DD205930A1 - PROCESS FOR THE PREPARATION OF OXYTETRACYCLIN - Google Patents

Abstract

THE INVENTION CONCERNS THE PRODUCTION OF OXYTETRACYCLIN ON FERMENTATIVE PATHS USING SACCHAROSE AS A SUBSTRATE. THE PURPOSE OF THE INVENTION IS TO EXCLUDE THE EXCLUSIVITY OF SUBSTRATE RELIABILITY AND TO ENABLE OXYTETRACYCLINFERMENTATION WITH COMPARABLE PERFORMANCE ALSO ON AN ALTERNATIVE SUBSTRATE. IN THE PURPOSE OF THE INVENTION, THE TASK IS HAVE BEEN ESTABLISHED THAT THE SUBSTRATE OF SACCHAROSE BEING PREVENTED IN ITS FRUIT AND GLUCOSE COMPONENTS IS PREPARED.

Description

The invention "relates to the production of oxytetracyoline by fermentation using sucrose as a substrate.

Characteristic of the known technical solutions

Oxytetracycline production is known to be by cultivating Streptomyces rimosus on nutrient media containing as carbohydrate source starch or a hydrolyzed product thereof. This substrate is digested by enzymes that are formed by the strain during the cultivation and fed to the metabolism. The use of amylase with a temperature optimum of at least 60 ° C. allows the use of high starch concentrations in the medium, so that high yield strains can be supplied with sufficient substrate without viscosity problems complicating the fermentation process.The principle of fermentation on medium with polymeric carbohydrate substrates is based thereon to direct the metabolism in the direction of an intensive biosynthesis of the antibiotic by a delayed substrate feeding as a result of the decomposition.This can be achieved in the same way by a suocesive substrate dosage, however, requires a mineral or enzymatic cleavage of the starch outside the fermentation system.

- & JI1L1982 * O2O8Ö2-.

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The wax part of the described prior art is that starch must be available natively or in hydrolyzed form in order to make a Oxytetraoyclinherstellung can.

Object of the invention

The twofold aspect of the invention is to eliminate the likelihood of substrate dependence and to allow for oxytetraoyolin fermentation with comparable performance even on an alternative substrate.

Explanation of the essence of the invention

The invention has for its object to develop a regime that allows the use of non-starchy media in the Oxytetraoyclinfermentation. According to the invention the object is achieved in that as a substrate sucrose is used, which is previously broken down into its components fructose and glucose. It has surprisingly been found that in addition to the known usability of fructoso by Streptomyoes rimosus during the product formation phase fructose and glucose are metabolized in parallel and the biosynthesis auoh Hoohleistungsstämmen no limitation. In the application of sucrose it is necessary to provide a mineral or enzymatic hydrolysis of the fermentation, whereby the applied technology must be adapted to the possibilities. If appropriate corrosion-resistant containers are available, a 40% Saooharoselösung with sulfuric acid is added so that a concentration of 0.5-1 N is present in the reaction mixture, Duroh standing at temperatures of 20 to 30 ⁰ C, the substrate within 24 hr completely decomposed into glucose and fructose. The same result is achieved when said solution of saooharose is passed through a tubular reactor containing invertase fixed to a support. The invertase is purified in a known manner and applied to the support with glutaraldehyde.

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braolit. The resulting hydrolyzate is 30 min. sterilized at 120 C and stored in an equally sterilized container. The use in the fermentation process is carried out by means of a Substratdosierungsregimes. For this purpose zwisohen the storage tank and the fermenter aseptisoh working pumping system is inserted, whose flow rate is such that 0.5 to 2.0 ml / h per 1 1 of the Reaktornutzvolumens can be dosed.

The rate of rotation is determined by a program. If no carbohydrate source is contained in the medium as a starting substrate, the feed is 0.6 to 0.8 ml / 1. h from beginning to end. If, on the other hand, a concentration of at most 2 $ of starch is initially applied, then the beginning and intensity of the dosage follow the pH movement of the fermentation batch. The dosage sets at a rate of 0.6 to 0.8 ml / 1. h, as soon as the maximum of the medium alkalization is exceeded. It is at 1.2 to 1.7 ml / 1. h, if analytically no free glucose can be detected after 72 hours in the medium. In the course of the fermentation, it is necessary to register the dissolved oxygen concentration and, by changing the air quantity or the stirring rate, to ensure that the spirit oxygen concentration does not fall below the saturation value of $ 40. Under these conditions, at least 14,000 IU / ml are achieved in 200 hours fermentation time.

The economy of the solution principle results sioh especially from securing the production of antibiotics in case of loss of starch as a carbohydrate raw material, without the use of the alternative substrate sucrose, with its use in the conventional batch process, no OTC biosynthesis feasible, an impairment of the fermentation results.

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Ausführunffsbeispiele example 1

Clay of a lyophil preserve of the strain Streptomyces rimosus submerse cultivation passages are placed in Sohüttelkolben containing media with peanut meal, starch, inorganic salts and CaCO ,, in tap water. After culturing at 28 ⁰ C on Rundsohwingtischen to the 24th hour is carried out harvesting and transfer to a second Impfpassage which is gezüohtet in a stirred reactor. Also this medium contains peanut meal and starch, as well as inorganic salts and CaCO ₅ . The inoculum is mature after 24 hours and serves as a seed for the main culture. This is carried out in a 2.5 liter fermenter, which has a stirring (600 rpm), aeration (30 l / h) and pH and temperature control. The starting volume is 1.8 1 fermentation medium. This consists of: (g / l) peanut meal - 7jO, corn steep liquor (100 # TS) - 2,3, (NH ₄ J ₂ SO ₄ - 5,0, ZnSO ₄ 0,025, MnSO ₄ - 0,17, CaCO ₅ - .5, corn oil - 0.9 The sterilization is 60 min at 120 ⁰ C, the pH after sterilization 7.0 to 7.3.

