Academic literature on the topic 'Penicillium sclerotiorum'

Abstract Purpose Sclerotiorin, an azaphilone produced by some filamentous fungi including Penicillium sclerotiorum, is a pigment with variety of biological activities including lipoxygenase inhibition, reduction of cholesterol levels, and anti-cancer properties. Sclerotiorin has potential use in pharmaceutical as well as food industries. In this context, the purpose of this study was to provide a simple and robust procedure for optimised production of sclerotiorin by P. sclerotiorum using a central composite design developed through response surface methodology (RSM) and to identify the molecule(s) involved in the signalling mechanism in P. sclerotiorum. Methods The optimisation of sclerotiorin production was carried out using RSM in shaken flasks and the obtained results were then replicated using a 2-L stirred tank bioreactor. Penicillium sclerotiorum ethyl acetate culture extract was analysed using thin layer chromatography (TLC) and potential signalling molecules were identified using Gas chromatography-mass spectrometry (GC-MS). Results The experimental studies suggested an increase in the sclerotiorin production by 2.1-fold and 2.2-fold in shaken flasks and stirred tank bioreactors respectively. Further analysis of P. sclerotiorum ethyl acetate culture extract reported the presence of ricinoleic acid, an oxylipin, belonging to a family of signalling molecules tentatively involved in the enhancement of sclerotiorin production. Conclusion This paper has highlighted the positive effect of the optimal supplementation of P. sclerotiorum culture extracts for enhanced production of sclerotiorin. It has also examined potential molecules involved in the signalling mechanism in P. sclerotiorum culture extract for the overproduction of sclerotiorin.

Four new azaphilones, sclerotiorins A–D (1–4), as well as the dimeric sclerotiorin E (5) of which we first determined its absolute configuration, and 12 known analogues (5–16) were isolated from the fermentation broth of Penicillium sclerotiorum OUCMDZ-3839 associated with a marine sponge Paratetilla sp.. The new structures, including absolute configurations, were elucidated by spectroscopic analyses, optical rotation, ECD spectra, X-ray single-crystal diffraction, and chemical transformations. Compounds 11 and 14 displayed significant inhibitory activity against α-glycosidase, with IC50 values of 17.3 and 166.1 μM, respectively. In addition, compounds 5, 7, 10, 12–14, and 16 showed moderate bioactivity against H1N1 virus.

To discover the new medical entity from edible marine algae, our continuously natural product investigation focused on endophytes from marine macroalgae Grateloupia sp. Two new azaphilones, 8a-epi-hypocrellone A (1), 8a-epi-eupenicilazaphilone C (2), together with five known azaphilones, hypocrellone A (3), eupenicilazaphilone C (4), ((1E,3E)-3,5-dimethylhepta-1,3-dien-1-yl)-2,4-dihydroxy-3-methylbenzaldehyde (5), sclerotiorin (6), and isochromophilone IV (7) were isolated from the alga-derived fungus Penicillium sclerotiorum. The structures of isolated azaphilones (1–7) were elucidated by spectrometric identification, especially HRESIMS, CD, and NMR data analyses. Concerning bioactivity, cytotoxic, anti-inflammatory, and anti-fibrosis activities of those isolates were evaluated. As a result, compound 1 showed selective toxicity toward neuroblastoma cell line SH-SY5Y among seven cancer and one fibroblast cell lines. 20 μM of compounds 1, 3, and 7 inhibited the TNF-α-induced NFκB phosphorylation but did not change the NFκB activity. Compounds 2 and 6 respectively promoted and inhibited SMAD-mediated transcriptional activities stimulated by TGF-β.

Abstract Sclerotinia sclerotiorum (Lib.) de Bary and Botrytis cinerea Pers. were highly pathogenic to celery stored at 0° to 1°C in normal air (21% O2). Alternaría dauci (Kuhn) Groves & Skolko, Rhizopus nigricans Ehrenb., Penicillium sp., and Fusarium oxysporum Schlecht, were nonpathogenic. An atmosphere of 7.5% CO/1.5% O2 was more suppressive to disease caused by B. cinerea and S. sclerotiorum than low 1.5% O2 atmosphere alone. The 4% CO2/1.5% O2 and 0.0003% C2H4/1.5% O2 atmospheres were slightly suppressive to disease caused by S. sclerotiorum only. The 7.5% CO/1.5% O2 atmosphere also was consistently suppressive to mycelial growth, spore germination, and germ tube elongation of B. cinerea.

