DD231949A3 - METHOD FOR THE FERMENTATIVE MANUFACTURE OF NOURSEOTHRICIN - Google Patents

Abstract

There is described a fermentative nourseothricin recovery process which accumulates high levels of nourseothricin using nutrient media containing hexose, dihydrogen, oligomer or polymer containing carbohydrate sources and appropriately suitable excipients over approximately 140 hours of fermentation with controlled acidity.

Description

Field of application of the invention

The invention relates to the fermentative production of the antibiotic nourseothricin. It has ergotropic effects and therefore has great economic importance for agriculture and the pharmaceutical industry.

Characteristic of the known technical solution

Nourseothricin is an antibiotic isolated from the culture of Streptomyces noursei JA 3890 b, which is active against Gram-positive and against Gram-negative bacteria and mycobacteria and also has antiviral properties (Bradler, G., and Thrum, H., 1963: Nourseothricin A and B, two new antibacterial antibiotics of a Streptomyces noursei variant, Z. Allg. Mikrobiol., 3, 105-112). Nourseothricin is a mixture of streptothricin Fund D (Gräfe, U., Bocker, H., Reinhard, G., and Thrum, H., 1974: Regulatory influence of n-ocherothricin biosynthesis by o-aminobenzoic acid in cultures of Streptomyces noursei JA 3890b Microbiol., 14, 659-673).

A method has already been proposed according to which nourseothricin is produced with this strain in an aerobic submersed process. A disadvantage of this method is the use of inhibitors of the respiratory chain of the type of Alkaliazide, Brenzkatechins and / or Amytals and inhibitors of amino acid transport. In a further proposal, in one embodiment, a fermentative nourseothricin production process is specified in which the constant presence of glucose in the culture solution is ensured by periodic addition. The disadvantage is that this larger quantities of glucose, which must be prepared only from polymeric carbohydrates containing raw materials are needed.

In a publication (Gräfe, U., Bocker, H., Reinhard, G., and Thrum, H., 1974: Regulatory influence on nourseothricin biosynthesis by o-aminobenzoic acid in cultures of Streptomyces noursei JA 3890b, Z. AIIg. Microbiol , 659-673) describes a nutrient medium in which maize starch is used as carbon source. In this process, using the parent strain of Streptomyces noursei JA 3890 b also with the addition of inhibitors of the above types and inhibitors, described in the patent WP A61 K / 223321, such a low yield of nourseothricin obtained that an economical production is not possible is.

Object of the invention

The invention includes the goal of producing nourseothricin in a sufficiently effective manner on hexose-poor, in particular low-glucose nutrient media.

Explanation of the essence of the invention

The invention has for its object to provide a technically advantageous method which allows the industrial production of nourseothricin by means of a hexose-poor, di-, oligo- or polymer-containing carbon source in the nutrient medium. It has been found that certain nourseothricin-producing strains having sufficient amylolytic activity, with starch as the primary low-hex, polymeric carbohydrate source, accumulate high levels of nourseothricin in the nutrient solution. Surprisingly, it was found that these strains accumulate in these large amounts even without the addition of inhibitors of the respiratory chain or of the amino acid transport nourseothricin. These findings resulted, for example, for those under no. ZIMET43700, ZIMET 43701 and ZIMET 43708 deposited in the depository of the ZIMET deposited nourseothricin-producing strains.

As a result of this finding, the method according to the invention is carried out as follows:

Vegetative inoculum of the nourseothricin-producing strain used, which has been cultured from mycelium in a manner known per se in several stages, is used to inoculate fermentation broths of the following basic composition:

Starch, in particular corn starch, as main carbohydrate source in an amount of 50 to 150 grams per liter

- Complex nitrogen sources in the form of peanut extraction or soybean meal in an amount of 5 to 25 grams per liter and / or corn steep liquor in an amount of 1 to 10 grams per liter

Inorganic nitrogen source, for example, ammonium sulfate in an amount of 5 to 15 grams per liter

- Calcium carbonate, for example in the form of chalk, in an amount of 4 to 20 grams per liter

Defoamers, for example defoamers based on silicone or polyethylene glycols and / or vegetable fats.

