FACULTY OF SCIENCE DEPARTMENT OF BIOCHEMISTRY Ph.D. Dissertation Biosensors for analysis of drinks Miodrag Milovanović Supervisor: Assoc. Prof. Dr. Petr Skládal Brno, 2019 Masaryk University Biosensors for analysis of drinks Bibliographic Entry Author: Miodrag Milovanović, MSc. Faculty of Science, Masaryk University Department of Biochemistry Title of Thesis: Biosensors for analysis of drinks Degree programme: Biochemistry Field of study: Biochemistry Supervisor: Assoc. Prof. Dr. Petr Skládal Academic Year: 2018/2019 Number of Pages: XIII + 247 Keywords: Wine, Wine analysis, Organic acids, Bioelectronic tongue, Lactate oxidase, Sarcosine oxidase, Fumarase, Capillary electrophoresis, Principal components analysis, Self-organizing map, BioCarboxylic units, Quick Response wine classification, Smart wine classification Author Miodrag Molovanović |Acknowledgements II Masaryk University Biosensors for analysis of drinks Bibliografický záznam Autor: Mgr. Miodrag Milovanović Přírodovědecká fakulta, Masarykova univerzita Ústav biochemie Název práce: Biosenzorová analýza nápojů Studijní program: Biochemie Studijní obor: Biochemie Vedoucí práce: Doc. RNDr. Petr Skládal, CSc. Akademický rok: 2018/2019 Počet stran: XIII + 247 Klíčová slova: Víno, Analýza vína, Organické kyseliny, Bioelektronický jazyk, Laktát oxidasa, Sarkosin oxidasa, Fumarát oxidasa, Kapilární elektroforéza, Analýza hlavních komponent, Kohonenova samoorganizační mapa, BioKarboxylová jednotka, QR klasifikace vína, Smart klasifikace vína Author Miodrag Molovanović |Acknowledgements III Masaryk University Biosensors for analysis of drinks A long time ago, when I was a kid, my father said to me: “Dear son, imagination does not cost anything, therefore free your mind and the rest will follow.” Milan Milovanović Today, thirty years later… Thanks to his words, from many ideas that I had, the one idea is about to be realized. Author Miodrag Molovanović |Acknowledgements IV Masaryk University Biosensors for analysis of drinks Acknowledgements First and foremost, I would like to thank GOD for giving me the strength, knowledge, ability and opportunity to undertake this research study and to complete it satisfactorily. Without his blessings, this achievement would not be possible. Also, I owe huge gratitude to the Priests at the helm of the Orthodox Church in Brno. Throughout the writing of this dissertation, I have received a great amount of support and assistance. I would like to thank my supervisor Assoc. Prof. Petr Skládal, whose expertise was invaluable in formulating of this research topic and methodology and I am grateful for your consistent support and guidance during the running of this project. I would also like to thank my consultant and ex-colleague Dr Jiří Žeravík, for the excellent cooperation and for all of the opportunities I was given to conduct my research and my dissertation to the end at the University of Masaryk. I would like to acknowledge all my colleagues at Masaryk University for their wonderful collaboration, especially Matěj Pastucha. All members of our Biosensor group supported me greatly and were always willing to help me. In addition, I would like to thank my parents for their wise counsel, encouragement, patience and moral support. You are always there for me. Special thanks to my friends for showing their true friendship, care, understanding and support when I needed them the most: Nataša Paunović, Marko Stojanović, Stefan Mihailović, Siniša Petrik and Roman Majstorović. They were more like a family to me apart from being my close friends. Miodrag Author Miodrag Molovanović |Acknowledgements V Masaryk University Biosensors for analysis of drinks Abstract This thesis shows the connection between the geographical and social aspects of wine in one hand and the profile of carboxylic acids from the other hand. The role of Sun and “Terroir” and their influence on the quality of grapes and wines are deeply presented. The term “Terroir” includes the soil, topography and climate, expressing a significant influence on grapes, wine and quality of the wine. There are many options for how producers can control the environment in order to enhance the wine quality. When the grapes have already matured the social aspect starts to influence the wine. The first social aspect which has a direct influence on wine is technology. There are many types of procedures on how to produce the wine because the technology is the first fingerprint of producers on wine. The tourism and economy as further social aspects have a significant influence on wine, as well. All these influences on wine have a direct link to the created carboxylic acids in wine. Due to this, the approach for the determination of carboxylic acids is focusing on the biosensor as an innovative tool for