UNIVERSIDAD DE SALAMANCA - FACULTAD DE FARMACIA Departamento de Química Analítica, Nutrición y Bromatología CARACTERIZACIÓN QUÍMICA Y PROPIEDADES BIOACTIVAS DE HONGOS SILVESTRES PORTUGUESES COMESTIBLES CHEMICAL CHARACTERIZATION AND BIOACTIVE PROPERTIES OF PORTUGUESE WILD EDIBLE MUSHROOMS Lillian Bouçada de Barros 2008 ii Acknowledgements ACKNOWLEDGEMENTS To all the people that has been fundamental in providing me with help, direction and support throughout this PhD journey and in particularly to: Doctor Celestino Santos Buelga, my supervisor, thank you for an invaluable and rewarding experience, your support and encouragement has been a source of motivation. Doctor Isabel Cristina Fernandes Rodrigues Ferreira, my co-supervisor, thank you for not only being a fabulous supervisor and a fantastic mentor, but also for being a friend. I will never forget all your efforts in helping me achieve this goal. You will always be a source of inspiration. Doctor Paula Baptista, thank you for giving me the opportunity to discover the amazing world of fungi and for kindly collecting and identifying all the wild mushroom species. Thank you to all the my colleagues and friends in the LQBA (Laboratório de química e Bioquímica Aplicada, Bragança) and also to all within the department of Química analítica, Nutrition y Bromatología, Facultad de Farmacia of Salamanca, you have all contributed in making this journey an enjoyable one. A particularly thank you to Montserrat Dueñas, this last year would not be possible without your support and encouragement. A special thanks to Daniela Correia, for the love, encouragement and support that you have given me throughout this process. To Soraia Falcão and João Barreira, thank you for the invaluable good times we had throughout this journey and rewarding experience. I send my love and thanks to all my wonderful friends, those friends I have known for a long time and those who I have made along the way. Thank you for being so patient. iii Acknowledgements A grateful thank you to the Foundation for Science and Technology (PPCDT/AGR/56661/2004) for financial support of this work. My dear family, Mom, Dad, Diana, Rodrigo and in particularly to my parents in law, Manuel and Teresinha and grandmother Inocência, Thank you for your encouragement and for being so proud of my efforts. Your love and excitement has helped me believe in myself. Finally, I would like to thank Pedro Bastos, my husband, the most supporting and beautiful person, someone can have. Your constant love, patience and understanding have been my strength throughout this long journey. To my unborn daughter, Inês…... iv Abstract ABSTRACT Nineteen different mushroom species (Agaricus arvensis, Agaricus bisporus, Agaricus romagnesii, Agaricus silvaticus, Agaricus silvicola, Cantharellus cibarius, Hypholoma fasciculare, Lactarius deliciosus, Lactarius piperatus, Lepista nuda, Leucopaxillus giganteus, Lycoperdon molle, Lycoperdon perlatum, Macrolepiota mastoidea, Macrolepiota procera, Ramaria botrytis, Sarcodon imbricatus, Tricholoma acerbum and Tricholoma portentosum) from Northeast of Portugal, one of the European regions with higher wild edible mushrooms diversity, were evaluated for their chemical composition, nutritional value and bioactive properties (antioxidant and antimicrobial activities), in order to valorise mushrooms as a source of nutrients and nutraceuticals. The analysis of nutrients included determination of proteins, fats, ash, and carbohydrates, particularly sugars by HPLC-RI. The analysis of nutraceuticals included determination of fatty acids by GC-FID, and other phytochemicals such as tocopherols, by HPLC-fluorescence, phenolic compounds by HPLC-DAD-ESI/MS, carotenoids and ascorbic acid, by spectrophotometric techniques. The antioxidant activity was screened through chemical and biochemical assays. The chemical assays allowed an evaluation of their reducing power, radical scavenging activity and inhibition of -carotene bleaching, while biochemical assays evaluated the lipid peroxidation inhibition capacity, using erythrocytes and brain cells as models. The antimicrobial activity was evaluated using clinical isolates or collection microorganisms (Gram positive and Gram negative bacteria and fungi). The macronutrient profile in general revealed that the wild mushrooms were rich sources of protein and carbohydrates and had low amounts of fat. The analysis of fatty acid composition allowed the quantification of twenty three fatty acids. Unsaturated fatty acids and, in particular, oleic and linoleic acids, were predominant. Mannitol and trehalose were the most abundant sugars. The analysed mushrooms also contain very useful phytochemicals such as phenolic compounds, tocopherols, ascorbic acid, and carotenoids. Particularly, four