In the course of a 72- to 196-hour fermentation at temperatures of 28 to 3OX with and without replenishment of liquefied, concentrated polymeric carbohydrate sources using enzyme preparations m.t amylolvtischer activity, for example, brewery enzyme, in a conventional manner, and be. Constant maintenance of the acetic acid in the formulation By adding 25% ammonium hydroxide solution at a pH of 5.0-8.0, but preferably at pH 5.5-6.5, a high concentration of nourseothricin is obtained reached in the Kulturlosung. The workup of Nourseothricins done in a conventional manner.

embodiments

The invention will be explained in more detail by way of examples:

example 1

Example Ί

From a mycelium preserve of the strain Streptomyces noursei ZIMET 43700 pre-cultures are created. These contain 100 ml each of 500 ml steep bottles or 400 ml each of the culture medium in 2.5 liter Erlenmeyer flasks of the following composition: peanut extraction 15 g per liter of tap water, corn steep liquor 40g, ammonium sulphate 5g, calcium carbonate 6g , The pH is adjusted to pH 6.5 to 6.8 before sterilization. Sterilization is carried out at 121 ^° C. for 35 minutes. . ,,. _nnn ._ "

The first cultivation stage is cultivated for 48 hours at 29 ° C to 3OX on a rotary vibrating table. After the presence of a sufficient amount of mycelium, the transfer of each 20 ml of preculture solution as inoculum is carried out on the entire cultivation phase, which is cultured under the same conditions for 24 hours. "^" H "" "

For the inoculation of a 15 l fermentor with 101 nutrient solution, the content of two culture flasks of the second growing phase

Composition of the nutrient solution: per liter of tap water corn starch 80g, peanut extraction meal 25g, Maieque water 10g, ammonium sulphate 10g, sunflower oil 3g, Rügen chalk 5g. The pH is adjusted to 6 ⁸ b, s7 0 before sterilization. Sterilization is carried out for 30 minutes at 121 ° C. During the fermentation, 300 g of starch (after treatment with brewing enzyme in a known manner) are added twice, and the acidity of the fermentation solution is adjusted to pH 6.2 by means of ammonium hydroxide At the end of a 144 hour fermentation at a speed of 360 rpm and aeration rate of 0.75VVM, 7.2 grams of nourseothricin per liter of culture solution were obtained

certainly.

Example 2.

Two shake flaps (2.5 l) each containing 400 ml of nutrient solution are inoculated with 0.5 ml each of a mycelium preserving sponge of the strain Streptomyces noursei ZIMET 43701 and cultured for 48 hours at 29 ° C. on a round-shake. Composition of the nutrient solution: per liter of tap water corn starch 15g, soybean meal 15g, potassium dihydrogen phosphate 0.3g, sodium chloride 5g, calcium carbonate 1g. The pH of the solution is adjusted to pH 6.5 to 6.8 before sterilization. Sterilization is carried out at 121 ^° C. for 30 minutes.

The combined contents of the first precultures are used to inoculate a 250 l tank with 1501 Nährlosung the 9 oak composition as in the first preculture. The cultivation is carried out at 29 ° C for 40 hours be. a speed of 240 rpm and a ventilation rate of 0.5VVM. , ,

451 of this seed culture are used as the inoculum for 400 L broth adjusted to an acidity of pH 6.8 to 7.0 in a 720 L fermentor. The cultivation is carried out at a ventilation rate of 1.0WM and a speed of 320UPM at 29 ° C. The acidity of the culture solution is automatically kept constant at a pH of 6.2 by addition of 25% ammonium hydroxide solution.

The nutrient solution has the following composition: per liter of tap water corn starch 60g, peanut extraction meal 10g corn steep liquor 2g, ammonium sulphate 8g, zinc sulphate 0,05g, manganese sulphate 0,03g, sunflower oil 0,5g, chalk 3,75g.

The acidity of the nutrient solution is adjusted to a pH of 6.8 to 7.0 before sterilization. Sterilization is carried out with direct steam at 12 OX for 30 minutes. In the course of the fermentation, 6 times 6 kg of maize starch liquefied in a manner known per se with brewing enzyme as concentrated solution (about 30%), for the 30th, 45th, 54th, 70th, 100th and

120th hour added. , , ,

After 120 hours of fermentation, a culture solution of 5501 was harvested, the concentration of Nourseothr.cin being 6.53 grams per liter.

Example 3

For Impfamtanzucht a lyophil dried on glucose gelatin mycelium (gross weight of the canned contents about 300mg) of the strain Streptomyces noursei ZIMET 43708 with 3ml physiological saline solution aufqgeschwämmt. 0.5ml of this suspension is used as the inoculum of 400 ml first-stage pre-breeding medium. The pre-breeding medium contains the following starting materials per liter of tap water: glucose 40 g, soybean meal 15 g, potassium dihydrogen phosphate 0.3 g, sodium chloride 5 g and calcium carbonate 3 g. . ",.,. , ·,

