Fusafungine

Locabiotal,Servier,France,1963

In a 5-liter round flask provided with two tubes, one of which is adapted for subsequent connection to a source of sterile air, 2 liters of fermentation medium are prepared according to the following formulation:
Both openings of the flask are stopped with cotton wool and the medium is sterilized by placing it in an autoclave for 30 minutes at 120°C. The flask is then cooled to 29°C to 30°C and a small sample is taken to check the sterility and the pH value which should be approximately 5.
The spores from an inclined culture of Fusarium lateritium Wr, CSB 119.63 on a gelose medium are extracted with sterilized distilled water to obtain a suspension containing about 600,000 spores per ml. This suspension is then used to seed the medium prepared as earlier described. The contents of the flask are left to incubate at 27°C. Sterile air is injected into the liquid to effect thorough agitation and uniform supply of oxygen into the medium.
After 55 hours of fermentation, the contents of the round flask is transferred under aseptic conditions into a metal reactor of about 100 liters capacity containing 60 liters of sterile medium prepared as follows:
The culture is incubated at a temperature of 28°C in the reactor for 60 hours with mechanical agitation and constant aeration. The resulting broth is seeded into 600 liters of a sterile culture medium contained in a metal fermenting vat 1,800 liters in capacity and prepared according to the following formulation:
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The culture is incubated for 55 hours at 28°C with constant forced aeration and agitation, and the broth is seeded into the production medium. In a fermentation vat 12 cubic meters in capacity provided with suitable stirring means, a temperature control jacket, sterile air-injecting and dispersing means, and means for automatically injecting sterile antifoaming agent if required, there are prepared 6 cubic meters of a culture medium of the following formulation:
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The medium is sterilized by heating it at 120°C for 40 minutes and is then cooled to 30°C. After seeding, the medium is incubated for about 60 hours, the temperature being maintained at 30°C. Throughout the period of fermentation, agitation is maintained at a rate of 20-40 rpm and sterile air is injected into the bottom of the vat at a rate of 4.8 cubic meters per minute by means of the air dispersing device. Fermentation is arrested when about 90% of the carbohydrates have been consumed. The average Fusafungine content in the fermentation broth is then found to be about 0.5 to 0.8 grams per liter. The fermented broth is filtered under pressure and the content of the filterpress frames is washed with 2 cubic meters of water, then the filter cake is partially dried in a blast of compressed air. The mycelium is then dried in a ventilated oven at 70°C for 30 hours, dried and ground.
The yield obtained is 88 kilograms of dry product, containing 5.71% of Fusafungine. This is extracted from the crude product as follows: the dry powder is suspended in 836 liters of methanol, and 44 liters of an acetic buffer at pH 4.25 (0.05 M) is added. The mixture is agitated for one hour at ordinary temperature, then drained to separate the exhausted powder from the methanol solution. This solution is transferred into an evaporator in which its volume is reduced to 200 liters. 100 liters of hexane are added, followed by 200 liters of water with agitation. After 15 minutes agitation, the mixture is allowed to stand for 30 minutes and the underlying phase is drawn off. The hexane extract is exhausted with three 25-liter batches of a methanol/water mixture, 3/1 by volume. The methanol mixture is then concentrated to 12.5 liters under reduced pressure. In this concentration step, the methanol is evaporated so that the water content of the residue increases regularly end the Fusafungine precipitates.
The resulting suspension is placed in a round flask equipped with a scraperagitator device, and agitation is effected for 48 hours in an ice water bath. The antibiotic is isolated from the mother liquor by filtration through a Buchner filter. The filter cake is washed with 5 liters of a methyl alcohol and water mixture (1/2.5 by volume) cooled to 4°C. After drying in an oven at reduced pressure, 2.805 kilograms of a greyish-yellow crude product is obtained.
This crude product is dissolved in 140 liters anhydrous undenatured methyl alcohol, then 100 grams of discoloring carbon black, and 100 grams of a filtering aid are added. The mixture is agitated 30 minutes. The carbon black, filtering agent and insoluble impurities are filtered out. The filter cake is washed with 14 liters of methyl alcohol. The filtrate is placed in a receiving vessel, and 280 liters of distilled water at 70°C temperature are poured in with agitation. While continuing to agitate slowly, the mixture is allowed to cool gradually to a temperature of about 35°C. Crystallization is then initiated by adding a few crystals of pure Fusafungine, and agitation is continued for another 12 hours. The crystallization is allowed to proceed for 48 hours at +4°C. The pure Fusafungine crystals are collected by filtration. The filter cake is washed with 10 liters of methanol/water (1/2 by volume) mixture preliminarily cooled to +4°C and then with 20 liters of distilled water. The crystals are dried in an oven at 40°C under reduced pressure. A yield of 2.110 kilograms of pure Fusafungine antibiotics has thus been obtained.

Antibacterial