times in succession, to obtain a strain that has a consistent ability to solidify milk, d. H. a high Shows viability in milk medium. Then the strain thus obtained was placed in a medium cultured containing about 18% skimmed milk. After 96 hours of cultivation, the lactic acid bacterium had grown sufficiently, and the culture was pulverized by instant spray drying at a temperature of 70 to 100 ° C. After Pulverization can be carried out using a carrier substance or different types of enzymes, Mix vitamins and flavorings into the powdered preparation.

Claims (1)

Claim: Process for the production of an active lactic acid bacterial preparation with antibiotic resistance through successive cultivation of Streptococcus faecalis in a nutrient medium containing the respective antibiotic, stabilizing the Antibiotic resistance by growing the thus obtained Streptococcus faecalis several times in a milk medium containing the appropriate antibiotics together, and spray drying the culture thus obtained, characterized in that it is called Streptococcus faecalis a sulfonamide-resistant strain of Streptococcus faecalis according to the substance gradient agar method and by successive transfer to media with subinhibiting concentration in the order streptomycin, Chloramphenicol, tetracycline, erythromycin and aminobenzoylpenicillin are selected and after Stabilization and further cultivation in a skimmed milk medium, the culture is spray-dried.