DE829936C - Continuous process for the production of penicillin by biological means - Google Patents

Description

Continuous process for the production of penicillin on biological Ways This I, .rfindung relates to a continuous method of implementation microbiological processes.

It relates to the manufacture of antibiotics by such microbiological process. In lresondex-en, it relates to manufacturing from penicillin to penicillin-producing molds.

Preferably \\ - ground liquid nutrients for breeding l'enicillitim notatuin used with the production of penicillin in such a nutrient liquid. The technique of immersion culture is preferably used.

It is known to breed Penicillium notatuin and to breed penicillin in to produce liquid culture media according to the immersion culture technique. With this technique the mold mass is immersed in the nutrient liquid, which is stirred and one Is subjected to ventilation.

Such microbiological processes were previously based on the C'hargenmet'hode carried out taking a batch of the nutrients with the appropriate mold or another microorganism and fermentation in the said batch has been completed, during which the batch has been stirred and: vented until the maximum Generation of penicillin e.g. in the mentioned batch was achieved. 1? Ine such batch method is slow and costly and has many other 'disadvantages, especially with regard to the production of antibiotics and in particular the Production of penicillin by the action of 1'enicilliutn notatum.

In the batch method, \\ ground the sterile liquid nutrients exposed to infection each time it is tapped. the one from your vaccine or comes from the free atmosphere as a result of technical errors during inoculation.

The mold becomes ßeinipfuiig with everyone. which are produced separately for each batch, are subject to an undesirable change which reduces its ability to produce penicillin. Therefore (Ii (# Atlsbellt (# im, every new batch is subject to an undesirable fluctuation.

In the manufacture of full penicillin one batch from such medium The (ieschivin- (littleness of the cultivation of the Schimniell) grows after a certain Time to a maxinitiin. (It depends on the breeding conditions. The l @ esch (@irt <ligkeit the development then decreases from this maximum speed all. \\ 'etiti the development speed is on your Z-Taxirntim, generates the continued fermentation reaction substances that strive. the 1'etlicilliii in to destroy the liquid batch. Such harmful substances include an excess all certain organic acids or ammonia. (read through I) amamination of amino acids is formed.

If the inoculation mass zti (#incr »etl (#il batch of nutrients is inflicted, there is a long hurry: \ ntruntime of the incubation, which is the time and the cost of production increases. When the fermentation of the batch is complete, it is necessary to empty the fermentation tank or tank, empty fermentation tank or cleil Sterilize the tank, then refill all nutrients with a new batch and finally sterilize the nice batch of nutrients in your container. This logical procedure increases time and effort.

According to the present Erfitidting a kc) titititiierlic'lie Generation is created and the disadvantages mentioned are eliminated. Vs a plurality of fermenters is used. two more men who are in series Application kmitinen.

The mold or other microorganisms will be in the first Fermentation tanks, bred. ulid there will be a fermentation in (learn first (@: irl> eliülter maintained below the level which produces harmful (substances or a Supersports of substances which are toxic in high concentrations. It will a part of the fermented nutrients and the fungus or other microorganisms from (learn first fermentation container in Additional precautions and steps are followed, discussed later will. Zalilrei # 7lie additional facts tin (1 advantages of the present (leii invention are described in the following description uii (1 of the schematic drawing. The invention is illustrated using an example for the production of penicillin atis T'enicillinnt notatum set out in more detail.

Example of process stage i The first fermentation tank or tank .T is closed and provided with sterilized inlets. CItirc 1i. Which it contains with an inoculation tank C, (lci- the inoculant, and finite two storage tanks D and D ', which contain the sterilized nutrients. I) as I # assun @ s ability of the first (iiit-1) eli-tank _ 1 is 22 7 1o 1. This tank _9, which rushed only (# Giirutrgszo ii (# ( is partially filled with a first _mount v (») i 3 ido 1 liquid nutrients. I) ics (# 1,5 ido 1 liquid nutrients consist of 1 4 383 I water, 5931 g Standardniaiswasclifliissigl <(# it tiii ( 1 geciglletc 296 kg milk sugar Any nutrients can be used.

