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Phytases in Yeast and Lactic Acid Bacteria Isolated from Grain-Based Food

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Phytases in Yeast and Lactic Acid Bacteria Isolated from Grain-Based Food DEPARTMEnt OF FOOD SCIEncE LINA NUOBARIENE faculty of science QUALITY And TEchnOLOGY university of copenhagen PHD thESIS 2012 · ISBN 978-87-7611-530-2 LINA NUOBARIENE Phytases in Yeast and Lactic Acid Bacteria Isolated from Grain-based Food Phytases in Yeast and Lactic Acid Bacteria Isolated from Grain-based Food and Lactic Bacteria from Acid Isolated Yeast in Phytases Nowadays, the consumption of whole-grain breads is gaining popular- ity across the world due to increasing awareness of its nutritional benefits. Phytases in Yeast Whole grains contain wide range of nutrients, such as dietary fibre, vitamins and Lactic Acid Bacteria and minerals. Despite that whole grains contain 20-70% of the daily require- ments of the minerals, their absorption in human gut are very low due to Isolated from antinutritional phytic acid salt (phytate), and this may lead to mineral defi- Grain-based Food ciencies. To increase the bioavailability of minerals, enzymatic degradation of phytate and its dephosphorylated isomers is needed. PHD thESIS 2012 The aims of the thesis were therefore: (i) to isolate and identify phytase active yeasts and lactic acid bacteria strains to be used in bread making to increase the content of bioavailable minerals in bread; Lina Nuobariene (ii) to purify and characterize the phytase isolated from the most prominent yeast strain for phytase production. The highest specific extracellular phytase activities were observed in Saccharomyces cerevisiae L1.12 (10.6 U/1010 CFU), S. cerevisiae L6.06 (8.2 U/1010 CFU), Lactobacillus frumenti 2.1 (13 U/1010 CFU), and L. frumenti 2.5 (16 U/1010 CFU). Furthermore, Saccharomyces pastorianus, Candida humilis, and L. fru- menti have for the first time been recognized as phytase positive strains. Phytase purification resulted in 8.1-fold purification with a yield of 3.6% to a maximum specific activ- ity of 42.9% U/mg. The optimum pH and temperature were found to be 4.0 and 35°C, respectively. FACULTY OF SCIENCE UNIVERSITY OF COPENHAGEN PhD thesis Lina Nuobariene Phytases in yeast and lactic acid bacteria isolated from grain-based food Quality and Technology ▪ Department of Food Science Faculty of Science ▪ University of Copenhagen Title Phytases in yeast and lactic acid bacteria isolated from grain-based foods Submission th July 12 2012 Defence October 5th 2012 Supervisors Associate Professor Åse Solvej Hansen - To my Mother and Father Faculty of Science, Department of Food Science, Section for Quality and Technology University of Copenhagen - Mamai ir T ėveliui Associate Professor Nils Arneborg Faculty of Science, Department of Food Science, Section for Food Microbiology University of Copenhagen Opponents Associate Professor Charlotte Bjergegaard (Chairman) Department of Food Science, Faculty of Science University of Copenhagen, Denmark Associate Professor (Docent) Anders Thomas Andlid Department of Chemical and Biological Engineering Food Science, Chalmers University of Technology, Sweden Senior Scientist Henrik Brinck-Pedersen Institute for Molecular Biology and Genetics, Science and Technology University of Aarhus, Denmark Cover illustration by Lisbeth Kløcker Petersen PhD thesis 2012©Lina Nuobariene ISBN 978-87-7611-530-2 Printed by SL grafik, Frederiksberg C, Denmark (www.slgrafik.dk) Title Phytases in yeast and lactic acid bacteria isolated from grain-based foods Submission th July 12 2012 Defence October 5th 2012 Supervisors Associate Professor Åse Solvej Hansen - To my Mother and Father Faculty of Science, Department of Food Science, Section for Quality and Technology University of Copenhagen - Mamai ir T ėveliui Associate Professor Nils Arneborg Faculty of Science, Department of Food Science, Section for Food Microbiology University of Copenhagen Opponents Associate Professor Charlotte Bjergegaard (Chairman) Department of Food Science, Faculty of Science University of Copenhagen, Denmark Associate Professor (Docent) Anders Thomas Andlid Department of Chemical and Biological Engineering Food Science, Chalmers University of Technology, Sweden Senior Scientist Henrik Brinck-Pedersen Institute for Molecular Biology and Genetics, Science and Technology University of Aarhus, Denmark Cover illustration by Lisbeth Kløcker Petersen Preface This PhD thesis has been accomplished to fulfil the requirements for obtaining a PhD degree at the Quality and Technology Section, Department of Food Science, Faculty of Life Science, University of Copenhagen. This PhD project was financed by a scholarship from faculty of LIFE Science, University of Copenhagen therefore grateful acknowledge goes to them for the funding source that made my PhD work possible. PhD project was carried out under the supervision of Associate professor Åse Solvej Hansen and Associate Professor Nils Arneborg. Åse, thank you for your valuable The doctor of the future will give no medicine, knowledge and for always keeping your door open for scientific discussions. The But will interest his patients