Producation and Characterization of colored metabiolites and Pigments of microbial Isolates

Abstract

Among different microbes, fungi have been found as an important source of various exogenous natural color metabolites and pigments of immense industrial importance in sectors of food, dyeing, pharmaceutical and cosmetic. Based upon these facts, the present study evaluated the potential of selected fungal isolates towards production of color metabolites. Besides, the color metabolites were biochemically characterized in addition to their biological activities.

Colored secondary metabolites production and their bioactive potential were evaluated in three different newly isolated fungi. Two of these fungi were isolated from Kala pani soil and identified as Penicillium verruculosum SG (KC698959) and Aspergillus fumigatus SG4 (JX863917) whereas, the endophytic fungus isolated from Taxus baccata was Chaetomium strumarium. The pigment production was initially screened out on solid state fermentation conditions using five different media however, Potato dextrose agar (PDA) proved to be the best culture medium. In liquid state fermentation conditions, Potato dextrose broth (PDB) was selected as the best culture medium. After selection of the culture media, different operational parameters viz, pH, temperature, carbon source and concentration, nitrogen source and C: N ratios were optimized. Overall, the production was most efficient at pH 5.5, with 20-40g/L glucose (C source) and yeast extract (N source), C: N ratio was 4:1 for Penicillium verruculosum; 10:1 for Chaetomium strumarium and 8:1 for Aspergillus fumigatus under mesophilic temperature (25-30˚C) in PDB. Standard qualitative and quantitative screening test of the colored extracts of fungi indicated presence of important classes of compounds viz. phenols, flavonoids, anthraquinones, terpenoids, alkaloids and coumarins, showing some similarity between plant and fungal pigments whereas, tannins were absent in them. These compounds were considerably stable in a pH range of 5-8 and ≤100˚C with red pigment of Penicillium verruculosum being stable at even acidic conditions i.e., pH 3. Pigmented filtrates of the fungi revealed a significant antimicrobial activity in terms of zone of inhibition (mm) against test pathogenic bacteria (0-24) and fungi (0-30). Besides, they showed varying antioxidant (EC50 = 7-21 µg/mL) and free radical scavenging activities [EC50 (DPPH) = 10-38, (ABTS) = 95-134 µg/mL]. Howover, the pigmented filtrates of fungi showed a low level of phytotoxicity (Seed germination and shoot length) and cytotoxicity (EC50). Such potential characteristics indicated the likelihood of their utilization in food and pharmaceutical industries.

Fungal isolates viz. Penicillium verruculosum SG, Aspergillus fumigatus SG4 and Chaetomium strumarium SG1 were evaluated for the production of bioactive color metabolites on optimized potato dextrose broth (25˚C). Logical fragmentation pattern following column chromatography, thin layer chromatography (TLC) and liquid chromatography and mass spectrometry (LCMS) of crude culture filtrate of fungus Penicillium verruculosum SG demonstrated presence of polyketide pigments (Monascorubrin, Monascin, Glutamyl Monascorubrine and analogue of Monascorubrine) and other different bioactive compounds viz. Pyripyropene, Orevactaene, N-De methyl Calcimycin and Citrinadin. Cytotoxicity against five different cell lines i.e., KA3IT, MDCK, HSCT6, NIH3T3 and HEK293 of the selected colored fractions of fungal filtrate containing different compounds revealed IC50 (µg/mL) values ranging from 5-100. It was comparatively higher in case of Orevactaene (5+0.44) [(non-significantly differed with Monascorubrine)] followed by Pyripyropene (8+0.63) against cancer cell line KA3IT. Overall, these compounds significantly showed less toxicity towards normal cell lines. X ray crystallography of yellow crystals of a pure compound (224.21 m/z) confirmed its 3 dimensional structure and was identified as Phenazine 1 carboxylic acid (C13H8N2O2), which is previously known as a broad spectrum antibiotic, antifungal and bio-control agent. Some unknown compounds not found in libraries were also detected in the culture filtrate.

In case of Chaetomium strumarium, logical fragmentation pattern following column chromatography, TLC and LCMS of crude culture filtrate revealed the presence of colored compounds i.e., Monascorubramine (purple red), Alizarin and Cochliodinol (Purple). Besides, bioactive colorless compounds Dihydromaltophytin, Nivalenol and Paxilline were also detected. Overall, cytotoxicity against all five cell lines i.e., KA3IT, MDCK, HSCT6, NIH3T3 and HEK293 of the selected colored fractions of fungal filtrate containing different compounds revealed IC50 (µg/mL) values ranging from 1-73 µg/mL. It was comparatively higher in case of Nivalenol/Paxilline (10.6±9.76) followed by crude filtrate (28± 20.4) [(Non-significantly differed with Cochliodinol, Monascorubramine and Alizarin)] > Dihydromaltopytin (45.2±24.4). Overall, these compounds significantly showed less toxicity towards normal cell lines. Whereas in case of cancer cell line KA3IT, Cochliodinol containing fraction was most effective (1IC50=0±0.44) [(Non significantly differed from crude culture filtrate)] followed by Nivalenol/Paxilline containing fraction (20±0.34) [(Non significantly different from Dihydromaltophytin and Alizarin)] >Monascorubramine (50±0.89). A pure orange compound was isolated and its proton NMR and LCMS confirmed it to be anthraquinone compound. Besides, many unknown compounds were also detected in LCMS profile of the selected fungi.

In Aspergillus fumigatus, logical fragmentation pattern following column chromatography, TLC and LCMS of crude culture filtrate revealed the presence of colored compounds i.e., PPR Monascorubramine and Fusarubin. Other colorless bioactive compounds were also detected in the fungal filtrate i.e., Pyrrocidine b, Communosine g, Teritrem and Tryptoquialanine A and B. Overall, the cytotoxicity against the five cell lines viz. KA3IT, MDCK, HSCT6, NIH3T3 and HEK293 shown by these compounds ranged from 1-200 µg/ml. All the fractions of this fungus showed quite low cytotoxicity (IC50) value i.e., 1-1.5 µg/ml against cancer cell line (KA3IT) which differed significantly from crude culture filtrate (2.8±0.44). Overall, the pigmented filtrate of this fungus was more cytotoxic as compared to the other 2 fungi showing its higher anticancer potential.

Fungi proved to be an important source of colored metabolites of polyketide nature. Production of these metabolites helped under mesophilic temperature, pH (5.5-7), glucose and yeast extract. Stability of the metabolites was considerably high at temperature < 100˚C though it varied a little with varying pH clearly predicting their industrial applications. Furthermore, detailed evaluation of different compounds in various fractions of fungal filtrates typically indicated their non-toxic nature and role as biomedical agent in treating cancer. The study concludes that optimized yield of aforementioned compounds by manipulating different biotechnological procedures can further help their large scale application in pharmaceutical, cosmetic and food industries. However, large scale production and application is still needed to be explored.