O mofo branco causado por Sclerotinia sclerotiorum pode inviabilizar o cultivo de olerícolas em ambiente protegido. Para elaborar-se um programa de controle biológico desse patógeno, necessita-se de antagonistas adequados. Este trabalho objetivou selecionar antagonistas fúngicos eficazes no controle de S. sclerotiorum em pepineiro (Cucumis sativus) cultivado em estufa, bem como, analisar a interferência dos antagonistas no crescimento vegetal. Foram utilizados um isolado de S. sclerotiorum obtido de pepineiro e 112 isolados fúngicos de quatro gêneros: Trichoderma, Fusarium, Penicillium e Aspergillus. Em experimento in vitro, foi utilizada a técnica do papel celofane e selecionados oito isolados de Trichoderma virens, os quais promoveram maior inibição no crescimento do patógeno (94 a 100%). Dois experimentos in vivo foram desenvolvidos em estufa utilizando-se substrato autoclavado e não autoclavado, em copos plásticos, e substrato não autoclavado, em sacos plásticos; o substrato foi infestado com S. sclerotiorum e foram utilizados oito isolados de T. virens como antagonistas. Todos os isolados testados controlaram o tombamento de mudas, mas o efeito sobre o crescimento vegetal variou de acordo com os isolados e o tratamento do substrato.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Enzimas degradadoras de xilana, principal componente da hemicelulose, têm sido utilizadas em várias aplicações biotecnológicas, sendo que em alguns processos é necessário o uso de enzimas purificadas. Aplicações comerciais para as enzimas xilanolíticas envolvem a hidrólise enzimática da xilana, que está presente nos resíduos agrícolas e agroindustriais, sendo convertido a xilose e outros açúcares, que podem ser utilizados como substratos em processos fermentativos para a obtenção de proteínas celulares, combustíveis líquidos e outras substâncias químicas. A utilização destas enzimas também diminui a liberação de agentes poluentes em determinados efluentes, como da indústria de polpa de celulose. Xilanases e β- xilosidases são produzidas principalmente por bactérias e fungos, sendo que em geral, os fungos as produzem em níveis mais elevados. O gênero Penicillium apresenta espécies já caracterizadas como boas produtoras destas enzimas. Uma linhagem deste gênero, isolada de solo brasileiro, na região da Mata Atlântica e identificada como Penicillium sclerotiorum destacou-se por produzir xilanase em níveis elevados. O objetivo deste trabalho consistiu na avaliação da influência das condições de cultivo sobre a produção do complexo xilanolítico produzido por P. sclerotiorum, na caracterização físico-química desse sistema, bem como purificação e caracterização bioquímica de seus principais componentes. Por meio da determinação das condições ótimas de produção e da caracterização deste complexo enzimático foi possível estabelecer metodologias eficientes de purificação de xilanases e uma β-xilosidase. Através da caracterização físico-química das enzimas purificadas, foi possível avaliar seu potencial biotecnológico, visando futuras aplicações em processos industriais.
Xylan degrading enzymes, the main component of hemicellulose, have been used in various biotechnological applications, and in some cases the use of purified enzymes is necessary. Commercial applications of xylanolytic enzymes involve the enzymatic hydrolysis of xylan, which is present in agricultural and agro-industrial wastes, and can be converted to xylose and other sugars, which can be further used as substrates in fermentation processes to obtaining cellular protein, liquid fuels and other chemicals. The utilization of these enzymes also decreases the release of certain pollutants in wastewater, as in the pulp and paper industry. Xylanases and β-xilosidases are mainly produced by bacteria and fungi, and in general, the fungi produce them at higher levels. The genus Penicillium presents species already characterized as good producers of these enzymes. One strain of this genus isolated from Brazilian soil in the Mata Atlântica region and identified as Penicillium sclerotiorum attracted attention by producing xylanase in high levels. The objective of this study was to evaluate the influence of culture conditions on the production of the xylanolytic complex produced by P. sclerotiorum to characterize physical and chemical properties of this system as well to purify and biochemical characterize its main components. By determining optimal conditions for production and by characterizing this enzymatic complex it was possible to establish efficient methodologies for purification of xylanases and one β-xylosidase. Through their physical and chemical characterization, it was possible to evaluate their biotechnological potential for future applications in industrial processes.