The nutrients are sterilized by heating in the closed containers.-1, D and 1) under a pressure of about 2 atm. The pipe l) in (lungs between the lighterA and the containers ß, C. I) utid /) 'siti (T closed during the heating period. This overheating also sterilizes the interior of the borrowed filter .l. The closed container .- 1 and its hilialt i @ (#ril (#ii then allcooled to 24 °, while the IZ () lii) itidations are closed. Since the drawing is purely schematic, it does not show the i (@tweildigcli valves in the connection ports The passage between the individual containers should be fed through the gravity, pumps can be used provided that all parts are kept absolutely sterile and are protected against poisoning by accidentally present microorganisms. Procedure stage 2 After the sterile nutrients have been removed. 1 were cooled to 2d °, the ('-karge \ ciii 1-51401 with 3781 standard bowls from (learn) ic- container ('inoculated. The standard) felt in the Nutrients in the container-1 for a time bred for 48 hours, while di (# "hemperatnr Maintained at 2-1 during this development period will. 3781 inoculations immediately become equally finite fell] 15 rdo l 'nutrients mixed. I n this 1 »ui- The diving culture technique will play I #: iitt \ ickelli of the mold. The batch in (I (#ni 13e'holder A is stirred vigorously, and (las \\ 'ac @ is- The growth of the mold is determined by passing it through it of 70791 sterile air per minute through the batch in the container. = l by means of the usual Rülir- tii (I ver promoted t iltingsvorrichtung wherein diu up 1.tift egg ? ' downwards through this first mass in the usual way (# pearls while ensuring a continuous and even 13eliifting of the batch. This ent- speaks at full speed (#tw (t 4. + 9 ccnl Air per liter of nutrient solution in (no first approach. As mentioned above. is (li (° I? developing time a Container A 48 hours. At the end of this period, the 1), 1 value of the batch in container A 7.0 lüs 7.2 corresponds to your processed mold strain.

At the end of this 4.9 hour period, the mold content is o.5 to o.6 g (dry matter) per l <10 ccm nutrients.

\\ 'hen the nutrients in (learn container. = I up to their \ Istxiiiiiiiii fermented by penicillitiin notatum, the generation of mold is about 0: 7 to 0.8 g (dry matter) per 100 ccm of nutrients, and the pH value will be be about 7.3. At the end of this development period of 48 hours in <learn (r . 1 the development - "(# speed is at its maximum, but (fermentation has not yet formed any harmful substances or has an (excess of substances that are harmless in low concentrations. This harmful stage of fermentation that Ammonia or a ('tl) elimination of organic acids would produce. follows the Level of maximum development speed after a short period.

Immediately at the end of the mentioned period of 48 hours, the fermented liquid in the container. -I is continuously transferred to a second fermentation container and tank B at a rate of about 3121 per hour. The tank is sterile and forms the second Fermentation zone. The liquid that is fed into container h contains unused nutrients, mushroom balls and penicillin. The mass in container A is continuously kept at a constant volume of t 5 i 40 1 by adding fresh, sterilized nutrients <learn storage container I) or I) 'in the lieli: iltei-. -f at the same speed of about 312 l per hour. The fresh nutrient liquid has the composition of: - \ starting liquid in container A. Such fresh, sterilized nutrients are supplied to container A at a temperature of 2d ", which is maintained in container .9. The addition of the mass in the container A by has fresh and completely unfermented nutrients Effect that prevents fermentation in the container. that it reaches the dangerous point or stage, where undesirable substances or excessive contamination concentrations of substances can be achieved that are ringer concentrates ir are harmless. At the same Time is the fermentation speed of the Melgum mass in the container. -1 on their liveliest get the most active level. The lieliiilter / @ hat eiii Uasstnigsverniiigen -oii 7570 1. The mass from your container .I is poured into the Reefer h in a filling time of 12 to 1fl hours hastily fed, so that (read volume of the \ lasse in your Container B is maintained below about 568o I. the Mass in the container / i from your container.-I is supplied, has an activity of at least l, 50 to 200 1? inlieiteil per cubic centimeter, depending from your special stanini read penicillin produced- the mushrooms. During the filling period, the mixed nutrients, molds and penicillin, which are fed from container A to container B, are kept in container B at a temperature of 2d °. This temperature of 2.f ° is maintained in container B all the time. The development rate of the mold in the container B is reduced to below the development rate in the container A, thereby preventing the formation of ammonia, excessive organic acids, and so on.

Process level 4 After the mass in the container I> 'has the required Volume of 568o 1 'has been reached, the volume of this mass is obtained by subtracting The mass of this is constant at the feed rate, in Italy 3t21 per hour held. The procedure is thus after the mentioned initial fill time of 12 up to 16 hours continuously. [) per mass, which continuously from the container B withdrawn is the final broth or product. This one has a penicillin content from at least 250 to 35o units per cubic centimeter. Its pn value is about 7.3. While the continuous process is in operation, the rapid winding speed of the mold in the container I) sufficiently below the speed in the container A held to prevent the formation of ammonia, excess organic acids, etc. to prevent.