After transfer of the seed culture (200 ml), the conditions are set on the fermenter and the fermentation is carried out. The sucrose solution for the dosage is prepared as follows:

500 g of sucrose in 500 ml of 0.5 N H ₂ SO ₄ contribute to let solution to stand at room temperature for 24 hours, neutralized with solid NaOH and 3N NaOH and 1.2 Auffülung 1. Sterilization: 30 minutes at 120 ⁰ C. Der.Fermentationsverlauf and the dosage rates are given in Table 1.

Example 2

The cultivation of the parent material proceeds under analogous conditions, for example 1. As a fermentation ersystem a 30 1 reactor is used, which is fed with 20 nutrient medium.

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This "consists of (g / l): Erdnußsohrot - 21, corn steep liquor (100 # TS) - 9.2, (NH _{4) 2} S0 ₄ - 10.0, ZnSO ₄ - 0.05 MnSO ₄

- 0.04, corn starch - 20.0, CaCO ₅ - 12.0, corn oil - 0.9. Sterilization conditions are: 40 min at 120 C (using direct steam). The saooharose solution is prepared by means of an enzyme reactor by homogenizing 50% sucrose solution over a column filled with purchased or self-prepared carrier fixed invertase. The technique of this working sound corresponds to the known state of the art. The sucrose hydrolyzate is sterilized at 120 ° C for 30 minutes. The fermentation conditions and the course of fermentation are given in Table 2. Foaming is controlled by programmed addition of corn oil (5 ml / hr). The Siehe-, tion of the dissolved oxygen concentration (40 fo of saturation) is carried out by changing the stirrer speed in accordance with the following numerical values: 1 to 16 h - 150 U / min until 40 h

- 200 U / min, to 48 h - 300 U / min, to 96 h - 350 U / min and from 97. Ii - 400 U / min.

Table 1 dosage PH 0 g / l 0 pi Trockeniaasse -.0,9 OTC emotion t ml / 1. H 0 0 g / l ε / ι IE / nil U / min H sucrose 0 0 .m7,7 hydrolyzate 40? ig 0 99 0 21 ,; 0.60 6.5 0 »9 <- · 0 025 25.7 650 O 0.00 b, 9 0 97 0 024 29.7 It 11 0.73 b, O 0 80 0 023 28.6 η 22 Il 6.7 0 25 0 022 32.7 4-00 ti 31 Il 6.4 0 16 0 0: 8 34.7 1525 Il 46 Il 6.3 0 'Il 0 Ο16 £ 000 Il 55 Il 6.2 0 "I 0 Ο16 3400 Il 70 It 6.3 0 , 08 0 0 .6 4000 Il 79 It 6.4 0 , 07 0 013 , 4C00 Il 95 Il 6.4 , 08 013 5400 Il 103 Il 6.3 , 08 Oi 3 69ΟΟ Il vi 8 " 6.4 , 09 0i3 7200 I! • 30 Il 6.4 0.3 , 8_400. Il .42

Table 2 ' O dosage pH ° / aig Nik-N 0 pi dry matter OTC Bührung t (h) 16 ml / 1. H 6.4 s / i 0 g / l g / l IU / ml U / min 40 sucrose 7.5 0 48 Hydro lys at 40 6.5 64 • 6.3 2.02 0.023 • OEO 88 6.2 2, i5 200  WC. 6.3 1.40 30.4 1000 300 136 6.4 0.67 , 03 26.3 - 350  : 60 0.72 6.3 0.48 , 03 27.3 GOOO 350 184 0.62 6.3 0.42 , 02 29.1 7200 400 208 1.6 0 6.5 0.32 33.9 8400 400 1.82 6.8 0.42 4i, 7 10800 400 1.62 0.27 39.1 1: 200 400 1.67 0.38 51.6 12800 400 1.82 0.73 50.9 14000 400

Claims (4)

Erfindungsanspruoh A process for the production of oxytetracycline by fermentation using streptomycetes, characterized in that a carbohydrate source
Hydrolyzate is used by Sacoharose and its
Feed to the fermentation process in the form of a programmed dosage is done. 2. The method according to item 1, characterized in that in carbohydrate-free substrates from the beginning Ms to the end
the fermentation 0.6 to 0.8 ml hydrolyzed sucrose / h. 1 Reactor Nutzvolumen be supplied. 3. Procedure under point 2, characterized daduroh that when using a maximum of $ 2 containing carbohydrates. Substrate from the time of maximum medium analysis 0.6 to 0.8 ml of hydrolyzed sucrose / h. 1 reactor working volume and from the 72nd hour of the fermentation time or from the time of lack of free glucose 1.2 to 1.7 ml / 1. h be fed « 4. Verfahren.nach point 1 and 2 or 3, characterized in that hydrolyzed sucrose is supplied using carrier-fixed invertase. For this. JL pages tables