Acidity is adjusted to pH 6.5 before sterilization. Per 400ml of this medium are filled in 2.5I, closed with cotton stopper Steil Breast Bottles. Sterilization is carried out at 12 OX in an autoclave for 35 minutes. The inoculated cultures are incubated on swing tables at 29X for 48 hours. M'tJOOml of the culture solution of the first preemergence stage thus obtained is inoculated as a second stage in a 1801 steam-sterilized medium (45 minutes at 120 ° C.) of the same composition as the stainless steel fermentor filled in the first pre-breeding stage (gross volume 250I). The culture is carried out over a period of 40 hours (pH 5.1 to 5.3) at 28X b.s 29X, a speed of 240 rpm and a ventilation rate of 1.0WM. For the main culture serves a Edelstahlfermentor (gross volume 42001), which with

2500I nutrient solution of the following / on 1 liter of tap water related composition is filled. Maize starch 62.5, glucose Z, 5g, soybean meal 12g, ammonium sulphate 11g, magnesium sulphate 2g, sodium chloride 1g, calcium carbonate 6g, zinc sulphate 0 5q polypropylene glycol 0.5g. Acidity is adjusted to pH 6.2 before sterilization. The sterilization of the nutrient medium (without glucose) takes place in situ by preheating with jacket steam to 75X and subsequent regeneration by means of direct steam at 120X for 15 minutes. After cooling to 29X, the solution is added to a 40% aqueous solution by heating for 30 minutes on 12OX separately sterilized glucose under aseptic conditions. Prior to inoculation, the starting acidity of the broth is adjusted to pH 6.8 ± 0.1 with sulfuric acid.

The fermentation batch of the main culture inoculated with 4% inoculum from the second preemergence stage is maintained at 29 ⁰ C for 140 hours, a ventilation rate of 0.5VVM (0 to 20 hours) and 1.0WM (20 to 14O.Outs) at 0 , 03MPa overpressure with stirring cultured at 180 rpm. The acidity of the culture solution is controlled to a pH of 6.0 ± 0.1 with 25% ammonium hydroxide solution after physiologically induced slow drop from the starting pH. In the biological test, the following amounts of product were determined in the culture solution: Fermentation hour Nourseothricin (h) (gr ¹ )

68th 3.9 92nd 5.2 116th 7.5 140th 9.7

Example 4

The precultivation is carried out as described in Example 3; The strain ZIMET 43708 is also used. To the main culture, a stainless steel fermentor (gross volume 7201) is used, which is filled with 500 liters of nutrient solution following 1 liter of tap water: corn starch 62.5g, glucose 2.5g, soybean meal 12g, ammonium sulphate 11g, magnesium sulphate 2g, sodium chloride 1g, calcium carbonate 6g , Iron (III) sulphate 0.1g, polypropylene glycol 0.5g. The acidity of the solution is adjusted to pH 6.2 before sterilization. The sterilization of the nutrient medium (without glucose) takes place in situ by preheating with jacket steam to 75 ° C and subsequent heating by direct steam to 12O ⁰ CfUr 15 minutes. After cooling to 29 ⁰ C, the strength in the form of a 50% aqueous solution by 30 minutes heating at 120 ° C separately sterilized glucose is added under aseptic conditions. Prior to inoculation, the starting acidity of the broth is adjusted to pH 6.8 ± 0.1 with sulfuric acid.

The main fermentation batch inoculated with 4% inoculum from the second preemergence stage is run at 29 ⁰ C for 116 hours, a ventilation rate of 0.5VVM (0 to 20 hours) and 1.0WM (20 to 116 hours) at 0.03MPa Overpressure cultured with stirring at 320 rpm. The acidity of the culture solution is constantly controlled after physiologically induced slow decrease from the starting pH to a pH of 6.0 ± 0.2 with 25% ammonium hydroxide solution. In the biological test, the following product concentrations were determined in the culture solution: Fermentation hour Nourseothricin (h) (gr ¹ )

44th 2.2 68th 4.0 92nd 5.2 116th 6.5

Example 5

The pre-cultivation is carried out as described in Example 3; The strain ZIMET 43708 is also used. To the main culture, a stainless steel fermentor (gross volume 450I) is used which is filled with 300I nutrient solution of 1 liter of tap water composition: corn starch 120g, glucose 5g, soybean extraction 24g, ammonium sulphate 11g, magnesium sulphate 2g, sodium chloride 1g, calcium carbonate 6g, zinc sulphate 0, 5g and polypropylene glycol 0.5g.

