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Kozarski Maja
07/2012Dissertation
Polysaccharides from mushrooms, plants and microorganisms have received considerable attention due to their biological activities, such as antitumor, antiviral, antioxidation, anticomplementary, anticoagulant, hypolipidemic, immunostimulant and immunological activities, which made them possible to be used in many fields including food, cosmetics, biomedicine, agriculture, environmental protection and wastewater management. Oxidative deterioration in natural and processed foods is a large economic concern in the food industry because it affects many quality characteristics such as flavor, color, texture, and the nutritive value of foods. In addition, its products are potentially toxic. Also, reactive oxygen species have been implicated in the pathophysiology of various clinical disorders, including ischemia, reperfusion injury, myocardial infarction, rheumatoid arthritis, neurodegenerative processes, atherosclerosis, acute hypertension, hemorrhagic shock and diabetes mellitus. Food spoilage and food poisoning are among the most important issues facing the food industry. Foodborne illness is a global problem. Development of novel antibacterial compounds for resistant organisms is becoming critically important. The present study was focused on the researching of possible antioxidant and antimicrobial properties of polysaccharide extracts from the selected Basidiomycetes mushroom species: Agaricus bisporus, Agaricus brasiliensis, Ganoderma applanatum, Ganoderma lucidum, Lentinula edodes, Phellinus linteus and Trametes versicolor. Based on the analysis, the extracts contained a mixture/complex of polysaccharides, proteins and polyphenols, which were still present in the extracts after hot water treatment, ethanol precipitation and dialysis. The prevalent monosaccharide in all extracts was D-glucose, while D-galactose, D-mannose and L-fucose/D-xylose were present to a lesser or greater quantities. Uronic acid was found in the G. applanatum polysaccharides. Samples contained varying amounts of both α- and β-glucans. Total glucan contents varied considerably, from 35 % for to 87 %. Glucopyranosyl units were primarily connected by β-glycosidic linkages (57-97%), except in the commercial G. lucidum spore extract, where percentage of α-glucan compared to the total glucan content was 94,2%. FT-IR analysis showed strong aromatic and ester peaks next to the different sugar and polysaccharide signals. The FTIR spectra of all investigated samples showed characteristic absorption bands related to the residual proteins, phenols and pigments. The results of elemental analysis indicated presence of non protein nitrogen in the polysaccharide extracts. The scanning electron microscopy was observed different surface morphology of polysaccharide extracts. The results of the study indicated that polysaccharide extracts of selected Basidiomycetes mushrooms are antioxidative and possess antibacterial properties. The antioxidant and antimicrobial effects of the extracts were resistant to high temperatures (121ºC), at which they have been exposed during the extraction process. Extracts obtained by hot water extraction and ethanol precipitation were effective as additionally dialyzed and with ethanol purified extracts. For measuring antioxidant activity in vitro, 4 different methods corresponding to different levels of antioxidant action were used: free radical-scavenging ability by using the DPPH assay, inhibition of lipid peroxidation, ferric reducing antioxidant power assay and chelating ability on ferrous ions. EC50 values of the DPPH scavenging activity of the polysaccharide extracts from commercial G. lucidum spore, P. linteus, G. applanatum and L. edodes fruiting bodies were found to be particularly low and were comparable with scavenging ability of α- tocopherol and ascorbic acid, EC50 < 0,1 mg/mL. G. applanatum extract showed the highest inhibition of lipid peroxidation (EC50 = 2,07 mg/mL). Ascorbic acid displayed an EC50 value of 1,64 mg/mL in the prevention of the lipid peroxidation. G. applanatum extract also had the highest reducing power (EC50 = 0,18). The EC50 values of the extracts in the reducing power assay ranged from 0,47 to > 20 mg/mL. The high reducing power of most extracts was in correlation with the non polysaccharide components present in the extracts. Positive correlation was found between EC50 values of the reducing power abilities and the amount of total glucans, β-glucans and polysaccharides content in the non dialyzed polysaccharide extracts. Strong negative correlation was observed between total phenol content and EC50 values of the reducing power, as moderate negative correlation with total protein content. For all polysaccharide extracts, which were additionally purified by dialysis and ethanol precipitation, a decrease of EC50 values of the reducing power correlated with higher phenol and α-glucan contents, i. e. increase of the reducing power correlated with higher phenol and