Orientador: Eleonora Cano Carmona
Banca: Aline Aparecida Pizzirani Kleiner
Banca: Helia Hamuri Sato
Banca: Rosa dos Prazeres M.F. Inocentes
Banca: Marcia Regina Brochetto Braga
Resumo: Enzimas degradadoras de xilana, principal componente da hemicelulose, têm sido utilizadas em várias aplicações biotecnológicas, sendo que em alguns processos é necessário o uso de enzimas purificadas. Aplicações comerciais para as enzimas xilanolíticas envolvem a hidrólise enzimática da xilana, que está presente nos resíduos agrícolas e agroindustriais, sendo convertido a xilose e outros açúcares, que podem ser utilizados como substratos em processos fermentativos para a obtenção de proteínas celulares, combustíveis líquidos e outras substâncias químicas. A utilização destas enzimas também diminui a liberação de agentes poluentes em determinados efluentes, como da indústria de polpa de celulose. Xilanases e β- xilosidases são produzidas principalmente por bactérias e fungos, sendo que em geral, os fungos as produzem em níveis mais elevados. O gênero Penicillium apresenta espécies já caracterizadas como boas produtoras destas enzimas. Uma linhagem deste gênero, isolada de solo brasileiro, na região da Mata Atlântica e identificada como Penicillium sclerotiorum destacou-se por produzir xilanase em níveis elevados. O objetivo deste trabalho consistiu na avaliação da influência das condições de cultivo sobre a produção do complexo xilanolítico produzido por P. sclerotiorum, na caracterização físico-química desse sistema, bem como purificação e caracterização bioquímica de seus principais componentes. Por meio da determinação das condições ótimas de produção e da caracterização deste complexo enzimático foi possível estabelecer metodologias eficientes de purificação de xilanases e uma β-xilosidase. Através da caracterização físico-química das enzimas purificadas, foi possível avaliar seu potencial biotecnológico, visando futuras aplicações em processos industriais.
Abstract: Xylan degrading enzymes, the main component of hemicellulose, have been used in various biotechnological applications, and in some cases the use of purified enzymes is necessary. Commercial applications of xylanolytic enzymes involve the enzymatic hydrolysis of xylan, which is present in agricultural and agro-industrial wastes, and can be converted to xylose and other sugars, which can be further used as substrates in fermentation processes to obtaining cellular protein, liquid fuels and other chemicals. The utilization of these enzymes also decreases the release of certain pollutants in wastewater, as in the pulp and paper industry. Xylanases and β-xilosidases are mainly produced by bacteria and fungi, and in general, the fungi produce them at higher levels. The genus Penicillium presents species already characterized as good producers of these enzymes. One strain of this genus isolated from Brazilian soil in the Mata Atlântica region and identified as Penicillium sclerotiorum attracted attention by producing xylanase in high levels. The objective of this study was to evaluate the influence of culture conditions on the production of the xylanolytic complex produced by P. sclerotiorum to characterize physical and chemical properties of this system as well to purify and biochemical characterize its main components. By determining optimal conditions for production and by characterizing this enzymatic complex it was possible to establish efficient methodologies for purification of xylanases and one β-xylosidase. Through their physical and chemical characterization, it was possible to evaluate their biotechnological potential for future applications in industrial processes.
Doutor

The fungus Penicillium sclerotiorum produces a wide variety of secondary metabolites, among them the sclerotiorin, which has many uses in food and pharmaceutical industries. Due to its strong orange color, its antioxidant activity and other functions related to cholesterol reduction and treatment of diabetes, this substance has been subject of extensive studies in food science. In this project, substances were produced by esclerotiorin biotransformation with the fungus Beauveria bassiana. Five new products with strong red color were obtained. The change in coloration of the molecule was due to the exchange of the oxygen heteroatom attached to sclerotiorin carbon 1 by a nitrogen atom. The biotransformation experiment was repeated using, as substrate, 1-methyl-sclerotiorin, a semi-synthetic substance, but the same results were not obtained. After these results, it was performed a screening with 14 different fungi, besides Beauveria bassiana, to evaluate their capacity to biotransform sclerotiorin. The extracts were analyzed by HPLC and showed that, from these 14 fungi, nine were able to transform sclerotiorin molecule in one or more substance with red color.
O fungo Penicillium sclerotiorum produz uma grande variedade de metabólitos secundários, entre eles, a esclerotiorina, que tem tido aplicações nas indústrias alimentícia e farmacêutica. Devido à sua forte coloração alaranjada, sua capacidade antioxidante, potencial atividade biológica de redução do colesterol plasmático e no tratamento da diabetes, esta substância tem sido alvo de diversos estudos na área de alimentos. Neste projeto, foram produzidas substâncias através da biotransformação da esclerotiorina com o fungo Beauveria bassiana, obtendo-se cinco produtos de forte coloração avermelhada. A mudança na coloração da substância aconteceu devido à troca de um heteroátomo de oxigênio ligado ao carbono 1 da molécula de esclerotiorina por um átomo de nitrogênio. O experimento de biotransformação foi repetido utilizando, como substrato, a 1-metil-esclerotiorina, uma substância semi-sintética, porém os mesmos resultados não foram obtidos. Após a obtenção destes resultados, foi feita uma triagem com 14 diferentes espécies de fungos, além de Beauveria bassiana, para verificar a capacidade destes fungos em biotransformar a esclerotiorina. Os extratos foram analisados por CLAE e mostraram que, destes 14 fungos, nove foram capazes de biotransformar a molécula de esclerotiorina em uma ou mais substâncias que apresentaram coloração avermelhada.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES#
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Microorganisms have a high potential in the production of several essential metabolites for various industrial sectors. Among these metabolites are the pigments and biosurfactants that are important molecules used in the food, textile, cosmetic and pharmaceutical industries. In this context, this study was carried out to identify the species of Penicillium sp. Isolated from the soil of the caatinga of Pernambuco, besides producing, isolating and characterizing the pigment and biosurfactant produced. Penicillium sp. isolated were identified by morphological and molecular analyzes (PCR), while pigment and biosurfactant production was performed using medium containing different concentrations of whey and barley according to the Delineation central composite (CCD) 22. Pigment extraction was performed from biomass, purification by thin layer chromatography (TLC) and column chromatography, pigment was preliminarily identified by high performance liquid chromatography (HPLC). While the biosurfactant was extracted from the metabolic liquid and proved the extracellular production by the surface tension. The results obtained from the classic identification for Aspergilloides indicate that the fungus belongs to the genus and species Penicillium sclerotiorum. This fungus was able to produce high yield of biomass (15.4g/L) and pink pigment (2.23 g/L) in the medium consisting of 8% whey and 6% barley in condition four of the planning. In this medium, the data showed high carbon content (42.56%) and nitrogen (6.28%), resulting in a C / N ratio of 15: 1. The pigment had a pink band with a value of Rf = 0.961, retention time of 2.828 at 247 nm and inhibitory activity for gram-negative bacteria (Staphylococcus aureus and Klebsiella pneumoniae) and Candida glabrata yeast. Still, the maximum biosurfactant production occurred in the medium consisting of 4% whey and 2% barley with a reduction in surface tension from 70 to 27 mN/m. Emulsion index of 68% using engine burned oil, dispersion index of 33.15 cm2 and reduced viscosity from 279.6 to 48.5 cP. Penicillium sclerotiorum demonstrated the ability to produce pigment and biosurfactants from an economic environment consisting of whey and barley, thus presenting a viable alternative for the substitution of synthetic dyes and chemical surfactants meeting the essential requirements for sustainability.
