Dissociation of a Candida tropicalis culture and its capacity to stimulate levorin synthesis when cultured together with Actinomyces levoris

Dissociation of the yeast-like fungi C. tropicalis used for mixed cultivation with the levorin-producing organism was studied. It was shown that colonies of 2 types were grown on wart-agar. They differed in the morphology and capacity for stimulation of the antiobiotic synthesis in mixed fermentation with 2 organisms. The colonies of the S-form increased the antibiotic production by 50--60 per cent, while the R-forms increased the antibiotic production only by 25--30 per cent. The strains of C. tropicalis differed in the ratio of the S- and R-forms in both the initial cultures and the cultures stored for prolonged periods of time. Addition of KH2PO4 to the agarized in the amount of 0.5 per cent promoted preservation of the S-forms in the population. Repeated subsequent transfers of the S-variants (10 generations) on wart-agar did not decrease the stimulating effect of C. tropicalis on the actinomycete. An increase in the amount of the inoculum of the yeast-like fungi promoted a decrease in the time of their preliminary cultivation.     

Effect of different pH values on the medium on the synthesis of antibiotics in the joint cultivation of Actinomyces levoris with yeasts]

Changes in the pH level of the fermentation medium used for preliminary cultivation of C. tropicalis were studied with respect to its initial aciditv or alkalinitv. When C. tropicalis was grown on the medium used for levorin fermentation with ph 5.1--10.3, the yeast changed it in 24 hours to the level of 6.2--7.9. As dependent on the initial pH values for cultivation of C. trophicalis, production of levorin on subsequent inoculation of Act. levoris changed. The antibiotic activity increased and ranged within 120--178% of the control. Synthesis of levoristatin, a non-polyenic antibiotic equally increased under such conditions and ranged within 153--163% of the control. The pH values of 9.4--10.3 of the initial fermentation medium were optimal for mixed cultivation of Act levoris and C. tropicalis and maxium production of levorin and levoristatin.     

The role of succinic acid in the biosynthesis of levorin

The influence of succinic acid as a component of media for biosynthesis of levorin, a polyenic antibiotic was studied. It was shown that with the use of the soybean-corn medium supplemented with succinic acid (0.05-0.4 per cent) the antibiotic content in the fermentation broth was higher than that in the control. The highest stimulating effect (135 per cent) was observed with addition of 0.1 per cent of succinic acid. For providing optimal antibiotic production in the synthetic medium supplemented with succinic acid (0.4 per cent) addition of acetic acid (0.05 per cent) was required. Studies with the soybean-corn medium with and without succinic acid revealed differences in the level of p-aminoacetophenone, an aromatic fragment of the levorin molecule. Under the conditions of the medium with succinic acid the content of p-aminoacetophenone in the mycelium was higher by 10 to 18 per cent as compared to that in the control and depended on the fermentation period. The role of succinic acid in biosynthesis of levorin is discussed.     

Determining the conditions for stimulation of levorin biosynthesis in the presence of succinic acid

Conditions promoting the stimulating effect of succinic acid on biosynthesis of levorin were determined. The optimal concentration of succinate was shown to be equal to 0.05-0.3 per cent and the best time for the addition was before inoculation. The most pronounced stimulating effect was observed when the initial pH value of the fermentation medium was equal to 7.0. The important role of ammonium nitrogen in manifestation of the succinic acid stimulating effect on biosynthesis of levorin was suggested.     

Sensitivity of fungi of the genus Candida to antifungal drugs

Seventy two Candida strains isolated from patients with candidiosis of the oral mucosa were studied with respect to their sensitivity to nystatin, levorin, decamine, ethonium, sanguiritrin and clotrimazole. At concentrations of 0.5 to 5 micrograms/ml all the Candida species i.e. C. albicans, C. tropicalis, C. krusei and C. quilliermondii were highly sensitive to clotrimazole. Fungistatic action of levorin, nystatin and sanguiritrin was observed in 91, 67 and 38 per cent of the strains respectively. The Candida strains were resistant to decamine and ethonium used in the above concentrations.     