in the care of human frame, in diet, discussions with you always were eyes opening, productive and inspiring; the advices And in the cause and prevention of disease and comments were always interesting and stimulating. Nils, thank you for guiding me in the complex world of microbiology. To both, Åse and Nils, I send you my gratitude for the patience and the encouragement you have given me. – Thomas Alva Edison I am thankful to all my colleagues at Q&T for contributing to a pleasant, welcoming and professionally fruitful working environment. Special thanks go to Marta Jolanta Popielarz and my officemate Solveig Warnecke. Marta, thank you for your time we have spent together. We have shared many ups and downs in our lives and you always been there for me no matter what occasion. Solveig, I am grateful for your attentive listening and for guiding me along the Danish society. Furthermore, special thanks go to Associate Professor Finn Kvist Vogensen for his support and kind help in the world of lactic acid bacteria. I also wish to thank Lisbeth and Abdel from Q&T, Basheer and Janne from Food Microbiology for the assistance with laboratory work. Warm thanks go to all my friends outside work, for supporting me, having fun with me and listening when I need to talk. Special thanks go to Mohsen Rafi Moussa. Thank you so much for always supporting me and always being there for me. i Preface This PhD thesis has been accomplished to fulfil the requirements for obtaining a PhD degree at the Quality and Technology Section, Department of Food Science, Faculty of Life Science, University of Copenhagen. This PhD project was financed by a scholarship from faculty of LIFE Science, University of Copenhagen therefore grateful acknowledge goes to them for the funding source that made my PhD work possible. PhD project was carried out under the supervision of Associate professor Åse Solvej Hansen and Associate Professor Nils Arneborg. Åse, thank you for your valuable The doctor of the future will give no medicine, knowledge and for always keeping your door open for scientific discussions. The But will interest his patients in the care of human frame, in diet, discussions with you always were eyes opening, productive and inspiring; the advices And in the cause and prevention of disease and comments were always interesting and stimulating. Nils, thank you for guiding me in the complex world of microbiology. To both, Åse and Nils, I send you my gratitude for the patience and the encouragement you have given me. – Thomas Alva Edison I am thankful to all my colleagues at Q&T for contributing to a pleasant, welcoming and professionally fruitful working environment. Special thanks go to Marta Jolanta Popielarz and my officemate Solveig Warnecke. Marta, thank you for your time we have spent together. We have shared many ups and downs in our lives and you always been there for me no matter what occasion. Solveig, I am grateful for your attentive listening and for guiding me along the Danish society. Furthermore, special thanks go to Associate Professor Finn Kvist Vogensen for his support and kind help in the world of lactic acid bacteria. I also wish to thank Lisbeth and Abdel from Q&T, Basheer and Janne from Food Microbiology for the assistance with laboratory work. Warm thanks go to all my friends outside work, for supporting me, having fun with me and listening when I need to talk. Special thanks go to Mohsen Rafi Moussa. Thank you so much for always supporting me and always being there for me. i * * * * * Mama ir Tėti, šį darbą skiriu Jums. Jūs patys nuostabiausi. Silvija ir Aivarai, Jūs esat mano šviesa. Ačiū sesytėm Virginijai ir Vilandai už Jūsų palaikymą ir buvimą šalia Abstract kai man to labiausiai reikėdavo. Aš Jus visus labai myliu. Nowadays, consumption of whole-grain breads is gaining popularity across the world due to increasing awareness of its nutritional benefits. Whole grains contain wide range of nutrients, such as dietary fibre, vitamins and minerals. Despite that whole Lina Nuobariene grains contain 20-70% of the daily requirements of the minerals, their absorption in human gut are very low due to antinutritional phytic acid salt (phytate), and this may Copenhagen, 2012 July lead to mineral deficiencies. To increase the bioavailability of minerals, enzymatic degradation of phytate and its dephosphorylated isomers is needed. The main aim of this thesis was therefore to isolate and identify phytase active strains of yeasts and lactic acid bacteria to be used in bread making. The use of selected combination of yeast and lactic acid bacteria with high phytase activity could find use as starter cultures to improve mineral bioavailability in whole-grain bread fermented in short time as alternative to sourdough bread and/or yeast fermented bread. All experiments were carried out at conditions optimal for leavening of bread (30°C and pH 5.5). Another aim of the study was to isolate and identify phytase active yeasts and lactic acid bacteria from commercial sourdoughs. In addition, comparison between sourdoughs from different bakeries and countries on yeast strain distribution and their phytase activities was done.
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