Os micro-organismos apresentam elevado potencial na produção de diversos metabolitos essenciais para diferentes setores industriais. Dentre estes metabolitos estão os pigmentos e biossurfactantes que são importantes biomoléculas utilizadas na indústria alimentícia, têxtil, cosmética e farmacêutica. Neste contexto este estudo foi realizado com objetivo de identificar a espécie de Penicillium sp. isolado do solo da Caatinga de Pernambuco, além de produzir, isolar e caracterizar o pigmento e biossurfactante produzidos. O Penicillium sp. isolado foi identificado por análises morfológicas e molecular (PCR), enquanto a produção do pigmento e biossurfactante foi realizada utilizando meio contendo diferentes concentrações de soro de leite e cevada de acordo com o planejamento Delineamento Composto Central (DCC) de 22. A extração do pigmento foi realizada a partir da biomassa, a purificação através de cromatografia de camada delgada (CCD) e cromatografia em coluna, pigmento foi preliminarmente identificado por cromatografia líquida de alta eficiência (CLAE). Enquanto o biossurfactante foi extraído a partir do liquido metabólico e comprovado a produção extracelular pela tensão superficial. Os resultados obtidos a partir da identificação clássica para Aspergilloides indicam o fungo como pertencente ao gênero e espécie Penicillium sclerotiorum. Este fungo foi capaz de produzir elevado rendimento de biomassa (15,4g/L) e pigmento rosa (2,23 g/L) no meio constituído de 8% de soro de leite e 6% de cevada na condição quatro do planejamento. Neste meio, os resultados demonstraram elevado teor de carbono (42,56%) e nitrogênio (6,28%), resultando na razão de C/N de 15:1. O pigmento apresentou banda de coloração rosa intenso com valor de Rf=0,961, tempo de retenção de 2,828 a 247 nm e atividade inibitória para as bactérias (Staphylococcus aureus e Klebsiella pneumoniae) e na levedura Candida glabrata. Por outro lado a máxima produção de biossurfactante ocorreu no meio constituído por 4% de soro de leite e 2% de cevada com redução da tensão superficial de 70 para 27 mN/m. Um índice de emulsificação de 68% utilizando óleo queimado de motor, índice de dispersão de 33,15 cm2 e viscosidade reduzida de 279,6 para 48,5 cP. Penicillium sclerotiorum demonstrou habilidade de produzir pigmento e biossurfactantes a partir de meio econômico constituído de soro de leite e cevada, apresentando desta forma, uma alternativa viável para substituição dos corantes sintéticos e surfactantes químicos atendendo aos requisitos essenciais para sustentabilidade.

博士
國立臺灣大學
農業化學研究所
92
In this study, a fungal strain with high quantity of yellow pigment was isolated from an orchard soil from Pingtung in our laboratory. This strain was identified as Penicillium sclerotiorum and was designated as Penicillium sclerotiorum NTU-FC-13. A yellow needle crystal was obtained by silica-gel column chromatography from the crude pigments, and was identified as sclerotiorin through MS, IR, and NMR spectra. The results showed that the potato agar medium was the best for producing pigments among natural mediums. Maltose could significantly increase the pigment production. Neither organic nor inorganic nitrogen source would influence the pigments production but could increase the volume of biomass. Adding 0.1% sodium chloride could promote the productivity. P. sclerotiorum NTU-FC-13 produces sclerotiorin yellow pigment under submerged fermentation in potato maltose medium (15% potato, 2% maltose, 0.1% NaCl ), the sclerotiorin yield is 633 mg L-1 culture fluid and 39 mg g-1dry cell weight. In comparison with the open literature, we have shown that sclerotiorin produced by P. sclerotiorum has the highest yield with more than 7-9 times output per liter of culture fluid so far. A quantitative analysis of sclerotiorin from P. sclerotiorum was established by using HPLC/ Colorimetry combination method at the condition of unavailability of authentic sclerotiorin compound. Mycelium was extracted with methanol, then analyzed by HPLC ( a C18 reverse phase column with acetonitrile/water＝65/35, v/v as mobile phase and detected at 370 nm). The extract was also measured with the OD370 at the same time by spectrophotometers. The result could be obtained by using this method with less than 30 minutes. The safety test with ICR mice showed that the oral LD50 for the sclerotiorin is greater than 1g/kg body weight. The sclerotiorin was showed no cytotoxicity with human cell assay. The Antibacterical activity of 0.5% sclerotiorin to gram (+) was showed positive and no effect to gram (-). The properties of the sclerotiorin yellow pigment are quite stable during heat and acid situations, but changed to wine-red color during alkaline situation.

