Synopsis Final

Studies on the production and dyeing properties of water soluble pigments from filamentous fungi Introduction In recent years, pigments are envisaged to have extensive application in food, dyeing, and pharmaceutical industries for coloring the products. Some of the plant species such as Rubia tinctorum L (Angelini et al., 1997, Moresi et al., 2001), Isatis tinctoria L, (Kokubun et al., 1998) and Reseda luteola L (Cerrato et al., 2002) were found to be good sources of red (alizarin), indigo (indigotin) and yellow (luteolin) dyes respectively. In fact, all three dyes were extensively exploited until the commercial success of their synthetic analogues (Ball, 2002). Naturally derived colorants are also extracted from fruit skins, seeds or roots and the manufacturers are usually dependent on the availability of raw materials for the colour extraction. Further pigment profile in the natural sources is prone to variation and it is influenced by the extraction procedures employed. Thus, the chemical composition, including the presence of minor components, and the colour stability of naturally derived colour additives vary significantly among different suppliers and from batch to batch (Downham and Collins, 2000). The main disadvantage of these natural dyes lies in the order of magnitude of their yield (a few grams of pigment per kg of dried raw material). The red colorant carmine for instance, is a product of the female cochineal insect and to extract 100 g carmine colorant, approximately 14000 insects are required (Wissgott and Bortlik, 1996). This makes their current market price high, thus limiting their application. Though plant and animals are good sources for production of color pigment, the inability of them to meet the world demand has led to increased interest in synthetic colorant. Moreover, a great deal of concern has been raised by the effects of some synthetic dyes on human health as sources of skin cancer, disorders and allergic reactions (Francalanci et al.,2001). To overcome this limitation, it was suggested to exploit the potentiality of other biological sources such as fungi, bacteria and algae with modern biotechnological techniques to improve significantly the pigment production.

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Micro algae are known to produce a wide range of water-soluble pigments, but the low productivity of algal cultures is a significant bottleneck for their commercialization (Hejazi and Wijffels, 2004). Pigments of basidiomycetous fungi have been used in the past for dyeing wool and silk (Bessette and Besette, 2001). The appropriate use of fermentation physiology together with metabolic engineering (Nielsen et al., 2002) could allow the efficient mass production of colorants from fungi. The present investigation is focused on fungi that produce industrially important pigments.

Review Like plants, filamentous fungi synthesize natural products because they have an ecological function and are of value to the producer (Firn and Jones, 2003). Depending on the type of compound, they serve different functions—varying from protection against lethal photo-oxidations (carotenoids) to environmental stress (melanins) and acting as cofactors in enzyme catalysis (flavins). Filamentous fungi produce several characteristic of carotenoid and non- carotenoid pigments (Baker and Tatum, 1998; Medenstev and Akimenko, 1998; Adrio and Demain, 2003). Anthraquinone (octaketide) pigments like catenarin, chrysophanol, cynodontin, helminthosporin, tritisporin and erythroglaucin are produced by Eurotium spp., Fusarium spp., Curvularia lunata and Drechslera spp (Duran et al., 2002). Yellow pigments-epurpurins A to C were obtained from Emericella purpurea (Hideyuki et al., 1996) and azaphilone derivatives (hexaketides), falconensins A–H and falconensones A1 and B2, were produced both by Emericella falconensis and Emericella fructiculosa (Ogasawara et al., 1997). The red colorant is an extracellular metabolite of the anthraquinone class and is claimed to be produced by a variety of Penicillium oxalicum (Sardaryan et al., 2002; Sardaryan, 2004). Monascus pigments, which are water-soluble, are of great biotechnological interest. Monascus is a typical Ascomycete that produces a cleistothecium, a closed fruiting body containing eight ascospores, but reproduces asexually by the formation of conidiospores and a vegetative mycelium. The pigments may be produced both in the mycelium and liberated to the fermentation broth (Margalith, 1992). However,

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Monascus-fermented rice has been found to contain the mycotoxin citrinin (Liu et al., 2005). The production of citrinin together with pigments clearly limits the use of Monascus as a producer of natural food colorants. Optimization of growth medium is one of the essential steps to maintain a balance between the medium components to minimize the amount of unutilized components at the end of fermentation, and to have cost effective metabolic yield (Kumar et al., 1999; Prakasham et al., 2005). Pigment production is often influenced by different carbon and nitrogen sources. Lilly and Barnett (1962) reported that the biomass production of Monascus purpureus was found to be increased in carbon source like fructose, glucose and sucrose. McHan and Johnson (1970) showed that Monascus purpureus grew better in glucose-peptone -yeast extract broth than in any other complex medium. Zinc and a special combination of glycine, leucine and tryptophan yielded more growth (McHan and Johnson, 1979). The overwhelming majority of extraction procedures use the traditional liquid–liquid extraction (Bohm, 1999; Lacker, et al., 1999). However, the successful applications of modern extraction techniques such as solid-phase micro extraction (Emenhiser, et al., 1996) and microwave-assisted extraction (Csiktusnadi et al., 2000) have also been reported. The review clearly indicates that not much work has been undertaken on fungal pigments, although variety of pigmented fungi are present in nature. Hence the present work was planned with the aim of searching of novel water soluble pigments from filamentous fungi. The work was designed as given below. 1. Isolation of pigment producing fungi from diverse ecosystems like agricultural soils, forest soils and rhizoshpere of plants. 2. Selection of production medium and optimization of nutrient conditions for pigment production. 3. Characterization of the pigments. 4. Genetic improvement of the selected fungi through mutagenesis for enhanced pigment production