Action of the products of the vital activity of yeastlike fungi on the biosynthesis of levorin, levoristatin and fatty acids by a Streptomyces levoris culture

The data on the effect of the products of vital activity of Candida tropicalis, a yeast-like fungus, on the biosynthesis of levorin, levoristatin and fatty acids by Streptomyces levoris are presented. It was shown that the effect of the biostimulators was not specific with respect to production of levorin, since in the presence of the products of vital activity of C. tropicalis an increase in the synthesis of levoristatin and fatty acids was also observed. The qualitative and quantitative composition of the fatty acids of the mycelium of S. levoris was studied. Interrelation between the biosynthesis of levorin and synthesis of unsaturated fatty acids and branched chain fatty acids was noted.     

Effect of inorganic phosphate on levorin biosynthesis and on the mycelial makeup of Streptomyces levoris]

The effect of inorganic phosphate on biosynthesis of the polyenic antibiotic levorin by Streptomyces levoris and composition of the culture mycelium was studied. It was found that the synthetic medium with 0.4 mM of phosphate was optimal for growth of Str. levoris. When the concentration of phosphate was higher, the biomass increased, while the synthesis of levorin appeared to be inhibited and morphological changes in the culture were observed. Phosphate had a significant effect on the mycelium composition. When its concentration was increased 10 times as compared to the optimal one, the amounts of protein, RNA, total phosphorus and polyphosphates increased 1.3--1.4, 1.6--1.7, 2--3 and 10 times respectively, while the synthesis of levorin decreased 5 times. Changes in the lipid component of the mycelium were also observed. In the absence of inorganic phosphate in the medium the acetone precipitating fraction of the lipids contained 20--40 per cent of the phosphoruless compounds. During cultivation their portion increased up to 70--77 per cent. However, in the presence of its excess the polar lipids were represented only by phospholipids during the whole life cycle. The fatty acid spectrum of the lipids did not depend on the phosphate concentration and was represented mainly by saturated fatty acids with a branched chain of a series of iso- and anteiso-structures containing 14--18 carbon atoms.     

Nature of the stimulating substances found in the vital activity products of yeastlike fungi

The cultural broth of Candida tropicalis was shown to contain an active compound which stimulated the synthesis of levorin, a polyene antibiotic Succinic acid was found to stimulate the antibiotic synthesis. The stimulating effect of the active compound increased in proportion to the content of succinic acid in it and became maximal at the same concentration of succinic acid as in a pure preparation. However, succinic acid was not an only compound responsible for the elevated synthesis of the antibiotic since the biostimulating effect was higher than that of pure succinic acid.     

Effect of a modifying factor on the sensitivity of Crithidia oncopelti to levorin

The paper deals with possible discovery of ways for increasing sensitivity of trypanosomides to polyenic antibiotics. The following substances were tested: sodium pyruvate and acetate, calcium salts, ascorbic acid and 1-valine. The total number of the cells and the number of the viable cells in the culture and their morphological characteristics were used as the criteria for estimation of the C. oncopelti sensitivity. It was shown that sodium acetate most actively modified the levorin effect on C. oncopelti. Its addition in a concentration of 40 mg/ml to the cultivation medium with levorin in a concentration of 1 microgram/ml induced a trypanocidal effect. With the use of levorin alone such an effect was observed when the antibiotic was used in a concentration of 10 micrograms/ml. The growth rate of the protozoon was decreased by 60-80 per cent as compared to the control. The number of the viable cells was lowered 4 times. The morphology of the culture markedly changed. This indicates that the presence of sodium acetate as a modifier in the culture medium allowed one to decrease 10 times the dose of levorin and to preserve the trypanocidal effect.     

Effect of a biostimulant formed by yeastlike fungi on the dynamics of the accumulation of CoA, biotin and levorin in the process of growth of S. levoris

Regularities of the effect of a biostimulator produced by years-like fungi on accumulation of CoA, biotin and levorin in a developing culture of S. levoris were studied. It was shown that addition of the biostimulator to the fermentation medium resulted in increased accumulation of CoA and biotin in the mycelium of the levorin-producing culture within the first 48 hours of the growth and in their more intensive consumption at the final stages of the fermentation process. The rate of the levorin synthesis in the medium with the biostimulator markedly exceeded that in the control.     