Blood coagulation is a process that occurs normally in an animal during bleeding, to prevent the loss of blood from the body and is assisted by two key players -blood platelets and coagulation factors particularly thrombin. When this process becomes extensive, it leads to thrombosis, a condition which can lead to severities such as cardiac failure, brain hemorrhage,renal failure and deep vein thrombosis. Anti-thrombotic drugs which are administered to combat thrombosis can be classified into three categories - anticoagulants, antiplatelet drugs (also known as blood thinners) and fibrinolytic drugs. The anticoagulant and antiplatelet drugs constitute the anti-thrombotic drugs which ensure the prevention of thrombosis whereas fibrinolytic drugs are administered after the thrombotic plaque is formed in order to dissolve the clot. The present day human society has been plagued by cardiovascular disease (also known as coronary heart disease) due to sedentary lifestyle and unhealthy food habits. The major fear in case of this disease is the blockage of the coronary artery by theatherosclerotic plaque, which then results in decreased blood supply to the cardiac tissue. When the plaque completely blocks the artery,owing to the blood pressure, the atherosclerotic plaque ruptures resulting in the formation of a thrombus (blood clot), which cuts off the blood supply to the cardiac tissue and can finally lead to a cardiac arrest. Therefore, anti-thrombotic drugs play a pivotal role in combating heart disease. Amongst these, the administration of anticoagulant and antiplatelet drugs is highly crucial to prevent the formation of the thrombus in patients suffering from heart disease. There have been many drugs in the market at present but they can cause side effects such as unwanted bleeding, allergy and non-specific reactions. Also, the current need is to ensure that the anti-thrombotic drugs administered do not hamper haemostasis, although they prevent blood coagulation. Hence, there is a constant need to discover newer anti-thrombotic drugs from unexplored microbial sources. This thesis describes the identification of compounds from endophytic fungi that inhibit thrombin and platelet aggregation. We have screened forty eight endophytic fungi isolated from the plants Datura metel and Cassia fistulafor thrombin inhibitory activity. Two isolates from D. metel -Dm 3.3, Dm 14.2 and three isolates from C. fistula - C2, C3, C4 tested positive for thrombin inhibitory activity. When each of these five endophytic fungal isolates was further tested for their potential to inhibit thrombin and platelet aggregation separately; Dm 3.3, C2 and C3 showed potent anti-thrombotic properties. The present thesis work describes the identification of anti-thrombotic metabolites of two of these three endophytic fungal isolates namely - Dm 3.3 and C2. Based on ITS sequence analysis, Dm 3.3 was identified taxonomically to be Colletotrichum gloeosporioides. The hexane extract of endophytic fungal isolate,C. gloeosporioides showed higher inhibition of purified thrombin (Sigma thrombin) and plasma thrombin as well as the potential to inhibit platelet aggregation induced by ADP, when compared to the ethyl acetate extract of the fungus. When the hexane extract of C. gloeosporioides was analysed by GC-MS, two major metabolite peaks were identified by the NIST library data -retinoic acid and 6β-hydroxytestosterone. There have been several reports on the presence of retinal in fungi such as Mucormucedo and have suggested its role as an intermediate in the biosynthesis of the morphogen, trisporic acid (a retinoid derivative) with a vital role in the sexual reproductive phase of fungi in inducing the formation of zygophores (gametes) from the mycelium. There have also been reports which speculate that the homologs of the enzyme RALDH (retinaldehyde dehydrogenase) in fungi could possibly convert retinal to retinoic acid. The presence of steroids in fungi has been extensively reported to play a key role to induce meiosis in the antheridials and oogonium of the fungus -Saprolegniaferax. Therefore, both retinoid compounds and steroids play a crucial role in the sporulation of fungi, thus justifying the presence of the two metabolites, retinoic acid and 6β-hydroxytestosterone, in C. gloeosporioides. Since, we had observed two major metabolite peaks in the hexane extract of C. gloeosporioides which corresponded to retinoic acid and 6β-hydroxytestosterone, we investigated the anti-thrombotic properties of both the standards - retinoic acid and 6β-hydroxytestosterone. We observed that retinoic acid, but not 6β-hydroxytestosterone, showed potent inhibition of both thrombin and platelet aggregation.The three forms of vitamin A namely - retinoic