By continuing fermentation in container B without aeration, the growth rate of the mold in container B can be kept below the rate at which harmful substances are formed. The mixture in container B can, if desired, be mechanically stirred without aeration during the filling time and while the continuous process is in operation. There can also be ventilation of the material ini Container B take place. Then can if the second Mass here on a volume full 568o 1 will keep sterile air to ventilate this mass by means of the usual stirring and distribution devices at a rate of 566 liters per minute be used (lh at a speed of about 98 ccm per minute and per liter of nutrient fabrics in the second mass. The air bubbles upwards through the second mass in the usual way \ "Extension of a conventional stirrer. Some of the important advantages of the invention are the following: It will mass from your container A peeled off when the mold on its living the most active and active stage and before acid or ammonia in the N: their- substances are generated. By l: iitferiiuiig of liquid from your love li <iiter _! and \\ 'iederanfi = fill the -lasse im 13c- container. -I will be the finite fresh nutrients Fermentation products in the mass in container A thins. This is desirable because an over- concentrated concentration of such fermentation Products prevents the mold from remaining young, vigorous and healthy with the desired environmental activity.

The risk of infection is greatly reduced or eliminated.

The amount of inoculation is greatly reduced compared to the batch process. If inoculation is added in (part of the supply tank, such inoculation cannot be repeated sterilized and must be used immediately when they are ready for use is. In contrast to this, the sterile nutrient medium in the ile 'containers P and D' can re-sterilized as soon as desired. hs can take days or weeks stored after it has been sterilized before it is used.

The risk of an undesired mutation of the strain is greatly reduced, if not eliminated, (la by uninterrupted hoarding under identical Conditions the same mold mass is used continuously.

The aging of the sc'hiinin mushroom mass, which is characteristic for batch production is eliminated. Such an aging is at risk (read already produced penicillin. The stability of the penicillin in the final broth that is withdrawn from the container P is greatly increased.

The continuous process is cheaper and scores a much higher rate Production as (read batch process. Numerous changes and additions Lind Omissions' can be made in the preferred - %% iron version without to exceed the scope of the invention. You can use more than two fermentation containers. The flow of material into or through the Svstein brauc'lit is not continuous to be. The volumes in the various containers do not need to be kept constant to \ verden. The invention comprises the individual stages and intercom combinations of the entire procedure.

In general, the nutrients in container A are only partially fermented by means of a penicillin-producing mold, which is defined as the first fermentation zone. Some of the partially fermented liquid nutrients and the Schiniin mushrooms are extracted from the first fermentation zone. The fluid is replenished with sterile nutrients. The fermentation of the withdrawn and partially @crgoreu (nnutrients is continued at a slower rate than the fermentation speed in the first fermentation zone, which is limited by the fermentation tank A tle. H: s it is preferred to use the relevant liquid quantities in the first fermentation zone of the lielliil part. s. 1 mid in each of the following in the form of the @iirzone o (l @ r (lürrcnlen on correspondingly constant volumes zti lialten-It is preferred to keep the chal-gc of the nutrients and molds ini lehä lter. The partially completed fermentation can be continued at a lower rate than in the container A until the pH value of the end product is 7.2 his 7.4.

By replenishing the first _ \ Iasse with fresh nutrients at a speed, N \ -i (, ole mentioned, the l? ntwiaklti-iig in the created _Nlasse kept at their inaximaleii speed and prevented. (let the nutrients of the created mass are completely fermented.

Claims (1)

PATE \ TA \ S1'I;('CIII :: i. Method of making full I'eilicillill by fermentation of a flii; si "en \ iilirine (liuins with penicillin-producing molds, (la- characterized by, (let the sewage liquid during the subinersen / iiclitation (ler, '#, chiiniliel- mushrooms is aerated, breeding 1111 essential at their basic speed due to only partial @ 'replenishment of the nutrient Maintain quality, (read partially fermented Mediunt all drawn, by fresh nutrient (, crgälizt and the (ää rulig of the partially fermented and withdrawn medium in a special fermentation tank with less ventilation and slower @ äl-ullg zli Klide is performed, being the penicilliil gel in the said Let-e to at least 1.50 1> 1s 25o units Penicillin is kept per hullil <centinieter. 2. The method according to claim i, characterized in that indicates that the partially fermented and allgezo "eiieti \ <ilirstotte additionally: itally in a so @ whose C @ ärllehzilter llis at least 35oEill- lleitell Penicillili 1e Kul> i! kzelitinietei- fermented will. where the additional (;; irting without toilet- rope ventilation (lui-cli-efiilii-t will.