Differential spectrophotometric method of analysing levorin in the culture broth and in the mycelium]

It was shown on model experiments that the microbiological method was not applicable for determination of levorin content in industrial intermediate products containing in addition levoristatin, since the presence of the latter made higher the results of the microbiological assay. Because of this till to the present date the quantitative content of levorin in the industrial intermediate products was determined photometrically using alcohol (pure solvent) as the reference solution. Still, this method also made higher the results of the assay, especially when the content of levorin was determined in the fermentation broth. In the solid phase levorin is contained in the mycelium which occupies only 1 to 2 per cent of the fermentation broth, while the liquid phase or the fermentation broth filtrate occupies 98 to 99 per cent. It was found that the fermentation broth filtrate contained protein admixtures which coagulated on addition of alcohol to the fermentation broth and formed fine colloid solutions. As a result the absorption values became higher. In the present study not the pure solvent but an extract of the fermentation broth filtrate containing neither levorin, nor levoristatin was used as the reference solution. Such a differential method provided elimination of all errors due to the presence of various metabolites in the fermentation broth filtrate which varied both qualitatively and quantitatively.     

Effect of amigluracil and levorin on membrane permeability and protein synthesis in Candida albicans protoplasts]

It was shown that amigluracyl, a water soluble derivative of methacyl which decreased the nephrotoxic effect of polyens activated the membrane permeability in Candida albicans for a mixture of 14C-amino acids but had no significant effect on protein synthesis in this microorganism. The level of inhibition of the membrane permeability in C. albicans for the amino acids and protein synthesis in the fungus by levorin did not practically depend on the presence of amigluracyl in the incubation medium. The minimum levorin concentration inhibiting the growth of Candida albicans in the presence or absence of levorin was 0.039 gamma/ml. Therefore, amigluracyl may be used in combination with polyenic antibiotics for the treatment of mycoses.     

Biosynthesis of levorin and activity of several enzyme systems of Streptomyces levoris depending on culture conditions

The regularities of changes in the main oxidation-reduction enzymes of the tricarboxylic acid cycle (TAC) and the pentose cycle were studied under different cultivation conditions: with the use of the control soybean-corn-hydrol medium and the medium with addition of a biostimulant produced by C. tropicalis. It was shown that the activity levels of the dehydrogenase systems of the TAC and the pentose cycle of S. levoris grown in the presence of the biostimulant were higher. The increase in the production levels of levorin due to addition of the biostimulant was connected with the activity of the systems responsible for regeneration of NADP.H2.     

Physiological characteristics of the strains of Cryptococcus deffluens--producers of penicillin acylase]

A fermentation medium balanced by the main components was developed for Cryptococcus diffluens strains producing penicillin-V-acylases (PA). It was shown that the culture needed for production of the enzyme was inductor, which was phenoxyacetic acid (POAA). Additional introduction of ethanol to the medium provided an increase in production of PA by 36 per cent and the culture growth by 25 per cent. Introduction of one of the following substances to the medium with POAA and ethanol i.e. (CN3COO)2Ca, FeSO4, proline or asparagine provided an additional increase in the production level of PA by 24 to 94 per cent. The use of the medium varieties will permit one to isolate highly productive cells of the culture.     

Physiological difference during ethanol fermentation between calcium alginate-immobilized Candida tropicalis and Saccharomyces cerevisiae

Calcium alginate-immobilized Candida tropicalis and Saccharomyces cerevisiae are compared for glucose fermentation. Immobilized C. tropicalis cells showed a slight morphological alteration during ethanol production at 40 degrees C, but their fermentation capacity was reduced by 25%. Under immobilization conditions, the two species demonstrated two different mathematical patterns when the relationship between growth rate, respiration rate, and ethanol tolerance was assessed. The interspecific difference in behavior of immobilized yeast cells is mainly due to their natural metabolic preference. The production of CO(2) by calcium alginate-immobilized C. tropicalis, as well as the lower supply of oxygen to the cells, are the major factors that reduce ethanol production.     

Realization of the maximum specific growth rate of yeasts on optically heterogeneous media

The method of controlling the rate of the nutrient medium flow was developed with respect to the level of heat release during yeast cultivation on the optically heterogeneous nutrient medium. This method of cultivation allowed attainment of the maximum specific rate of growth of Candida tropicalis which was estimated to be 0.45 hr-1 in the experiments.     