convenient characterization. The subject of investigation beside the grapes of wine, the determination of the antioxidant activity of fruit wine is presented in this thesis as a side activity. Various types of fruits such as raspberry, blackberry, chokeberry, blueberry, apple and cherries are prepared for vinification process with sugar and without sugar. The 14 fruit wine samples were analyzed by standard and alternative techniques for measurement. The standard methods were applied for quality and quantity detection and the equipment and servicing of these techniques are not cheap. These techniques are a reverse phase (RP) HPLC and means of the mass spectrometer (MS) with triple quadrupole (TQ) analyzer. The alternative methods for measuring the antioxidant activity are the ferric reducing/antioxidant power (FRAP), tha stability of free radical molecul 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Folin–Ciocalteu assay. These methods are faster and cheaper than standard methods. The correctness of results in comparison with standard and alternative methods was satisfactory. The antioxidant activity of each fruit was expressed in the expected range. Author Miodrag Molovanović |Abstract VI Masaryk University Biosensors for analysis of drinks Sensors serving as a substitute of the human nose a tongue – electronic and bioelectronic nose and tongue are described. The designs, principles and the methods of (bio)electronic construction (nose and tongue, respectively) are mentioned. The direct applications in various fields are presented, as well. Moreover, the perspectives of both sensors are presented. The focus of this research was on developing the enzyme-based bioelectronic tongue for measurement of carboxylic acids by sarcosine oxidase enzyme inhibition effect. This focus is presented through the chapter of enzyme application in wine. The procedure of bioelectronic tongue preparation consists of creating the copolymer sieve (an equimolar mixture of o-phenylenediamine and resorcinol) with enzyme layer of sarcosine oxidase. This approach is used for preparing other bioelectronic tongue sensors such as lactate oxidase, sarcosine oxidase, and sarcosine oxidase / fumarase in the three sensing channels. The measurement is carried out by amperometric detection of hydrogen peroxide from the enzyme reactions. The 31 samples of grape wine were tested. Also, the standard method capillary electrophoresis (CE) and alternative (developed a bioelectronic tongue) was used for detection of carboxylic acids levels (indirectly related to malolactic fermentation). Both methods for detection of carboxylic acids were coupled to chemometrics methods as principal component analysis (PCA) and self-organizing maps (SOM). Both chemometrics methods have shown a good correspondence in presentation of the clusters of wine samples. The SOM treatment showed a better resolution of the generated patterns of wine samples than the PCA method for both CE and bioelectronic tongue data. The combination of bioelectrical tongue and SOM can be considered as a novel method in the classification of wine. Author Miodrag Molovanović |Abstract VII Masaryk University Biosensors for analysis of drinks Abstract Práce popisuje spojení mezi geografickými a společenskými aspekty vína na jedné straně a profilem zastoupených karboxylových kyselin na straně druhé. Široce je rozpracována role slunce a „terroir“ na kvalitu hroznů a vína. Pojem „terroir“ zahrnuje půdu, topografii a klima a má významný vliv na kvalitu vinných hroznů a vína. Vinaři mají mnoho možností, jak pěstebními podmínkami zvyšovat kvalitu vína. Po dozrání hroznů začínají vznikající víno ovlivňovat společenské aspekty, prvním z nich s přímým vlivem na víno je technologie. Existuje mnoho postupů výroby vína, a tak je volba výrobní technologie hlavním bodem, kde se na víně jeho výrobce podepíše. Dalšími společenskými faktory s významným vlivem na víno jsou turistika a ekonomie. Všechny tyto faktory přímo ovlivňují složení karboxylových kyselin ve víně. Práce se proto zaměřuje na jejich stanovení pomocí biosensoru jako inovativního nástroje vhodného pro jejich charakterizaci. Jako vedlejší výstup práce je prezentováno také stanovení antioxidační aktivity v ovocných vínech. Pro přípravu ovocných vín s či bez přidaného cukru jsou využívány různé ovocné poldy – maliny, ostružiny, aronie, borůvky, jablka a třešně. Standardními i alternativními metodami bylo analyzováno 14 vzorků ovocných vín. Standardní metody jako je vysokoúčinná kapalinová chromatografie s reverzní fází (RP-HPLC) a hmotnostní spektrometrie (MS) s trojitým kvadrupólem (TQ) sloužily ke kvalitativní i kvantitativní analýze ale významným faktorem jsou zde náklady na potřebnou instrumentaci. Alternativními metodami použitými pro měření antioxidační