DE1074824B - Process for the biological production of metabolic products of microorganisms containing vitamin B 12 and an antibiotic - Google Patents

Description

The invention relates to a method for the production of vitamin B ₁₂ and an antibiotic-containing metabolic products of microorganisms in the usual biological way.

According to the latest findings in animal nutrition, it is extremely advantageous _{to add small amounts of vitamins, in particular vitamin B 12} , and an antibiotic to the feed. It was also recognized that the addition of vitamins and an antibiotic greatly increases the effectiveness of pesticides against microbial pests.

It now seems obvious to simply use the metabolic products of such microorganisms as such additives to feed and pesticides, which _{produce both antibiotics and vitamins of the B 12} group. However, this is not possible because the metabolic products of these microorganisms contain _{far too small amounts of vitamin B 12 in relation to the antibiotics.} It has not yet been possible to influence the metabolism of these microorganisms in such a way that the proportion of vitamin B ₁₂ in the metabolic products is significantly greater or even to control the metabolism so that antibiotics and vitamin B ₁₃ in sufficient quantities and in the correct ratio are present to each other. _{If you wanted to achieve an optimal ratio of vitamin B 12} and antibiotics in the production of a polyvalent additive to feed and pesticides, you previously had to _{produce vitamin B 12} separately and add it to the metabolic product containing the antibiotics in the form of a concentrate.

The method according to the invention opens up the possibility _{of producing vitamin B 12} and an antibiotic-containing metabolic products of microorganisms in one operation, in which the two parts are present in sufficient quantities and in an optimal ratio to one another. The method according to the invention consists in that a _{microorganism which produces vitamin B 12 is} cultivated simultaneously with a microorganism which produces an antibiotic of the tetracycline series in a suitable nutrient medium under submerged conditions. This method can be carried out in such a way that either both microorganism strains are inoculated into the nutrient medium at the same time and the growth is regulated by the gradual addition of further nutrients or that the microorganism producing the antibiotic is only inoculated when the microorganism producing the vitamin shows maximum growth, whereby the nutrient medium is enriched with substances that promote the growth of the first-mentioned microorganism. In both cases the growth of both microorganisms is controlled by changing the sugar content, e.g. B. the glucose or sucrose content of the nutrient medium regulated.

Method of manufacture
of vitamin B ₁ <> and an antibiotic

'12

containing metabolic products
of microorganisms
by biological means

Applicant:

Eduard Belik, Roztoky,

Dr.-Ing. Milos Herold, Prague,

and RNDr. Jiri Doskocil, Roztoky

(Czechoslovakia)

Representative: Dipl.-Ing. A. Spreer, patent attorney,
Göttingen, Groner Str. 35

Claimed priority:
Czechoslovakia from May 26, 1955

Eduard Belik, Roztoky,
Dr.-Ing. Milos Herold, Prague,

and RNDr. Jiri Doskocil, Roztoky (Czechoslovakia), have been named as inventors

Methods are known per se for the simultaneous cultivation of two or more strains of microorganisms. In these processes, however, in general only bacteria of different types should be cultivated and not vitamins and antibiotics at the same time. There are also known methods in which different strains of e.g. B. Actinomycetes can be used to produce streptomycin, with vitamin B _{12 being} produced at the same time. Here too, vitamin B _{12 is} only produced in very small amounts that are not in proportion to the amount of antibiotic.

These known methods by no means allow the conclusion that it is possible, by suitable cultivation of two different microorganisms, to obtain metabolic products which _{contain vitamin B 12} in sufficient quantities and in an optimal ratio to the antibiotic. It was rather to be feared with previous experience that the production of vitamins from one strain would impair the production of antibiotics from the other strain or vice versa.

909 728/492

3 4

Moreover, these known methods were only used for example 1
superficial cultivation of strains applied in the laboratory. In contrast, with the composition of the nutrient solution (501): glucose \ * experienced according to the invention, the agricultural 0.25%, sucrose 2.5%, soy flour 2%, CaCO ₃ 0.6%, preparations by an industrial deep-tank fermentation 5 NaCl 0.25%, ammonium sulfate 0.3%, molasses 0.2%, fabricated, wherein there is also the possibility of the corn-steep 0.5%, CoCl ₂ 0.05%, p _H 6.9. To carry out the nutrient solution fermentation under non-sterile conditions, as in a wooden vat with heating and cooling coil, even those strains can be used that contain an antibiotic with a broad spectrum of bacteriostatic agents for 10 minutes with a ventilation ring and stirrer. After cooling to 28 ° C, the nutritional table and fungistatic effect, such as the chlorotetra- io solution with 2% 30-hour vegetative culture of the cyclin and the like Protect infection. solution of the following composition was prepared:

In the most advantageous implementation of the invented glucose 2 ⁰ I ₀ , corn steep 1%, ammonium sulfate 0.2%, the method according to the invention, the common fermentation CaCO ₃ 0.4%, CoCl ₂ 0.05% and 3% is a 36-hour period tation of Streptomyces olivaceus, which is the vitamin 15 vegetative culture of the strain S. aureofaciens Bg 16-3. B ₁₂ produced with the Streptomyces aureofaciens. This last culture was preferred on the medium following which produces the chlortetracycline. It is prepared composition: sucrose 3%, soy flour is possible, the growth of both organisms by 2%, CaCO ₃ 0.5%, NaCl 0.35%, ammonium sulfate 0.2%, determination of the sugar concentration in the corn steep nutrient solution B 54 0.6%. To control and regulate the inoculum of the strain, since the glucose contained the opti- 20 S. aureofaciens 250 y / ml chlorotetracycline. The male environment for a rapid growth of S. olivaceus fermentation was formed at 28 ° C with stirring for 300 rpm, while S. aureofaciens carried out the sucrose for its aeration and aeration 0.3 to 0.5 vol./min. Requires growth. At the beginning of the fermentation is useful.After 60 hours of fermentation, the production reaches the rich growth of S. olivaceus in the first row 810 y / ml chlorotetracycline and 1.8 y / ml vitamin B ₁₂ . Which take out all the glucose. In the further course of the determination of chlorotetracycline colorimetric fermentation is carried out the constant growth of the S. oli- according to Le vine, Garlock and Fischbach, vaceus is already maintained, while the S. aureofaciens The vitamin B ₁₂ was microbiologically determined by L. Leich later very quickly multiplied, with chlortetracycline manii 313 carried out. The breeding ground was formed on one. Filter press filtered off. The filtered mycelium, which

An alternative embodiment of the present 30 practically all of the chlorotetracycline as well as vit-

Invention is based on the preparation of a nutrient solution _{containing amine B 12} , was in a vacuum drum

With a low content of glucose (about 0.25 to ^{drying cabinet at 80 =} C dried for 2 hours. The

0.5%), which is sufficient for the growth of S. olivaceus. finished preparation was pulverized. The yield made

After a 30-hour fermentation, when the wax- 2 kg of the preparation with 20 g kg of chlorotetracycline and

turn of this strain is basically finished, one gives 35 35 mg / kg vitamin B ₁₂ from.

Sucrose or other nutrients too, e.g. B. ammonium. .

sulphate, which the growth of S.aureofaciens under-. Example λ

support. In this way, a favorable ratio is obtained. The nutrient solution had the following composition

made of antibiotic to vitamin. At the beginning (501): glucose 0.25%, soy flour 2%, CaCO ₃ 0.5%,

A sufficient amount of chlorotetracycline is maintained - ammonium sulfate 0.25%, NaCl 0.25%, molasses 0.2%,

which is sufficient to avoid infection, corn steep 1374 0.7%, CoCl ₂ 0.05%.

but which do not yet interfere with the growth of S. olivaceus. The nutrient solution was in a wooden tub as in the

can. Full production of chlorotetracycline is achieved in Example 1 boiled for 10 minutes, cooled to 28 ° C

one only in a later phase, when S. olivaceus already and with 3% of a 30-hour vegetative inoculum

is strongly grown and has not been inoculated by the antibiotic of the S. olivaceus strain, which on the same

can be more threatened. The growth and production manner as prepared in Example 1, and on

The activity of S. aureofaciens is very favorable in this phase with 4% 36-hour vegetative inoculum of the strain

influenced, namely by the use of the stick S. aureofaciens Bg 16-3 inoculated, that also on the same

Sources of the degradation substances of the S. olivaceus mycelium, wel- way as in Example 1 was prepared. The Fermen

ches already begins to autolyze in this phase. 50 tation was at 28 ° C with stirring 300 L ^T / min and

However, the practice of this invention is not carried out on the aeration 0.3 to 0.5 vol / min. To

The above mentioned antibiotic-producing strains decreased the glucose content for 30 hours of fermentation

limited. Also other strains that put the antibiotics down to 0.06%. Then 1.25 kg were added to the nutrient medium

Produce tetracycline series, e.g. B. Streptomyces (2.5%) sucrose, which was previously dissolved in 10 1 water.

rimosus (oxytetracycline) or Streptomyces viridifaciens 55 was added. After 72 hours of fermentation

(Tetracycline), can be applied as in the was a concentration of 790 y / ml chlortetracycline

Examples are given. and 2.4 y / ml vitamin B ₁₂ reached.