phenolic acids (protocatechuic, p-hydroxybenzoic acid, p- coumaric and cinnamic acid) and two vanillic acid isomers were detected, identified and quantified, as also three of the tocopherols vitamers (F-, G-, H- tocopherol); no tocotrienols were detected. All the species proved to have antioxidant properties, Chemical Characterization And Bioactive Properties Of Portuguese Wild Edible Mushrooms v Abstract namely radical scavenging activity and lipid peroxidation inhibition capacity. Ramaria botrytis was the most efficient species presenting the lowest EC50 values in the chemical and biochemical assays, which can be related to their higher content in bioactive compounds. The majority of the species revealed antimicrobial activity selectively against Gram + bacteria, in some cases, with lower minimum inhibitory concentration than the standards. Processing and cooking practices had a determining influence on chemical composition and antioxidant properties. Cooked samples showed lower nutrients concentrations and lower antioxidant activity than either dried or frozen samples. Nevertheless, for fatty acids and sugar individual profiles, only cooking procedures seemed to be relevant, the cooked samples presenting higher MUFA, and lower PUFA and sugars contents. The fruiting body maturity stage proved to have influence on chemical composition and bioactivity of the wild mushrooms; mature carpophorus with mature spores is not recommended for nutritional and medicinal proposals. In addition to dried mushrooms, alternative or substitute mushroom products are mycelia that could also be used as food and food-flavouring material, or in the formulation of nutraceuticals and functional foods. In order to explore it, mycelium of Leucopaxillus giganteus was produced using different pH, carbon and nitrogen sources in the culture medium, and evaluated for their bioactive properties. The antioxidants concentration increased along the growth time as a response to the oxidative stress and subsequent free radicals production. The aldohexose glucose and diammonium phosphate proved to be the most appropriate carbon and nitrogen sources to increase antioxidant activity, leading to the highest phenols content and lowest EC50 values. Public health authorities consider prevention and treatment with nutraceuticals/phytochemicals a powerful instrument in maintaining and promoting health, longevity and life quality. The beneficial effects of nutraceuticals will undoubtedly have an impact on nutritional therapy; they also represent a growing segment of today’s food industry. Mushrooms might be used directly in diet and promote health, taking advantage of the additive and synergistic effects of all the bioactive compounds present. Therefore, the ongoing research will lead to a new generation of foods, and will certainly promote their nutritional and medicinal use. Chemical Characterization And Bioactive Properties Of Portuguese Wild Edible Mushrooms vi Resumen RESUMEN Diecinueve especies diferentes de setas silvestres (Agaricus arvensis, Agaricus bisporus, Agaricus romagnesii, Agaricus silvaticus, Agaricus silvicola, Cantharellus cibarius, Hypholoma fasciculare, Lactarius deliciosus, Lactarius piperatus, Lepista nuda, Leucopaxillus giganteus, Lycoperdon molle, Lycoperdon perlatum, Macrolepiota mastoidea, Macrolepiota procera, Ramaria botrytis, Sarcodon imbricatus, Tricholoma acerbum and Tricholoma portentosum) del Nordeste de Portugal, una de las regiones europeas con mayor diversidad hongos silvestres comestibles, fueron evaluados con relación a su composición química, valor nutritivo y propiedades bioactivas (actividades antioxidante y antimicrobiana), con vistas a su valorización como fuente de nutrientes y nutracéuticos. El análisis de nutrientes incluyó la determinación de proteínas, grasas, cenizas, hidratos de carbono, y particularmente azúcares por HPLC-RI. El análisis de nutracéuticos incluyó la determinación de ácidos grasos por GC-FID y otros fitoquímicos, como tocoferoles por HPLC-fluorescencia, compuestos fenólicos por HPLC-DAD-ESI/MS, carotenoides y ácido ascórbico por espectrofotometría. La actividad antioxidante se valoró mediante ensayos químicos y bioquímicos. Los ensayos químicos consistieron en la evaluación del poder reductor, inhibición del radical libre DPPH e inhibición de la decoloración del G-caroteno, mientras que los ensayos bioquímicos evaluaron la capacidad de inhibición de la peroxidación lipídica, utilizando como modelos eritrocitos y células del cerebro. La actividad antimicrobiana fue evaluada utilizando aislados clínicos y microorganismos de colección (bacterias Gram positivas y Gram negativas y hongos). El perfil de macronutrientes reveló que, en general, los hongos silvestres son fuentes ricas de proteínas