To liquefy the high proportion of polymeric carbohydrate source brewing enzyme is used in a conventional manner. After the enzymatic hydrolysis of the starch, the remaining nutrient medium components are filled. The sterilization of the nutrient medium is carried out by heating to 120 ° C for 15 minutes. After cooling to operating temperature, the starting acidity of the nutrient medium is adjusted to the pH 6.8 ± 0.1 with sulfuric acid prior to inoculation. The main fermentation batch inoculated with 4% inoculum from the second preemergence stage is maintained at 29 ° C for 124 hours, a ventilation rate of 0.5WM (0 to 20h) and 1.0WM (20 to 124h) at 0.03MPa Overpressure with stirring with 400UPM cultivated.

At the 16th fermentation hour takes place in the addition of 12mg · I ~ ¹ phosphate-phosphorus (as potassium dihydrogen phosphate) in the form of a sterilized aqueous solution. The acidity of the culture solution is constantly controlled after physiologically induced slow decrease from the starting pH to a pH of 6.0 ± 0.2 with 25% ammonium hydroxide solution. In the biological test, the following product concentrations were determined in the culture solution. Fermentation hour Nourseothricin (h) (gr ¹ )

44th 2.6 68th 6.3 92nd 9.4 116th 12.4 124th 15.1

Example 6

The pre-cultivation is carried out as described in Example 3; the strain 43708 is used.

The nutrient solution and its preparation also correspond to that shown in Example 3. Two stainless steel fermenters (gross volume 720I), each filled with 500I nutrient solution, are used.

The inoculum amount and the process regime are also the same as in Example 3. In a fermentation batch, an addition of 10 mg.l ^{-1 of} phosphate-phosphorus (as potassium dehydrogen phosphate) in the form of a sterilized aqueous solution is carried out at the 12th fermentation hour. The other fermentation batch receives no addition.

In the biological test, the following different rapidly increasing nourseothricin concentrations were determined in the culture solutions:

fermentation hours nourseothricin r ¹ ) With (H) (G· Phosphate-phosphorus additive 4.1 without 5.6 3.8 7.4 68th 4.9 7.5 92nd 6.1 116th 7.1 140th

Example 7

The pre-cultivation is carried out as described in Example 3; The strain ZIMET 43708 is also used. To the main culture, a stainless steel fermentor (gross volume 720I) is used, which is filled with 500I nutrient solution of 1 liter of tap water composition: corn starch 62.5g, glucose 2.5g, soybean meal 16g, ammonium sulphate 11g, magnesium sulphate 2g, sodium chloride 1g, calcium carbonate 6g , Aluminum sulfate hydrate 0.13g, polypropylene glycol 0.5g. The acidity of the solution is adjusted to pH 6.2 before sterilization. Sterilization is carried out by heating to 120 ° C for 15 minutes. After cooling to 29 ° C, the starting acidity of the nutrient medium is adjusted to the pH 6.8 ± 0.1 with sulfuric acid before inoculation.

The main fermentation batch inoculated with 4% inoculum from the second preemergence stage is at 0.03 for 116 hours to 29 ° C, an aeration rate of 0.5WM (0th to 20th hour) and 1.0WM (20th to 116th hour) MPa overpressure with stirring cultivated at 320 rpm. The acidity of the culture solution is kept constant at a pH of 6.0 ± 0.3 with 25% ammonium hydroxide solution after physiologically induced slow decrease from the starting pH. In the biological test, the following product concentrations were determined in the culture solution: fermentation hour nourseothricin <h) (gr ¹ )

44th 2.5 68th 4.9 92nd 7.2 116th 9.3

Claims (4)

Invention claim: 1. A method for the fermentative production of Nourseothricin with strains of Streptomyces noursei under submerged aerobic culture conditions in a medium with suitable carbon and nitrogen sources and sufficient concentrations of mineral salts at a temperature of 28 ° ^{C to} 32 ° C while maintaining the acidity of the culture solution at a fixed limit in the Range of pH 5.0 to 8.0, characterized in that power mutants or selectants of Streptomyces noursei with sufficiently high amylolytic activity are used and culturing for 72 to 196 hours using low-hexose, especially low-glucose, di- and / or oligomeric and / or polymer-containing carbohydrate sources, but also without the presence of specific inhibitors of the respiratory chain or the amino acid transport takes place. 2. The method according to item 1, characterized in that are used as strains of Streptomyces noursei with sufficiently high amylolytic activity, the strains ZIMET 43700, ZIMET 43701 or ZIMET 43708. 3. The method according to item 1 and 2, characterized in that is used as a hexose, polymer-containing carbohydrate source starch, preferably corn starch. 4. The method according to item 1, 2, and 3, characterized in that the starch in a manner known per se treatment to increase the fluidity of their aqueous suspensions in full amount at the start of fermentation and / or in portions or continuously during of the fermentation process is added to the culture medium.