The whole production of such a polyvalent nutrient solution was made on a rotating drum feed additive is very simple and cheap, as the filter is filtered off, which was covered with a layer of potato biological agents always in the mycelium or in the 60 starch as a filter aid material. The fermented nutrient solution in the form of insoluble filtered mycelium was contained in salts together with the starch. It is therefore possible to dry a very rotating vacuum drying cabinet at 40 to 80 ° C effective combined polyvalent feed additive and pulverize the dry preparation simply by filtering, drying and pulverizing. The yield made 3.1 kg of the preparation that was obtained . 6g 12 g / kg chlortetracycline and 32 mg / kg vitamin B ₁₂

The following examples show various content.

Forms of implementation of the new process for fermentative Beistdel 3
Production of polyvalent feed additives. the

However, examples are not intended to encompass the invention in any direction of the nutrient solution of the same composition as

limit. 70 in example 2 were also like in a wooden tub

cooked in example 1. After cooling to 28 ³ C, 150 mg of chlortetracycline-H Cl and 100 mg of Actidion were added and the nutrient solution was inoculated with a 36-hour vegetative inoculum of the S. which was prepared in accordance with Example 1. The fermentation was carried out at 28 ° C. with stirring at 300 rpm and aeration at 0.3 to 0.5 vol / min. After 30 hours, 1.25 kg of sucrose were added to the nutrient solution, which had previously been boiled with 101% of water and then cooled. This was followed by inoculation with 3% of a 30-hour vegetative culture of the S. aureofaciens Bg 16-3 strain in accordance with Example 1. The fermentation lasted 72 hours. After this period, the nutrient solution reached a concentration of 980 μg / ml chlortetracycline and 2.68 μg / ml vitamin B ₁₂ . The fermented nutrient solution was processed into a dry powdery preparation in the same manner as in Example 2 at pH 7.6. The yield was 4.2 kg of a preparation with the content of 11 g / kg chlortetracycline and 28 mg / kg vitamin B ₁ .

12th

Example 4

The composition of the nutrient solution (5001): glucose 1.5%, starch 1.5%, CacO ₃ 0.5%, corn steep 0.7%, ammonium sulfate 0.3%, NaCl 0.25%, CoCl ₂ 0 .05%. The nutrient solution was boiled in a stemware tub with a stirrer, a ventilation ring and a cooling coil using direct steam for 10 minutes. After cooling to 28 ° C., 2.5% of a 30-hour vegetative culture of the S. olivaceus strain, prepared as in Example 2, and a 36-hour culture of the S.rimosus strain, prepared on a nutrient medium described above, were inoculated. The fermentation was carried out for 68 hours at the temperature of 30 ° C and stirring 200 rpm and aeration 0.3 to 0.5 vol / min. After 72 hours of fermentation, a concentration of 950 μg / ml oxytetracycline and 2.38 μg / ml B _{12 was} reached.

Example 5

The composition of the nutrient solution (5001): glucose 1%, sucrose 2 ¹ I ₂ ⁰ I ₀ , soy flour 2%, CaCO ₃ 0.5%, NaCl 0.5%, ammonium sulfate 0.35%, molasses 0.2%, Corn steep 0.3%, CoCl ₂ 0.05%. The nutrient solution was boiled in an earthenware tub as in Example 4 with direct steam for 10 minutes. After cooling to 28 ° C., 3% of a vegetative inoculum of a strain Nocardia sp., Which produces vitamin B ₁₂ , and 3% of a vegetative inoculum of the strain S. aureofaciens Bg 16-3 were inoculated. The inoculum S. aureofaciens was prepared as in Example 1 and contained 250 μg / ml chlorotetracycline. The fermentation was carried out at 28 ° C, stirring 300 rpm and aeration ίο 0.5 vol./min. After 70 hours of fermentation, a production of 850 μg / ml chlorotetracycline and 1.5 μg / ml vitamin B _{12 was} achieved. The fermented nutrient solution was processed into a technical concentrate in the manner described in Example 2.

Claims (4)

Patent claims: 1. A process for the production of vitamin B ₁₂ and an antibiotic-containing metabolic products of microorganisms in the usual biological way, characterized in that a _{microorganism producing vitamin B 12 is} grown simultaneously with a microorganism producing an antibiotic of the tetracycline series in a suitable nutrient medium under submerged conditions .ird. 2. The method according to claim 1, characterized in that both microorganism strains simultaneously be inoculated into the nutrient medium and the growth by gradually adding further nutrients is regulated. 3. The method according to claim 1, characterized in that the microorganism producing the antibiotic is only inoculated when the microorganism producing the vitamin is at its maximum Shows growth, with the nutrient medium promoting the growth of the first-mentioned microorganism Substances is enriched. 4. The method according to claim 2 and 3, characterized in that the growth of both microorganisms by changing the sugar content, e.g. B. the glucose or sucrose content of the nutrient medium, is regulated. Considered publications:
German patent specifications No. 288 375,607 205,831130, 647. © 909 728/492 1.60