α-glucan contents. FT-IR spectrum of G. applanatum extract showed the presence of a free uronic acid or esterified C=O group of the uronic acids as the structural component of at least one polysaccharide in this extract. Among the non dialyzed polysaccharide extracts the lowest EC50 values of the chelating activity of ferrous ions were found for commercial G. lucidum spore extract, 0,59 mg/mL and for P.linteus fruiting bodies extract, 0,91 mg/mL. Positive correlation was found between EC50 values of the chelating ability and the amount of total polysaccharides, glucans content in the extracts. Negative and strong correlation was observed with total phenol contents, as negative and moderate correlation with total protein content. Citric acid was not a good chelating agent for ferrous ions in this assay, EC50 > 20 mg/mL. Among dialyzed polysaccharide extracts, G. lucidum showed the best chelating ability (EC50 = 3,58 mg/mL). The study of antimicrobial properties of polysaccharide extracts showed that the Gram-positive bacteria were generally more sensitive to the tested extracts compared with Gram-negative bacterial species (Pseudomonas aeruginosa ATCC 27853, Pseudomonas aeruginosa ATCC 35032, Escherichia coli ATCC 25922, Salmonella enteritidis ATCC 13076, Proteus hauseri ATCC 13315, Shigella sonnei ATCC 29930, Yersinia enterocolitica ATCC 27729, Escherichia coli (0157:H7) ATCC 12900, Escherichia coli (0157:H7) ATCC 35150, Klebsiella pneumoniae ATCC 27736). Almost all extracts were acting microbicidal on bacteria E. faecalis ATCC 29212 and E. faecalis ATCC 49532. MIC values for polysaccharide extracts from G. lucidum, G. applanatum, P. linteus, T. versicolor, A. brasiliensis fruiting bodies and commercial and in laboratory prepared G. lucidum spore extract were between 1,25-2,5 mg/mL. The effect of extracts on the other Gram-positive bacteria: Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 6538, Bacillus cereus ATCC 10876, Geobacillus stearothermophylus ATCC 7953, Listeria monocytogenes ATCC 19115, Listeria monocytogenes ATCC 19112 were microbiostatic. Extracts expessed MIC values between 2,5-5 mg/mL on bacteria Bacillus cereus ATCC 10876 and Geobacillus stearothermophylus ATCC 7953. MIC values for G. lucidum and G. applanatum fruiting bodies extracts were 2,5 mg/mL in relation to Staphylococcus aureus ATTC 25923 and Staphylococcus aureus ATTC 6538. Extracts had no effect on the growth of yeasts Candida albicans ATCC 24443, Candida albicans ATCC 10259, Candida albicans ATCC 10231 and Cryptococcus neoformans ATCC 76484. Polisaharidi gljiva, biljaka i mikroorganizama privlače veliku pažnju zbog svojih interesantnih bioloških svojstava kao što su: antitumorno, antivirusno, antioksidativno, antikomplementarno, antikoagulaciono, hipolipidemijsko dejstvo, kao i imunomodulatorska i imunostimulativna aktivnost, što ih sve čini pogodnim za primenu u mnogim oblastima, među kojima su i prehrambena industrija, kozmetika, biomedicina, poljoprivreda, zaštita životne sredine i otpadnih voda. Oksidativna oštećenja prirodnih i industrijski pripremljenih namirnica predstavljaju veliki ekonomski problem u prehrambenoj industriji, jer direktno utiču na karakteristike kvaliteta kao što su ukus, boja, tekstura i nutritivne vrednosti. Pored toga, može doći do nastanka potencijalno toksičnih proizvoda. Reaktivne vrste kiseonika učestvuju u patofiziologiji različitih kliničkih poremećaja, kao što su: ishemija, reperfuzione povrede, infarkt miokarda, reumatoidni artritis, neurodegenerativni poremećaji, ateroskleroza, hipertenzija, hemoragički šok i dijabetes. Mikrobiološko kvarenje hrane i trovanje hranom su jedan od najvažnijih problema sa kojima se suočava prehrambena industrija. Alimentarne intoksikacije su danas globalni problem. Porast rezistentnosti patogena na poznate lekove stvara potrebu za razvojem novih antimikrobnih agenasa. U ovom radu su proučavana antioksidativna i antimikrobna svojstva polisaharidnih ekstrakata odabranih vrsta gljiva iz klase Basidiomycetes: Agaricus bisporus, Agaricus brasiliensis, Ganoderma applanatum, Ganoderma lucidum, Lentinula edodes, Phellinus linteus i Trametes versicolor. Na osnovu dobijenih rezultata može se zaključiti da polisaharidni ekstrakti, dobijeni nakon ekstrakcije vrelom vodom, alkoholne precipitacije i dijalize, predstavljaju smešu/komplekse polisaharida, proteina i fenola. Analizom monosaharidnog sastava utvrđeno je da svi ispitivani ekstrakti sadrže D-glukozu kao preovlađujuću monosaharidnu komponentu. U većini ekstrakata su u manjoj ili većoj koncentraciji prisutni D-galaktoza, D-manoza i L-fukoza/D-ksiloza. Prisustvo uronskih kiselina potvrđeno je u polisaharidima ekstrakta gljive G. applanatum. Ekstrakte karakterišu različiti sadržaji α- i β-glukana. Ukupan sadržaj glukana, u odnosu na ukupan sadržaj polisaharida u ekstraktima nalazio se u opsegu od 35% do 87%. Glukopiranozne monosaharidne jedinice su međusobno prevashodno povezane β-glikozidnim vezama (57-97%), osim u komercijalnom ekstr
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