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5. Optimization of the pigment production in solid substrate and cost effective production of pigments. 6. Testing for mutagenesis, antioxidant, antimicrobial activities and toxicity evaluation by seed germination 7. Determining the dyeing capacity on fabric and leather and to assess the colorfastness. Work undertaken: Soil samples were collected from 51 different locations. Pigment producing filamentous fungi were isolated. Potent and promising four isolates (Pacilomyces farinosus-red,

Emericella

nidulans-yellow,

Fusarium

moniliforme-Brown

and

Penicillium pupurogenum- thick red pigment) were selected on the basis of color and type of pigment (intracellular or extracellular). Culture conditions were optimized using different sources of carbon, nitrogen, temperature, pH and light. Effects of various heavy metals on the production of pigment were tested. Antimicrobial, MIC, antioxidant and mutagenic activity were determined. Mutants were developed and tested for their pigment production. Purification and characterization of pigment was done using column chromatography, UV-VIS, IR, NMR and GC-MS spectrum. The compounds identified were anthraquinone (Pacilomyces farinousus), di-o-acetyl-lanugon-j (Emericella nidulans),

Heptacosonic

acid

25-methyl

ester

(Fusarium

moniliforme)

and

chrysanthemum hydrolyzed (Penicillium pupurogenum). Dyeing property was tested on to cotton fabric and leather and the quality of the dyed specimen was determined by employing various tests. The results are presented.

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The thesis contains six chapters. The first chapter gives general introduction of the study. The chapter two covers literature review. The third chapter describes various methodologies adopted to study the effect of culture conditions on pigment production, purification, characterization, and the application of the pigment on fabric and leather. The chapter four deals with the results and chapter five focuses on the overall discussion of the above study. The sixth chapter highlights the results as summary, followed by the list of references.

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Cerrato, A., D. De Santis and M. Moresi, 2002. Production of luteolin extracts from Reseda luteola and assessment of their dyeing properties. J.Sci. Food Agric., 82:1189–1199. Duran ,N., M.F.S. Tixeira, R. de Conti and E. Esposito, 2002. Ecological-friendly pigments from fungi. Crit. Rev.Food. Sci Nutr., 42:53-66. Downham, A. and P.Collins, 2000. Coloring our foods in the last and next millennium. Int. J. Food. Sci. Technol ., 35:5-22. Emenhiser, K., N. Simunovic,L.C. Sander and S.J Schwartz,1996. Separation of geometrical carotenoid isomers in biological extracts using a polymeric C30 column in eversed-Phase Liquid Chromatography. J. Agric. Food Chem., 44, 3887–3893. Firn, R.D. and C.G. Jones, 2003.Natural products — a simple model to explain chemical diversity. Nat. Prod. Rep., 20:382-391. Francalanci, S., S. Giorgini,C. Brusi and A. Sertoli, 2001. L’impiegodi tessuti ecologici nella prevenzione della dermatiteallergica da contatto con coloranti. Proc 1st Health and Textile Int Forum, Biella [Online]. Available:www.tessileesalute.it/flex/cm/files/99aa8709ac2f35a8941e.pdf. Hejazi, M.A. and R.H Wijffels, 2004. Milking of micro algae. Trends Biotechnol., 22:189-194. Hideyuki, T., N. Koohei and K. Ken-ichi, 1996. Isolation and structures of dicyanide derivatives, epurpurins A to C, from Emirecella purpurea. Chem Pharm Bull (Tokyo)., 44:2227-2230. Kokubun, T., J. Edmonds and P. John, 1998. Indoxyl derivatives in woad in relation to medieval indigo production. Phytochemistry.,49:79–87. Kumar,C.G., M.P. Tiwari and K.D. Jany,1999. Novel alkaline serine protease from alkalophilic Bacillus spp.; purification and some properties. Process Biochem., 34:441 –449. Liu, B.H., T.S. Wu, M.C. Su, C.P. Chung and F.Y. Yu, 2005. Evaluation of citrinin occurrence and cytotoxicity in Monascus fermentation products. J Agric Food Chem., 53:170-175. Lilly, V.G. and H. L. Barnett, 1962. The utilization of sucrose and its constituents sugar by Monascus pupurus. Proc.W.Va .Acad.Sc., 24: 27 – 32.

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