Oleandomycin content of mycelium and fermentation solution during cultivation of Streptomyces antibioticus

The time-course of the oleandomycin content in the mycelium and fermentation broth-filtrate was studied by the microbiological assay at different periods of cultivation of strains 471 and 961 in fermenters and flasks containing a rich soybean-corn medium. It was shown that centrifugation of the mycelium over the sucrose density gradient induced a 25-80 per cent decrease in its moist weight at the expense of removal of the admixture components of the rich medium. Addition of glucose (2 per cent) to the culture-grown in a lactose medium by the 72nd hour of fermentation had no effect on further increase of the cell biomass. However, it lowered the content of the mycelium-fixed and excreted antibiotic at all the subsequent fermentation periods. The content of oleandomycin in the untreated mycelium was only 0.36 per cent of its content in the fermentation broth filtrate. After centrifugation of the mycelium over the sucrose density gradient and its intensive washing with distilled water the content of the mycelium-fixed antibiotic decreased still more. The time-course of the content of the mycelium-fixed and excreted oleandomycin was characterized by the presence of two activity peaks; by the 80-110th and by the 140-170th hour of cultivation.     

Radioprotective and antineoplastic activity of polyene antibiotics combined with dimethyl sulfoxide]

Radioprotective and antineoplastic activity of polyene, its derivatives and combinations with dimethyl sulfoxide (DMSO) was studied. The most potent radioprotective effect was demonstrated by methylated levorin, original levorin and by its isomer--isolevorin. Survival rate of the animals on 12th day after X-ray exposure was 100, 60, 60 per cent, at the control group 33.6, 20 and 0 per cent consequently. Levorin and alkyl derivatives of amphotericin B--methamphocin and buthamphocin inhibited growth of ascites and solid tumors to 46.3-79.0 per cent when compared to control group. Polyen antibiotics combined with DMSO also demonstrated antineoplastic activity at the animals treated with carcinogenic agent--diethyl nitrosoamine (DENA). 5-month survival of the animals was 76 per cent at nystatin and levorin group and 35.7 per cent at the control group (animals treated with DENA only).     

口腔黏膜真菌鉴定方法的比较

比较常见用于黏膜真菌菌种鉴别的多种方法,探寻最佳的鉴别方法。采集230例普通人群口腔黏膜样本,分别用玉米吐温-80培养观察厚膜孢子法、糖发酵生化反应法、CHROMagar假丝酵母菌显色培养基法、ITS基因的PCR-RFLP(聚合酶链反应-限制性片段长度多态性)法、ITS测序菌种鉴定法,鉴别真菌各菌株。结果显示:有56例菌株至少通过1种方法检出真菌;玉米吐温-80分离培养假丝酵母菌37株;50例菌株ITS基因测序共鉴定出8个菌种,白假丝酵母菌(C.albicans)29株,近平滑假丝酵母菌(C.parapsilosis)10株,热带假丝酵母菌(C.tropicalis)5株,Candida metapsilosis 1株,Lodderomyces elongisporus 1株,克柔假丝酵母菌(Candida krusei)1株,乙醇假丝酵母菌(C.ethanolica)1株,季也蒙毕赤酵母菌(Pichia guilliermondii)2株;CHROMagar假丝酵母菌显色培养基法鉴定出3种菌株,分别是白假丝酵母菌、热带假丝酵母菌、近平滑假丝酵母菌;PCR-RFLP法检出5种菌株,分别是白假丝酵母菌、热带假丝酵母菌、近平滑假丝酵母菌、季也蒙毕赤酵母菌、克柔假丝酵母菌,与基因的测序鉴定一致率为91%;糖发酵生化反应法阳性标本占被检出真菌例数的46.4%(26/56)。结果表明:ITS基因的测序法可以准确鉴定真菌各个菌种;PCR-RFLP法能鉴定常见的菌种,但操作繁琐;CHROMagar假丝酵母菌显色培养基法能快速准确鉴别3种常见假丝酵母菌菌种;玉米吐温-80可以准确培养鉴别白假丝酵母菌;糖发酵生化反应法,缺乏足够的敏感度和特异性,难以准确鉴别各个菌种。     

Production of cytokinin-like substances by mycorrhizal fungi of pine (Pinus sylvestris L.) in cultures with and without metabolites of actinomycetes

Studies on the effect of post culture liquids of actinomycetes on cytokinin-like substances production by mycorrhizal fungi have revealed that actinomycete metabolites inhibited or stimulated the synthesis of these compounds. The results of chromatographic analyses suggest, that substances stimulating the soybean callus are likely to be: riboside 6 (gamma, gamma-dimethylallylamino) purine and riboside zeatin. Using gas chromatography it was confirmed that both substances are produced by Rhizopogon luteolus. Paxillus involutus synthesizes probably besides the two substances also zeatin, as appears from the data obtained by column chromatography.