acid, retinal and retinol, showed potent inhibition of thrombin and platelet aggregation.Retinoic acid showed the maximum inhibition of thrombin (IC50values: Sigma thrombin – 67µg/ml; plasma thrombin – 49µg/ml), while retinol showed the maximum inhibition of platelet aggregation (97% inhibition at 120µg/ml). Therefore, we can infer that retinoic acid to be the active anti-thrombotic secondary metabolite present in C. gloeosporioides. In parallel to the work on the anti-thrombotic properties of the D. metel endophytic fungus –C. gloeosporioides (Dm 3.3), we have also investigated the thrombin and platelet aggregation inhibitory metabolites of C2, the endophytic fungus isolated from the plant Cassia fistula. The molecular taxonomic method of ITS PCR enabled us to identify the endophytic fungus C2 as Penicillium sclerotiorum. The ethyl acetate extract of the mycelium of Penicillium sclerotiorumwhen grown in potato dextrose broth showed the highest thrombin inhibitory activity. Hence, this extract was subjected to a bio-assay guided column chromatographic fractionation using silica gel column. Three fraction pools were obtained, each of which was found to possess a unique anti-thrombotic active principle (AP). Active principle 1 (AP1) was purified from fraction pool I by preparative TLC, while active principle 2 (AP2) was purified from fraction pool II by column chromatography (sephadex LH20 column followed by silica gel column) and preparative TLC. The compound AP1 showed inhibition of both Sigma thrombin (IC50 value – 58µg/ml) and plasma thrombin (IC50 value – 89µg/ml) as well as the inhibition of platelet aggregation (70% inhibition at 120µg/ml).AP2 showed inhibition of Sigma thrombin (IC50 value – 104µg/ml) and plasma thrombin (IC50 value – 107µg/ml) as well as the inhibition of platelet aggregation (41% inhibition at 120µg/ml). Amongst these metabolites, AP1 showed higher anti-thrombotic potential as compared to AP2. Thus, AP1 and AP2 are the two active metabolites purified from P. sclerotiorum which show potent anti-thrombotic properties. The structure elucidation of the active principles – AP1 and AP2 purified from the Cassia fistula endophytic fungus, Penicillium sclerotiorum, was performed using state-of-the art analytical techniques such as IR spectroscopy, UV spectroscopy, LC-MS, NMR spectroscopy and micro-analysis. The mass of AP1 was analysed by mass spectrometry to be 415 m/z and the structure characterization of this metabolite is awaited. The structure of AP2 was elucidated to be 5-(2,4-dinitrophenyl)hydrazono-2-bornanol, which has a mass of 390 m/z (as analysed by mass spectrometry) and belongs to the class of monoterpenoid derivatives called camphenes. Thus, the thesis work describes the identification of anti-thrombotic compounds from two endophytic fungi – Colletotrichum gloeosporioides and Penicillium sclerotiorum. Since these compounds have a microbial origin, there is a potential advantage to scale up their production by exploiting the tools of media optimization and environmental stress application. Structure of the anti-thrombotic secondary metabolites which were identified in this study

El presente trabajo evaluó el potencial biotecnológico del hongo Penicillium sclerotiorum UCP 1040, aislado del suelo del Estado de Pernambuco, para la produción de biosurfactante usando residuos agroindustriales como fuentes alternativas de carbono y nirógen. Las fermentaciones se realizaon durante 144 h, 28ºC y 150 rpm, en medios consistos por diferentes concentraciones de aceite post fritura y licor de lavado de maíz, de acuerdo a un diseño factorial 2 2. Los efectos de los sustratos en la producción del surfactante se evaluaron mediante análisis estadístico, utilizando la tensión superficial como variable de respuesta. Las propiedades emulsionantes y dispersantes del biosurfactante se investigaron mediante el índice de emulsificación (IE24) y el ensayo de dispersión, respectivamente. Los resultados obtenidos mostraron que P. sclerotiorum fue capaz de producir un compuesto surfactativo en presencia de sustratos renovables, observándose la mayor reducción de la tensión superficial (de 72,0 a 42,77 mN/m) en la condición 2 del diseño (medium compuesto por 3% aceite post fritura y 5% liquor de lavado de maíz). Aunque el biosurfactante producido en esta condición no mostró buenas propiedades emulsantes, demostró excelentes propiedades dispersantes, con un área de dispersión de aceite (ADA) de 44,18 cm2. El diagrama de Pareto demostró la influencia significativa de la interacción de los residuos en la producción del biossurfactante, constituyendo sustratos alternativos y de bajo costo, que hacen que el bioproceso sea económico y, por lo tanto, atractivo para diferentes industrias.