Fungicidally active antibiotic from streptomyces tendae Ettlinger et al. Tu 901

#The present invention relates to a new antibiotic, a microbiological process for its preparation from strains of Streptomycetes and its use as a plant protection agent.

#It has already been disclosed that polyoxines have found broad application as fungitoxic agents in plant protection. However, their disadvantage is that they are labile to alkali and furthermore that they only occur as mixtures of varying composition and are therefore difficult to dose precisely, and to standardize.

#It has now been found that the new antibiotic Nikkomycin can be obtained by aerobic culture of micro-organisms of the order Actinomycetales, especially of the family of the Streptomycetaceae, and in particular of strains of the genus Streptomyces, in an aqueous nutrient medium, followed by isolation therefrom.

#(2)b. The UV spectrum and the IR spectrum have been recorded (compare the corresponding representations in FIGS. 1 and 2). The UV spectrum of Nikkomycin, recorded in (a) 1 N hydrochloric acid and (b) in 0.1 N sodium hydroxide solution, is recorded in FIG. 1. The ordinate shows the extinction and the abscissa shows the wavelength (nm units). The IR spectrum of Nikkomycin, recorded in potassium bromide, is shown in FIG. 2. The ordinate shows the transmission in % and the abscissa shows the wave number (cm.sup.-1) and wavelength (.mu.m units).

#c. Uracil, an amino-hexuronic acid and a new amino acid containing a pyridine ring were detectable by means of mass spectrometry and of chemical degradation by acid hydrolysis.

#d. Paper electrophoresis: the antibiotic is amphoteric. Around a pH value of 6 the migration distance is small and accordingly this is probably the isoelectric point. The behavior of Nikkomycin on electrophoresis is shown in the Table which follows, in which the migration distances in paper electrophoresis are given as a function of the pH values of the buffer system.

#The nutrient solution in which the producer strain Streptomyces tendae Tu 901 is cultured is composed of 2% of soya flour and 2% of mannitol; the pH is adjusted to pH 7.5 before sterilizing. 10.times.500 ml Erlenmeyer flasks with 1 lateral intrusing, each containing 100 ml of nutrient solution, are inoculated with the producer strain and incubated for 48 hours at 27.degree. C. on a rotary shaking machine at 120 revolutions/minute. A 10 l fermenter ("New Brunswick"), which contains 10 l of nutrient solution, is inoculated with this pre-culture and incubated at 220 revolutions/minute and 27.degree. C., with an air supply of 4 l/minute, for 48 hours. A 100 l fermenter ("New Brunswick"), which contains 100 1 of nutrient solution, is inoculated with this pre-fermenter and incubated at 150 revolutions/minute and 27.degree. C., with an air supply of 450 l/minute, for 78 hours.

#The culture, with the addition of 2% of a filter aid (Hyphlo Supercel.RTM. , Johns Mansville), is pressed out by means of a filter press, first through a pre-clarifying filter (C 150, Schenk) and then through a post-clarifying filter (U 1,000, Schenk). The clear culture filtrate is acidified to pH 4.0 with acetic acid and charged onto a column (100 .times. 450 mm) filled with Dowex 50 W X 4.RTM. cation exchange resin (50-100 mesh, Na.sup.+ form). The flow rate is 10 l/hour. The column is washed with deionized water until the liquid issuing from the column is completely colorless. The antibiotic is eluted with 30 l of 0.01 N ammonia and 30 l of 0.05 N ammonia. The biologically active eluate is freed from ammonia on a rotary evaporator, acidified to pH 4.0 with acetic acid and applied to a column (70 .times. 900 mm) filled with "Amberlite 252".RTM. cation exchange resin (Na.sup.+ form). The flow rate is 5 l/hour. The column is washed with deionized water until the liquid issuing from the column is completely colorless. The antibiotic is eluted with 15 l of 0.05 N ammonia. The biologically active eluate is freed from ammonia on a rotary evaporator, concentrated to a small volume (1 l), acidified to pH 4.0 with acetic acid and charged onto a column (25 .times. 850 mm) filled with SP-Sephadex C-25 dextran. The flow rate is 100 ml/hour. The column is washed with deionized water until an adsorption is no longer shown in the UV continuous flow detector at 280 nm (Uvicord II, LKB). Impurities are eluted with 0.01 M pyridine acetate buffer (pH 4.7) and the antibiotic is eluted with 0.02 M pyridine acetate buffer (pH 4.7). The biologically active fractions are combined, freed from the buffer on a rotary evaporator and concentrated to a very small volume. The concentrate (10 ml) is charged onto a column (25 .times. 1,500 mm) filled with Bio-Gel P 2 cellulose (200-400 mesh) and eluted with deionized water. The flow rate is 100 ml/hour. The purity is checked by detection at 280 nm using a UV continuous flow detector. The biologically active fractions are combined and lyophilized in a freeze-drying unit.

#In the foregoing Example, the sources of the trademarked auxiliaries and technical instruments were as follows: