Biotechnology – Research and Industrial Applications · Ewa Huszcza Tomasz Boruczkowski Filip Boratyński Zbigniew Lazar Michał Piegza Monika Grzegorczyk Mateusz Kropiwnicki Joanna

Biotechnology – Research and Industrial Applications

17–18.09.2015, Wrocław – POLAND

Wroclaw Centre of Biotechnology 2014-2018

The Leading NationalResaerch Centre

Ministry of Scienceand Higher Education

The Faculty of Food Science

Biotechnology – ReseaRch and industRial applications

17–18.09. 2015 WRocłaW – poland













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scientiFic coMMitteeGeorge Aggelis (Greece)

Kurt Faber (Austria)Jean-Marc Nicaud (France)Helene Kautola (Finland)

Józefa Chrzanowska (Wrocław)Małgorzata Darewicz (Olsztyn)Włodzimierz Grajek (Poznań)

Waldemar Rymowicz (Wrocław)Maria Wojtatowicz (Wrocław

oRganiZing coMMitteeMałgorzata RobakBarbara Żarowska

Ewa HuszczaTomasz Boruczkowski

Filip BoratyńskiZbigniew LazarMichał Piegza

Monika GrzegorczykMateusz KropiwnickiJoanna Koniuszewska

Edyta WoźniakPaweł Mituła

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SponSorS

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TimeTable

thuRsday 17.09.20158.00–9.00 REGISTRATION

9.15–9.30 Opening Ceremony and Welcome – Józefa Chrzanowska

9.30–10.00 Plenary lecture No 1

Patterns of lipid accumulation in oleaginous microorganisms and biotechnological perspectives of single cell oil – George Aggelis (Greece)

10.00–10.30 Plenary lecture 2

Bioactive peptides from food proteins – methodological approaches and results – Małgorzata Darewicz (Poland)


Decarboxylases and Sulfatases: Biocatalytic Equivalents to the Kolbe-Schmitt – and the Mitsunobu-Reaction – Kurt Faber (Autrich)

11.00–11.30 Coffee Break

Session 1: MICROORGANISMS(Chair: Waldemar Rymowicz & George Aggelis)


Laboratory evolution in action: improving the growth properties of Bacillus subtilis – Akos Tibor Kovacs (Germany)

12.00–13.30 Short Communications (1.1–1.6)

13.30–14.30 Lunch

Session 2: ENZYMES & PEPTIDES(Chair: Małgorzata Darewicz & Marcin Drąg)


Hybrid Combinatorial Substrates Library (HyCoSuL) as a tool in design of specific and sensitive probes for endopeptidases – Marcin Drąg (Poland)



16.30–17.00 Coffee Break

POSTER SESSION(Chair: Małgorzata Robak & Akos Tibor Kovacs & Anne-Marie Crutz Le Coq)

16.45–18.30 MICROORGANISMS ENZYMES BIOSYNTHESIS & BIODEGRADATIONS BIOTRANSFORMATIONS

20.00–02.00 Banquet

Hotel Mercury, 1 Dominikanski Sq.

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FRiday 18.09.20159.00 Day 2 – opening:

(Chair: Włodzimierz Grajek & George Aggelis)


Yarrowia lipolytica, a model for lipid metabolism and a platform for lipid production – Jean-Marc Nicaud (France)

Session 3: BIOSYNTHESIS & BIODEGRADATIONS(Chair: Helena Kautola & Jean-Marc Nicaud)


Sugar utilisation for biosynthesis of valuable products by Yarrowia lipolytica and related species – Anne-Marie Crutz Le Coq (France)


Challenges and possibilities in using biodegradable raw materials – Helena Kautola (Finland)


11.30–12.00 Coffee Break & POSTERS EVALUATION – End of poster session

Session 4: BIOTRANSFORMATIONS(Chair: Kurt Faber & Czesław Wawrzeńczyk)


Microbial syntheses and transformations of lactones – Czesław Wawrzeńczyk (Poland)


13.30–13.45 Closing of the conference

13.45–14.45 Lunch

15.00–18.00 City sightseeing with guide

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ConTenTS

PLENARy LECTuRES (PL)PL 1 George Aggelis

PATTERNS OF LIPID ACCUMULATION IN OLEAGINOUS MICROORGANISMS AND BIOTECHNOLOGICAL PERSPECTIVES OF SINGLE CELL OIL . . . . . . . . . . . . 15

PL 2 Małgorzata Darewicz, Anna Iwaniak, Piotr MinkiewiczBIOACTIVE PEPTIDES FROM FOOD PROTEINS – METHODOLOGICAL APPROACHES AND RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

PL 3 Silvia Glueck, Katharina Plasch, Tamara Reiter, Verena Resch, Kurt FaberDECARBOxYLASES AND SULFATASES: BIOCATALYTIC EqUIVALENTS TO THE KOLBE-SCHMITT- AND THE MITSUNOBU-REACTION . . . . . . . . . . . . . 18

PL 4 Felix Blei, Jan Schwitalla, Derek Matern, Gergely Maróti, Axel A. Brakhage, Ákos T. KovácsLABORATORY EVOLUTION IN ACTION: IMPROVING THE GROWTH PROPERTIES OF BACILLUS SUBTILIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

PL 5 Marcin Drąg, Marcin Poręba, Paulina Kasperkiewicz, Wioletta Rut, Scott Snipas, Heather Parker, Christine Winterbourn, Guy S. Salvesen

HYBRID COMBINATORIAL SUBSTRATES LIBRARY (HYCOSUL) AS A TOOL IN DESIGN OF SPECIFIC AND SENSITIVE PROBES FOR ENDOPEPTIDASES . . . . . . . . . . . . . . 21

PL 6 Jean-Marc Nicaud, Athanasios Beopoulos, Anne-Marie Crutz-Le Coq, Remi Dulermo, Thierry Dulermo, Rodrigo Ledesma-Amaro, Zbigniew Lazar, Cecile Neuveglise, Heber Gamboa-Meléndez, Tristan Rossignol

Yarrowia lipolYtica, A MODEL FOR LIPID METABOLISM AND A PLATFORM FOR LIPID PRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

PL 7 Zbigniew Lazar, Thierry Dulermo, Cecile Neuvéglise,Tristan Rossignol, Malgorzata Robak, Jean-Marc Nicaud, Anne-Marie Crutz-Le Coq

SUGAR UTILISATION FOR BIOSYNTHESIS OF VALUABLE PRODUCTS BY Yarrowia lipolYtica AND RELATED SPECIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

PL 8 Helena KautolaCHALLENGES AND POSSIBILITIES IN USING BIODEGRADABLE RAW MATERIALS . 24

PL 9 Czesław Wawrzeńczyk, Filip Boratyński, Anna Gliszczyńska, Witold Gładkowski, Radosław Gniłka, Małgorzata Grabarczyk, Aleksandra Grudniewska, Agnieszka Leśniak, Marcelina Mazur, Teresa Olejniczak, Małgorzata Smuga

MICROBIAL SYNTHESIS AND TRANSFORMATIONSOF LACTONES . . . . . . . . . . . 26

SHORT COMMuNICATIONSLectures (L)Session 1: MICROORGANISMSL 1.1 Wojciech Łaba, Wiesław Kopeć, Dorota Chorążyk, Anna Pudło, Adam Kurzawa, Anna Kancelista, Magdalena Wróbel-Kwiatkowska, Michał Piegza

Bacillus cereus B5ESZ IN BIODEGRADATION OF PRETREATED PIG BRISTLES . . 31L 1.2 Kinga Bondarczuk, Anna Markowicz, Zofia Piotrowska-Seget

ANTIBIOTIC RESISTANCE IN A SEWAGE SLUDGE-FERTILIZED SOIL . . . . . . . . . . 32L 1.3 Adam Waśko, Magdalena Polak-Berecka, Dominik Szwajgier, Patrycja Boguta, Jolanta Cieśla, Tomasz Skrzypek

lactoBacilli AS CD2+ BIOSORBENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

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L 1.4 Magdalena Czołpińska, Anna Zofia Roszak, Katarzyna BuczkowskaANTIBACTERIAL POTENTIAL OF LIVERWORTS . . . . . . . . . . . . . . . . . . . . . . . . 34

L 1.5 Anna Kancelista, Regina Stempniewicz, Wojciech Łaba, Marta Pasławska, Michał Piegza, Aleksandra Wilczak, Monika Grzegorczyk, Magdalena Szczech, Danuta Witkowska

THE BIOLOGICAL ACTIVITY OF trichoderma atroviride SK80 IN BIOPREPARATIONS PRESERVED BY FOUNTAIN DRYING OR LYOPHILIZATION . 35

L 1.6 Zbigniew Lazar, Piotr Hapeta, Paweł Mituła, Małgorzata RobakMODIFICATIONS OF N-GLYCAN STRUCTURES SYNTHESIZED BY Yarrowia lipolYtica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Session 2: ENZyMES & PEPTIDESL 2.1 Paweł Serek, Iwona Bednarz-Misa, Magdalena Mierzchała-Pasierb, Agnieszka Bronowicka-Szydełko, Małgorzata Krzystek-Korpacka, Andrzej Gamian

ISOLATION AND PURIFICATION OF CYTOSOLIC ENOLASE FROM salmonella enterica SEROVAR TYPHIMURIUM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

L 2.2 Anna Dąbrowska, Konrad Babij, Marek Szołtysik, Łukasz Bobak, Aleksandra Zambrowicz, Józefa Chrzanowska

CASEINATE HYDROLYSATE OBTAINED WITH SERINE PROTEASE FROM Yarrowia lipolYtica YEAST AS GROWTH STIMULATOR OF PROBIOTIC BACTERIA . . . . . . 38

L 2.3 Ewelina Celińska, Monika Borkowska, Wojciech BiałasHETEROLOGOUS ExPRESSION OF A RAW-STARCH-DIGESTING INSECT AMYLASE IN Yarrowia lipolYtica – INTRODUCTION TO THE PRODUCTION PROCESS’ ENHANCEMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

L 2.4 Piotr Minkiewicz, Anna Iwaniak, Małgorzata Darewicz, Krzysztof Sieniawski, Piotr StarowiczBIOPEP DATABASE OF SENSORY PEPTIDES AND AMINO ACIDS . . . . . . . . . . . . . 40

Session 3: BIOSyNTHESIS & BIODEGRADATIONSL 3.1 Monika Grzegorczyk, xymena Połomska, Piotr Regiec, Maria Wojtatowicz, Barbara Żarowska

THE PRODUCTION OF KILLER TOxIN FOR PLANT PROTECTION BY deBarYomYces hansenii YEAST IN VARIOUS CULTURE MEDIA . . . . . . . . . 41

L 3.2 Magdalena Wróbel-Kwiatkowska, Waldemar Rymowicz, Jan SzopaPOTENTIAL APPLICATION OF GM FLAx . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

L 3.3 Anna Markowicz, Kinga Bondarczuk, Zofia Piotrowska-SegetASSESSING THE IMPACT OF SEWAGE SLUDGE APPLICATION ON THE MICROBIAL ACTIVITY, FUNCTIONAL DIVERSITY AND COMMUNITY STRUCTURE IN SOIL . . . 43

L 3.4 Tomasz Janicki, Jerzy DługońskiTHE ABILITY OF THE FILAMENTOUS FUNGUS IM 833 TO GROWTH IN THE PRESENCE OF HEAVY METALS AND xENOBIOTICS . . . . . . . . . . . . . . . . 44

L 3.5 Patrik R. Lennartsson, Mohammad J. TaherzadehINTEGRATION OF EDIBLE FILAMENTOUS FUNGI IN THE ETHANOL BIOREFINERY AND THE IMPORTANCE OF BY-PRODUCTS . . . . . . . . . . . . . . . . . . . . . . . . . . 45

L 3.6 Aleksandra M. Mirończuk, Magdalena Rakicka, Anna Biegalska, Waldemar Rymowicz, Adam Dobrowolski

A NOVEL TWO-STAGE FERMENTATION PROCESS OF ERYTHRITOL PRODUCTION BY YEAST Yarrowia lipolYtica FROM MOLASSES . . . . . . . . . . . . . . . . . . . . 46

Session 4: BIOTRANSFORMATIONSL 4.1 Beata Szmigiel, Ewa Żymańczyk-Duda

BIOCATALYZED SYNTHESIS OF TYROSOL DERIVATIVES . . . . . . . . . . . . . . . . . 47L 4.2 Paulina Walczak, Filip Boratyński, Teresa Olejniczak

MICROBIAL HYDROLYSIS OF BICYCLIC LACTAMS . . . . . . . . . . . . . . . . . . . . . . 48

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L 4.3 Witold Gładkowski, Anna Chojnacka, Anna Gliszczyńska, Natalia Niezgoda, Czesław Wawrzeńczyk

ENZYMATIC MODIFICATION OF PHOSPHOLIPIDS . . . . . . . . . . . . . . . . . . . . . 49L 4.4 Bartłomiej Potaniec, Mirosław Anioł

SYNTHESIS AND BIOTRANSFORMATION OF ANALOGUES NATURAL OF FLAVONIOIDS AND CHALCONES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

Posters (P)Session 1: MICROORGANISMSP 1.1 Miroslava Kačániová, Margarita Terentjeva, Marek Astaloš, Jana Petrová, Attila Kántor, Macej Kluz, Lukáš Hleba

ANTIBACTERIAL ACTIVITY OF SELECTED TEA SUBSTANCES, TEAS AND MEDICINAL HERBS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

P 1.2 Joanna Koniuszewska, Dominika Ciurko, Barbara Żarowska ANTIFUNGAL ACTIVITY OF SACCHAROMYCES CEREVISIAE YEASTS . . . . . . . . . 54

P1.3 Barbara Żarowska, Michał Piegza, Katarzyna Jaros-Koźlecka, Tomasz Koźlecki, Małgorzata Robak

ANTIMICROBIAL ACTIVITY OF SILVER NANOPARTICLES . . . . . . . . . . . . . . . . . 55P 1.4 Daria Kaczmarczyk, Stanisław Lochyński

MICROORGANISMS PRESENT IN FERMENTED GREEN TEA . . . . . . . . . . . . . . . . 56P 1.5 Michał Piegza, Joanna Koniuszewska, Anna Kancelista, Wojciech Łaba, Danuta Witkowska

BIOREACTOR CULTURES OF trichoderma MOULDS FOR BIOMASS AND SPORE PRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

P 1.6 Aleksandra Han, Anna Ropa, Joanna Koniuszewska, Wojciech Łaba, Michał Piegza trichoderma citrinoviride C1 AND trichoderma harzianum T33 AS ANTY-PHYTOPATHOGENIC AGENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

P 1.7 Monika Grzegorczyk, xymena Połomska, Barbara Żarowska CHARACTERISTICS OF KILLER YEASTS deBarYomYces hansenii AII4B SEGREGANTS: AII4BS AND AII4BR IN TERMS OF MORPHOLOGY AND KILLER ACTIVITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

P 1.8 Mateusz Kropiwnicki, Małgorzata Robak COMPARISON OF Yarrowia lipolYtica STRAINS BYRAPD . . . . . . . . . . . . . . . 60

P 1.9 Xymena Połomska, Magdalena Sznurowska, Joanna Matyjasz, Wojciech Pusz, Barbara Żarowska

VI1 – THE NEW PCR STARTER WHICH TOGETHER WITH ITS4 ALLOWS SPECIFIC IDENTIFICATION OF venturia inaequalis PHYTOPATHOGENIC FUNGI . . . . . 61

P 1.10 Attila Kántor, Miroslava KačániováSUITABLE MEDIUM FOR GluconoBacter oxYdans CULTIVATION . . . . . . . . . 62

P 1.11 Wojciech Łaba, Maja Pezała, Barbara ŻarowskaISOLATION OF KERATINOLYTIC BACTERIA FROM POULTRY . . . . . . . . . . . . . . . 63

P 1.12 Natalia Kmiecik, Ewa Żymańczyk-DudaAPPLICATION OF penicillium minioluteum AS A BIOCATALYST WITH HYDROLYTIC ACTIVITY FOR THE ACIDIC AMINOPHOSPHONIC ACID SYNTHESIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

P 1.13 Agata Głąb, Ewa Żymańczyk-Duda CYANOBACTERIA AS BIOCATALYSTS IN REDOx REACTIONS . . . . . . . . . . . . . . . 65

Session 2: ENZyMES & PEPTIDESP 2.1 Małgorzata Darewicz, Monika Pliszka, Justyna Borawska, Anna Iwaniak, Piotr Minkiewicz, Gerd Elisabeth Vegarud

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PROTEOLYTIC HYDROLYSIS OF OAT PROTEIN FRACTIONS ENABLES RELEASING PEPTIDES WITH ANGIOTENSIN I-CONVERTING ENZYME INHIBITING ACTIVITY . 66

P 2.2 Anna Iwaniak, Monika Protasiewicz, Małgorzata Darewicz, Piotr MinkiewiczFOOD PROTEINS AS THE PRECURSORS OF PEPTIDES WITH CARDIOPROTECTIVE AND BITTER TASTE PROPERTIES – AN in silico PREDICTION . . . . . . . . . . . . . . 67

P 2.3 Paulina Worsztynowicz, Włodzimierz Grajekin silico ANALYSIS AS A TOOL FOR EVALUATION OF BIOACTIVITY PEPTIDES DERIVED FROM FOOD PROTEINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

P 2.4 Paulina Worsztynowicz, Włodzimierz GrajekBIOSYNTHESIS AND IDENTIFICATION OF PROTEASE OF enterococcus faecalis ISOLATED FROM ARTISAN POLISH CHEESE . . . . . . . . . . . . . . . . . . . . . . . . . . 69

P 2.5 Agnieszka Szmyt, Anna Dąbrowska, Marek Szołtysik, Józefa ChrzanowskaHETEROLOGOUS ExPRESSION OF CHICKEN YGP40 PROTEIN . . . . . . . . . . . . . . 70

P 2.6 Marek Szołtysik, Anna Dąbrowska, Marta Pokora, Aleksandra Zambrowicz, Konrad Babij, Barbara Buda, Agnieszka Szmyt, Józefa Chrzanowska

Yarrowia lipolYtica LIPASES AND THEIR APPLICATION FOR MILK FAT HYDROLYSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

P 2.7 Ewa Szczepańska, Filip Boratyński, Zbigniew Lazar, Magdalena Rakicka, Ewa Tomaszewska-Ciosk, Jean-Marc Nicaud, Małgorzata Robak

LIPASE OF Yarrowia lipolYtica JMY329 . . . . . . . . . . . . . . . . . . . . . . . . . . . 72Session 3: BIOSyNTHESIS & BIODEGRADATIONSP 3.1 Marta Domżał, Marcin Łukaszewicz

OPTIMIZATION OF CULTURE CONDITIONS AND SEPARATION AND PURIFICATION PRODUCTS IN THE PROCESS OF SIMULTANEOUS PRODUCTION OF LEVAN AND POLY(γ-GLUTAMIC ACID) . . . . . . . . . . . . . . . . . 73

P 3.2 Krzysztof Cybulski, Anita RywińskaDETERMINATION OF THE THIAMINE CONCENTRATION IN SEED AND PRODUCTION CULTURE MEDIUM FOR EFFECTIVE BIOSYNTHESIS OF PYRUVIC ACID FROM GLYCEROL BY Yarrowia lipolYtica YEAST . . . . . . . 74

P 3.3 Magdalena Rakicka, Waldemar Rymowicz, Anita RywińskaPRODUCTION OF ERYTHRITOL BY Yarrowia lipolYtica IN CHEMOSTAT CULTURE USING PURE AND CRUDE GLYCEROL . . . . . . . . . . . . . . . . . . . . . . . 75

P 3.4 Ewelina Celińska, Włodzimierz GrajekHIGH-THROUGHPUT PROTEOME PROFILING OF L-PHE CATABOLISM AND DERIVATIVE AROMA PRODUCTION IN Yarrowia lipolYtica – MAPPING MOLECULAR IDENTITIES OF THE PATHWAY . . . . . . . . . . . . . . . . . . 76

P 3.5 Joanna Chmielewska, Józef Sowiński, Barbara Foszczyńska, Ewelina Szydełko-Rabska, Joanna Kawa-Rygielska

SORGHUM AS A RAW MATERIAL FOR DISTILLERY . . . . . . . . . . . . . . . . . . . . . . 77P 3.6 Witold Pietrzak, Patrik Lennartsson, Mohammad J. Taherzadeh, Joanna Kawa-Rygielska

LOW-TEMPERATURE PHOSPHORIC ACID PRETREATMENT TO ENHANCE WHEAT STRAW ENZYMATIC HYDROLYSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

P 3.7 Joanna Chmielewska, Joanna Kawa-Rygielska, Ewelina Dziuba,Józef SowińskiETHANOL PRODUCTION FROM LIGNOCELLULOSIC RAW MATERIALS . . . . . . . . 79

P 3.8 Sławomir Czabaj, Joanna Kawa-Rygielska, Jarosław Kliks, Ewelina DziubaTHE INFLUENCE OF MEAD WORT PREPARATION ON YEAST FERMENTATION ACTIVITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

P 3.9 Katarzyna Pielech-Przybylska, Maria Balcerek, Urszula Dziekońska, Piotr Patelski ETHANOL FERMENTATION OF DISTILLERY MASHES OBTAINED FROM MALTED AND UNMALTED CEREALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

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P 3.10 Katarzyna Pielech-Przybylska, Joanna Berłowska, Maria Balcerek, Urszula Dziekońska, Piotr Patelski, Marta Dutkiewicz, Halina Kalinowska

EFFECT OF AERATION AND NITROGEN SUPPLEMENTATION ON THE FERMENTATION EFFICIENCY OF SUGAR BEET PULP HYDROLYSATES . . . 82

P 3.11 Józef Błażewicz, Joanna Kawa-Rygielska, Witold PietrzakENZYMATIC PREPARATIONS IN THE PROCESS OF OBTAINING WORTS FOR FOOD-TYPE MALT CONCENTRATES PRODUCTION. . . . . . . . . . . . . . . . . . 83

P 3.12 Maria Balcerek, Joanna Berłowska, Katarzyna Pielech-Przybylska, Piotr Patelski, Urszula Dziekońska, Marta Dutkiewicz, Halina Kalinowska

ETHANOL FERMENTATION OF SUGAR BEET PULP HYDROLYSATES BY saccharomYces cerevisiae AND pichia stipitis YEAST STRAINS . . . . . . . 84

P 3.13 Maria Balcerek, Katarzyna Pielech-Przybylska, Piotr Patelski, Urszula Dziekońska, Ewelina Strąk

EFFECT OF YEAST ON THE VOLATILE PROFILE OF "WęGIERKA ZWYKŁA” PLUM DISTILLATES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

P 3.14 Sławomir Jabłoński, Maciej Sygit, Tadeusz Beutel, Marcin ŁukaszewiczTUBULAR BIOREACTOR FOR THE ANAEROBIC DIGESTION OF FOOD INDUSTRY LIqUID WASTE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

P 3.15 Katarzyna Zawadzka, Aleksandra Felczak, Katarzyna LisowskaMICROBIAL DEGRADATION OF DRUGS WITH A CARBAZOLE RING IN THEIR STRUCTURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

P 3.16 Anna K. Żołnierczyk, Begoña Ayuda-Durán, Susana González-Manzano, Ana M. González-Parámas, Mirosław Anioł, Celestino Santos-Buelga

INFLUENCE OF SPENT HOP ExTRACTS ON STRESS RESISTANCE AND THE AGEING PROCESS IN caenorhaBditis eleGans 88

P 3.17 Mateusz Kropiwnicki, Magdalena Wróbel-Kwiatkowska, Waldemar RymowiczPROPAGATION in vitro AND REGENERATION OF APPLES . . . . . . . . . . . . . . . . 89

P 3.18 urszula Gawlik-Dziki, Dariusz Dziki, Michał ŚwiecaINFLUENCE OF SPROUTING AND ELICITATION ON ANTIOxIDANT ACTIVITY OF WHEAT SEEDLIGS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

P 3.19 Michał Świeca, Barbara Baraniak, Urszula Gawlik-Dziki, Dariusz Kowalczyk THE METABOLOMIC AND BIOCHEMICAL CHANGES IN YOUNG BUCKWHEAT SPROUTS AFFECTED BY ELICITATION AND ELICITATION SUPPORTED BY THE PHENYLPROPANOID PATHWAY FEEDING 91

Session 4: BIOTRANSFORMATIONSP 4.1 Tomasz Tronina, Agnieszka Bartmańska, Jarosław Popłoński, Sandra Sordon, Ewa Huszcza

MICROBIAL TRANSFORMATION AS AN EFFICIENT TOOL FOR OBTAINING PHASE I AND PHASE II METABOLITES OF BIOACTIVE COMPOUNDS . . . . . . . . . . 92

P 4.2 Anna Panek, Mariusz Kozakiewicz, Alina Świzdor, Natalia Milecka-TroninaMICROBIAL TRANSFORMATIONS LEADING TO BIOLOGICALLY ACTIVE AND USEFUL STEROID COMPOUNDS: THE USE OF FUNGAL GENERA asperGillus AND circinella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

P 4.3 Alina Świzdor, Natalia Milecka-Tronina, Anna Panek, Paula Maj, Ewa RomaniukMICROBIAL TRANSFORMATION OF SOME B-NORSTEROIDS BY Beauveria Bassiana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

P 4.4 Małgorzata Grabarczyk, Katarzyna Wińska, Wanda Mączka, Barbara Żarowska, Mirosław Anioł

CHEMOENZYMATIC SYNTHESIS OF HYDROxYLACTONE WITH TRIMETHYLCYCLOHExENE SYSTEM AND THEIR BIOLOGICAL ACTIVITY . 95

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BIOTRANSFORMATION OF HALOLACTONES WITH GEM-DIMETHYLCYCLOHExANE SYSTEM . . . . . . . . . . . . . . . . . . . . . . . 96

P 4.6 Jakub Pannek, Filip Boratyński, Teresa OlejniczakSYNTHESIS, BIOTRANSFORMATION AND BIOLOGICAL ACTIVITY OF BICYCLIC LACTONES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97


LACTONES WITH METHYLCYCLOHExANE SYSTEM – OBTAINING AND BIOLOGICAL ACTIVITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

P 4.8 Monika Dymarska, Tomasz Janeczko, Edyta Kostrzewa-SusłowENANTIOSELECTIVE CONVERSION OF CERTAIN DERIVATIVES OF 7-HYDROxYFLAVANONE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

P 4.9 Sandra Sordon, Anna Madej, Jarosław Popłoński, Agnieszka Bartmańska, Tomasz Tronina, Ewa Huszcza

IMPROVEMENT OF ANTIOxIDANT ACTIVITY OF NATURAL FLAVONOIDS BY BIOTRANSFORMATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

P 4.10 Anna K. Żołnierczyk, Dagmara Baczyńska, Joanna Kozłowska, Małgorzata Grabarczyk, Wanda Mączka, Katarzyna Wińska, Edyta Woźniak, Mirosław Anioł

xANTHOHUMOL ACYL DERIVATIVES AS ANTIPROLIFERATIVE AND ANTIOxIDANT AGENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

P 4.11 Agnieszka Bartmańska, Tomasz Tronina, Jarosław Popłoński, Sandra Sordon, Ewa Brzezowska

BIOTRANSFORMATION OF ISOxANTHOHUMOL CONDUCTED IN VARIOUS MEDIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

P 4.12 Marta Czarnecka, Anna Gliszczyńska, Marta Świtalska, Joanna Wietrzyk, Czesław Wawrzeńczyk

CHEMOENZYMATIC SYNTHESIS AND ANTIPROLIFERATIVE ACTIVITY OF PHOSPHOLIPIDS CONTAINING CINAMMIC AND P-ANISIC ACIDS . . . . . . . . 103

P 4.13 Jarosław Popłoński, Sandra Sordon, Eliza Turlej, Joanna Wietrzyk, Tomasz Tronina, Agnieszka Bartmańska, Ewa Huszcza

NAPHTHOFLAVONES – METABOLISM, CYTOTOxICITY AND AROMATASE INHIBITION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

P 4.14 Monika Serafin, Ewa Żymańczyk-Duda BIOTRANSFORMATION OF 2-BUTYRYLOxY-2-(ETHOxY-P-PHENYLPHOSPHINYL)ACETIC ACID WITH IMMOBILIZED FUNGAL CELLS . . . . . . . . . . . . . . . . . . . . 106

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Plenary lectures

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Plenary lecture 1

patteRns oF lipid accuMulation in oleaginous MicRo- oRganisMs and Biotechnological peRspectives

oF single cell oil

George Aggelis

laboratory of microbiology, division of Genetics, cell Biology & development, department of Biology, university of patras, patras 26504, Greece

Lipid accumulation in oleaginous microorganisms growing on sugars or similarly metabolized substrates is a secondary anabolic process, occurring after essential nutrient (usually nitrogen) depletion in the me-dium. Specifically, under nitrogen starvation, citric acid is accumulated in the mitochondria, then excre-ted in the cytoplasm and converted into acetyl-CoA, which is channeled to lipid biosynthesis. The above model, introduced during the ‘80s for lipid accumulation in oleaginous microorganisms is similar to that proposed for mammalian cells. However, divergent biochemical behaviors have been identified in the va-rious oleaginous microorganisms having a biotechnological interest.

The growth of Yarrowia lipolytica on glycerol is characterized by three distinct phases, namely bio-mass production, lipogenic and citric acid production phases. High activity of NAD+–isocitrate dehydro-genase (ICDH) is detected during biomass production phase but this activity is significantly decreased afterwards inducing lipogenesis. Although transcription of acl1 and acl2, encoding for ATP-citrate ly-ase (ATP:CL), enzyme which cleaves citric acid (CA) into oxaloacetate and acetyl-CoA, occurs even under non-oleaginous conditions, high activity of ATP:CL is only detected under oleaginous conditions. A fur-ther drop in NAD+–ICDH activity and a decrease in glycerol kinase activity are observed during the CA production phase. Surprisingly, CA production is accompanied by storage (neutral) lipid turnover, along with remarkable biosynthesis of glycolipids, sphingolipids and phospholipids (mainly phosphatidylcholi-ne). Degradation of intracellular lipids is observed, even when significant amounts of glycerol are found in the growth media, indicating low uptake of glycerol. The low activity of malic enzyme (ME), which parti-cipates in the production of NADPH, indicated that ME may not be implicated in lipid biosynthesis, while the NADPH required for this process may be provided by alternative sources (i.e. pentose phosphate pa-thway) the activation of which requires nitrogen in the growth medium. Actually, in Yarrowia lipolytica lipid accumulation is favored in the presence of nitrogen (especially organic) in low concentrations in the growth medium.

In cryptococcus curvatus interplay between the biosynthesis of intra-cellular polysaccharides (ICP) and storage lipid occurs under nitrogen-limited conditions in media having as carbon source common su-gars (i.e. lactose or sucrose). Specifically, high quantities of ICP are accumulated under balanced growth conditions (despite the presence of nitrogen in the medium), being progressively decreased afterwards si-multaneously with a lipid accumulation. A very similar pattern was identified in the microalgae chlorella sp. and nannochloropsis salina. These organisms cultivated in a photobioreactor produce biomass rich in storage lipids and sugars, which are degraded when the cell density increases resulting in carbon and energy

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(light) limiting conditions. During sugars degradation, high activity of ATP:CL and low or zero activity of ICDH are observed, indicating that microalgae are able to synthesize lipids via storage sugars catabolism using citrate as donor of acetyl-CoA.

The yeast debaryomyces etchellsii grown under nitrogen limiting conditions reproduces asexually by buds, when nitrogen is available in the growth medium, or sexually by ascospores after nitrogen exhau-stion. During ascosporogenesis, an important increase in the cellular lipid content per unit of dry cell mass occurs. On the contrary, during transition from batch cultures, in which ascospore formation had been in-duced and maximum lipid accumulation achieved, to continuous cultures using dilution rates 0.026 and 0.019 h-1, a shift from sexual to asexual reproduction was observed since the number of asci was gradually decreased and only budding cells were observed at steady-states. It seems that nitrogen starvation induces both lipid accumulation and ascoporogenesis and therefore these processes coincide in d etchellsii. Bud-ding cells contain low quantities of lipids that are rich in oleic and linoleic acids while ascospore forming cells synthesize more saturated lipids. Stimulation of ascospore formation by other than nitrogen limita-tion means may lead to a significant improvement of single cell oil (SCO) production by oleaginous yeasts and/or make possible SCO production in continuous culture working at high dilution rates.

SCOs have been in the forefront of biotechnological products for many years. Their production cost, however, is higher than that of the common plant oils, thus only the high-added value SCOs could be economically produced. These include the oils containing polyunsaturated fatty acids, such as gamma linolenic, arachidonic and docosahexanoic acids and those having a special structure i.e. similar to that of the cocoa butter. Besides, fuel production from micro-organisms has gained much interest during the last years due to the escalating prices of petroleum fuels which intensified the search for alternative fuel so-urces. Thus, many researchers have turned to microbial oils (as alternatives to plant oils), as feedstock for biodiesel production. Although the productivity of oleaginous microorganisms has recently significantly increased by using genetically modified strains it is questionable whether SCO could be economically used in the biodiesel manufacture.

ReferencesArous et al., unpublished data; Bellou and Aggelis, 2012. J. Biotechnol., 164, 318–329. Bellou et al., 2014. Microbiology-SGM, 160, 807–817.Bellou et al., 2014. Biotechnol. Adv., 32, 1476–1493 and unpublished data.Makri et al., 2010. Biores. Technol., 101, 2351–2358; Papanikolaou and Aggelis, 2011. Eur. J. Lipid Sci. Technol., 113, 1031–1051 and 1052–1073.Tchakouteu et al., 2015. Eur. J. Lipid Sci. Technol., in press.

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Plenary lecture 2

Bioactive peptides FRoM Food pRoteins – Methodological appRoaches and Results

Małgorzata Darewicz, Anna Iwaniak, Piotr Minkiewicz

chair of food Biochemistry, faculty of food science, university of warmia and mazury in olsztyn, pl cieszyński 1, 10-726 olsztyn-Kortowo, poland, darewicz@uwm edu pl

Food proteins possess a wide range of nutritional properties. A growing body of scientific evidence in the past decadehas revealed that many food proteins and peptides exhibit specific biological activities in ad-dition to their established nutritional value. Biologically active peptide sequences are encrypted within a protein and become active when released. In foods they are usually released via gastrointestinal dige-stion, fermentation processes with proteolytic microorganisms or in vitro enzymatic hydrolysis and thus may have an influence on human health. A large range of bioactive peptides have been isolated from food protein sources including among the others angiotensin converting enzyme (ACE) inhibitory pepti-des, antioxidant, antimicrobial, antiamnestic, opioid, sensory, mineral binding. Human health unfavorable peptides i.e. coeliac-toxic are also studied. Likewise, currently research is being carried out to find the new sources of bioactive peptides as well as study their means of obtaining, bioavailability, biological proper-ties and mechanisms of action. An improved understanding of the molecular aspects of bioactive peptides has provided an insight into progress in their application as components of food preventing diet-related diseases. This presentation will be an overview of various in silico, in vitro, ex vivo and in vivo models that are used to study the bioactive properties of food peptides. In particular, it will be focused on bioinforma-tics, chemometrics and digestion simulation methods designed to evaluate the molecular and biological aspects of peptides originating form food proteins. Research concerning food proteins and peptides incre-asingly relies on in silico methods and tools. One of the most popular in silico tools are databases of bioacti-ve food proteins and peptides like e.g. BIOPEP (www.uwm.edu.pl/biochemia), EROP Moscow (www.erop.inbi.ras.ru) and PepBank (www.pepbank.mgh.harvard.edu). The most popular examples of chemometric methods practiced the analysis of bioactive peptides include artificial neural networks (ANNs), principle component analysis (PCA), and linear regression analyses like partial least squares regression (PLS) and/or multivariate linear regression (MLR). Until now there are many reports in the literature on digestion of food proteins using commercial digestive enzymes of non-human origin with further identification of ge-nerated bioactive peptides during in vitro digestion. However, relatively few studies on digestion of foods using human gastrointestinal enzymes have been conducted. Thus, our methodology relying on simula-tion of the gastrointestinal digestion of food proteins to obtain the biologically active hydrolysates seems to be an innovative approach for studying biopeptides derived from various food sources.

Work supported form Warmia and Mazury University in Olsztyn funds.

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Plenary lecture 3

decaRBoxylases and sulFatases: Biocatalytic equivalents to the KolBe-schMitt- and the MitsunoBu-Reaction

Silvia Glueck1,2, Katharina Plasch2, Tamara Reiter1,2, Verena Resch2, Kurt Faber1,2

1austrian centre of industrial Biotechnology c/o 2department of chemistry, university of Graz, a-8010 Graz, austria,

Kurt faber@uni-Graz at

Recent advances in molecular biology enable the production of enzymes of increasing complexity at redu-ced costs and shorter development times, which allows to substitute or enable catalytic processes, which are inefficient or impossible by traditional methods, by biocatalytic equivalents.

In this context, two biological solutions for chemical problems are demonstrated:(i) The Kolbe-Schmitt-reaction is a classic process for the production of aromatic carboxylic acids, which

requires harsh reaction conditions (120–300°C, 5–100 bar CO2) and often suffers from incomplete o/p-regioselectivities. In contrast, the biocatalytic equivalent employs decarboxylases running in the reverse (carboxylation) direction at physiological conditions and uses bicarbonate as CO2 source [1]. The carboxylation of phenols proceeds with excellent regioselectivity in the o- or p-position and also the side-chain carboxylation of styrenes is possible [2].

(ii) The inversion of secondary alcohols is commonly achieved via the Mitsunobu-protocol, which shows desastrous atom economy and is therefore not suitable for large scale. A biocatalytic equivalent is ba-sed on inverting alkyl sulfatases, which hydrolyse sulfate esters of sec-alcohols with strict inversion of configuration [3]. Combination of an inverting with a retaining sulfatase possessing matching (oppo-site) enantiopreference allows the production of a single enantiomeric sec-alcohol in 100% theoretical yield as the sole product from the corresponding racemic sulfate ester [4].

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Funding by the Austrian Science Fund (FWF) within the DK Molecular Enzymology (projects W9, P26863 and I1637) and the Austrian BMWFW, BMVIT, SFG, Standortagentur Tirol and ZIT through the Austrian FFG-COMET-Funding Program is gratefully acknowledged.

References[1] Glueck S.M., Gümüs S., Fabian W.M.F., Faber K., 2010. Chem. Soc. Rev., 39, 313; Wuensch C., Schmidt

N., Gross J., Grischek B., Glueck S.M., Faber K., 2013. J. Biotechnol., 168, 264; Wuensch C., GrossJ., Steinkellner G., Lyskowski A., Gruber K., Glueck S.M., Faber K., 2014. RSC Adv. , 4, 9673.

[2] Wuensch C. , Pavkov-Keller T., Steinkellner G., Gross J., Fuchs M., Hromic A., Lyskowski A., Fauland K., Gruber K., Glueck S.M.,. Faber K., 2015. Adv. Synth. Catal. , 357, DOI 10.1002/adsc.201401028.

[3] Toesch M., Schober M., Faber K., 2014. Appl. Microbiol. Biotechnol. , 98, 1485; Knaus T., Schober M., Kepplinger B., Faccinelli M., Pitzer J., Faber K., Macheroux P., Wagner U., 2012. FEBS J., 279, 4374; Schober M. , Knaus T., Toesch M., Macheroux P., Wagner U., Faber K., 2012. Adv. Synth. Catal. , 354, 1737.

[4] Schober M., Toesch M., Knaus T., Strohmeier G.A., van Loo B., Fuchs M., Hollfelder F., Macheroux P., Faber K., 2013. Angew. Chem. Int. Ed., , 52, 3277.

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Plenary lecture 4

laBoRatoRy evolution in action: iMpRoving the gRoWth pRopeRties oF Bacillus suBtilis

Felix Blei1, Jan Schwitalla1, Derek Matern2, Gergely Maróti3, Axel A. Brakhage2, Ákos T. Kovács1

1terrestrial Biofilms Group, institute of microbiology, friedrich schiller university Jena, Germany 2department of molecular and applied microbiology, leibniz institute for natural product research

and infection Biology – hans Knöll institute, Jena, Germany 3institute of Biochemistry, Biological research center, hungarian academy of sciences, szeged, hungary

Growth and metabolism of microbes define their advantage in a variety of biotechnological applications. To improve the yield of microbial growth or the production of certain metabolites, selected microbial tra-its are modified in the target organism. However, the use of genetically modified organisms is opposed in several EU member countries. Therefore, there is an urgent need to obtain microbial cell factories with im-proved properties using alternative methodologies. Laboratory evolution offers a promising methodology to create microbes with desired excellent traits. Here, a novel approach will be presented that focuses on enhancing growth properties and secondary metabolite production of the Gram positive bacterium, Ba-cillus subtilis.

During co-cultivation of B subtilis with aspergillus niger, bacterial cells attach to the fungal hyphae resulting in altered metabolism and secondary metabolite production in both partners [1]. Using labora-tory evolution approach, B subtilis was co-evolved with a niger. After several rounds of co-cultivation, the co-evolved B subtilis isolates showed remarkable phenotypic differentiation. Certain isolates showed enhanced surfactin production, others had improved resistance at low pH or increased spreading on agar medium that depended on the presence of the fungus. Interestingly, isolates from distinct co-evolved li-nages showed heterogeneity in these improved properties. In parallel, B subtilis evolved under similar conditions but in the absence of a niger showed different adaptation mechanism, the isolated strains had improved colony biofilm development. Finally, the impact of co-evolved B subtilis isolates on the growth properties of a niger will be presented, focusing on the effect of enhanced production of the antifungal compound, surfactin.

Genome resequencing and targeted mutagenesis will help us to identify the genetic changes that re-sult in improved growth properties. This approach highlights the potential of laboratory evolution to select for excellent microbial strains for biotechnological applications.

References[1] Benoit I., van den Esker M.H., Patyshakuliyeva A., Mattern D., Blei F., Zhou M., Dijksterhuis J., Bra-

khage A.A., Kuipers O.P. de Vries R.P., Kovács Á.T., 2015. Bacillus subtilis attachment to aspergillus ni-ger hyphae results in mutually altered metabolism. Environmental Microbiology (doi: 10.1111/1462-2920.12564).

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Plenary lecture 5

hyBRid coMBinatoRial suBstRates liBRaRy (hycosul) as a tool in design oF speciFic and sensitive pRoBes FoR endopeptidases

Marcin Drąg1, Marcin Poręba1, Paulina Kasperkiewicz1, Wioletta Rut1, Scott Snipas2, Heather Parker4, Christine Winterbourn4, Guy S. Salvesen2

1division of Bioorganic chemistry, faculty of chemistry, wrocław university of technology, wybrzeże wyspiańskiego 27, 50-370 wrocław, poland

2sanford-Burnham medical research institute, 10901 north torrey pines road, la Jolla, usa 3department of Biochemistry and molecular and structural Biology, Jozef stefan institute, ljubljana,

slovenia; 4university of otago, christchurch, new zealand marcin drag@pwr edu pl

Proteases are known to participate in many cellular processes due to their ability to process the peptide bond. They are key players in several cascades taking place in the cell with apoptosis, fibrinolysis, blood clotting, complement fixation, gastrulation or general cell cycle being only a few examples. However, pro-teases can be also bad guys and participate in the cellular events, which lead to severe diseases such as can-cer, diabetes, pathogens infections or hypertension [1].

Each protease recognizes only substrates, which can fit into the recognition pockets. There are seve-ral methods of determination of substrate specificity [2]. One of the most versatile is Positional Scanning Substrate Combinatorial Library approach, which allows fast and reliable determination of substrate pre-ferences for most of the proteases. To date used libraries contained only natural amino acids and only very occasionally unnatural amino acids were incorporated into the structure of synthesized substrates [3]. To get better insight into substrate specificity, we have applied several different tailored to certain type of pro-tease approaches using broad range of unnatural amino acids. This allowed us to obtain for several diffe-rent endopeptidases much better substrates comparing to natural amino acids derivatives. Full strategy for profiling of substrate specificity for serine and cysteine endopeptidases will be presented [4, 5].

References[1] Drąg M., Salvesen G.S., 2010. Nature Reviews Drug Discovery, 9 (9), 690–701.[2] Poręba M., Drąg M., 2010. Current Medicinal Chemistry, 17(33), 3968–3995.[3] Kasperkiewicz P., Gajda A.D., Drąg M., 2012. Biological Chemistry, 393 (9), 843–851.[4] Kasperkiewicz P., Poręba M., Snipas S.J., Parker H., Winterbourn C.C., Salvesen G.S., Drąg M., 2014.

Proc. Natl. Acad. Sci. USA, 111(7), 2518–2523.[5] Poręba M., Kasperkiewicz P., Snipas S.J., Fasci D., Salvesen G.S., Drąg M., 2014. Cell Death & Differen-

tiation (Nature Press), (9), 1482–1492.

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Plenary lecture 6

Yarrowia lipolYtica, a Model FoR lipid MetaBolisM and a platFoRM FoR lipid pRoduction

Jean-Marc Nicaud1,2, Athanasios Beopoulos1,2, Anne-Marie Crutz-Le Coq1,2, Remi Dulermo1,2, ThierryDulermo1,2, Rodrigo Ledesma-Amaro1,2, Zbigniew Lazar1,2,3,

Cecile Neuveglise1,2, Heber Gamboa-Meléndez1,2, Tristan Rossignol1,2

1inra, umr1319, micalis, domaine de vilvert, f-78352 Jouy-en-Josas, france 2agroparistech, umr micalis, Jouy-en-Josas, france2, department of Biotechnology

3department of Biotechnology and food microbiology, wrocław university of environmental and life sciences, chełmońskiego 37/41, 51-630 wrocław, poland

Jean-marc nicaud@grignon inra fr

Yarrowia lipolytica is a non-conventional oleaginous yeast model for fundamental and applied studies on lipid metabolism. Currently, the most popular oleaginous yeast species for lipid production are lipomyces starkeyi, rhodosporidium toruloides and Yarrowia lipolytica Among these species, Y lipolytica is the only yeast for whom a large outfit of tools is available: a well-curated genome, efficient genetic tools, a recent li-pid metabolism model of fatty acid transport and activation, and several genome scale models.

Since thatY lipolytica emerge as an efficient host for the production of usual and unusual lipids. It was important to understand the fatty acid transport and activation mechanisms in this yeast compared to that of s cerevisiae. We found that Y lipolytica has homologous genes involved in fatty acid transport and activation similar to those of s cerevisiae (faa1, fat1, pxa1, pxa2, ant1 …). However, our cur-rent model for fatty acid transport and activation, which will be presented, differs significantly from the one of s cerevisiae [1].

Production of biofuels using microorganisms is one of the most promising alternative to petroleum-based chemistry. Several groups have focused on both, the genetic improvement of the Y lipolytica ability to accumulate high amounts of biolipids and on the modification of its fatty acid profile.

Key genes involved in lipid synthesis and remobilization as well as in sugar utilization and central metabolic pathways have been characterized. A review on these key genes allowing to increase lipid accu-mulation and to alleviate the competitive pathways will be presented, including genes involved in glycerol-3-P synthesis, genes governing citric acid and fatty acid synthesis, as well as the ones responsible for TAG synthesis, TAG remobilization and fatty acid degradation. In addition, recent studies revealing the impor-tant roles of the glucokinase (GlK1, YALI0E15488 g) and hexokinase (hxK1, YALI0B22308 g) for impro-ving lipid production on glucose and fructose media will be presented [2].

References[1] Dulermo R., Gamboa-Melendez H., Ledesma R., Thevenieau F., Nicaud J.M., 2015. Unraveling fatty

acid transport and activation mechanisms in Yarrowia lipolytica. BBA Molecular and Cell Biology of Lipids, in press.

[2] Lazar Z., Dulermo T., Neuvéglise C., Crutz Le Coq A.M., Nicaud J.M., 2014. Hexokinase – A limiting factor in lipid production from fructose in Yarrowia lipolytica, Metabolic Engineering, 26, 89–99.

Key words: yeast, metabolic engineering, lipid production, biolipid, lipid metabolism.

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Plenary lecture 7

sugaR utilisation FoR Biosynthesis oF valuaBle pRoducts By Yarrowia lipolYtica and Related species

Zbigniew Lazar1,2,3, Thierry Dulermo1,2, Cecile Neuvéglise1,2,Tristan Rossignol1,2, Małgorzata Robak3, Jean-Marc Nicaud1,2, Anne-Marie Crutz-Le Coq1,2

1inra, umr1319, micalis, domaine de vilvert, f-78352 Jouy-en-Josas, france 2agroparistech, umr micalis, Jouy-en-Josas, france


Yarrowia lipolytica is a well-known biotechnological yeast producing lipids and organic acids from various carbon sources. Use of sugars deriving from co-products of sugar industry or raw materials as lignocellu-loses is a valuable trait for industrial applications. However, Y lipolytica has a restricted range of sugar sub-strates. For instance, strain W29 (‘French’ line) from which current strains optimized for lipid production derived, does not grow well in fructose. Hexose metabolism and upake, with a particular focus on fructose, was analyzed in this fastidious strain in comparison to two other natural isolates growing more efficiently on fructose (‘German’ and ‘Polish’ strains). This allowed us to identify hexokinase as a key component of fructose metabolism and a limiting factor for lipid production. A W29 derivative efficiently using fructose and sucrose was obtained. Hexose transporters were characterized for the ‘French’ and ‘German’ strains of Y lipolytica. Six functional hexose transporters were identified among the 24 members of the Y lipolytica Sugar Porter family. Based on the genome sequence of 5 different species of the genus Yarrowia and the basal species candida hispaniensis, an evolutionary scenario was reconstructed for this transporter fami-ly, which appeared to be dynamic (duplication and loss of genes), with a family expansion in Y lipolytica. One transporter exhibited a significant difference in its range of substrate between the two strains, sugge-sting transporters as another source of phenotypic diversity for sugar utilization. Yet, only a subset of he-xose transporters appeared to be physiologically active in glucose and fructose in Y lipolytica, the same for the two strains. Modified strains expressing only one transporter at a time were constructed and led to the conclusion that gowth and fructose consumption but also citric acid production are dependent on both hexokinase and transport.

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Plenary lecture 8

challenges and possiBilities in using BiodegRadaBle RaW MateRials

Helena Kautola

häme university of applied sciences, hämeenlinna, finland

The two main challenges the world is facing is energy crisis and environmental pollution. These challen-ges produce more side challenges in modern economy and quality of life [1]. Biodegradation is a process by which microbial organisms transform metabolically or enzymatically the structures of chemicals into the environment [2].

Renewable energy sources are the most important components for solving the bioenergy demand as most of it can be traced back to sunlight. The bioenergy can be produced from agricultural crops and its residues, processing wastes from agro industries, forest, and food industry and urban wastes etc. The bio-energy like bioethanol feedstock can be of sugar-based (sugarcane and beet sugar), starchy (potato, corn, rice) or lignocellulosic (wood, straw, grasses etc.) raw materials [1, 3].

The standards have also been developed for specifying the biodegradability, disintegration during biological treatment, minimal effect on the biological treatment process and minimal effect on the quality of the resulting compost. The measurement of the packaging material (plastic) degraded will give the CO2 emitted [4].

The next economic wave is bio-economy. Finland has plenty of natural resources [5]. The Finnish wood industry is moving towards 2nd generation bioenergy [6]. The waste streams of forestry-based indu-strial processing has been of great interest during the last years. The value-added products like polyhydro-xyalkanoate biopolymers (polyester family) have industrially relevant thermoplastic and elastomeric pro-perties (like in plastics), and they are pure, nontoxic, biodegradable, insoluble in water and produced from renewable resources [7].

The potato industry is also seeking for new products isolated from the starch production process. Dairy industry has plenty of wastewater and the treatment of it has a great challenge to get bio-components for reuse (8). Dairy industry in Finland is looking for value added milk chain with protein feed from silage and efficient use of sludge by fractionation and water removal [9].

The biogas industry network is getting larger, because of the new legislation of not having any orga-nic wastes in waste stations [10]. The recent work has studied the process behavior, biogas formation and its composition with also the economic aspects [11].

References[1] Mekala N.,K., Potumathi R., Baadhe R.R., Gupta V.K., 2014. Current Bioenergy Researches: Strengths

and Future Challenges, Bioenergy Research: Advances and Applications, Elsevier 2014, 1–21, http://dx.doi.org/10.1016/B978-0-444-59561-4.00001-2.

[2] http://toxics.usgs.gov/definitions/biodegradation.html[3] Tuomela M., Vikman M., Hatakka A., Itävaara, M., 2000. Biodegradation of Lignin in a Compost En-

vironment: a Review. Bioresource Technology, 72, 169–183.

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[4] Greene J., 2014. Degradation and Biodegradation Standards for Starch-Based and Other Polymeric Materials. Starch Polymers, Elsevier, 321–356.

[5] Manninen J., Implementation of Finnish Bioeconomy Strategy /Views from Ministry of Employment and the Economy, Biotechnology as an Enabler in Bioeconomy, ChemBio Seminar, 18.–19.3.2015, Helsinki.

[6] Biofore 3/2014, 13.[7] Laylock B., Pratt S., Halley P., Werker A., Lant P., 2014. Biodegradable Polymers from Pulp and Paper

Wastewater Streams – a Critical Review. Appita, 67(4), 1–7. [8] Porwal H.J., Mane A.V., Velhal S.G., 2015. Biodegradation of Dairy Effluent by Using Microbial Iso-

lates Obtained from Activated Sludge. Water Resources and Industry,9 (2015) 1–15. http://dx.doi.or-g/10.1016/j.wri.2014.11.002.

[9] Harju M., Opportunities of Biotechnology in the Milk Chain, Biotechnology as an Enabler in Bioeco-nomy, ChemBio Seminar, 18.–19.3.2015, Helsinki.

[10] Kauppalehti 19.3.2015.[11] Kymäläinen M., Lähde K., Arnold M., Kurola J., Romantschuk M., Kautola H., 2011. Biogasification of

Biowaste and Sewage Sludge – Measurement of Biogas quality, Journal of Environmetal Management 2011 YJEMA2652. http://dx.doi.org/10.1016/j.jenvman.2011.01.003.

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26

Plenary lecture 9

MicRoBial synthesis and tRansFoRMations oF lactones

Czesław Wawrzeńczyk, Filip Boratyński, Anna Gliszczyńska, Witold Gładkowski, Radosław Gniłka, Małgorzata Grabarczyk, Aleksandra Grudniewska,

Agnieszka Leśniak, Marcelina Mazur, Teresa Olejniczak, Małgorzata Smuga

department of chemistry, faculty of food science and technology, wrocław university of environmental and life sciences, norwida 25, 50-375 wrocław, poland

Whole cells of microorganisms are very useful biocatalysts in the synthesis of many chiral compounds in the form of optically pure isomers. They are also applied in the industrial scale for the production of phar-maceuticals [1, 2] and food ingredients [3].

In our laboratories the whole cells of many filamentous fungi strains and yeasts for synthesis of many biologically active compounds have been applied. In this lecture the synthesis of bioactive γ- and δ-lactones, mainly insect antifeedants and odoriferous compounds, will be presented. They were obta-ined as optically pure or enantiomerically enriched isomers. The microbial lactonization of γ,δ-epoxy esters (Scheme 1) [4], reduction of keto acids (Scheme 2) [5] and Baeyer-Villiger oxidation of ketones (Scheme 3) [6] were the crucial steps of these syntheses.

O

CO2C2H5

OO

OHH

H

H

Rhodotorula rubra AM82

Scheme 1

ee = 76%trans-( )- -hydroxy- -lactone

RRS

OH

O

O

OO

Scheme 2

Chaetomium sp. KCH6670Saccharomyces cerevisiae AM464

trans-(+)-whisky lactoneee = 99%

RS

OFusarium oxysporum AM21

Scheme 3

O

O

(+)- -thuja "abnormal" lactone

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27

Many natural biologically active lactones possess usually additional functional groups, like: hydroxy, carboxy and epoxy. For this reason it was rational to subject the lactones synthesized in our group to the additional microbial functionalization. The biohydroxylation and epoxidation were the most often trans-formations carried out by applied microorganisms (Scheme 4) [7].

O

O

R2

R1

O

O

O

R1, R2 = CH3 or H

O

O

OH

Absidia cylindrospora AM336Absidia cylindrospora AM336

Scheme 4

The biotransformations of γ-halolactones by whole cells of microorganisms afforded also very inte-resting products. For this group of substrates the hydroxylation and dehalogenation were the most often transformations observed [8].

References[1] Zaks A., Dodds D.R., 1997. Drug Discovery Today, 2, 513–531.[2] Gao F., Zhang J-M., Wang Z-G., Peng W., Hu H-L., Fu C-M., 2013. Asian Pac. J. Cancer Prev, 14, 5599–

5608.[3] Wache Y., Aguedo M., Nicaud J.M., Belin J.M., 2003. Appl. Microbial. Biotechnol., 61, 393–404.[4] Olejniczak T., Gawroński J., Wawrzeńczyk C., 2001. Chirality, 13, 302–307.[5] Boratyński F., Smuga M., Wawrzeńczyk C., 2013. Food Chem., 142, 419–427.[6] Gniłka R., Szumny A., Białońska A., Wawrzeńczyk C., 2012. Phytochemistry Lett., 5, 340–345.[7] Gładkowski W., Grabarczyk M., Wińska K., Ratuś B., Białońska A., Ciunik Z., Wawrzeńczyk C., 2007.

J., Mol. Catal. B-Enzym, 49, 79–87.[8] Gładkowski W., Mazur M., Białońska A., Wawrzeńczyk C., 2011. Enzyme Microb. Technol., 48,

326–333.

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short communications

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session 1 microorganisms lecture 1.1

Bacillus cereus B5esZ in BiodegRadation oF pRetReated pig BRistles

Wojciech Łaba1, Wiesław Kopeć2, Dorota Chorążyk2, Anna Pudło2, Adam Kurzawa3, Anna Kancelista1, Magdalena Wróbel-Kwiatkowska1, Michał Piegza1

1 department of Biotechnology and food microbiology 2department of animal products technology and quality management, wrocław university of envi-

ronmental and life sciences, chełmońskiego 37, 51-630 wrocław, poland 3 institute of production engineering and automation, wrocław university of technology, Łukasiewicza

3/5, 50-371 wrocław, poland

The management over the pig bristle waste generated in slaughterhouse processing remains a problematic issue due to the exceptional resilience of this keratinic appendage to bioconversion processes. In order to exploit this specific protein source a distinctive technological approach must be employed. Bacillus cereus B5esz was described previously as capable of effective decomposition of keratins, mainly feathers, in liquid culture conditions. Its crude keratinase was readily capable of efficient digestion of chicken feathers, and the yield of the process could be further enhanced by substrate pretreatment. To develop an environment-friendly bioconversion process of minimum effluent output, moderate substrate pretreatment, incorporat-ing minimal amounts of auxiliary chemicals, should be developed.

The objective of the study was to optimize the substrate pretreatment and the enzymatic digestion of pretreated pig bristles with a concentrated crude keratinase of B cereus B5esz. At first, the effect of thermo-chemical pretreatment of bristles was evaluated in relation to the activity of the tested enzyme prepara-tion. In addition, the biodegradation rate of bristles was determined in both, 24-hour enzymatic reactions and in 10-day bacterial culture. Afterwards, a Box-Behnken experimental design was applied to estimate the effect of three influential parameters: substrate content, sulfite concentration during substrate pretre-atment and reaction temperature, on the yield of free amino acids and protein recovery, at a constant en-zyme load. The amino acid composition of hydrolysates was determined and the deterioration of keratin fibres was visualized using SEM microscopy.

Among tested compounds for bristle pretreatment, 100 mM sulphite allowed for the highest degree of biodegradation in the enzymatic reaction (24% substrate loss). Similarly, the same substrate pretreat-ment facilitated degradation in bacterial culture conditions, which resulted in the accumulation of over 8 mg ml-1 protein at 31% substrate loss.

Two separate regression models were developed for the process of hydrolysis, considering the rele-ase of amino acids and protein recovery as an outcome. According to ANOVA results, a high suitability and precision of both models was obtained. An optimum value of the parameters was found for the rele-ase of amino acids and the most influential factor was bristle concentration (linear relationship) and reac-tion temperature (linear and quadratic). The protein recovery depended mainly on the linear influence of bristle concentration and the pretreatment rate. After enzymatic digestion, the hydrolysates were rich in branched chain amino acids (valine, isoleucine and leucine).

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antiBiotic Resistance in a seWage sludge-FeRtiliZed soil

Kinga Bondarczuk, Anna Markowicz, Zofia Piotrowska-Seget

department of microbiology, university of silesia in Katowice, faculty of Biology and environmental protection, 40-032 Katowice, Jagiellońska 28, poland,

kkrysta@us edu pl

The production of sewage sludge is increasing and using this by-product as a fertilizer is an excellent so-lution for its disposal. However, despite its indisputable richness in nutrients, sewage sludge bears hazar-dous contents including antibiotics and antibiotic-resistant bacteria that may pose a risk for human and environmental health. Since antibiotic resistance phenomenon threatens modern therapy risk assessment of sewage sludge land application in this context is needed. Moreover, results revealed that the persistence of antibiotic resistance among bacteria may be enhanced by the presence of heavy metals in the environ-ment as a result of co-selection.

The aim of the project is to study the persistence of antibiotic resistance in a sewage sludge-fertilized soil with an emphasis on antibiotic resistance determinants and their possible co-selection. To study the horizontal transfer of antibiotic resistance genes plasmid DNA was isolated and its further characteriza-tion was performed. To monitor antibiotic- and metal-resistant bacteria in the fertilized soil classical plate counts were applied.

Preliminary results from the first monitoring period of the experiment including the characteriza-tion of isolated plasmids and culturable fraction of resistant bacteria will be presented.

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lactoBacilli as cd2+ BiosoRBent

Adam Waśko1, Magdalena Polak-Berecka1, Dominik Szwajgier1, Patrycja Boguta2, Jolanta Cieśla2, Tomasz Skrzypek3

1 department of Biotechnology, human nutrition and science of food commodities, university of life sciences in lublin, skromna 8, 20-704 lublin, poland 2 institute of agrophysics pas, doświadczalna 4, 20-290 lublin, poland

3 laboratory of confocal and electron microscopy, interdisciplinary researchcenter, John paul ii catholic university of lublin, poland

The aim of this work was assessment of the ability of eleven lactobacillus species to absorb Cd2+. After the selection of species that effectively removed Cd2+ from water solution, the cell surface properties of mi-croorganisms were determined. Metal resistance of selected Lactobacilli against Cd2+ were determined by measuring minimum inhibitory concentrations (MICs) and minimal bactericidal concentration (MBC). Results show that seven species (lb acidophilus, lb salivarius, lb agilis, lb johnsonie, lb crispatus, lb rhamnosus and lb helveticus) exert the highest resistance to Cd2+ (at the level of 31.25 μg/ml Cd2+). Select-ed bacteria were used to study the removal of metal ions (MRE). lb galinarum and lb crispatus showed the highest ability to remove Cd2+ from water solutions. The electrical properties of cell surfacesof lb gali-narum and lb crispatus were assessed by microelectrophoresis. The most negative values of zeta potential were obtained for lb galinarum, but differences between zeta potentials of lb galinarum and lb crispa-tus were generally statistically insignificant. Fourier Transform Infrared Spectroscopy analysis revealed that in the binding of Cd2+, mainly carboxyl groups, phosphate and polysaccharides were involved. Energy Dispersive Spectroscopy on the Scanning Electron Microscopy confirmed the presence of high amount of Cd2+ on the cell surface of examined bacteria after the biosorption experiment.

The findings of this study clearly demonstrate that lb galinarum and lb crispatus showed high abil-ity to remove Cd2+ from water solutions. The interaction of bacteria with Cd2+metal ions occurs mainly through the binding to carboxyl groups and polysaccharides present on the cell surface. The present results also indicate that lb galinarum and lb crispatus are a promising tool for removing of heavy metal ions from the food products consumed by humans and animals.

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34


antiBacteRial potential oF liveRWoRts

Magdalena Czołpińska, Anna Zofia Roszak, Katarzyna Buczkowska

department of Genetics, faculty of Biology, institute of experimental Biology, adam mickiewicz university poznań, poland

Many species of plants produce compounds, which exhibit various biological properties. These compo-unds are antibacterial, antiviral and antifungal. Their chemical structure allows qualify them to terpeno-ids, phenol derivatives and alkaloids. The evolutionarily oldest land plants – liverworts – has an ability to synthesizing chemical compounds belonging to mentioned earlier groups. So far, published data confirm content, i.a. terpenoids and phenolic compounds in liverworts and give us acknowledgment about their antimicrobial activity. This antimicrobial activity is variable and depends not only on species or region of occurrence but also on method of extraction or distillation or part of plant from which we obtain extracts. Recently we can observe increased interest in use of substances of natural, plant origin in the fight against microorganisms. The using of plant extracts is extremely promising, because many strains become resi-stant to commonly used antibiotics which are produce by pharmaceutical companies. Therefore, we have to find other opportunities to fight with pathogens.

In the present contribution was tested antibacterial activity of extracts from selected liverworts spe-cies. Different species of these plants were examined, i.a. Bazzaniatrilobata, trichocolea tomentella, aneura pinguis, preissia quadrata, plagiohila asplenioides and marchantia polymorpha Different organic solvents were used, i.a. ether, isopropanol, acetone, ethanol (50%). We observe differences in biological activity and it depends on plant specie and solvent. In our research proteins from plants were also isolated and electro-phoresis was done. The aim of electrophoresis was examined which proteins are responsible for this anti-microbial activity.

These results showed that liverworts are source of some chemical compounds which quantity and biological activity should be examined in further researches.

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the Biological activity oF trichoderma atroviride sK80 in BiopRepaRations pReseRved By Fountain dRying

oR lyophiliZation

Anna Kancelista1, Regina Stempniewicz1, Wojciech Łaba1, Marta Pasławska2, Michał Piegza1, Aleksandra Wilczak1, Monika Grzegorczyk1, Magdalena Szczech3,

Danuta Witkowska1

1department of Biotechnology and food microbiology 2institute of agricultural engineering, wrocław university of environmental and life sciences, poland

3research institute of horticulture, skierniewice, poland

Biopreparations were obtained by fountain drying or lyophilization of biocontrol agent trichoderma atro-viride SK80 cultured on two types of lignocellulosic medium in solid state fermentation (SSF). The obta-ined biopreparations were vacuum sealed and stored at room temperature for 12 months. The effect of the biomass preservation method and storage time of bioproducts on the viability of the strain SK80 was exa-mined. Biomass yield, expressed as the number of cfu/g dm, in SSF cultures and in biopreparations was determined. Furthermore, the ability of t atroviride SK80 strain to biosynthesis of selected hydrolytic en-zymes directly by the SSF cultures and after 12 months of storing was also examined.

Both applied lignocellulosic media were suitable materials for the production and preservation of biomass of the tested trichodermastrain.However, the highest number of cfu/g dm after SSF cultures was achieved on tri-component media. After the preservation process the tested microorganism has retained a high survival rate in the range of 48.60–65.37% and 15.43–100.00% in the fountain and freeze-drying, re-spectively. The survival rate was dependent on the carrier type and the presence or absence of the protecti-ve agent (freeze-drying).The highe fficiency of biopreparations also results from the strains vitality during storage, and in the present study it depended primarily on the preservation method. After 12 months of storage the survival of SK80 strain in dried biopreparations was in the range of 8.44–19.16%, correspon-ding to a biomass yield 108 cfu/g dm, while in freeze-dried biopreparations the number of live conidia was significantly reduced to a level of 103cfu/g dm. It was also shown that the tested strain maintained the abi-lity for the biosynthesis of lytic enzymes after 12 months of storage.

In conclusion, the obtained results indicate the potential of t atroviride SK80 strain as a prospective biocontrol agent in the production of new biopreparations and fertilizers used in plant protection.

The study was a part of the project „Polish trichoderma strains in plant protection and organic waste management”, under Priority 1.3.1, subject area “Bio”, co-financed by The European Union through Euro-pean Regional Development Fund within the Innovative Economy Operational Programme, 2007–2013. Project No UDA-POIG.01.03.01-00-129/09-08.

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36


ModiFications oF n-glycan stRuctuRes synthesiZed By Yarrowia lipolYtica

Zbigniew Lazar1, Piotr Hapeta1, Paweł Mituła2, Małgorzata Robak1

1department of Biotechnology and food microbiology, 2department of chemistry, wrocław university of environmental and life sciences,

chełmońskiego 37, 51-630 wrocław, poland

N-linked protein glycosylation is very important for protein folding, subunit oligomerization, sorting and transport of secreted and membrane bound proteins [1]. Popper glycan profile is a problem in heterolo-gous protein expression, especially those with pharmacological interest. Application of glycoproteins with non-human oligosaccharide chains can increase immunological respons of patient [2].

The following work was focused on N-glycans occurring in glycoproteins produced by Y lipolytica A-101 strain and its suc+ transformants. All analysis were carried out on membrane proteins which were an example of homologous proteins and on heterologous invertase (suc2 gene from s cerevisiae) expres-sed by this yeast [3]. Additionaly putative galactosyltansferase and UDP-galactose transporter were iden-tified and investigated.

Analysis of N-glycans was carried out by MALDI-TOF-MS and GC-MS. Overexpression of Y lipo-lytica genes was carried out by random chromosomal integration using zeta sequences. All the genes were overexpressed under strong, constitutive TEF promoter. Gene deletion was performed by double crossing-over using promoter and terminator as homology regions.

Predominant structures of N-glycans found both on membrane proteins as well as on heterologous invertase were composed mainly of GlcNAc2Man8 (37–44%) and GlcNAc2Man9 (20%). The longest oli-gosaccharide chains contained up to 14 mannose residues (~1%). Furthermore, glycosidase treatment revealed N-glycan modification which turned out to be galactose, modification so far not described for Y lipolytica. Oligosacharide chains were composed in 84% from mannose, 15% from galactose and 1% from N-acetylglucosamine. Candidate genes responsible for galactosyltransferase activity and for UDP-galactose transporter activity were identified. These genes were overexpressed as well as deleted in Y lipo-lytica A-101 background. Preliminary analysis of obtained transformants showed differences in yeast phy-siology.

References[1] Helenius A., Aebi M., 2004. Annu., Rev., Biochem., 73, 1019–1049. [2] Jacobs P.P., Callewaert N., 2009. Curr., Mol., Med., 9, 774–800. [3] Walczak E., Robak M., 2009. Acta Sci. Pol. Biotechnol., 8, 25–36.

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37

session 2 enzymes & PePtides lecture 2.1

isolation and puRiFication oF cytosolic enolase FRoM salmonella enterica seRovaR typhiMuRiuM

Paweł Serek, Iwona Bednarz-Misa, Magdalena Mierzchała-Pasierb, Agnieszka Bronowicka-Szydełko, Małgorzata Krzystek-Korpacka, Andrzej Gamian

department of medical Biochemistry, wrocław medical university, chałubińskiego 10, 50-332 wrocław, poland

Enolase is one of the key of enzymes of glycolytic pathway located in cytoplasm of prokaryotes and euka-ryotes. Enolase-like proteins were identified in the cell wall outer membrane of some Gram-negative bac-teria, but transport mechanism from the cytoplasm to outerbacterial membrane is still unknown.

The aim of the research was isolation and purification of cytosolic enolase from salmonella serovar Typhimurium. The specific activity of membrane enolase on the surface of intact bacterial cells was also evaluated.

In order to obtain electrophoretically homogeneous protein, after ultrasonic disintegration of the bacterial cells and ultracentrifugation, the proteins were precipitated with ammonium sulphate and dia-lyzed. Purification of cytosolic fraction was performed using fast protein liquid chromatography (FPLC) with use of ion-exchange chromatography on Sephadex DEAE A-50 and gel filtration on Sephadex G-100. In the end of purification, we used preparative electrophoresis on Prep-cell 491 system (Bio-Rad). The ho-mogeneity of protein was evaluated using electrophoresis in native conditions. At each stage enzymatic activity of enolase was measured spectrophotometrically in reaction of dehydratation 2-phosphoglycerate (2PGA) to phosphoenolpyruvate (PEP).

The same enzyme reaction allowed to examine the activity of outer membrane enolase, showing the dose-dependent activity catalyzed by the intact bacterial cells.

Obtaining pure protein allows for investigate surface enolase properties and the role which this en- pure protein allows for investigate surface enolase properties and the role which this en-zyme plays in the pathogenesis of diseases.

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caseinate hydRolysate oBtained With seRine pRotease FRoM Yarrowia lipolYtica yeast as gRoWth stiMulatoR

oF pRoBiotic BacteRia

Anna Dąbrowska, Konrad Babij, Marek Szołtysik, Łukasz Bobak, Aleksandra Zambrowicz, Józefa Chrzanowska

department of animal products technology and quality management, wrocław university of environmental and life sciences, chełmońskiego 37/41, 51-630 wrocław, poland

The effect of sodium caseinate hydrolysate (SCH) addition on growth of standard yoghurt cultures and Bi-fidobacterium adolescentis during co-fermentation and its viability during storage at 4ºC in yoghurts has been evaluated. SCH was obtained with the use of extracellular serine protease from Y lipolytica yeast. Stirred probiotic yoghurts were prepared by using whole milk standardized to 16% of dry matter with the addition of either skim milk powder (SMP), whey protein concentrate, SCH-SMP (ratio 1:1), SCH. The hydrolysate influenced on increase the yoghurt culture counts at the initial stage of fermentation and sig-nificantly inhibited the decrease of bacteria population throughout the storage at 4ºC in comparison to the control. The post-fermentation acidification was also retarded by the addition of SCH. The hydrolysate did not increase significantly the Bifidobacterium adolescentis counts at the initial stage of fermentation pro-cess. However, the SCH significantly improved its viability. After 21 days of storage, in the yogurts supple-mented with SCH, the population of these bacteria oscillated around 2.91 log10 CFU/g, while in samples where SMP or whey protein concentrate was used, the bacteria were no longer detected.

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heteRologous expRession oF a RaW-staRch-digesting insect aMylase in Yarrowia lipolYtica – intRoduction

to the pRoduction pRocess’ enhanceMent

Ewelina Celińska, Monika Borkowska, Wojciech Białas

department of Biotechnology and food microbiology, poznań university of life sciences, wojska polskiego 48, 61-627 poznań, poland

As we have previously reported [Celińska et al., 2015, Appl. Microbiol. Biotechnol, 99(6), 2727–2739], an insect-derived gene encoding alpha-amylase could be efficiently expressed in a non-conventional yeast species Yarrowia lipolytica. It has been demonstrated that the enzyme exerts its amylolytic activity also to-wards raw, non-pretreated starch granules. Such a characteristics is of great importance for industrial ap-plications. Here we present preliminary studies towards improvement of the enzyme production.

Fed-batch culture mode is known to be an efficient strategy pursued towards improvement of hete-rologous proteins production. Fed-batch cultures were carried out in 5 L bioreactors, in rich culture me-dium, with two portions of the concentrated medium fed after 24 and 48 h of culturing. The obtained re-sults demonstrated that the fed-batch mode of culturing could improve production of the heterologous protein by more than eight fold, when compared to our previous results obtained in batch cultures. Further studies need to be carried out to enhance the process.

Project number: 2012/05/N/NZ1/00002, financially supported by the National Science Center, Poland.

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Biopep dataBase oF sensoRy peptides and aMino acids

Piotr Minkiewicz1, Anna Iwaniak1, Małgorzata Darewicz1, Krzysztof Sieniawski2, Piotr Starowicz1

1chair of food Biochemistry, faculty of food science, university of warmia and mazury in olsztyn, pl cieszyński 1, 10-726 olsztyn-Kortowo, poland, minkiew@uwm edu pl

2Krzysztof sieniawski, ul stanisława Żurawskiego 17/19, 10-689 olsztyn, poland

One of the classes of compounds which affect the food taste are peptides. They may reveal all basic tastes. Peptides are annotated in specialized databases utilizing amino acid sequences (such as BIOPEP) or che-mical databases using codes such as SMILES, InChI or InChIKey (e.g. ChemSpider).

The aim of this work was to create a database of sensory peptides and amino acids. Information abo-ut compound taste was based on sensory studies, described in literature. Bioactivity data was taken from the BIOPEP database of bioactive peptides and from other databases (e.g. ChemSpider or PubChem). Data was inserted into a database using BIOPEP form. Sequences of peptides were converted into chemical co-des by use of Open Babel program. The resulting annotations were verified and corrected by means of Ket-cher program available at ChemSpider website.

The information annotated in the BIOPEP database includes: peptide sequence written in one-let-ter code, information about taste (“Activity” tab), reference („References” tab), structure written by use of chemical codes, bioactivity data, if necessary („Additional information” tab), ID numbers from other da-tabases („Database reference” tab).

Database contains the tools allowing to determine the location of peptides in a protein sequences, comparison of protein sequences as a precursors of sensory peptides using quantitative descriptors, prote-olysis simulation and calculation of parameters for quantitative description of simulated proteolysis.

BIOPEP database is available at the website: http://www.uwm.edu.pl/biochemia/index.php/pl/biopep.

Work supported form Warmia and Mazury University in Olsztyn funds.

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session 3 Biosynthesis & Biodegradations lecture 3.1

the pRoduction oF KilleR toxin FoR plant pRotection By deBarYomYces hansenii yeast in vaRious cultuRe Media

Monika Grzegorczyk1, xymena Połomska1, Piotr Regiec2, Maria Wojtatowicz1, Barbara Żarowska1

1department of Biotechnology and food microbiology, 2 department food storage and technology faculty of food science, wrocław university

of environmental and life sciences, chełmońskiego 37, 51-630 wrocław, poland

The biological control of phytopathogenic fungi is an alternative to commonly used chemical fungicides. Among different groups of microorganisms, yeasts producing killer toxins seem to be very promising bio-logical control agents.

debaryomyces hansenii AII4b killer yeast strain used in the study exhibits antagonistic activity to-wards certain phytopathogenic filamentous fungi, such as: Botrytis cinerea, fusarium linii, venturia ina-equalis and others. Earlier studies showed that culturing of that yeast in semi-synthetic YPG medium of pH 4.6 stabilized by citric-phosphate buffer result in the highest activity of killer protein, up to 1550 aU. Unfortunately, the cost of such medium is also very high. Thus, the aim of the study was to lower the cost of culturing medium in order to enable industrial production of killer toxin preparation.

The first step was to substitute citric-phosphate buffer by inorganic acid. Studies showed that HCl is the most suitable pH-adjuster. Therefore, it was used in all further media formulations. Other YPG com-pounds were replaced by several low-cost materials, such as: serolat, beet molasses, waste glycerol, corn steep liquor and Yarrowia lipolytica yeast extract. The highest killer activity (891 aU) was obtained in BC10 medium (beet molasses 20 g/L, corn steep liquor 10 g/L). The cost of initial medium is around 16,000 PLN/ton (3,800 euro/ton), while the cost of the BC10 is only 214 PLN/ton (51 euro/ton).

This work was supported by reasearch grant POIG.01.03.01-02-080/12, co-financed by the Euro- pean Union from the European Regional Development Found

Key words: killer yeast, Debaryomyces hansenii, low-cost materials, phytopathogenic fungi

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42


potential application oF gM Flax

Magdalena Wróbel-Kwiatkowska1, Waldemar Rymowicz1, Jan Szopa2

1 department of Biotechnology and food microbiology, wrocław university of environmental and life sciences, chełmońskiego 37/41, 51-630 wrocław, poland

2department of Genetics, plant Breeding and seed production, wrocław university of environmental and life sciences, pl Grunwaldzki 24a, 53-363 wrocław, poland

Flax (linum usitatissimum L.) is an annual plant cultivated in the temperate climate. There is growing in-terest in improving flax plant’s quality by metabolic engineering. Recent progress in this area is a good re-ason for the generation of flax plants with new features.

The purpose of our research was to improve the quality of flax fibre designed for medical and indus-trial applications.

The main conclusions that can be drawn from the studies is that flax fibres supplied with phenylpro-panoids are useful for preparing wound dressing and those enriched with PHB (poly-β-hydroxybutyrate) might be the suitable component of the composites. Two kinds of the composites have been prepared based on PLA (polylactide) or alternatively PCL (poly-�-caprolactone) matrix. Both composites were rein-�-caprolactone) matrix. Both composites were rein--caprolactone) matrix. Both composites were rein-forced with fibres contained polyhydroxybutyrate.

Our results showed that composites with polylactide might be the source of new scaffolds for tissue engineering (bone and muscle engineering), while composites based on polycaprolactone might serve as entirely biodegradable agricultural items.

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43


assessing the iMpact oF seWage sludge application on the MicRoBial activity, Functional diveRsity

and coMMunity stRuctuRe in soil

Anna Markowicz, Kinga Bondarczuk, Zofia Piotrowska-Seget

department of microbiology, university of silesia, Jagiellońska 28, 40-032 Katowice, poland, anna markowicz@us edu pl

The use of sewage sludge as a fertilizer is one of the most recommended method in waste management. It is rich in organic matter and other valuable substances which may improve soil fertility and productivity. However, sewage sludge may also be a source of toxic compounds such as heavy metals, hydrocarbons and antibiotics. Since indigenous microbial communities play a major role in the proper functioning of soils, there is a need for assessing the response of microbial communities to sewage sludge application.

The purposes of this study was to evaluate the activity, structural and functional diversity of micro-bial communities in soil enriched with sewage sludge.

The study was carried out on two experimental fields: one with the sewage sludge application at a dose 10 t/ ha and the second without amendment. 20 composite soil samples were collected from the plots just after sewage sludge application and after a month. In all investigated samples enzymatic activities of dehydrogenase, urease, and phosphatase were evaluated. The microbial community composition were characterized using the Phospholipid Fatty Acids (PLFA) and substrate utilization pattern using the Bio-log® method. The presentation will include preliminary results of the project.

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the aBility oF the FilaMentous Fungus iM 833 to gRoWth in the pResence oF heavy Metals and xenoBiotics

Tomasz Janicki, Jerzy Długoński

department of industrial microbiology and Biotechnology, faculty of Biology and environmental protection, university of Łódź, Banacha 12/16, 90-237 Łódź, poland

xenobiotics belonging to the group of EDCs have a adverse impact on living organisms by modulating the-ir immune system. They get into the environment mainly as result of industrial processes. The ongoing de-velopment of the industry enhances this negative process. Technical nonylphenol, 4-tert-octylphenol and 4-cumylphenol are a compounds exhibiting structural similarity to natural hormones. This xenoestrogens are widely used as surfactants, plasticizers and polycarbonate plastics. Some heavy metals such as Zn, Mn, Ni and Pb similarly to the above mentioned substances have the ability to interfere with the proper func-tioning of the endocrine system.

The aim of this study was to evaluate the ability to grow of IM 833 in the presence of heavy metals and xenobiotics. The culture of IM 833 was conducted in Sabouraud medium. Tested fungus has the ability to growth in the presence of 25 mg/l of tested xenobiotics with addition of heavy metals.

The work was supported by the National Center for Science, Poland (Project No. UMO-2011/01/B/NZ9/02898)

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45


integRation oF ediBle FilaMentous Fungi in the ethanol BioReFineRy and the iMpoRtance

oF By-pRoducts

Patrik R. Lennartsson, Mohammad J. Taherzadeh

swedish centre of resource recovery, university of Borås, sweden

Many filamentous fungi have the ability to utilize complex substrates that are not fully used in current etha-nol biorefineries. However, since most starch-based ethanol plants sell the fermentation residues as animal feed, any new microorganisms have to be food-grade. The best strain fulfilling all criteria found so far is neurospora intermedia. By incorporating the strain in an ongoing starch-based process to utilize some of the fermentation residues (thin stillage) the ethanol production could be increased by 5%. The fungal bio-mass also has a high protein content (>50%) with a good amino acid and lipid composition, which can in-crease the quality of the animal feed. The fungal cultivation also decreased the total solid load in the thin stillage by 34%, which can improve the following evaporation and drying processes. The process has been confirmed to yield close to identical results when scaled up in an 80 m3 bioreactor. Whole stillage could also be utilized by the fungi, with a potential to increase the ethanol production by 10% after addition of 1 FPU cellulase/g suspended solid. However, there is still potential for improvement since the larger par-ticles in the whole stillage only offered a minor contribution to the ethanol production.

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46


a novel tWo-stage FeRMentation pRocess oF eRythRitol pRoduction By yeast Yarrowia lipolYtica FRoM Molasses

Aleksandra M. Mirończuk, Magdalena Rakicka, Anna Biegalska, Waldemar Rymowicz, Adam Dobrowolski

department of Biotechnology and food microbiology, wrocław university of environmental and life sciences, chełmońskiego 37/41, wrocław, 51-630, poland

adam dobrowolski@up wroc pl

Yarrowia lipolytica is a proper hos for erythritol production. In present study, a novel two-stage fermenta-tion process of erythritol production based on molasses and glycerol was investigated. First, the biomass of Y lipolytica was grown on medium containing sucrose as a sole carbon source. Next, after utilization of the sucrose by yeasts, glycerol was added into medium. In order to use molasses as a substrate for eryth-ritol synthesis, sucrose utilization was established by expressing the saccharomyces cerevisiaesuc2 gene. Molasses, as a main by-product of sugar industry is a suitable substrate for high biomass production. Gly-cerol is also the by-product derived from the diesel production process, moreover it was shown, as a cru-cial factor for an efficient erythritol synthesis by Y lipolytica In our study, during the cultivation yeast Y lipolytica was able to produce 52–142 g l-1 of erythritol, with the productivity oscillated from 0.3 to 0.85 g l h-1 and yield 0.24–0.72 g g-1. During these processes, the biomasses oscillated from 25 to 41 g l-1. This work presents genetically modified strains of Y lipolytica as suitable tools for direct conversion of industrial mo-lasses and glycerol into value added product erythritol.

This work was financed by the polish National Centre for Research and Development under Project LIDE -R/010/207/L-5/13/NCBR/2014 “Improving the biosynthesis of natural sweeteners from renewable feed-stock by the yeast Yarrowia lipolytica”.

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47

session 4 Biotransformations lecture 4.1

BiocatalyZed synthesis oF tyRosol deRivatives

Beata Szmigiel, Ewa Żymańczyk-Duda

department of Bioorganic chemistry, faculty of chemistry, wrocław university of technology, wybrzeże wyspiańskiego 27, 50-370 wrocław, poland

Antioxidants are compounds having ability to neutralize free radicals - reactive molecules or atoms, which stabilizing their structures via interaction with different types of biological molecules, causing their degra-dation. That activity leads to damage viable human cells, resulting in many diseases. Antioxidants provide positive effect in the treatment and prevention of such illnesses, which manifest as: cancer, cardiovascu-lar diseases, diabetes, brain stroke, skin diseases and delay the process of aging. That knowledge about de-structive effect of free radicals induces to constantly seek for new substances able to prevent organism da-mages or for novel modification of the known ones [1].

There is an interest in the use of biotechnological methods for obtaining various compounds using environmentally friendly methods. Some of them are already introduced into the pharmaceutical and co-smetic companies, which successfully applied biotransformation methods with whole cells biocatalysts or isolated enzymes for the defined compounds synthesis.

The aim of the study is the use of microorganisms for the synthesis of antioxidants – namely tyro-sol derivatives, searching for natural biocatalysts with specific activity. The crucial idea is to elaborate the method of conversion of not expensive starting materials (2-phenylethanol) into desired products (tyro-sol and hydroxytyrosol) in as simple as possible manner, ending by obtaining of the expected products of expected biological properties. Preliminary results shows that biohydroxylation of 2-phenylethanol ca-talyzed by rhizopus oryzae leads to the tyrosol [2-(4-hydroxyphenyl)ethanol ] and then to hydroxytyro-sol [2-(3,4-dihydroxyphenyl)ethanol ]. These compounds are known from their unusual antioxidant ac-tivities and are usually derived from the natural sources, such as olive oil viaexpensive extractive methods [2, 3]. That is why the pursuit of the alternative methods of their preparation is still under consideration. Already performed experiments confirm that rhizopus oryzae strain is able to hydroxylate the 2-phenyle-thanol to desired antioxidants within the very short time period - one hour biotransformation.

The results were evaluated using HPLC method. Further studies are intended to optimize the bioca-talytic protocol, to increase the efficiency and the scale of the process.

References[1] Andreassi M., Andreassi L., 2004. Journal of Cosmetic Dermatology, 2, 153–160.[2] Ting Hu., xiao-Wei He., Jian-Guo Jiang, xi-Lin xu., 2014. J. Agric. Food Chem., 62, 1449−1455.[3] Nieves Franco M., Galeano-Díaz T., López Ó., 2014. Food Chemistry, 163, 289–298.

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48


MicRoBial hydRolysis oF Bicyclic lactaMs

Paulina Walczak, Filip Boratyński, Teresa Olejniczak

department of chemistry, wrocław uniwersity of environmental and life sciences, norwida 25, 50-375 wrocław, poland

Nowadays, there is growing interest in new compounds that inhibit growth of filamentous fungi. The most attractive are the ones of low toxicity and high biodegradability, or the ones of high selectivity of action, not affecting the growth of saprophytic fungi. Such compounds may include natural lactams biosynthe-sized by plants. Plants have evolved many secondary metabolites involved in their defense against phyto-pathogens 10–13. Compounds with lactam moiety display wide spectrum of biological activities, such as fungistatic. Additionally they are part of the structure lactam antibiotics. They are used for the production of polyamides and as solvent, for example a polymer of 1-vinyl-2-pyrrolidone – as artificial blood plasma, the gel dressing.

In this aspect of our interest to bicyclic lactams, which are analogs of naturally occurring in plants of the family apiaceae (including ligusticum officinale, ligusticum chuanxiong, l wallichii, angelica sinen-sis, apium graveolens).

We planned to obtain the optically active lactams biotechnological methods using microorganisms. Among the advantages of carrying out the reaction biotransformation replace the substrate specificity, re-gioselectivity and stereospecificity mild processing conditions. This is the method recommended by the Green Chemistry, and compounds obtained by this method are treated as natural.

References[1] Liu q., Shao C., Gu Y., Blum M., Gan L., Wang K., Chen M., Wang C., 2014. Antifouling and fungici-

dal resorcylic acid lactones from the sea anemone-derived fungus Cochliobolus lunatus. J. Agric. Food Chem.. DOI: 10.1021/jf500248z.

[2] Peretto G., Du W., Avena-Bustillos R.J., Berrios J., Sambo P., McHugh T.H., 2014. Optimization of an-timicrobial and physical properties of alginate coatings containing carvacrol and methyl cinnamate for strawberry application. J. Agric. Food Chem., 62, 984−990.

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enZyMatic ModiFication oF phospholipids

Witold Gładkowski, Anna Chojnacka, Anna Gliszczyńska, Natalia Niezgoda, Czesław Wawrzeńczyk

department of chemistry, wrocław university of environmental and life sciences, norwida 25, 50-375 wrocław, poland

Phospholipids are widely applied in foods, pharmaceutical and cosmetic products where they function as emulsifiers, stabilizers and antioxidants1. Modified phospholipids could have much better surface active properties and nutritional value than the original ones2.

Lecithin from egg yolk contains mainly saturated fatty acids and there are only 16% of linoleic acid (n-6 PUFA) and no n-3 polyunsaturated fatty acids3. Incorporation of PUFA and conjugated fatty acids (CFA) into PLs is of great interest because of the possible medical applications and using them as functio-nal food additives.

To modify phosphatidylcholine (PC) from egg yolk, we decided to incorporate such acids in the sn-1 position by lipase-catalyzed interchange reaction with the natural plant oils rich in PUFA and CFA. Three different lipases were investigated as biocatalysts in the interesterification processes: Lipozyme RM IM (lipase from Mucor miehei), Lipozyme TL IM (lipase from Thermomyces lanuginosa) and Novozym 435 (candida antarctica lipase B).

The fatty acid composition in modified PC clearly reveals that mainly saturated fatty acids which are predominant in sn-1 position of PC from egg yolk, were replaced by the unsaturated ones.

It has been found that the most effective enrichment was achieved in processes with application of Novozyme 435 as biocatalyst.

AcknowledgementsThis work was financed by National Science Center Project no. 2012/05/B/NZ9/03358

References[1] Guo Z. , Vikbjerg A.F., xu x., 2005. Biotechnol. Adv., 23, 203.[2] Jones M.N., 1995. Adv. Colloid Interface Sci., 54, 93.[3] Haraldsson G.G., Thorarensen A., 1999. J. Am. Oil. Chem. Soc., 76, 1143.

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synthesis and BiotRansFoRMation oF analogues natuRal oF Flavonioids and chalcones

Bartłomiej Potaniec, Mirosław Anioł


Chalcones, (e)-1,3-diphenyl-2-propen-1-ones, belong to a large group of bioflavonoids occurring in plants. They are precursors in the biosynthesis of all classes of flavonoid compounds. They have structure of the α, β-unsaturated natural bicyclic ketones, show broad spectrum of action, depending on the nature of the substituents attached to the aromatic rings. They have antibacterial activity, anti-tumor and anti-inflammatory properties. The high biological activity of the naturally occurring chalcones encouraged the research community to obtaining new derivatives with trans-chalcone skeleton both by chemical synthesis and by the biotransformations. We present preliminary current studies of chemical synthesis of aza- and phenyl derivatives of some natural chalcones and flavonoids.

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Posters

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session 1 microorganisms Poster 1.1

antiBacteRial activity oF selected tea suBstances, teas and Medicinal heRBs

Miroslava Kačániová1, Margarita Terentjeva2, Marek Astaloš1, Jana Petrová1, Attila Kántor1, Macej Kluz3, Lukáš Hleba1

1faculty of Biotechnology and food sciences, slovak university of agriculture, nitra, slovak republic 2institute of food and environmental hygiene, faculty of veterinary medicine,

latvia university of agriculture, Jelgava, latvia 3faculty of Biology and agriculture, university of rzeszów, rzeszów, poland

The aim of this study was to determine in vitro the antimicrobial activity of selected medicinal herbs (melissa officinalis, mentha piperita, tilia platyphylla, sambus nigra, equisetum arvense), tea substances (Mathé: substance from ilex paraguariensis, Rooibos: tea substance aspalathus linearis) and teas (Assam: Indian black tea from camellia sinensis, Pu-erh: darkpu-erh (shu) from camellia sinensis, Sencha: Japanese green tea from camellia sinensis) against five species of pathogenic microorganisms. Firstly, we determi-ned the total number of microorganisms (CPM). Then colonies were identified after cultivation by MAL-DI-TOF MS Biotyper. Evaluation of the antimicrobial activity was performed by disc diffusion method, well diffusion method and detection of minimum inhibitory concentration (MIC). For antibacterial activi-ty, bacteria escherichia coli CCM 2024, Yersinia enterocolitica CCM 5671, Klebsiella pneumonie CCM 2318, staphylococus aureus CCM 2461 and Bacillus thurigiensis CCM19 were used. The inhibition zones were measured in mm in disc diffusion method and well diffusion method. The MIC of the individual extracts was measured spectrophotometrically. Mass spectrometry revealed seven Gram positive bacteria Bacil-lus cereus, B mycoides, B pumilus, enterococcus durans, staphylococcus epidermis, s hominis, s warneri, four Gram negative bacteria acinetobacter junii, hafnia alvei, Klebsiella pneumoniae, sphingomonas spp. and two yeast candida glabrata, cryptococcus albidus. The results show that certain tea extracts are parti-cularly active against various pathogenic bacteria. Tea extracts (Sencha, Rooibos, Mate, Assam) and plants extract (tilia platyphyllos, sambucus nigra, melissa officinalis) were found to have the strongest antibacte-rial activity against bacteria staphylococcus aureus CCM 2461.

Key words: tea substance, tea, medicinal plants, bacteria, antimicrobial activity

This work was co-funded by VEGA 1/0611/14.

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54


antiFungal activity oF saccharomYces cerevisiae yeasts

Joanna Koniuszewska, Dominika Ciurko, Karolina Banach, Maria Wojtatowicz, Barbara Żarowska

department of Biotechnology and food microbiology, wrocław university of environmental and life sciences, poland

It is estimated that losses in the production of fruits and vegetables caused by harmful microorganisms are about 20–30% per annum over the world. The most numerous group of diseases are diseases caused by fungi. Mycosis appears during the cultivation and during the storage of raw materials. So far, the most effective agents used to fight the fungal diseases were chemical plant protection products. Because of the threat posed by them, they are no longer so popular. Current trends are going in the direction of restricting the use of chemical products in favor of biological protection methods. These methods are aimed at redu-cing the growth of harmful pathogens by using other microorganisms or their metabolites.

The present work focused on evaluating the ability of saccharomyces cerevisiaeyeasts to reduce the growth of filamentous fungi and examining the mechanism of their action. The study used the following filamentous fungi: alternaria alternota, aspergillus niger, Botrytis cinerea, chaetomium sp , fusarium ave-nacum, fusarium linii, monilia fructigena, penicillium sp , and phylophtora cactorum

Pour plates with YPG medium and proper amount of yeast cells were prepared. The pieces of agar medium with tested fungi were put on the top of YPG medium. The co-cultures of microorganisms were incubated in 20 and 14°C. The experiment was carried out in two variants of pH: neutral and acidic.

Based on these results it can be concluded that saccharomyces cerevisiae yeasts as a result of compe-tition for nutrients and living space definitely limited the growth of the following fungi: a niger, B cinerea, chaetomium sp , and penicillium sp The best results were obtained at 20°C, for both the acidic as well as neutral pH. In other cases the growth inhibition was on a low level.

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55


antiMicRoBial activity oF silveR nanopaRticles

Barbara Żarowska1, Michał Piegza1, Katarzyna Jaros-Koźlecka2, Tomasz Koźlecki3, Małgorzata Robak1

1 department of Biotechnology and food microbiology, wrocław university of environmental and life sciences, chełmońskiego 37, 51-630 wrocław, poland

2tK nano, Gorlicka 64/50, 51-314 wrocław, poland 3 faculty of chemistry, wrocław university of technology,

wyspiańskiego 27, 50-370 wrocław, poland

The increasing importance of nanotechnology in many areas of life inspires to study the antimicrobial ac-tivity of some nanoparticles [1, 2]. The BioscreenC microbial analyzer gives the opportunity to check the growth of microorganisms in the medium added of different concentrations of nano particles at the same time, so in the same conditions [3].

The aim of that study was to establish the minimal concentration of silver nano-particles (SNP) which inhibits the growth of bacteria: escherichia coli, pseudomonas fluorescence, staphylococcus aureus andyeasts: saccharomyces cerevisiae, Yarrowia lipolytica, candida albicans in BioscreenC cultures on ap-propriated media Tested concentrations of SNP were 0–10.7 µg/mL for bacterial and yeast growth analy-sis and 0; 2.6; 5.1 and 21.4 µg/mL for filamentous fungi. The anti-fungal activity against aspergillus niger and aureobasidium pullulans was checked on agar medium.

Inhibition of microorganisms growth by SNP was species dependent. The highest growth inhibition was observed for yeasts. Growth of c albicans was inhibited in 100% at SNP concentration of 5.35 µg/mL (and higher). For Y lipolytica and s cerevisaie growth inhibition in 95–100% was noted at SNP concentra-tion of 10.7 µg/mL. Bacterial growth was less susceptible to SNP. e coli growth was inhibited in 78% at the highest concentration of SNP (10.7 µg/mL) but only during first 12 h of culture. At the same concentration the growth of p fluorescens was inhibited in 80–89% during 48 h of culture. The same level of inhibition as for p fluorescens was observed for s aureus.

Filamentous fungi growth was inhibited by SNP at the concentration of 5.1 µg/mL. The minimal concentration of SNP needed to inhibit the growth of bacteria determined it that study

was lower (6.5–14 times) that determined by others [4].

References[1] Yoon K.Y., Byeon J.H., Park J.H., Hwang J., 2007. Science of the Total Environment, 373, 572–575.[2] Tran q.H., Nguyen V.q., Le A-T., 2013. Adv. nat. Sci.: Nanosci. Nanotechnol., 4, 033001.[3] Grabarczyk M., Mączka W., Winska K., Żarowska B., Anioł M., 2013. Journal of the Brazilian Chemi-Journal of the Brazilian Chemi-

cal Society, 24 (12), 1913–1922. [4] Rai M., Yadav A., Gade A., 2009. Biotechnology Advances, 27, 76–83.

This work was financed by the NCBR project No PBS 3/B1/10/2015

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56


MicRooRganisMs pResent in FeRMented gReen tea

Daria Kaczmarczyk1,2, Stanisław Lochyński1,2

1department of Bioorganic chemistry, faculty of chemistry, wrocław university of technology, wybrzeże wyspiańskiego 27, 50-370 wrocław, poland

2institute of cosmetology, wrocław college of physiotherapy, Kościuszki 4, 50-038 wrocław, poland

Tea has been known and appreciated in many countries and cultures for millennia. Presently, it is one of the most popular drinks worldwide. In China, it has been used already five thousand years ago, mainly as stimulating and detoxicating elixir. Recently many scientific reports have shown that tea has beneficial in-fluence on human health and well-being.

Tea as a plant belongs to camelliaceae family. The leaves are picked up from evergreen shrubs and may be processed using different methods. Green tea leaves (Theae viridis folium) is obtained through the process of quick drying of the leaves after picking them, in order to inactivate enzymes.

Kombucha is acquired by fermentation process utilizing tea and sucrose and consists of a symbiotic colony of multiple nonpathogenic bacteria and yeast. The product of biotransformation occurs in form of slightly carbonated consumable broth. Its potential therapeutic properties have become the field of inte-rest and research.

Inoculations of Kombucha were performed using different solid and liquid media. Estimation of number of grown colonies as well as optimal conditions for stable growth of microorganisms present in Kombucha were the field of interest. Results show that the optimal time of microorganisms growth is aro-und 7th day of the cycle. In order to determine the kind of microorganisms present in Kombucha, a me-asurement series using media which are specific to the most expected strains were conducted. The biggest CFU number was recorded in 7th day of the cycle. Inoculations allowed to acquire strains, which were sent to Leibniz-Institut DSMZ to be identified. Research results will be presented.

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57


BioReactoR cultuRes oF tRichodeRMa Moulds FoR BioMass and spoRe pRoduction

Michał Piegza, Joanna Koniuszewska, Anna Kancelista, Wojciech Łaba, Danuta Witkowska


trichoderma fungi are the subject of constant interest in the broad range of agro-food industries, due to the antagonistic properties against phytopathogenic microorganisms and the decomposition of waste ma-terials using enzymatic properties. As a natural biofungicide it could be an alternative to chemicals pestici-des, simultaneously positively affecting the growth of crops. However, to be able to propose a bio-prepara-tion containing trichoderma fungus, the efficient method for producing and applying a particular strain of preferred characteristics must be determined at first. One of the easiest ways to obtain a preparation with a high density of fungal conidia is application of liquid cultures. Although most preparations use fungal cultures prepared in solid medium (SSF), incorporating spores in the mycelium-overgrown substrates, for the production of spores culturing in liquid media could also be employed, conducted on shakers (labora-tory scale) or in bioreactors.

The presented research focuses on the production of the spores by 2 strains of trichoderma fungi: t harzianum T33 and t citrinoviride C1 in liquid medium, in Biostat B Plus bioreactors on the basis of two media with different composition, particularly with varying ratio of carbon to nitrogen (GLU 10.5:1; GGB 3.8:1 of Ca ions). Media were sterilized directly in the bioreactors of the working volume 2000 ml. Cultures were carried out at constant aeration (2 m3/min, pH = 5.5, temp = 250C), changing only the stir-ring rate intensity in the range of 0–800 rpm. The total number of spores was evaluated, and their growth capacity with the use of PDA with Rose Bengal dishes method. At the end of each culture the total amount of biomass obtained containing living cells per 1 g dry weight was estimated.

The cultures of both tested strains resulted in the production of spores at 107 to 108/ml. This number was not correlated with the outcome of the spores condition because the number of colony forming units in 1 gram of wet biomass, obtained after centrifugation of culture, developed at the level at least two loga-rithmic orders lower.

The variation in the intensity of stirring in a small way affected the production of biomass (cfu/g s.m.) of both tested strains. t harzianum T33 in GLU medium at increased speed, produced slightly more biomass (6.0x 105–6.9x 105 cfu/g sm) in comparison with the culture without stirring (3.9 x 105 cfu/g sm), while in the GGB medium, the largest number of living cells (5.4 x 106 cfu/g sm) was obtained with a mechanical stirrer off, and only under the forced mixing by continuous aeration (2 m3/min) condition. t citrinoviride C1 efficiently produced biomass at the highest intensity of stirring (800 rpm/min) in GGB medium, reaching density of 1.1 x 106 cfu/g sm and exhibiting constant growth in the GLU medium, simi-lar to the case of the T33 strain.

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tRichodeRMa citrinoviride c1 and trichoderma harzianum t33 as anty-phytopathogenic agents

Aleksandra Han, Anna Ropa, Joanna Koniuszewska, Wojciech Łaba, Michał Piegza


As a result of an increasing number of studies concerning substances produced by trichoderma, individual strains of these fungi are increasingly applied in the process of biocontrol. trichoderma is extremely useful for creating vaccines immunising against plant pathogens.

In the present study we tested antagonistic effects of t citrinoviride c1 and trichoderma harzia-num t33 on the growth of 11 selected plant pathogens. The effect of tested strain on pathogen growth was analyzed: using plate method, activity of laminarinases and chitinases produced by the tested strain, the growth kinetics of particular pathogen strains in the presence of t citrinoviride c1 and t harzianum t33 metabolites, spot test and turbidimectric test with the use of Biocreen C.

The aim of the test was to established the influence of metabolites produced by the tested strain on the growth of pathogen strains. Selected parameters of growth kinetics were determined, such as maxi-mum specific growth rate, lag-phase duration and stationary phase time.

The results showed, that the metabolites produced by the tested trichoderma strains exhibit various intensity of inhibition or reducing the growth of phytopathogenic fungi. The thermally processed and con-centrated culture fluid did not pose any effect on the type of interactions between organisms. In all variants under study the tested strains stimulated the formation of conidia of aspergillus type fungi. After conduc-ting all the tests it was revealed that both t citrinoviride c1 and t harzianum t33 had an effect on redu-cing the growth of most tested pathogenic strains.

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chaRacteRistics oF KilleR yeasts deBarYomYces hansenii aii4B segRegants: aii4Bs and aii4BR in teRMs oF MoRphology

and KilleR activity

Monika Grzegorczyk, xymena Połomska, Barbara Żarowska


Killer phenomenon in yeast was first observed in saccharomyces cerevisiae in 1963 and it was described as the ability for synthesis of proteins exhibiting antagonist or lethal activity towards sensitive strains of yeasts and filamentous fungi. Such characteristic was further observed in many yeast strains from different ge-nera, such as: candida, debaryomyces, wickerhamomyces, metschnikowia and others. Killer activity differs among killer strains in spectrum and mechanisms of action and it can be determined either by: VLPs (Vi-rus like particles), dsDNA plasmids, or chromosomal genes. Some of killer yeasts strains and their toxins are described as potentially applicable treatment for fungal infections in fields, like: food industries, crop protection or even in medicine.

debaryomyces hansenii AII4b killer strain used in the study was isolated from Rokpol cheese and it exhibits antagonistic activity towards sensitive yeast strains belonging to Yarrowia lipolytica species and certain phytopathogenic filamentous fungi. Earlier studies showed thatculturing of AII4bin YPG medium of pH 4.6 at the temperature of 14°C,resulted inthe highestproductionand activity ofkillerproteins. While passaging the culture of AII4b in YPG medium the emergence of two segregants was noted.

The aim of this study was to characterize newly distinguished two yeast segregants of killer strain debaryomyces hansenii AII4b: S (smooth) and R (rough), in terms of cell and colonial morphology on va-rious media, and both: growth rate and cell biomass production, as well as the ability of synthesis and se-cretion of killer toxins in liquid cultures (YPG). The study showed AII4bS differed in colonial morphology and exhibited both: higher biomass yield and killer toxin activity in YPG cultures in comparison to AII4bR segregant. The study indicates that there are significant differences between AII4bS and AII4bR segregants and further studies, including molecular analysis shall be conducted in the future.

Key words: killer yeast, debaryomyces hansenii, segregants

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coMpaRison oF Yarrowia lipolYtica stRains By Rapd

Mateusz Kropiwnicki, Małgorzata Robak


Yeast Yarrowia lipolytica is highly valued species, best known for it’s ability to assimilate unusuall carbon sources and the ability to synthesize organic acids and extracellular enzymes, which makes this species often used in biotechnological processes and environmental protection [1, 2]. Strong interest in yeasts with potential applications in industry, entails the need for the rapid and reliable identification. The techniques based on the analysis of genetic material are particularly useful. Compared to traditional methods, which use evaluation of phenotypic traitsor carbon source assimilation profiles, they are generally faster and more reliable. Random Amplified Polymorphic DNA(RAPD) is one of the molecular biology methods, al-lowing organisms differentiation [3].

Each of 22strains tested in this study was previously subjected to PCR-RFLP analysis and assigned to Yarrowia lipolytica species. The isolated genetic material was subjected to RAPD analysis with four pri-mers: (GAC)5,(GTG)5,(GACA)4 and M13 oligonucleotide. Amplification reactions proceed under the fol-lowing conditions: initial denaturation in 94°C (3 min), 35 cycles 94°C (30s) 50°C (1 min), 72°C (1 min) and final elongation in 72°C (6 min). Reactions were performed in triplicate. Amplification products were separated on 1% agarose gel. Obtained electrophoretic profiles were used to generate similarity matrices in the BioGene software. Then, the similarity matrices were used to construct dendrograms.

Phylogenetic analysis allowed us to assign the tested yeast to six groups. The first group contained 2 strains isolated in Poland (Pludry), one strain from ATCC and one strain obtained in Germany. The se-cond group contained 5strains of the W29 family isolated from sewage in Paris. These 2 groups were simi-larin87%. The third group with only two strains from Pludry was similar to the rest in 83%. The last of the five strains from Pludry was in the fourth group, together with two strains isolated from soil and cheese (Poland). The similarity to the rest was 79%. Y lipolytica A101 isolated from soil in Wrocław and family strains, were in the fifth group similar to the others in 73%. The last, sixth group included 3 strains newly isolated from soil. This group was the most distant, with similarity level at 65%. Unfortunately the used set of primers did not allow us to differentiate all tested strains, however we were able to determined specific products for each primer.

References[1] Coelho M.A.Z., Amaral P.F.F., Belo I., 2010. Current research, technology and education topics in ap-Current research, technology and education topics in ap-

plied microbiology and microbial biotechnology, 2, 930–940.[2] Tomaszewska L., Rakicka M., Rymowicz W., Rywińska A., 2014. FEMS yeast research, 14(6), 966–

976.[3] Walczak E., Czaplińska A., Barszczewski W., Wilgosz M., Wojtatowicz M., Robak M., 2007. Food

microbiology, 24(3), 305–312.

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vi1 – the neW pcR staRteR Which togetheR With its4 alloWs speciFic identiFication oF ventuRia inaequalis

phytopathogenic Fungi

Xymena Połomska1, Magdalena Sznurowska1, Joanna Matyjasz1, Wojciech Pusz2, Barbara Żarowska1

1Biotechnology and food microbiology department, faculty of food science 2department of life protection, faculty of life sciences and technology

wrocław university of environmental and life sciences, wrocław, poland

Moulds belonging to v inaequalis species are the cause of most dangerous diseases of apple trees in the world. They infect leaves, flowers and fruits, what is the cause of large economic losses. Their identification is based mainly on macroscopic and microscopic morphological assessment. More and more work is de-voted to their molecular identification. However, the problem here is a small amount of DNA sequences available for that and other species of the genus venturia. Most information can be found about the ITS region and fragments encoding the small and large subunit of rRNA .

On the basis of these sequences, a new starter – Vi1 was developed. It is used in PCR reaction to-gether with the ITS4 universal primer. The PCR product of 606 bp appears only in the case of strains be-longing to v inaequalis species. Incomplete matching of primer Vi1 to the DNA template is detected in 3 other species of venturia genus. No similar sequences occur in case of the remaining species from men-tioned genus. In addition, the unique restriction profile after the digestion of the PCR product with ecoRI, hinfI and sacII is obtained.

The effectiveness of the method was confirmed by in silico analysis and practically on 9 v inaequ-alis strains from CBS culture collection. Twenty two environmental isolates from venturia genus were also analyzed. Nine of them turned out to belong to the v inaequalis species. In contrast, a standard PCR -RFLP of rDNA using ITS1 and ITS4 primers showed no difference between all tested isolates. The sequ-ence analysis confirmed that as many as 10 out of 24 species within the venturia genus gives the same or similar restriction profile to v inaequalis, precluding their diversification.

This work was supported by reasearch grant POIG.01.03.01-02-080/12, co-financed by the European Union from the European Regional Development Found.

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suitaBle MediuM FoR GluconoBacter oxYdans cultivation

Attila Kántor, Miroslava Kačániová

department of microbiology, tr a hlinku 2, 949 76 nitra, slovakia, faculty of Biotechnology and food sciences, slovak university of agriculture in nitra

The aim of this study was to select the best cultivation medium for growing Gluconobacter oxydans. Glu-conobacter oxydans was isolated from unfiltered tank wine (Welschriesling) with alcohol content 10.86%. Tank wine was aseptically collected into 200 mL plastic bottle and stored in the fridge at 8°C/24h for microbiological analysis. Gluconobacter oxydans grew on Wort agar and MEA+BG after 3–5 days at 28±2°C and after that was identified by MALDI-TOF MS Biotyper (Bruker Daltonics, Germany).

We selected 7 different agars for suitable growing of Gluconobacter oxydans. Acetic acid bacteria grow weakly on solid media, but yeasts grow very well on different agar media also suitable for acetic acid bacteria without added antibiotics. We used Yeast Peptone Dextrose agar (YPDA), Malt Extract agar with bromocresol green (MEA+BG), Acetobacter agar (AA), Tryptic Glucose Yeast agar (TGYA), Wort agar (WA), Dextrose Tryptone agar (DTA) and Glycerol Peptone Yeast extract agar (GPYA) in this study. Glu-conobacter oxydans isolated from white wine was streaked over into the new agar plates and incubated at 28±2°C for 3 days. After incubation we obtained results that show the best growing medium for this bac-terium. This species grew on 6 agar mediums, not grew on YPDA, and weakly grew on TGYA and AA. Good growing was obtained on MEA+BG, DTA and WA. The best cultivation medium for Gluconobacter oxydans growing was GPYA. This bacterium forms small transparent shiny flat colonies on AA and the medium was not clear around the colonies. Gluconobacter oxydans produce small, flat, punctiform, cre-amy-white colonies on TGYA and flat, punctiform, shiny yellow colonies on DTA. Larger shiny colonies grew on MEA+BG and WA. On MEA+BG the colonies were olive-green with dark green center, convex and circular. On WA the colonies were milky-white, punctiform to circular and also convex. Colonies on GPYA forms 0.2 to 0.5 mm large translucent raised colonies with irregular edge.

Key words: Gluconobacter oxydans, cultivation media, morphology

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isolation oF KeRatinolytic BacteRia FRoM poultRy

Wojciech Łaba, Maja Pezała, Barbara Żarowska

department of Biotechnology and food microbiology, wrocław university of environmental and life sciences,


Keratinolytic microorganisms constantly remain a vital subject of research due to their high applicatory value. Potential applications may be associated i.a. with production of keratin hydrolysates or feather me-als as feed components or contribute to environmental protection.

The aim of the study was isolation of keratinolytic microorganisms from skin surface or feathers of various poultry, including chicken, duck, turkey and goose, and further evaluation of their applicatory po-tential.

Swab samples from skin surface and feathers were collected from several birds and inoculated onto PCA, YCG and skim milk agar plates. 55 of the obtained isolates were capable of casein decomposition, while 9 of the bacterial isolates exhibited considerable caseinolytic activity. Selected proteolytic isolates were cultured in synthetic medium with feathers, as a main nutrient source. Keratinolytic and proteolytic activities were measured throughout the culture course and concentration of keratin hydrolysis products (proteins, amino and thiol groups of amino acids) was measured. Two Gram-positive strains were selected as capable of exceptionally effective biodegradation of feathers in liquid culture conditions and selected for further evaluation.

Two keratinolytic bacteria were identified as micrococcus luteus and Kytococcus sedentarius, accord-ing to BBL Crystal system. Using cell-free culture fluid of both bacteria, optimization of reaction condi-tions (pH and temperature) on casein and native feather keratin was performed. Optimization was con-ducted with two-factor surface response experimental design.

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application oF penicillium minioluteum as a Biocatalyst With hydRolytic activity FoR the acidic aMinophosphonic

acid synthesis

Natalia Kmiecik, Ewa Żymańczyk-Duda

department of Bioorganic chemistry, wrocław university of technology, wybrzeże wyspiańskiego 27, 50-370 wrocław, poland

Bioconversion procedure is currently common method of valuable products synthesis. Th e main advan-ioconversion procedure is currently common method of valuable products synthesis. The main advan-tages of whole-cells biocatalyst are diversity of enzymes and ability to adapt to the different environmental conditions. Biotransformations are successfully applied for gaining of the new chemicals and pharmaceu-ticals of defined absolute configuration [1].

Phosphonates compounds with stable carbon to phosphorus bond are known from their variable biological activities and important as industrial targets [2, 3]. It is necessary to stress, that biosynthesis of organophosphorous compounds is still not fully explored field of science.

The aim of work was the selection of the whole-cell biocatalysts with hydrolytic activity for prepa- for prepa-prepa-ration of phosphonate analogues of acidic amino acid. To obtain phosphonic analogue of aspartic acid via biohydrolysis of o,o-dimethyl-4-oxoazetidin-2-ylphosphonate, whole cells of p minioluteum were applied. Recorded NMR spectra allowed confirming the reaction progress and also postulating possible mechanism of microbial conversion, which appeared to be a multistep one. The bioconversion is initiated by sequential ester bond hydrolysis, aft er it is accomplished, amide bond hydrolysis is performed, what fi -sequential ester bond hydrolysis, aft er it is accomplished, amide bond hydrolysis is performed, what fi -ester bond hydrolysis, after it is accomplished, amide bond hydrolysis is performed, what fi-nally resulted in phosphonic analogue of aspartic acid synthesis over time. Such approach resulted in pro-duction of desired product with moderate yield and satisfactory optical purity. Elaborated procedure is promising for further scalling up the process.

References[1] Olmo M., Andreu C., Asensio G., 2011. Journal of Molecular Catalysis B: Enzymatic, 72, 90–94.[2] Żymańczyk-Duda E., 2008. Phosphorus, Sulfur and Silicon, 183, 369–382.[3] Naydenova E.D., Todorov P.T., Troev K.D., 2010. Amino Acids, 38, 23–30, 2010.

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cyanoBacteRia as Biocatalysts in Redox Reactions

Agata Głąb, Ewa Żymańczyk-Duda

department of Bioorganic chemistry, faculty of chemistry, wrocław university of technology wybrzeże wyspiańskiego 27, 50-370 wrocław, poland

Cyanobacteria are phototrophic microorganisms able to adapt and grow in almost every aquatic environ-ment. They produce various organic compounds from inorganic carbon source via their metabolic pa-thways. Some of biosynthesized products are bioactive, as among their metabolites are antibacterial, an-tifungal, antiviral, anticancer and immunosuppressive agents [1]. Cyanobacteria gained recently much attention due to their potential in production of third generation biofuels and because that the possibili-ty of genetic manipulation of their genome is quite easy to perform [2]. Although they are widely used in many fields of biotechnology, their potential as biocatalysts is still not thoroughly investigated. Reports show, that cyanobacteria possess biocatalytic potential, for example synechococcus sp. was found to reduce limonene [3] and enones [4] to corresponding alcohols.

The aim of the research is to evaluate the biocatalytic potential of the cyanobacteria basing on the reduction of acetophenone to the phenylethyl alcohol of defined absolute configuration and on the corre-sponding enantioselective oxidation of (rs)-1-phenylethanol used as a model compounds.

Among investigated cyanobacteria synechococcus sp. turned out to be the most efficient catalysts al-lowed obtaining 85% of ee via enantioselective reduction of the strating ketone. The best catalyst for the oxidation reaction is leptolyngbya foveolarum.

References[1] Abed R.M.M., Dobretsov S., Sudesh K., 2008. Applications of cyanobacteria in biotechnology. J. Appl.

Microbiol., 106, 1–12.[2] Machado I.M.P., Atsumi S., Cyanobacterial biofuel production. 2012, J. Biotechnol., 162, 50[3] Hamada H., Kondo Y., Ishihara K., Nakajima N., Kurihara R., Hirata T., 2003. Stereoselective biotrans-

formation of limonene and limonene oxide by cyanobacterium, Synechococcus sp. PCC 7942. J. Bio-sci. Bioeng., 96, 581.

[4] Shimoda K., Kubota N., Hamada H., Kajib M., Hirata T., 2004. Assymetric reduction of enones eith Synechococcus sp. PCC 7942. Tetrahedron Asymmetr., 15, 1677.

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session 2 enzymes & PePtides Poster 2.1

pRoteolytic hydRolysis oF oat pRotein FRactions enaBles Releasing peptides With angiotensin i-conveRting enZyMe

inhiBiting activity

Małgorzata Darewicz1, Monika Pliszka1, Justyna Borawska1, Anna Iwaniak1, Piotr Minkiewicz1, Gerd Elisabeth Vegarud2

1chair of food Biochemistry, faculty of food science, university of warmia and mazury in olsztyn, pl cieszyński 1, 10-726 olsztyn-Kortowo, poland, darewicz@uwm edu pl

2department of chemistry, Biotechnology and food science, norwegian university of life sciences, 1432 Ås, norway

Biologically active peptides derived from food proteins are considered as regulators of e. g. cardiovascular, immune, nervous or digestive system. The best known bioactive peptides with antihypertensive properties are angiotensin I-converting enzyme [EC 3.4.15.1] inhibitors. They act as crucial agents in lowering high blood pressure and regulating the cardiovascular system activity. Oat proteins may be the source of bio-active peptides. The nutritional value of globulins and prolamins derived from oat seeds is comparable to meat and milk proteins.

The aim of this study was to analyze the ACE inhibiting (ACEi) activity of oat protein hydrolysates as well as identify ACEi peptides obtained via simulation of human digestive tract conditions. The study also covered the in silico analysis involving the use of UniProt and BIOPEP databases, Fragment Ion Cal-culator and Sequence Specific Retention Calculator application. Digestion was carried out in three steps: chewing – 3 min, stomach with a gradual pH reduction from ~6.2 to 2.5 continuing for next – 2 hours, du-odenal – 1 hour (pH = 7). The sample treated with gastric and duodenal juices demonstrated the highest degree of ACE inhibition (84%, IC50=0.44 mg/ml). The proteins are only partially digested with pepsin in the stomach, so the sample digested by gastric juice was characterized by a lower degree of inhibition (74%, IC50=5.25 mg/ml) comparing to the sample treated with gastric and duodenal juices. The degree of ACE inhibition of untreated sample was the lowest (47%, IC50=27.62 mg/ml). Hydrolysed proteins were separated using RP-HPLC-MS method. Amino acid sequences were identified using LC-MS/MS method. The following ACEi sequences were identified in oat proteins hydrolysates: GDAP, LSP, LLP and VAV. To conclude, our studies documented ACEi effects of oat proteins hydrolysates obtained by human gastro-intestinal enzymes.

Work supported from Warmia and Mazury University in Olsztyn funds.

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Food pRoteins as the pRecuRsoRs oF peptides With caRdiopRo-tective and BitteR taste pRopeRties – an in silico pRediction

Anna Iwaniak, Monika Protasiewicz, Małgorzata Darewicz, Piotr Minkiewicz

chair of food Biochemistry, faculty of food science, university of warmia and mazury in olsztyn, pl cieszyński 1, 10-726 olsztyn-Kortowo, poland,

ami@uwm edu pl

Food proteins are the source peptides, which are beneficial for health. The beneficial properties of pepti-des include their cardioprotective effect (e. g. ACE inhibitors, cholesterol level reductants and antithrom-botic peptides).

There are three ways to produce bioactive peptides. The first one includes the hydrolysis of protein by digestive tract system enzymes. The second covers the peptides production by protein proteolysis using the plant-originating or bacterial enzymes. The third way relies on the fermentation processes involving proteolytic starter cultures. Enzymatically released peptides, due to their health beneficiary properties as well as impact on food taste (i. e. the bitterness), can positively or negatively affect the health and sensory quality of food. For example, some of the ACE inhibitory peptides were discovered to show a bitter taste.

Bioinformatic methods (in silico) become more popular among the food scientists dealing with the analysis of food components, including bioactive peptides. in silico analysis can support the studies carried out in laboratory conditions, particulary is found useful in the preliminary selection of the food material (protein) for the most effective prediction of potential bioproducts (i.e. biopeptides).

The aim of the study was to apply the in silico methods such as selected databases and computer pro-grams to show the procedure suitable to find proteins which are the source of peptides with cardioprotec-tive/bitterness activity. We applied the computer simulation of hydrolysis of the food protein sequences to predict the primary structure of released peptides with above-mentioned activities. The procedure applied showed that our results can be applicable for design of foods affecting their health and sensory quality.

Work supported form Warmia and MazuryUniversity in Olsztyn funds.

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in silico analysis as a tool FoR evaluation oF Bioactivity peptides deRived FRoM Food pRoteins

Paulina Worsztynowicz, Włodzimierz Grajek

department of Biotechnology and microbiology, poznań university of life sciences, poland

Bacteria of the genus enterococcus are present in many dairy food. Recent studies indicated that enterococ-cus strains share interesting biotechnological traits such as bioactive peptides and bacteriocin production [Foulquie´ Moreno et al. 2006]. The results of previous study together with literature information allowed us to identify the proteases responsible for the proteolytic activity of e faecalis and bacteria of the genus Lactobacillus, as coccolysin (EC 3.4.24.30) and proteinase P1 (EC 3.4.21.96), respectively.

The aim of this study was to investigate potential of coccolysin and proteinase P1 to generate bioac-tive peptides from bovine whey protein.

in silico analysis of enzymatic hydrolysis was carried out using BIOPEP database. in silico proteolysis of 4 selected whey proteins performed using coccloysin and proteinase P1 showed that both enzymes were able to generate bioactive peptides in one enzyme setup. It was determined that ACE – and DPP-IV – in-hibitor peptide fragments were the most obtained bioactive sequences in analysed hydrolysates.

Furthermore, a computer simulation of gastrointestinal digestion was performed. As a result of this study, treatment of coccolysin and the proteinase P1 hydrolysates with pepsin (EC 3.4.23.1) and trypsin (EC 3.4.21.4) increases the amount of released bioactive peptides.

This study suggests that in silico analysis is a good tool for initial screening of proteins and hydroly-sates for biological activity. The whey protein hydrolysates obtained by coccolysin and proteinase P1 dige-stion could be a source of peptides for applications in functional food production.

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Biosynthesis and identiFication oF pRotease oF enterococcus faecalis isolated FRoM aRtisan polish cheese

Paulina Worsztynowicz, Włodzimierz Grajek

department of Biotechnology and microbiology, poznań university of life sciences, poland

Enterococci belong to a group of lactic acid bacteria (LAB) and occur in many dairy fermented food. Stu-dies on the microbiota of dairy food have indicated that besides a rennin used for protein coagulation in dairy industry, proteinase and peptidase of LAB strains play an important role in milk protein degradation during cheese production [Wilkinson et al.1994].

The objective of this study were to isolate and identify the protease produced by enterococcus faeca-lis, which was isolated from Polish traditional cheese.

A cell envelope proteinase (CEP) and an extracellular – secreted (E) were isolated and enzyme acti-vity depending on bacterial growth phase was determined. The maximum activity of CEP was at the ini-tial stationary phase in approximately 25 h of milk fermentation (17,1 ± 0,42 U) while the highest protease activity of E was detected during the exponential growth phase (14,0 ± 0,20 U). CEP and E fractions were identify by zymography, SDS- PAGE and LC-MS-MS/MS analysis. As a result of this study, the key enzyme of proteolytic system of e faecalis is cocolysin.

This study suggests that e faecalis is also actively producing extracellular – secreted protease at the early stage of the cell growth while most of studies on the proteolytic system of LAB strains revealed only cell envelope proteinase.

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heteRologous expRession oF chicKen ygp40 pRotein

Agnieszka Szmyt, Anna Dąbrowska, Marek Szołtysik, Józefa Chrzanowska

department of animal products technology and quality management, wrocław university of environmental and life sciences,

chełmońskiego 37/41, 51-630 wrocław, poland

Vitellogenins are heterogenous group of phosphoglycoproteins that occure in all oviparous organisms: in-sects, fish, amphibians, reptiles, birds and prototheria (egg laying mammals). There are three types of hen (Gallus gallus) vitellogenins. Vitellogenin II (VtgII) is a precursor of main egg yolk proteins – lipovitellin, phosvitin and 40 kDa glycoprotein YGP40. The protein is produced by the liver under beta-17-estradiol stimulation, released into the bloodstream and internalized by the growing oocyte. Then VtgII has been reported to be proteolytically cleaved by cathepsin-D. Recent data showed that this protein, besides female specific function, plays an important role in host defense reactions, as the primary immune response.

YGP40 is a C-terminal fragment of VtgII. Data obtained by Polanowski et al. [2013], showed that YGP40 is a source of several peptides with immunomodulatory and immunoregulatory activities. These peptides of low molecular weight (1–35 kDa) complex was named yolkin, exhibits immunostimulating properties similar to mammalian colostrinin.

The egg yolk is the natural source of yolkin. The applied method allowed to isolate the peptides com-plex alongside IgY purification. Nevertheless, the purification yield is low and time- consuming, as it ta-kes 5 days to obtain no more than 1,7 mg of yolkin from one egg yolk. For this reasons, in our studies we focused on obtaining the YGP40 protein via escherichia coli BL21 expression system and the hydrolysis of recombinant protein.

ReferencesPolanowski A., Sosnowska A., Zablocka A., Janusz M., Trziszka T., 2013. Imunologically active peptides

that accompany hen egg yolk immunoglobulin Y: separation and identification. Biol. Chem., 394, 879–887.

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yaRRoWia lipolytica lipases and theiR application FoR MilK Fat hydRolysis

Marek Szołtysik, Anna Dąbrowska, Marta Pokora, Aleksandra Zambrowicz, Konrad Babij, Barbara Buda, Agnieszka Szmyt, Józefa Chrzanowska

department of animal products technology and quality management, wrocław university of environmental and life sciences, chelmońskiego str 37/41, 51- 630 wrocław, poland

The aim of this study was to obtain and characterize aroma preparations, for intensification and mimicking cheese flavor, with the use of the non-commercial lipase isolated from yeast Yarrowia lipolytica The cream and butter oil hydrolysis was conducted at temp. 37oC for 72 h. Substrates without enzymes were used as a control.Lipases (extra – and intracellular) were isolated from submerged cultures of yeast cultivated in bioreactor. The yeast were grown at pH 7.5 in complex medium supplemented with casein and the waste products of oil industry. After 48 h the culture was centrifuged and the obtained supernatant was used as the raw extracellular enzyme preparation. The cell pellet was washed three times with Sörensen buffer pH 7.0, centrifuged, suspended in 30 ml of the same buffer, sonicated at 4oC and centrifuged again. The obta-ined supernatant was the intracellular enzyme preparation. The degradation of milk fat was analyzed after 48 and 72 h of hydrolysis by the determination of free fatty acids release by gas chromatography combined with mass spectrometry. In obtained hydrolysates the volatile compounds were analyzed after solid phase microextraction combined by GC-MS.

The extra- and intracellular enzyme preparations isolated form yeast Y lipolytica used in the study showed the ability to degrade milk fat, preferentailly releasing long chained FFA- C14-C18 and unsatura-ted oleic acid C18:1.The enzyme coctail prepared as the mixture of extra- and intracellular lipases of Y li-polytica was more effective in cream fat degradation in comparison to butter oil. The use of enzyme coctail resulted in accumulation of volatile compounds among which butyric and capronic acids were detected in highest concentration.

This work was supported by reasearch grant POIG.01.03.01-02-080/12, co-financed by the Euro- pean Union from the European Regional Development Found.

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lipase oF Yarrowia lipolYtica JMy329

Ewa Szczepańska1, Filip Boratyński2, Zbigniew Lazar1, Magdalena Rakicka1, Ewa Tomaszewska-Ciosk3, Jean-Marc Nicaud4,5, Małgorzata Robak1

1department of Biotechnology and food microbiology, 2department of chemistry,

3department food storage and technology, wrocław university of environmental and life sciences, norwida 25, 50-375 wrocław, poland

4inra, umr1319, micalis, domaine de vilvert, f-78352 Jouy-en-Josas, france 5agroparistech, umr micalis, Jouy-en-Josas, france2, department of Biotechnology, france

Lipases are widely used in: oils and fats processing, formulations of detergents and cosmetics, synthesis of chemicals and pharmaceuticals, paper and leather industry. In the genome of Y lipolytica yeasts 16 genes encoding lipases are present: Lip2p, Lip4p, Lip5p, Lip7p-Lip19p [Fickers et al. 2011]. The most commonly synthesized and secreted to the medium is the isoform Lip2p [Pignède et al. 2000].

The aim of the study was to investigate biosynthesis of Lip2p by Y lipolytica JMY329, Y lipolytica W29and Y lipolytica A-101. The tests included biosynthesis of lipase, purification by ultra-filtration and enzyme immobilization, as well as gene expression level and gene copy number determination. The impact of free and immobilized lipase on the hydrolysis of phenylethyl acetate, bi- and tricyclic lactons and (e)-4-(2,5-dimethylphenyl)-but-3-en-2-ol biotransformation was investigated.

It was determined that Y lipolytica JMY329 has 25 copies of the Lip2 gene. The level of expression of this gene is four times higher than in parent strain Y lipolytica W29. The enzyme production in 2 L biore-actor led to lipase with activity of 23 U/mL and after concentration and separation of small proteins (<10 kDa) of 74 U/mL. Enzyme immobilization was successful but not effective in biotransformation. Only concentrated preparation of lipase gave some positive results. Lip2p of Y lipolytica JMY329 is specific to the (s)-enantiomer of phenylethyl acetate. The (s)-enantiomer of phenylethanol was obtained with yield of 44% and an enantiomeric excess of 98% (in 3 hours of reaction).

It will be important to continue the research on Y lipolytica lipase Lip2 by study leading to optimi-zation of culture, immobilization and biotransformation conditions.

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session 3 Biosynthesis & Biodegradations Poster 3.1

optiMiZation oF cultuRe conditions and sepaRation and puRiFication pRoducts in the pRocess oF siMultaneous

pRoduction oF levan and poly(γ-glutaMic acid)

Marta Domżał, Marcin Łukaszewicz

department Biotechnology, university of wrocław, Joliot-curie 14a, 50-383 wrocław, poland

Levan, a polymer composed of D-fructofuranose units joined by β-(2,6) linkages, can be obtained from both plants and microorganisms. Microbial levan is considered to have a wide range of applications in food, cosmetics, pharmaceuticals, and medicine. There are many investigations into levan formation, but unfortunately they all reported the disadvantages of low yield and impure products. Poly-γ-glutamic acid (γ-PGA) is a naturally-occurring biopolymer composed of units of L-glutamic acid, D-glutamic acid, or both. Due to its biodegradability, non-toxicity, and lack of immunogenicity, γ-PGA is used in the food in-γ-PGA is used in the food in--PGA is used in the food in-dustry, medicine, and environmental protection.

The aim of this research was to optimize the conditions of levan and γ-PGA production by B subti-lis natto in solid-state fermentation using rapeseed meal as the main substrate. Suitable methods of sepa-ration and purification of the products were also investigated to optimise process efficiency and the final product’s purity.

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deteRMination oF the thiaMine concentRation in seed and pRoduction cultuRe MediuM FoR eFFective Biosynthesis

oF pyRuvic acid FRoM glyceRol By Yarrowia lipolYtica yeast

Krzysztof Cybulski, Anita Rywińska



Yarrowia lipolytica is currently one of the most extensively studied ‘‘non-conventional’’ yeasts, capable of producing a wide range of metabolites from cheap carbon source such as glycerol.

The aim of the present study was to determinate the thiamine concentration in seed and production culture medium for effective pyruvic acid production from glycerol as an alternative to expensive chemi-cal synthesis.

The strain used in this study was Y lipolytica SKO6 originating from the collection of Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences. Two different seed cultures medium were tested. First medium was Yeast Nitrogen Base (YNB) without thiami-ne, which was supplemented with thiamine hydrochloride (1–2 µg/L) before its inoculation. Second seed culture medium was complete YNB containing thiamine in concentration of 0.4 µg/L. Seed cultures were performed in Erlenmayers flasks on a rotary-shaker at 30oC and 160 rpm. The production cultures were performed in 5-L stirred-tank bioreactor (Sartorius Stedim, Germany) at working volume of 2 L, at con-stant conditions (pH 4.0, stirrer speed – 800 rmp, temp. – 30oC) and supplemented with thiamine hydro-chloride in the range of 0.5–2.5 µg/L. The sole carbon source was glycerol (50 or 100 g/L).

In presented study yeast Y lipolytica SKO6 produced 11.9–46 g/L of pyruvic acid while the biomass concentration reached 4.0–12.9 g/L. Production of pyruvic acid was the most effective when complete YNB seed culture medium was used and the production medium was not extra supplemented with thia-mine. In those conditions yeast produced 46 g/L of pyruvic acid with co-production of 12 of α-ketoglutaric acid in 79 h. Above mentioned batch culture was characterized by yield of 0.44 g/gand volumetric acid pro-duction rate of 0.65 g/Lh.

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75


pRoduction oF eRythRitol By Yarrowia lipolYtica in cheMostat cultuRe using puRe and cRude glyceRol

Magdalena Rakicka, Waldemar Rymowicz, Anita Rywińska

department of Biotechnology and food microbiology,

wrocław university of environmental and life sciences, wroclaw, poland magdalena rakicka@up wroc pl

The commercial production of erythritol uses glucose as substrate and the yeast-like fungi- moniliella pol-linis in batch or fed-batch cultures. The batch, fed-batch and repeated batch cultures were used for the ery-thritol production by various Yarrowia lipolytica strains on the glycerol media. The continuous production of erythritol from glycerol by Y lipolytica in chemostat culture was not investigated yet.

The aim of this study was to compare kinetic of the parameters of continuous processes and to eva-luate the efficiency of erythritol biosynthesis from pure and crude glycerol by Yarrowia lipolytica Wrati-slavia K1. During 650-h of continuous culture, at a dilution rate 0.01 h−1, in a medium with pure glycerol (200 g/L), nitrogen and phosphorus sources, the concentration of erythritol and biomass as well as the vo-lumetric rate of erythritol production were 109.8 g/L, 20.0 g/L and 1.19 g/L/h, respectively. The conversion of glycerol into erythritol in steady state was 0.55 g/g. The concentration of erythritol and its yield from glycerol slightly decreased, 81.8 g/L and 0.41 g/g, respectively, when crude glycerol was used as carbon so-urce. The acetate-negative mutant Wratislavia K1 is an effective erythritol producer and preserves unchan-ged properties during the long term run of the chemostat process. Erythritol biosynthesis in this process was characterized by high purity, owing to the low concentrations of undesired metabolites, such as orga-nic acids and other polyols.

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76


high-thRoughput pRoteoMe pRoFiling oF l-phe cataBolisM and deRivative aRoMa pRoduction in Yarrowia lipolYtica –

Mapping MoleculaR identities oF the pathWay

Ewelina Celińska, Włodzimierz Grajek

department of Biotechnology and food microbiology, poznań university of life sciences,

wojska polskiego 48, 61-627 poznań, poland

The purpose of this study was to define molecular identities (proteins, and ultimately genes) directly and indirectly involved in catabolism of L-phenylalanine in Yarrowia lipolytica. Our previous research have demonstrated that Y lipolytica is able to produce considerable amounts of a valuable aroma compound, 2-phenylethanol (2-PE) of rose-like-odor (J Ind Microbiol Biotechnol. 2013:40(3-4):389-92); which con-stitute the main product of L-Phe catabolism. Although the general overview of the Ehrlich pathway in yeast and bacteria (leading from L-Phe to 2-PE) is commonly known, the particular genes involved in this pathway have not been earlier identified in Y lipolytica, to date. This in turn disables targeted genetic mo-difications with homologous genes aiming at the trait’s enhancement.

To this end, we adopted high-throughput proteome profiling (LC-MS/MS), followed by extensi-ve bioinformatic analysis. We succeeded in determination of the key proteins putatively involved in the Ehrlich pathway in Y lipolytica. Additionally, an interesting observations regarding hyphae-to-yeast cells transition are presented.

The results are shown in full in: Celińska et al. 2015; FEMS Yeast Res. 2015 Aug;15(5). doi: 10.1093/fem-syr/fov041. Project No 01.01.02-00-074/09.

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77


soRghuM as a RaW MateRial FoR distilleRy

Joanna Chmielewska1, Józef Sowiński2, Barbara Foszczyńska1, Ewelina Szydełko-Rabska2, Joanna Kawa-Rygielska1

1department of food storage and technology, chełmońskiego 37, 51-630 wrocław, 2department of crop production, pl Grunwaldzki 24 a, 50-363 wrocław

wrocław university of environmental and life sciences, poland

Among the crops currently being actively investigated for bioethanol production sweet sorghum is the most promising one. Sweet sorghum is a drought tolerant crop that requires lower inputs costs and grows in marginal lands.

The aim of this study was to evaluate the possibility of comprehensive utilization of sweet sorghum-grown in Poland to produce ethanol. The research material was consisted of variants sucrosorgo304 with different nitrogen fertilization method, in form of biomass from whole plants as well as juice extruded in a press and bagasse. The juice was fermented directly. Biomass from whole plants and bagasse were prepa-red by high-temperature acid (2% H2SO4) or high-temperature alkaline (NaOH) pretreatment and enzy-matic hydrolysis (Accellerase xC and Accellerase 1500). The fermentation process was conducted at 30oC with rehydrated sacccharomyce cerevisiae SIHA Active Yeast 6 (Eaton).

As a result, after fermentation of sorghum juices to 434 dm3 of 100% ethanol per ton of extract was achieved. Acid pretreatment proved to be a better way of treating the raw material in comparison with the alkaline prehydrolysis, which results in higher yields of ethanol. The yield of ethanol from plant biomass ranged to 340 dm3 of 100% ethanol per ton of dry matter, whereas to 297 dm3 of 100% ethanol per ton of dry matter of bagasse.

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78


loW-teMpeRatuRe phosphoRic acid pRetReatMent to enhance Wheat stRaW enZyMatic hydRolysis

Witold Pietrzak1, Patrik Lennartsson2, Mohammad J. Taherzadeh2, Joanna Kawa-Rygielska1

1department of food storage and technology, wrocław university of environmental and life sciences, poland

2swedish centre of resource recovery, university of Borås, sweden

Lignocellulosic residues represent an abundant and cheap raw material for biotechnological applications. However, their recalcitrant structure demands harsh pretreatment to remove lignin, dissolve hemicellulo-ses and degrade crystalline structure of cellulose to improve further hydrolysis and sugar yield. Most pre-treatment processes use high temperatures (above 100°C) and corrosive chemicals (like H2SO4) which cau-ses formation of inhibitors like HMF, furfural and organic acids.

In this research project the possibility of using low-temperature (95°C) pretreatment catalyzed by phosphoric acid (1; 2; 4 g H3PO4 100 g-1 straw) for 2; 4; 6; 8 and 10 h was conducted for wheat straw at 10% w/w solids prior to enzymatic hydrolysis (10 FPU g-1 solids) at 8% w/w solids. Pretreatments with H2SO4 in identical conditions, using high temperature (121°C) and hydrolysis of untreated straw were conducted for comparison as well. The resulting sugars and inhibitors yield were determined by HPLC. It was stated that pretreatment with phosphoric acid resulted similar glucose and xylose yield in comparison to high temperature pretreatment. Sulfuric acid pretreatment at low temperatures yielded the most sugars when long process times were applied (>6 h). Very low concentrations of inhibitors were found for phosphoric acid pretreatment (below 0.02 g L-1 furfural and 0.1 g L-1 HMF).

The obtained results shown that low-temperature pretreatment phosphoric could be a vital alterna-tive to high-temperature and pressure processes for lignocelluloses conversion.

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79


ethanol pRoduction FRoM lignocellulosic RaW MateRials

Joanna Chmielewska1, Joanna Kawa-Rygielska1, Ewelina Dziuba1, Józef Sowiński2

1department of food storage and technology, chełmońskiego 37, 51-630 wrocław 2department of crop production, pl Grunwaldzki 24 a, 50-363 wrocław

wrocław university of environmental and life sciences, poland

The objectives of energy policy for Poland assume that by 2020 total yield of energy from renewable energy sources should reach 15% in relation to the total energy production in the country. Lignocellulosic mate-rials are one of the major renewable resources for bioethanol production.

In this study, looking for alternative raw materials and effective methods of substrates pretreatment to enhance the efficiency of bioethanol production, selected fractions of quinoa (whole plants, small stems, shoots and leaves and seeds), selected types of straw (cereal, rape, corn and sorghum) and plants such as miscanthus, willow and spartina were used. All raw materials were prepared by microwave irradiation, high-temperature acid (170oC, 1.5h, 2% H2SO4) or high-temperature alkaline (170oC, 1.5h, 2% NaOH) pre-treatment and enzymatic hydrolysis (Accellerase xC and Accellerase 1500, 50oC,48h). The fermentation process was conducted at 30oC with rehydrated sacccharomyce cerevisiae SIHA Active Yeast 6 (Eaton).

The combined process of lignocellulose degradation involving microwave irradiation, acid- or alka-line pretreatment followed by enzymatic hydrolysis resulted in the productionof fermentable sugarsava-ilable toyeast.In addition,a significant effect of the kind of raw material as well as the type of pretreatment on final ethanol yield was showed.

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80


the inFluence oF Mead WoRt pRepaRation on yeast FeRMentation activity

Sławomir Czabaj, Joanna Kawa-Rygielska, Jarosław Kliks, Ewelina Dziuba

department of food storage and technology, faculty of food science, wrocław university of environmental and life sciences, wrocław 51-630, poland

slawomir czabaj@wnoz up wroc pl

The aim of this study was to determine the influence of mead wort preparation techniques on dynamics and final ethanol fermentation products. Triple mead worts (1:2 (honey:water)) were prepared from di-luted with spring water honeydew honey. The total wort extract was set to 36°Blg. Honey worts were heat treated under different thermal processing conditions such as: traditional gently boiling for 30 minutes, 10 minutes pasteurization under 65°C and without thermal treatment (control).

The fermentation was conducted under laboratory conditions with use of two industrial distillers yeast strains (saccharomyces cerevisiae): Safspirit Malt and Safspirit Fruit from Fermentis company. All worts were supplemented with diammonium hydrogen phosphate (0,4 g/l). The fermentation progressed at controlled temperature (18°C) until two subsequent weights of samples did not differ by more than 0,5 g and CO2 production dynamics were reported.

The highest yields of CO2 where noticed between 4th and 5th day of fermentation. Parameters such as: carbohydrate profiles, ethanol, glycerol and organic acids (lactic and acetic acid) content was determined by High Performance Liquid Chromatography (HPLC) analysis.

As a result of conducted research a six variants of meads where produced with the content of etha-nol ranging from 13 to 16% (v/v). Significant influence of used yeast strain was observed. In every mead variant fermented with the use of Safspirit Fruit yeast strain achieved best fermentation dynamics and final product quality.

The best fermentation dynamics and highest ethanol concentration was obtained in pasteurized wort fermented with Safspirit Fruit yeast strain.

Key words: mead, honey, thermal treatment, strain selection

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81


ethanol FeRMentation oF distilleRy Mashes oBtained FRoM Malted and unMalted ceReals

Katarzyna Pielech-Przybylska, Maria Balcerek, Urszula Dziekońska, Piotr Patelski

institute of fermentation technology and microbiology, faculty of Biotechnology and food sciences, Łódź university of technology, wólczańska 171/173, 90-924 Łódź, poland

The aim of the study was to evaluate the fermentation efficiency of distillery mashes prepared from the mixture of unmalted barley and malted wheat or barley, using the endogenous enzymes of the malts, as well as volatile compound concentration in distillates obtained.

The unmalted barley (DANKO, Poland), and malts – Oak Smoked Wheat Malt, Distillery Wheat Malt and Munich Malt from barley (Weyermann®, Germany) were used as the raw materials. Milled unmal-ted barley cereals were mixed with milled malts used in dosage of 20% and 50% to get final weight of 1 kg and then mixed with water (3.5 L). Starch liquefaction and saccharification conditions were as follows: pH 5.3–5.5; mashing time, 60 minutes; temperature, 53–56°C. The fermentations were carried out using Etha-nol Red yeast (Lesaffre, France), at 35°C by 72 hours. After the completion of fermentation, ethanol was di-stilled from the mashes. Obtained spirits were quantitatively analyzed for volatile compounds (GC-MS).

The mashes prepared from wheat and barley malts in dosage of 20% and 50% were characterised by ethanol yield 70–76% of the theoretical yield. Increasing dose of malts from 20% to 50% resulted in higher (by 32–92%) aldehydes concentration in distillates obtained. A simillar dependence was observed for es-ters concentration, but only for trials with using Distillery Wheat Malt. Moreover the concentration of the acetaldehyde diethyl acetal in distillates obtained from the mashes with a higher dose of malt (50%) was 2.5 fold higher (barley malt and wheat malt) and 1.5 fold higher (smoked wheat malt).

AcknowledgmentsThe research was financed by a grant from NCBiR (Polish National Centre for Research and Development) implemented within the framework of the Programme of Applied Research – PBS2/B8/9/2013 „Modern technologies of spirits production taking into account their accelerated maturation and improving of the physico-chemical stability.“

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82


eFFect oF aeRation and nitRogen suppleMentation on the FeRMentation eFFiciency oF sugaR Beet pulp hydRolysates

Katarzyna Pielech-Przybylska1, Joanna Berłowska1, Maria Balcerek1, Urszula Dziekońska1, Piotr Patelski1, Marta Dutkiewicz1, Halina Kalinowska2

1institute of fermentation technology and microbiology, 2institute of technical Biochemistry, faculty of Biotechnology and food sciences,

Łódź university of technology, wólczańska 171/173, 90-924 Łódź, poland

The sugar beet pulp is a waste product of the sugar industry, which could be an attractive raw material for ethanol and yeast production.

The aim of the study was to examine the fermentation efficiency of sugar beet pulp hydrolysates. Fermentation trials were carried out by using Kluyveromyces marxianus (NCYC, United Kingdom) and saccharomyces cerevisiae (Ethanol Red, Lesaffre, France) yeast strains in dosage of 2 g d.m./L. Worts were supplemented with (NH4)2HPO4 in dosage of 0,3 g/L, molasses (in dosage of 5 and 10% of total sugars) and Activit preparation (mix of amino acids, vitamins and minerals; in dosage of 0,3 g/L). 24 and 48 ho-urs aeration of hydrolysates during fermentation process (by using K marxianus) was also applied in some trials.

The results showed that fermentation dynamics was affected by molasses addition. Worts supple-mented with molasses fermented dynamically with higher (by 10 and 14%), ethanol yield in comparison with trials supplemented with (NH4)2HPO4 and Activit preparation respectively. The complexity and varia-bility of the molasses dry matter components affects positively the growth and propagation of yeast.

The aeration of sugar beet pulp hydrolysates by 24 hours influenced positively on the fermentation dynamics and ethanol yield with using K marxianus yeast, in comparison with trials without aeration. Higher oxygen concentration in the medium caused an increase in the efficiency of fermentation (calcula-ted to the total sugars before fermentation) by 28.5% in comparison with the appropriate control samples without aeration.

AcknowledgmentsThe research was financed by a grant from NCBiR implemented within the framework of the Programme of Applied Research – PBS1/B8/3/2012 “Biomass sugar beet pulp as a new raw material for the production of fermentation substrates”.

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83


enZyMatic pRepaRations in the pRocess oF oBtaining WoRts FoR Food-type Malt concentRates pRoduction

Józef Błażewicz, Joanna Kawa-Rygielska, Witold Pietrzak

department of food storage and technology, wrocław university of environmental and life sciences, poland

Enzymatic preparations are necessary in the process of worts production when the substitution of malt with unmalted adjunct exceeds 20%.

The aim of the study was the indication of the effect of the enzymatic preparations use in the obta-ining of worts with 10, 20, 30 and 40% share of unmalted barley grain on the content of extractive substan-ces in worts for malt concentrates production.

Research materials were: pilsner type malt, unmalted barley grain, enzymatic preparation Termamyl SC (1 ml kg-1 raw material dry matter) containing thermostable α-amylase and Ceremix 6x MG prepara-tion (2 g kg-1 raw material dry matter) containing α-amylase, protease and enzymes degrading non-starch polysaccharides. The worts were obtained using congress mashing method.

There were determined: filtration time and final wort volume, extract content, extractivity of malt-adjunct mixture, total nitrogen and FAN content.

The use of Termamyl SC preparation on the stage of unmalted adjunct gelatinization did not brought the expected results. Increasing the share of adjunct, without Ceremix 6x MG addition, caused reduction of all quality parameters of worts. The use of Ceremix 6x MG preparation allowed to obtain the extractivi-ty of raw material mixture of 2–3% lower than for pilsner malt and kept good filtration time (max. 75 min), final volume (>300 mL) and extract content (>8.8% w/w). The content of total nitrogen and FAN (min. 135 mg L-1) was lower than in all-malt worts.

The use of Termamyl SC and Ceremix 6x MG enzymatic preparations allowed to obtain worts su-itable for production of food-type malt concentrates with 10–40% share of unmalted adjunct in the form of barley grain.

Key words: enzymatic preparations, malt, wort, malt concentrate

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84


ethanol FeRMentation oF sugaR Beet pulp hydRolysates By saccharomYces cerevisiae and pichia stipitis Yeast strains

Maria Balcerek1, Joanna Berłowska1, Katarzyna Pielech-Przybylska1, Piotr Patelski, Urszula Dziekońska, Marta Dutkiewicz, Halina Kalinowska2

1institute of fermentation technology and microbiology, 2institute of technical Biochemistry, Łódź university of technology, 90-924 Łódź, wólczańska 171/173, poland

Sugar beet pulp which consists mainly of polysaccharides such as cellulose and hemicellulose is a promi-sing raw material for fuel ethanol production. The main components of these polysaccharides are fermen-table sugars – glucose, galactose, xylose and arabinose. The yeast saccharomyces cerevisiae are the mostfre-quently used miocroorganisms for ethanol biosynthesis. Unfortunatelly their inability to ferment pentose sugars limits their use in lignocellulosic ethanol production.

The aim of this study was to evaluate the efficiency of bioethanol production from sugar beet pulp hydrolysates and the effect of the type of yeast used on ethanol yield.

Sugar beet pulp was obtained from the sugar factory in Dobrzelin (Poland). Hydrolysis was carried out by using enzymatic preparations such as Viscozyme and Ultraflo Max (Novozymes, Denmark). Worts were supplemented with (NH4)2HPO4 and then fermented by Ethanol-Red (Fermentis–Lesaffre, France), (2 g d.m./L) and pichia stipitis (LOCK 105, Łódź), (1 g d.m./L) yeast strains, sequentially or simultane-ously. Process was conducted at 28–30°C.

The concentration of sugars determined in the hydrolyzate from the fresh pulp was as follows: glu-cose –12.4 g/L, galactose – 16.3 g/L, fructose – 2.7 g/L, mannose – 0.34 g/L, xylose – 2.1 g/L and arabinose – 15.3 g/L. The sequential application of p stipitis yeast to the worts previously fermented with s cerevisiae yeast resulted in an increase in ethanol concentration from 9.5 g/L to 12.2 g/L and fermentation efficiency from ca. 53 to 80% (yield calculated relatively to the utilized sugar). Also, the use of a mixed culture of the above mentioned yeast gave similar concentration of ethanolin comparison to the process that was carried out sequentially. The utilisation of hexose sugars varied between 85 and 90%, while pentose sugars were utilized in ca. 65%.

AcknowledgmentsThe research was financed by a grant from NCBiR implemented within the framework of the Programme of Applied Research – PBS1/B8/3/2012 “Biomass sugar beet pulp as a new raw material for the production of fermentation substrates”.

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85


eFFect oF yeast on the volatile pRoFile oF 'WęgieRKa ZWyKła' pluM distillates

Maria Balcerek, Katarzyna Pielech-Przybylska, Piotr Patelski, Urszula Dziekońska, Ewelina Strąk

department of spirit and Yeast technology, institute of fermentation technology and microbiology, Łódź university of technology, 90-924 Łódź, wólczańska 171/173, poland

The aim of this study was to evaluate the effect of yeast and a fermentation temperature on the chemical composition of plum distillates.The raw materials were plums var. Węgierka Zwykła purchased from Po-lish fruit processing factories. Fermentations were carried out: (1) spontaneously, (2) by addition of raisins (1.5 g/L) treating them as the source of microorganisms, (3) by using dry wine yeast saccharomyces bay-anus (Fermentis – Lesaffre, France), (0.3 g/L). Fermentations were conducted at 18°C and at 30°C. Chro-matographic analysis of volatile compounds was carried out using a GC apparatus (Agilent 7890A, USA) with mass spectrometer (Agilent MSD 5975C).

A chemical composition of the distillates was diversified and dependent on the yeast and a tempe-rature of process. The fermentation of plum mashes with the addition of raisins at 18°C resulted in the hi-ghest concentration of ethyl acetate and isoamyl acetate in the distillates in comparison with those obta-ined after fermentation with s bayanus yeast or spontaneously. On the other hand, the distillates obtained from spontaneously fermented fruits were characterized by a higher content of ethyl benzoate and ethyl caprylate. All of the plum spirits were rich in higher alcohols irrespectively of fermentation variant.

Plum distillates also contain methanol which is undesirable compound. According to Regulation (EC) no 110/2008, the concentration of this compound in plum brandies should not exceed 12 g/L alcohol 100% v/v. Its amounts in the tested spirits were higher which indicates the need to carry out a corrective distillation.

The results of sensory evaluation showed, that the best rated were distillates obtained after the fer-mentation carried out at 18°C, especially from mashes fermented with the addition of raisins.

AcknowledgmentsThe research was financed by a grant from NCBiR (Polish National Centre for Research and Development) implemented within the framework of the Programme of Applied Research – PBS2/B8/9/2013 “Modern technologies of spirits production taking into account their accelerated maturation and improving”.

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86


tuBulaR BioReactoR FoR the anaeRoBic digestion oF Food industRy liquid Waste

Sławomir Jabłoński1, Maciej Sygit2, Tadeusz Beutel1, Marcin Łukaszewicz1

1faculty of Biotechnology, university of wrocław, Joliot-curie 14a, 50-383 wrocław, poland

2sYGma sp z o o , ruska 61, 50-079 wrocław, poland

Anaerobic digestion in industrial applications typically occurs in reactors with a low wall surface-to-vo-lume ratio (such as CSTR or USAB). The concept of a tubular bioreactor was developed. This reactor re-sembles the intestinal tracks of animals, which are the natural environment in which anaerobic digestion occurs.

While the conditions differ between individual parts of the bioreactor, it is possible to promote par-ticular stages of anaerobic digestion. The result of such process organization is the different types of biogas collected from different parts of the bioreactor. For instance, the methane concentration in the first part of reactor is low (below 40% by volume),but biogas from the terminal part contains more than 70% methane. This situation facilitates the purification of methane before its storage or utilization.

The construction of a tubular reactor also enables the development of unique populations of mi-croorganisms specialized for certain types of biochemical reactions. The information obtained from the analysis of microorganism populations from different reactor compartments may be useful for developing high-rate digestion systems dedicated for the utilization of different types of wastes.

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87


MicRoBial degRadation oF dRugs With a caRBaZole Ring in theiR stRuctuRe

Katarzyna Zawadzka, Aleksandra Felczak, Katarzyna Lisowska

department of industrial microbiology and Biotechnology, faculty of Biology and environmental protection, university of Łódź, Łódź, poland

Carbazole is an N-heteryclic compound commonly used in the production of dyes, insecticides and drug. Carvedilol and ondansetron have a carbazole ring in their structure. Carvedilolis an on selective ß-blockerand α1-blockerused in the treatment of mild to severe congestive heart failure(CHF) andhigh blood pressure. Ondansetron is aserotonin 5-HT3receptor antagonistused to preventnauseaandvomiting-caused bycancer chemotherapy, radiation therapy, and surgery.

The aim of this study was to assess the ability to degrade carvedilol and ondansetron by carbazole-eliminating microscopic filamentous fungi.

qualitative and quantitative analysis of carvedilol and ondansetron elimination by ten strains of fi-lamentous fungi was studied using liquid chromatography and mass spectrometry (qTRAP 4500 LC/MS/MS System – AB Sciex).

Among tested microorganisms three filamentous fungi effectively eliminate of carvedilol from their growth environment, while a complete removal of ondansetron was observed in the cultures of two strains.

This study was supported by the National Centre for Science, Poland (project no. DEC-2013/09/N/NZ9/01668).

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88


inFluence oF spent hop extRacts on stRess Resistance and the ageing pRocess in caenorhaBditis eleGans

Anna K. Żołnierczyk1, Begoña Ayuda-Durán2, Susana González-Manzano2, Ana M. González-Parámas2, Mirosław Anioł1, Celestino Santos-Buelga2

1department of chemistry, The faculty of food science, wrocław university of environmental and life sciences,

50-375 wrocław, c K norwida 25, poland, agrot2@gmail com 2Grupo de investigación en polifenoles (Gip-usal), unidad de nutrición y Bromatología,

facultad de farmacia, universidad de salamanca, salamanca, spain

Spent hops, which are a by-product of the process of hop extraction in the beer brewing industry, are a very rich source of many flavonoids (namely chalcones and flavanones). Among them xanthohumol, a dietary prenylated flavonoid chalcone is the most important one. This compound has been indicated to possess health-promoting properties as an anti-oxidant, a cancer chemopreventive agent or an anti-micro-bial agent. In this study, acetone and methanol spent hop extracts have been prepared containing 32 and 21% of xanthohumol, respectively. The in vitro antioxidant activity (ABTS, FRAP) of the extracts has been investigated, as well as their effects on resistance against oxidative stress using c elegans as a model orga-nism. Studies were carried out using the wild type N2 strain of c elegans grown on NGM plates containing the studied extracts (50, 100, 200 µg/mL). It was found that spent hop extracts increased the survival rate in c elegans subjected to thermally-induced oxidative stress from 5 to 15%, compared to untreated worms. Furthermore, the methanol extracts reduced the levels of oxidation biomarkers, such as ROS levels (decre-ase of 10 to 25% for the different extract concentrations assayed) and protein-carbonyl levels (decrease of 5% only at the highest concentration), whereas acetone extracts did not show any significant effect on the-se markers. The obtained resulted supported the bioactive potential, especially to protect against oxidative stress, of compounds from humulus lupulus L., which can be extracted from waste material (spent hops).

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89


pRopagation in vitro and RegeneRation oF apples

Mateusz Kropiwnicki, Magdalena Wróbel-Kwiatkowska, Waldemar Rymowicz


chełmońskiego 37/41, 51-630 wrocław, poland

Apples (malus domestica Borkh.) are one of the most important fruit crops in the temperate climate and major fruit crop in Poland. Propagation of apples under in vitro conditions gives the possibility of pro-duction healthy plants with desired traits and quickly multiplication of them. On the other hand establi-shment of in vitro tissue cultures of plants allows to determine and optimize the transformation procedure of those plants.

Thus Polish cultivar of apple (malus domestica cultivar alva) was used for initiation of sterile tissue cultures.

Obtained plant explants of apples (cotyledons, hypocotyls and leaves) were used for regeneration process included callus and shoot induction stage. Conducted experiments showed that calli and shoots were successfully generated. This method can be applied for genetic engineering of apple in future.

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90


inFluence oF spRouting and elicitation on antioxidant activity oF Wheat seedligs

urszula Gawlik-Dziki1, Dariusz Dziki2, Michał Świeca1

1department of Biochemistry and food chemistry, university of life sciences, skromna 8, 20-704 lublin, poland

2Thermal engineering department, university of life sciences, doświadczalna 44, 20-280 lublin, poland

Three Polish winter wheat cultivars (triticum aestivum, ssp. vulgare): Bogatka, Mulan and Muszelka were studied. Seeds were placed in distilled water (control) and in distilled water contained 0.1% (w/v) of sac-cacharomyces cerevisiae (SC) extract, 0.1% (v/v) salix daphnoides (SD) bark extract and their combina-tions (1:1, SCD) for 6 h at 25°C. Seeds were germinated (20°C in darkness ) for 4 days in a controlled in-cubator. Powdered sprouted seeds were extracted witch MeOH: water (1:1, v/v, pH = 1) mixture (chemical extract, CE). Total phenolic content (TPC), antiradical activity (AA), reducing power (RP), chelating abi-lity (CHEL), reducing power (RED) and ability to prevent lipids against oxidation (LPO) were estimated. Both sprouting and elicitation significantly increased TPC (compared to dormant seeds, regardless of va-riety). Most effective elicitor seem to be SCD. Sprouting increased AA of compounds Bogatka and Mulan phytochemicals. Elicitation with all tested elicitors increased AA of Bogatka seedlings, whereas in the case of Muszelka and Mulan such impact was observed after treatment with SD and SCD. Sprouting signifi-cantly increased RP of seeds in respect of wheat variety. Significant increase of RP was observed after tre-atment Muszelka seeds with SCD and Mulan seeds with SD and SCD. Sprouting not influenced on CHEL activity of CE compounds from all wheat varieties. Most effective elicitor was SD (in respect of wheat va-riety). Influence of sprouting on LPO activity is not clear; in the case of Mulan the increase was observed whereas in the case of Bogatka activity decreased. Elicitation significantly increased this activity in respect of wheat variety, however the most effective elicitors seem to be SC and SCD. Both germination and elici-tation are simple techniques that may be used to improve the nutraceutical potential of wheat flour.

The study was financed by the Polish National Science Centre (Grant 2012/07/B/NZ9/02463).

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the MetaBoloMic and BiocheMical changes in young BucKWheat spRouts aFFected By elicitation and elicitation

suppoRted By the phenylpRopanoid pathWay Feeding

Michał Świeca, Barbara Baraniak, Urszula Gawlik-Dziki, Dariusz Kowalczyk

department of Biochemistry and food chemistry, university of life sciences, skromna 8, 20-704 lublin, poland

A metabolomic and biochemical analysis (including low-molecular antioxidants contents and activities of enzymes involved in phenolic synthesis and metabolism) was performed in the buckwheat sprouts. For improving the quality of the studied sprouts elicitation with salix bark infusion and elicitation supported by the phenylpropanoid pathway feeding were used.

Shikimic acid feeding (both elicited and non-elicited) was most effective in improving the to-tal phenolics (by about 10 and 20%, respectively), condensed tannins (by about 30 and 28%, respec-tively), and flavonoids (by about 46 and 70%, respectively). Significant increases of vitexin, rutin, chlorogenic acid and isoorientin contents were also observed. The treatments increased the ascorbic acid content too. The accumulation of these compounds was caused by the stimulation of two main enzymes of the phenylpropanoid pathway (tyrosine ammonia-lyase and phenylalanine ammonia-ly-ase). Tyrosine ammonia-lyase activities were effectively induced by feeding with tyrosine (about four times that of the control), whereas phenylalanine ammonia-lyase activity was the highest in elicited control sprouts and those fed with shikimic acid (an increase by 60% compared to the control). The used biotech treatments significantly increased the antioxidant activity of the sprouts. Total antioxi-dant capacity of sprouts was correlated with phenolic content and was found to be the most effective-ly increased by feeding with shikimic acid and further elicitation. The used biotech treatments signi-ficantly improved a pro-health quality of sprouts without any undesirable change in their metabolism e.g. decreasing a redox potential, an increase the activities of polyphenol oxidase and peroxidase.

In the light of presented results it may be concluded that elicitation and elicitation supported by the phenylpropanoid pathway are metabolic engineering tools effectively improving the functional quality of the sprouts.

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session 4 Biotransformations Poster 4.1

MicRoBial tRansFoRMation as an eFFicient tool FoR oBtaining phase i and phase ii MetaBolites

oF Bioactive coMpounds

Tomasz Tronina, Agnieszka Bartmańska, Jarosław Popłoński, Sandra Sordon, Ewa Huszcza

department of chemistry, wrocław university of environmental and life sciences, norwida 25, 50-375 wrocław, poland,

tomasz tronina@up wroc pl

xanthohumol-the major hopprenylated flavonoid found in resin, exhibits potent biological activities (such as: anticancer,antioxidant, anti-inflammatory, antibacterial, antiviral, and antifungal), was used as a sub-strate for obtaining its aurone. Due to desired biological properties, xanthohumol and its derivatives are considered potential candidates for the treatment of cancer and other ailments. Hence, it is crucial to in-vestigate their metabolism. Numerous fungi, yeasts and bacteria can transform xenobiotic compoundsin a manner similar to that of mammalian systems and therefore can be used to mimicmammalian drug me-tabolism and as a potentially useful tool for drugdesign.

Fifty six microorganisms (fungi and bacteria) were screened for (z)-6,4’-dihydroxy-4-methoxy-7- -prenyloaurone transformation activity. Five metabolites were obtained as a result of biotransformation. Their structures were established on the basis of their spectral data. The compounds have not been pre-viously reported in the literature. Phase I metabolites: (z)-2”-(2”’-hydroxyisopropyl)-dihydrofurano-[4”,5”:6,7]-4’-hydroxy-4-methoxyaurone and (z)-2”,2”-dimethyl-(2h)-pyrono[5”,6”:6,7]-4’-hydroxy-4-methoxyaurone were produced by fusariumoxysporum AM 13 and melogrammacamphylosporum AM 563 respectively. Microbial regioselective glycosylation andsulfation at the C-6 position led tophase II metabo-lites: two glucosides (6-o-β-D-glucopyranoside and 6-o-β-D-(4”’-o-methyl)-glucopyranoside) and one sulfate.

Obtained results demonstrate, that microorganisms can be used as an efficient tool in the study of drug metabolism andan alternative to synthetic chemistry for obtaining considerable amounts of a number of drugs.

This work was partially supported by Wroclaw Centre of Biotechnology, programme The Leading National Research Centre (KNOW) for years 2014–2018.

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MicRoBial tRansFoRMations leading to Biologically active and useFul steRoid coMpounds: the use oF Fungal geneRa

asperGillus and circinella

Anna Panek, Mariusz Kozakiewicz, Alina Świzdor, Natalia Milecka-Tronina


The presented investigations concern effective transformation of steroid substrates in the cultures of fun-gi belonging to the genera aspergillus and circinella, leading to the products of proven biological activity higher than the starting materials, as well as the products which, due to their structural analogy to the ac-tive compounds, may exhibit such an activity.

Our research focused on biotransformation of dehydroepiandrosterone, androsterone, epiandros-terone and androstenediol. The resulting products were formed through, among other pathways, hydroxy-lation (7α- and 7β-hydroxy derivatives), oxidation of the hydroxyl group (7-oxo-derivative) and lactoniza-α- and 7β-hydroxy derivatives), oxidation of the hydroxyl group (7-oxo-derivative) and lactoniza-- and 7β-hydroxy derivatives), oxidation of the hydroxyl group (7-oxo-derivative) and lactoniza-β-hydroxy derivatives), oxidation of the hydroxyl group (7-oxo-derivative) and lactoniza--hydroxy derivatives), oxidation of the hydroxyl group (7-oxo-derivative) and lactoniza-tion leading to the δ-lactone in the D-ring of the steroid molecule.

The obtained compounds possess (or may possess) neuroprotective, anti-inflammatory, immuno-stimulatory, anti-glucocorticoid, thermogenic and antitumor effects. The presented data include descrip-tion of the transformation conditions, intermediate products, as well as procedures of isolation and puri-fication.

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MicRoBial tRansFoRMation oF soMe B-noRsteRoids By Beauveria Bassiana

Alina Świzdor, Natalia Milecka-Tronina, Anna Panek, Paula Maj, Ewa Romaniuk

department of chemistry, wrocław university of environmental and life science, norwida 25, 50-375 wrocław, poland

Previous studies carried out by our group had demonstratedthat Beauveria bassianaKCH 1065 is a unique species of the Beauveria genusin its metabolism of steroids that results in Baeyer–Villiger (BV) oxida-tions of the carbonyl group to the corresponding 11α-hydroxylactones.The metabolic pathway present in thisstrain consists of two major steps that occur in a precise sequence:11α-hydroxylation of the substrate and then its subsequent BV oxidation. Only the 11α-hydroxy derivatives of 3-oxo-4-ene, 3β-hydroxy-5-ene as well as 3β-hydroxy- and 3-oxo-5α-saturated 17-ketosteroids underwent effective oxidative lac-tonization. In this way, the oxidation of DHEAled to 3β,11α-dihydroxy-17a-oxa-D-homo-androst-5-en-17-one (64%). Furthermore, the study using a series of 5α-saturated steroids has highlighted that a small change of the steroid structure can result in significant differences of the metabolic fate.

Due to the important bioactivity of steroidal lactonesas well as DHEA, we are investigating within the current study the transformation of B-nor-DHEA and its acetate.

Although the first stage of the transformation of steroid ester was a hydrolysis of the ester bond and the released alcohol underwent further conversion, we observed the difference in the metabolism of both substrates. It was found that only the ester was oxidized to lactone (28%) but not to 11α-hydroxylactone. B-nor-DHEA was converted into a mixture of 11α- and 15α-hydroxy derivatives. Some of these metaboli-tes were also reduced to 17β-alcohols and converted into 3-oxo-4-ene steroids. Remarkably, in this fungus a metabolism involving a 3β-HSD/isomerase enzyme(s) was not observed for “normal” 5-ene steroids.

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cheMoenZyMatic synthesis oF hydRoxylactone With tRiMethylcyclohexene systeM and theiR Biological activity

Małgorzata Grabarczyk1, Katarzyna Wińska1, Wanda Mączka1, Barbara Żarowska2, Mirosław Anioł1

1department of chemistry, wrocław university of environmental and life sciences, norwida 25, 50-375 wrocław, poland


Ionones are an important group of natural compounds. Structural analogues of ionone are responsible for the biological activity like: cytotoxic, ichthyotoxic, allelopathic, anti-ischemic, anti-inflammatory.

The commercially available racemic (±) α-ionone was used as a substrate for the four-step chemi-α-ionone was used as a substrate for the four-step chemi--ionone was used as a substrate for the four-step chemi- was used as a substrate for the four-step chemi-cal synthesis of three new γ-halolactones. These halolactones were converted into the hydroxylactone by microorganisms like (usarium species, syncephalastrum racemosum, Botrytis cinerea. Most of them trans-formed these lactones by hydrolytic dehalogenation into a hydroxylactone, mainly the (+) stereoisomer. It was found that hydroxylactone exhibits growth inhibition against some tested microorganisms. All halo-lactones inhibited growth of only some bacteria strains. An olfactory analysis of all the new compounds has proven that most of them were characterized by pleasant smell.

This project was financed by European Union from the European Regional Development Fund. Grant No. POIG. 01.03.01-00-158/09.

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BiotRansFoRMation oF halolactones With geM-diMethylcyclohexane systeM




Hydroxylactones are often met in nature mainly in plants and sea organisms like alga or sponges. They are known from their different properties like: antimalarial, antifungal, cytotoxic, antibacterial, anticancerous. These compounds can be obtained using chemical synthesis or biotransformation.

Three lactones with the gem-dimethylcyclohexane ring: chloro-,bromo- and iodolactone were used as substrates for the screening biotransformation by whole cells of nine fungal strains (fusarium species, syncephalastrum racemosum and cunninghamella japonica). Some of these microorganisms (mainly fu-sarium species) transformed all three lactones during the hydrolytic dehalogenation into hydroxylactone. Two microorganisms (fusarium culmorum and fusarium scirpi) converted iodolactone with very high enantioselectivity (75.1% and 91.6%, respectively). The (+) isomer of hydroxylactone was preferred in all cases. At the last step the hydroxylactone obtained during biotransformation was examined for its biolo-gical activity against bacteria, yeasts and fungi. It was found that this compound inhibits growth of some tested microorganisms.

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synthesis, BiotRansFoRMation and Biological activity oF Bicyclic lactones

Jakub Pannek, Filip Boratyński, Teresa Olejniczak

departament of chemistry, wrocław university of enviromental and life sciences, norwida 25, 50-375 wrocław, poland

Phthalides are bicyclic lactones produced by plants of the family Apiaceae Lindl.. Some of them are de-scribed in the literature [1]. It has been proved that (+)- and (–)- isomers may have different biological ac-tivity, aroma and taste. In this purpose we plan to synthesis both enantiomers of natural lactones and their derivatives.

Optically active compounds can be obtained by asymmetric chemical synthesis, as well as by quickly developing or more environmentally friendly biotechnological methods.

Herein, we present six analogues of natural trans-4-butyl-cis-3-oxabicyklo[4.3.0]nonan-2-one, which were synthesized as racemic mixtures in Grignard reaction in the presence of cadmium chloride. Enantiomers were obtained by microbial transformation of ketoacids or ketoesters.

O OXOCH3

CH3O O

Hmicrobiological reduction

X=H or -Me

Racemic mixtures and both enantiomers were tested as fungistatic agents against candida albi-cans, the major cause of human fungal disease worldwide [2,3].

References[1] Karmakar R., Pahari P., Mal D., 2014. Chem. Rev., 114, 6213–6284.[2] Dalle F., Wächtler B., L’Ollivier C., Holland G., Bannert N., Wilson D., Labruère C., Bonnin A., Hube

B., 2010. Cell Microbiol., 12, 248–271.[3] Ferreira A.V., Prado C.G., Carvalho R.R., Dias K.S., Dias A.L., 2013. Mycopathologia, 175, 265–272.

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lactones With Methylcyclohexane systeM – oBtaining and Biological activity




Eight new lactones (δ-chloro-, δ-bromo- and δ-iodo-γ-lactones), each with a methylcyclohexane ring, were obtained by chemical means from (4-methylcyclohex-2-en-1-yl) acetic acid or (6-methylcyclohex-2- -en-1-yl) acetic acid. Ten fungal strains (fusarium species, syncephalastrum racemosum and Botrytis cine-rea) were tested on their ability to convert these lactones into other products.

Some of the tested fungal strains transformed chloro-, bromo- and iodolactone with a methyl gro-up at C-5 into hydroxylactone during hydrolytic dehalogenation. In the case of lactones with the methyl group at C-3, no structural modifications of halolactones were observed. In most cases, the optical purity of the product was low or medium, with the highest rate for chlorolactone (45.4%) and iodolactone (45.2 % and 47.6%).

All of the obtained compounds were tested with reference to their smell. Seven halolactones and the hydroxylactone obtained during biotransformation of halolactones with 5-methylcyclohexane ring were examined for their antimicrobial activity. These compounds were capable of inhibiting growth of some bacteria, yeasts and fungi.

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enantioselective conveRsion oF ceRtain deRivatives oF 7-hydRoxyFlavanone

Monika Dymarska, Tomasz Janeczko, Edyta Kostrzewa-Susłow

department of chemistry, wrocław university of environmental and life sciences, 50-375 wrocław, poland, ekostrzew@gmail com

Potential application of flavonoid aglycones is limited by low solubility, low permeability through biologi-cal membranes and insufficient stability in lipophilic media. Therefore the alternative may be to use either esters of flavonoids or products of their biotransformation[1].

There is growing interest in microbial transformation of flavonoids as a green alternative to chemi-cal synthesis. By the use of biotransformation we can modify structures of compounds in order to improve their biological properties and to increase their hydrophilicity or bioaccessibility[2].

In the cultures of aspergillus niger, racemic flavonoid derivatives (7-methoxy-, 7-acetoxy-, 7-propiono-xyflavanone) underwent microbial transformations resulted in optically pure (+)-(r)-7-hydroxyflavanone. Enantiomeric excesses of products were determined by HPLC using chiral column. The absolute configu-ration at C-2 was established by CD analysis.

O

O

RO O

O

OHA. niger

3

2

45

6

7

ee=100%

R1 -CH3 (±)-7-methoxyflavanone (+)-(R)-7-hydroxyflavanoneR2 -C(O)CH3 (±)-7-acetoxyflavanoneR3 -C(O)CH2CH3 (±)-7-propionoxyflavanone

Poster 4.8

Enantioselective conversion of certain derivativesof 7-hydroxyflavanone

Monika Dymarska, Tomasz Janeczko, Edyta Kostrzewa–Susłow*

Department of Chemistry, Wrocław University of Environmental and Life Sciences, 50-375, Poland, *email: [email protected]

Potential application of flavonoid aglycones is limited by low solubility, low

permeability through biological membranes and insufficient stability in lipophilic media.

Therefore the alternative may be to use either esters of flavonoids or products of their

biotransformation[1].

There is growing interest in microbial transformation of flavonoids as a green

alternative to chemical synthesis. By the use of biotransformation we can modify structures of

compounds in order to improve their biological properties and to increase their hydrophilicity

or bioaccessibility[2].

In the cultures of Aspergillus niger, racemic flavonoid derivatives (7-methoxy-,

7-acetoxy-, 7-propionoxyflavanone) underwent microbial transformations resulted in optically

pure (+)-(R)-7-hydroxyflavanone. Enantiomeric excesses of products were determined by

HPLC using chiral column. The absolute configuration at C-2 was established by CD

analysis.

Scheme 1. Microbial transformations of 7-hydroxyflavanone derivatives

There are number of studies on 7-hydroxyflavanone as a potential therapeutic agent.

This compound has antimicrobial potential against Streptococcus pneumoniae and may be

used as a treatment against respiratory infections[3]. It is proven that

Scheme 1. Microbial transformations of 7-hydroxyflavanone derivatives

There are number of studies on 7-hydroxyflavanone as a potential therapeutic agent. This compo-und has antimicrobial potential against streptococcus pneumoniae and may be used as a treatment against respiratory infections[3]. It is proven that 7-hydroxyflavanone has antimetastatic effect in SCC-4 cells (hu-man oral squamous carcinoma)[4].

In light of the above evidence for a wide range of biological activities of the said compound it appe-ars to be important to develop a method to obtain optically pure 7-hydroxyflavanone and undergo a series of tests in vitro and in vivo.

References[1] Kostrzewa-Susłow E., Dymarska M., Janeczko T., 2014. J. Mol. Catal. B-Enzym, 102, 59–65.[2] Kostrzewa-Susłow E., Dymarska M., Janeczko T., 2014. Pol. J. Microbiol, 63 (1), 111–114.[3] Zampini I.C., Villena J., Salva S., Herrera M., Isla M.I., Alvarez S., 2012. J. Ethnopharmacol., 140, 287–292.[4] Yang S.-F., Yang W.-E., Kuo W.-H., Chang H.-R., Chu S.-C., Hsieh Y.-S., 2008. Arch. Oral. Biol., 53,

287–294.

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iMpRoveMent oF antioxidant activity oF natuRal Flavonoids By BiotRansFoRMation

Sandra Sordon, Anna Madej, Jarosław Popłoński, Agnieszka Bartmańska, Tomasz Tronina, Ewa Huszcza

department of chemistry, faculty of food science, wrocław university of environmental and life

sciences, norwida 25, 50-375 wrocław, poland

Flavonoids are a large group of naturally occurring polyphenolic compounds. They are widely distributed secondary metabolites with different metabolic functions in plants. Flavonoids exhibit a diverse spectrum of biological activities such as: antioxidant, antimicrobial, antitumor, anti-inflammatory, antiviral and es-trogenic.

Many studies suggest that flavonoids exhibit various biological activities related to humans. Most of the beneficial health effects of flavonoids are attributed to their antioxidant abilities. In general, it is consi-dered that a higher number of hydroxyl substituents in a flavonoid results in a higher antioxidant activity.

Biotransformation processes, in opposition to the classic chemical synthesis, are environmentally friendly, proceed under mild conditions and allow regio- and stereoselective modifications of the substra-tes. Moreover, according to the European Union Law, the products obtained by biotransformation of na-tural compounds are classified as natural ones (EU Directive 88/388/EEC).

In the course of our studies to generate bioactive compounds from natural flavonoids by biotransfor-mation, we report utilisation of red yeasts to obtain biotransformation products with greatly increased an-tioxidant activity compared to the starting substrates.The biotransformation of naringenin, luteolin, hespe-retin and chrysin led to hydroxylated products with catechol moiety which are strong radical scavengers.

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xanthohuMol acyl deRivatives as antipRoliFeRative and antioxidant agents

Anna K. Żołnierczyk1,*, Dagmara Baczyńska2, Joanna Kozłowska1, Małgorzata Grabarczyk1, Wanda Mączka1, Katarzyna Wińska1, Edyta Woźniak1,

Mirosław Anioł1

1department of chemistry, The faculty of food science, wrocław university of environmental and life sciences,

norwida 25/27, 50-375 wrocław, poland

2molecular techniques unit, department of forensic medicine, wrocław medical university, wrocław, poland

agrot2@gmail com

xanthohumol (1), prenylated flavonoid presented in the high concentration in the spent hops exhibits in-teresting biological properties like antioxidant, antibacterial, antiviral, antifungal and antitumor. Structu-ral modifications can change thephysico-chemical properties, activity and potency of the compounds and decide about its biological activity. For example, acyl derivatives are liphophilic compounds which possess very good permeability through the membrane. Therefore a series of mono- and diacyl- xanthohumol (1) de-rivatives was synthesized (yields 10–30%) to investigate their biological activity and compare with the acti-vity of the parent compound. All the compounds were tested against the antiproliferative activity of human cancer cell line of colon adenocarcinoma (HT-29) using SRB assay. The most active were three acylated compounds: 4-o-acetylxanthohumol (2) (IC50=11.84 μM), 4,4’-Di-o-acetylxanthohumol (3) (IC50=11.75 μM) and 4-o-decanoylxanthohumol (4) (IC50=10.04 μM) (for xanthohumol (1) IC50=9.74 μM). The antio-xidant activity was determined by the method of DPPH and ABTS and expressed as mM of Trolox equ-ivalent antioxidant capacity (TEAC) per gram. Only one of the tested compound, 4-o-acetylxanthohumol (2) (0,015mM TEAC for DPPH and 0,16 mM TEAC for ABTS methods) showed a lower antioxidant acti-vity compared to xanthohumol (1) (0,038 mM TEAC for DPPH and 0,19 mM TEAC for ABTS methods). Other tested compounds showed similar or higher antioxidant activity as xanthohumol (1).

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BiotRansFoRMation oF isoxanthohuMol conducted in vaRious Media

Agnieszka Bartmańska, Tomasz Tronina, Jarosław Popłoński, Sandra Sordon, Ewa Brzezowska


Isoxanthohumol is the major prenylflavonoid in beer, which is the most important dietarysource of this compound. Content of it depends on a type of beer and ranges from 40 (European lager) to 3440 (strong ale) µg/L. Isoxanthohumol has received much attention as a proestrogen, an antiviral agent, an antioxi-dant, and as a cancer chemopreventive agent. Although isoxanthohumol is such a valuable compound, lit-tle is it known about its metabolism.

Microbial transformations are a convenient way of receiving compounds that are particularly diffi-cult to synthesize in a classic way.

The aim of the present work was to isolate and characterize new fugal metabolites of isoxanthohumol. In order to select microorganisms capable of transforming isoxanthohumol screening tests on 51 fungal cultures were performed.

Several of the tested strains metabolized substrate. We observed that the ability to transform isoxan-thohumol does not depend on the taxonomical affiliation of the microorganisms.

fusarium oxysporum AM13 and mucor hiemalis AM450 were selected for scale-up studies in Sabouraud medium. mucor hiemalis converted isoxanthohumol into isoxanthohumol 7-o-β-D-glucopyranoside. 2″-(2″′-hydroxyisopropyl)-dihydrofurano [2″,3″:7,8]-4″,5-hydroxy-5-methoxy-flavanone was obtained in transformation of substrateby fusarium oxysporum.

No product was obtained by absidia glauca in transformation of isoxanthohumol conducted in a phosphate buffer. In the same medium Beauveria bassiana converted isoxanthohumol to isoxantho- -humol 7-O-β-D-4’’’-O-methylglucopyranoside. We isolated this metabolite in low yield.

We obtained phase II products. These conjugation reactions show that there are similarities between mammalian and microbial systems.

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cheMoenZyMatic synthesis and antipRoliFeRative activity oF phospholipids containing cinaMMic and p-anisic acids

Marta Czarnecka1, Anna Gliszczyńska1, Marta Świtalska2, Joanna Wietrzyk2, Czesław Wawrzeńczyk1

1department of chemistry, wrocław university of environmental and life sciences, wrocław, poland 2ludwik hirszfeld intitute of immunology and experimental Therapy, polish academy of sciences,

department of experimental oncology, wrocław, poland

In recent years there has been a significant increase in interest in the polyphenolic acids as biologically ac-tive molecules. These compounds can be used as the ingredients in the production of functional and nu-traceutical food which posses favorably impact on human health.

In this group of compounds cinnamic acid and p-anisic acid have a wide spectrum of biological properties, such as antioxidant[3], antibacterial[ ] and anti-inflammatory activity[4]. In many of the stu-dies, anti-tumor effect of cinnamic and anisic acids have been demonstrated in vivo assays. Ekmekcioglu et al.[4] showed that cinnamic acid inhibits the growth of tumor cells of human colon adenocarcinoma (CaCO-2), while p-anisic acid can be useful in the cancer prevention and treatment by affecting COx-2 activity[6].

In our study we focused on the synthesis of structured phospholipids containing phenolic carboxy-lic acids in their structure. We performed chemoenzymatic synthesis and obtained new derivatives with cinnamic and anisic acids moiety in the position sn-1 and sn-2 of lecithin. In next step of our project the inhibition of the proliferation of cancer cells line of human promyelocytic leukemia (MV11-4) exposed on obtained derivatives was investigated. The linkage the phenolic acids with polar molecule such PC increased their bioavailability in the comparison with used substrates. Our results indicates that this type of molecules may be used in the future as a dietary supplements and nutraceuticals in the treatment of ci-vilizations diseases.

O OH

OCH3

O

OH

Fig. 1. p-anisic acid Fig. 2. cinammic acid

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References[1] Avanesyan A.A., Pashkov A.N., Simonyan N.A., Simonyan A.V., Myachina O.V., 2009. Pharmaceutical

Chemistry Journal, 43, 249–250.[2] Heleno S.A., Ferreira C.F.RI., Esteves A.P., Ćirić A., Glamočlija J., Martins A., Soković M., queiroz

M.J.R.P., 2013. Food and chemical toxicology, 58, 95–100.[3] O. Sytar, Journal of King Saud University – Science, 2015, 27, 136–142.[4] L.P. Zhang, Z.Z. Ji, 1992. Acta Pharmaceutica Sinica, 27, 817–823.[5] Ekmekcioglu C., Feyertag J., Marktl W., 1998. Cancer Letters, 128, 137–144.[6] Tao L., Wang S., Zhao Y., Sheng x., Wang A., Zheng S., Lu Y., 2014. Phytomedicine, 21, 1473–1482.

This project was financed by National Science Center Project no. 2013/09/D/NZ9/02457.

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naphthoFlavones – MetaBolisM, cytotoxicity and aRoMatase inhiBition

Jarosław Popłoński1, Sandra Sordon1, Eliza Turlej2, Joanna Wietrzyk2, Tomasz Tronina1, Agnieszka Bartmańska1, Ewa Huszcza1

1 department of chemistry, wrocław university of environmental and life sciences, norwida 25, 50-375, wrocław, poland

2 neolek laboratory of experimental anticancer Therapy, ludwik hirszfeld institute of immunology and experimental Therapy, polish academy of sciences, department of experimental oncology,

weigla 12, 53-114 wrocław, poland jarek poplonski@gmail com

The α-naphthoflavone (7,8-benzoflavone) and β-naphthofl avone (5,6-benzofl avone) are synthet-β-naphthofl avone (5,6-benzofl avone) are synthet--naphthoflavone (5,6-benzoflavone) are synthet-ic compounds used in drug metabolism studies as modulators of cytochrome P-450 enzymes. The α-naphthoflavone is a potent aromatase inhibitor found as an ingredient of food supplements for body builders as, erroneously termed, a natural compound. There are no studies of pharmacokinetics for men, however α-naphthoflavone bioavailability, tested in rat model, is low (0,6–13,2%). The α-naphthoflavone may be used in breast cancer treatment being potent inhibitor of breast cancer resistance protein, al-though tested on chickens indicate that chronic oral administration of the compound can lead to abnormal development of the testes. Furthermore, to our best knowledge, there are no data concerning the cytotox-icity of this compound, nor its human or microbiome metabolites.

We would like to present the results of the in vitro cytotoxicity assay of α-naphthoflavone, its synthe-tic derivatives and microbial metabolites obtained by biotransformations. Cytotoxicity was evaluated by means of antiproliferative assay on both normal and cancer cell lines. Because some α-naphthoflavone me-tabolites are more potent aromatase inhibitors than α-naphthoflavone, we also tested the potency of these compounds as aromatase inhibitors using recombinant human enzyme.

This work was financed by National Science Centre, Grant No. DEC-2013/09/N/NZ7/01478.

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BiotRansFoRMation oF 2-ButyRyloxy-2-(ethoxy-p-phenylphosphinyl)acetic acid With iMMoBiliZed Fungal cells

Monika Serafin, Ewa Żymańczyk-Duda

department of Bioorganic chemistry, faculty of chemistry, wrocław university of technology, wybrzeże wyspiańskiego 27, 50-370 wrocław, poland

Due to different biological activity of stereoisomers there is a necessity to obtain optically pure compo-unds designed for further application as pharmaceuticals or agrochemicals [1]. enantioselective enzyma-tic synthesis is becoming a common method for obtaining active compounds with a defined configuration especially in the pharmaceutical industry [2]. Immobilized whole-cells are one of the biocatalyst form that can be used in biotransformation processes. This system allows enhancing the stability of the biocatalyst, reusing of biocatalyst, simplifying downstream processes and enabling continuous operation of enzyma-tic processes [3].

Whole-cell biocatalysts immobilized on polyurethane foams and in calcium alginate were used for the resolution of the racemic mixture of 2-butyryloxy-2-(ethoxy-P-phenylphosphinyl)acetic acid conta-ining two stereogenic centers. Biotransformation processes in shake flask and in recirculated fixed-bed batch reactor were carried out.

The activity of biocatalyst immobilized in calcium alginate was lower than in the case of process with biomass growing on polyurethane foams, when this negative effect was not observed. Moreover, applica-tion of such biocatalyst in recirculated fixed-bed batch reactor allows to scale-up the process.

References[1] Scriba G.K., 2002. Journal of Pharmaceutical and Biomedical Analysis, 27, 373–399.[2] Nestl B.M.,Nebel B.A., Hauer B., 2011. Current Opinion in Chemical Biology, 15, 187–193.[3] Homaei A.A., Sariri R., Vianello F., Stevanato R., 2013. Journal of Chemical Biology, 6, 185–205.

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parTiCipanTS liST

NO. NAME AND SURNAME INSTITUTION TOWN / ADDRESS COUN-

TRY1. Aggelis

GeorgeLaboratory of Microbiology, Division of Genetics, Cell Biology & Development, Department of Biology, University of Patras

Patras 26504 Greece

2. Anioł Mirosław

Department of Chemistry, Wrocław University of Environmental and Life Sciences

Norwida 25, 50-375 Wrocław

Poland

3. Astaloš Marek

Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture

Tr. A. Hlinku 2, 949 76 Nitra

Slovak Republic

4. Ayuda-Durán Begoña

Grupo de Investigación en Polifenoles (GIP-USAL), Unidad de Nutrición y Bromatología, Facultad de Farmacia, Universidad de Salamanca

37008 Salamanca Spain

5. Babij Konrad

Department of Animal Products Technology and quality Management, Wrocław University of Environmental and Life Sciences

Chełmońskiego 37,51-630 Wrocław

Poland

6. Baczyńska Dagmara

Molecular Techniques Unit, Department of Forensic Medicine, Wrocław Medical University

Mikulicza-Radeckiego 4, 50-345 Wrocław

Poland

7. Balcerek Maria

Institute of Fermentation Technology and Microbiology, Faculty of Biotechnology and Food Sciences, Łódź University of Technology

Wólczańska 171/173,90-924 Łódź

Poland

8. Banach Karolina

Department of Biotechnology and Food Microbiology Wrocław University of Environmental and Life Sciences

Chełmońskiego 37/41,51-630 Wrocław

Poland

9. Bartmańska Agnieszka

Department of Chemistry, Faculty of Food Science, Wrocław University of Environmental and Life Sciences


Poland

10. Bednasz- -Misa Iwona

Department of Medical Biochemistry at Wrocław Medical University

Chałubińskiego 10, 50-332 Wrocław

Poland

11. Beopoulos Athanasios

INRA, UMR1319, Micalis Domaine de Vilvert, F-78352 Jouy-en-Josas

France

12. Berłowska Joanna


Wólczańska 171/173, 90-924 Łódź

Poland

13. Beutel Tadeusz

Faculty of Biotechnology, University of Wrocław

Joliot-Curie 14a, 50-383 Wrocław

Poland

14. Białas Wojciech

Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences

Wojska Polskiego 48, 61-627 Poznań

Poland

15. Biegalska Anna

Department of Biotechnology and Food Microbiology, University of Environmental and Life Sciences


Poland

16. Błażewicz Józef

Departament of Food Storage and Technology, Wrocław University of Environmental and Life Sciences


Poland

17. Blei Felix

Terrestrial Biofilms Group, Institute of Microbiology, Friedrich Schiller University

Zwaetzengasse 3 07743 Jena

Germany

18. Bobak Łukasz



Poland

19. Boguta Patrycja

Institute of Agrophysics PAS Doświadczalna 4, 20-290 Lublin

Poland

20. Bondarczuk Kinga

Department of Microbiology, University of Silesia

Jagiellońska 28, 40-032 Katowice

Poland

21. Boratyński Filip



Poland

22. Borawska Justyna

Faculty of Food Science, Chair of Food Biochemistry, University of Warmia and Mazury in Olsztyn

pl. Cieszyński 1, 10-726 Olsztyn-Kortowo

Poland

23. Borkowska Monika


Wojska Polskiego 48,61-627 Poznań

Poland

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24. Brakhage Axel A.

Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute

Beutenbergstraße 11/a, 07745 Jena

Germany

25. Bronowicka- -Szydło Agnieszka



Poland

26. Brzezowska Ewa



Poland

27. Buczkowska Katarzyna

Department of Genetics, Faculty of Biology, Institute of Experimental Biology, Adam Mickiewicz University Poznań

Umultowska 89, 61-614 Poznań

Poland

28. Buda Barbara


Chełmońskiego 37, 51-630 Wrocław

Poland

29. Celińska Ewelina



Poland

30. Chmielewska Joanna

Department of Food Storage and Technology, Wrocław University of Environmental and Life Sciences


Poland

31. Chojnacka Anna



Poland

32. Chorążyk Dorota



Poland

33. Chrzanowska Józefa

Department. of Animal Products Technology and quality Management, Wrocław University of Environmental and Life Sciences


Poland

34. Cieśla Jolanta

Institute of Agrophysics PAS Doświadczalna 4, 20-290 Lublin

Poland

35. Ciurko Dominika

Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences


Poland

36. Crutz-Le Coq Anne-Marie

INRA, UMR1319, Micalis, AgroParisTech, UMR Micalis, Department of Biotechnology

Domaine de Vilvert, F-78352 Jouy-en-Josas,

France

37. Cybulski Krzysztof



Poland

38. Czabaj Sławomir

Department of Food Storage and Technology, Faculty of Food Science, Wrocław University of Environmental and Life Sciences


Poland

39. Czarnecka Marta

Department of Chemistry, Wrocław University of Environ-mental and Life Sciences


Poland

40. Czołpińska Magdalena

Department of Genetics, Institute of Experimental Biology, Faculty of Biology, Adam Mickiewicz University Poznań


Poland

41. Dąbrowska Anna

Department. of Animal Products Technology and quality Management, Wrocław University of Environmental and Life Sciences

Chełmońskiego 37, 51- 630 Wrocław

Poland

42. Darewicz Małgorzata

Faculty of Food Science, Chair of Food Biochemistry, University of Warmia and Mazury


Poland

43. Długoński Jerzy

Department of Industrial Microbiology and Biotechnology, Faculty of Biology and Environmental Protection, University of Łódź

Banacha 12/16, 90-237 Łódź

Poland

44. Dobrowolski Adam


Chełmońskiego 37/41, 51-630 Wrocław

Poland

45. Domżał Marta

Faculty of Biotechnology, University of Wrocław Biotransformation Department, University of Wrocław


Poland

46. Drąg Marcin

Division of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology

Wybrzeże Wyspiańskiego 27, 50-370 Wrocław

Poland

47. Dulermo Remi

INRA, UMR1319, MicalisAgroParisTech, UMR Micalis, Department of Biotechnology

Domaine de Vilvert 78352 Jouy-en-Josas

France

48. Dulermo Thierry



France

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49. Dutkiewicz Marta



Poland

50. Dymarska Monika



Poland

51. Dziekońska Urszula



Poland

52. Dziki Dariusz

Thermal Engineering Department, University of Life Sciences

Doświadczalna 44, 20-280 Lublin

Poland

53. Dziuba Ewelina



Poland

54. Faber Kurt

Austrian Centre of Industrial Biotechnology c/o Department of Chemistry, University of Graz

A-8010 Graz Austria

55. Felczak Aleksandra


Pilarskiego 14/16, 90-231 Łódź

Poland

56. Foszczyńska Barbara



Poland

57. Gamboa- -Meléndez Heber



France

58. Gamian Andrzej



Poland

59. Gawlik-Dziki Urszula

Department of Biochemistry and Food Chemistry, University of Life Sciences

Skromna 8, 20-704 Lublin

Poland

60. Gładkowski Witold



Poland

61. Gliszczyńska Anna


Norwida 25,50-375 Wrocław

Poland

62. Glueck Silvia


A-8010 Graz Austria

63. Głąb Agata

Department of Bioorganic Chemistry, Wrocław University of Technology


Poland

64. Gniłka Radosław



Poland

65. González- -Manzano Susana



66. González- -Parámas Ana M.



67. Grabarczyk Małgorzata



Poland

68. Grajek Włodzimierz



Poland

69. Grudniewska Aleksandra



Poland

70. Grzegorczyk Monika



Poland

71. Han Aleksandra



Poland

72. Hapeta Piotr



Poland

73. Hleba Lukáš,



Slovakia

74. Huszcza Ewa

Department of Chemistry, Faculty of Food Science, Wrocław University of Environmental and Life Sciences


Poland

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75. Iwaniak Anna

Faculty of Food Science, Chair of Food Biochemistry, University of Warmia and Mazury in Olsztyn

pl. Cieszyński 1, Olsztyn10-726 Olsztyn-Kortowo,

Poland

76. Jabłoński Sławomir



Poland

77. Janeczko Tomasz

Department of Chemistry, Wrocław University of Environmental and Life Sciences,


Poland

78. Janicki Tomasz


Banacha 12/16, 90-237 Łódź

Poland

79. Jaros- -Koźlecka Katarzyna

TK Nano Gorlicka 64/50, 51-314 Wrocław

Poland

80. Kačániová Miroslava

Slovak University of Agriculture in Nitra, Faculty of Biotech-nology and Food Sciences, Department of Microbiology


Slovakia

81. Kaczmarczyk Daria

1Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, 2Institute of Cosmetology, Wrocław College of Physiotherapy

1Wybrzeże Wyspiańskiego 27, 50-370 Wrocław,2Kościuszki 4, 50-038 Wrocław

Poland

82. Kalinowska Halina

Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Łódź University of Technology


Poland

83. Kancelista Anna



Poland

84. Kántor Attila

Department of Microbiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra


Slovakia

85. Kasperkie-wicz Paulina



Poland

86. Kautola Helena

Häme University of Applied Sciences Hämeenlinna Finland

87. Kawa- -Rygielska Joanna



Poland

88. Kliks Jarosław

Department of Food Storage and Technology, Faculty of Food Science, Wrocław University of Environmental and Life Sciences


Poland

89. Kluz Maciej

Faculty of Biology and Agriculture, University of Rzeszów Ćwiklińskiej 1, 35-601 Rzeszów

Poland

90. Kmiecik Natalia

Department of Bioorganic Chemistry, Wrocław University of Technology


Poland

91. Koniuszew-ska Joanna

Food Microbiology, Wrocław University of Environmental and Life Sciences


Poland

92. Kopeć Wiesław



Poland

93. Kostrzewa- -Susłow Edyta



Poland

94. Kowalczyk Dariusz

Department of Biochemistry and Food Chemistry, University of Life Sciences,


Poland

95. Kovács Ákos T.


Neugasse 23 07743 Jena

Germany

96. Kozakiewicz Mariusz



Poland

97. Koźlecki Tomasz

Faculty of Chemistry, Wrocław University of Technology


Poland

98. Kozłowska Joanna

Department of Chemistry, The Faculty of Food Science, Wrocław University of Environmental and Life Sciences


Poland

99. Kropiwnicki Mateusz



Poland

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111

100. Krzystek- -Korpacka Małgorzata

Department of Medical Biochemistry, Wrocław Medical University


Poland

101. Kurzawa Adam

Institute of Production Engineering and Automation, Wrocław University of Technology

Łukasiewicza 3/5, 50-371 Wrocław

Poland

102. Łaba Wojciech



Poland

103. Lazar Zbigniew



Poland

104. Ledesma Rodrigo

INRA, UMR1319, Micalis, AgroParisTech, UMR Micalis, Jouy-en-Josas, Department of Biotechnology

Domaine de Vilvert, F-78352 Jouy-en-Josas,

France

105. Lennartsson Patrik R.

Swedish Centre of Resource Recovery, University of Borås, Sweden

Allégatan 1, Borås Sweden

106. Leśniak Agnieszka

Department of Chemistry, Wrocław University of Environ-mental and Life Sciences


Poland

107. Lisowska Katarzyna


Pilarskiego 14/16, 90-231 Łódź

Poland

108. Lochyński Stanisław

1Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology 2Institute of Cosmetology, Wrocław College of Physiotherapy

1Wybrzeże Wyspiańskiego 27, 50-370 Wrocław2Kościuszki 4, 50-038 Wrocław

Poland

109. Łukaszewicz Marcin



Poland

110. Mączka Wanda



Poland

111. Madej Anna Faculty of Food Science, Department of Chemistry, Wrocław University of Environmental and Life Sciences


Poland

112. Maj Paula Department of Chemistry, Wrocław University of Environmental and Life Sciences


Poland

113. Markowicz Anna

Department of Microbiology,

University of SilesiaJagiellońska 28, 40-032 Katowice

Poland

114. Maróti Gergely

Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences

Temesvári krt. 62H-6726 Szeged

Hungary

115. Matern Derek

Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute

Neugasse 23 07743 Jena

Germany

116. Matyjasz Joanna



Poland

117. Mazur Marcelina



Poland

118. Mierzchała- -Pasierb Magdalena



Poland

119. Milecka-Tro-nina Natalia



Poland

120. Minkiewicz Piotr

Chair of Food Biochemistry, Faculty of Food Science, University of Warmia and Mazury in Olsztyn

pl. Cieszyński 1, 10-726 Olsztyn-Kortowo,

Poland

121. Mirończuk Aleksandra



Poland

122. Mituła Paweł



Poland

123. Neuveglise Cecile

INRA, UMR1319, MicalisAgroParisTech, UMR Micalis, Department of Biotechnology


France

124. Nicaud Jean-Marc



France

125. Niezgoda Natalia



Poland

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126. Olejniczak Teresa



Poland

127. Panek Anna



Poland

128. Pannek Jakub



Poland

129. Parker Heather

University of Otago Christchurch New Zealand

130. Pasławska Marta

Institute of Agricultural Engineering, Wrocław University of Environmental and Life Sciences


Poland

131. Patelski Piotr



Poland

132. Petrová Jana



Slovakia

133. Pezała Maja



Poland

134. Piegza Michał



Poland

135. Pielech- -Przybylska Katarzyna



Poland

136. Pietrzak Witold



Poland

137. Piotrowska- -Seget Zofia

Department of Microbiology,

University of SilesiaJagiellońska 28 Street, 40-032 Katowice

Poland

138. Plasch Katharina

Department of Chemistry, University of Graz

A-8010 Graz Austria

139. Pliszka Monika

University of Warmia and Mazury in Olsztyn, Faculty of Food Science, Chair of Food Biochemistry


Poland

140. Pokora Marta


Chełmońskiego 37, 51- 630 Wrocław,

Poland

141. Polak- -Berecka Magdalena

Department of Biotechnology, Human Nutrition and Science of Food Commodities, University of Life Sciences in Lublin


Poland

142. Połomska xymena



Poland

143. Popłoński Jarosław

Faculty of Food Science, Department of Chemistry, Wrocław University of Environmental and Life Sciences


Poland

144. Poręba Marcin



Poland

145. Potaniec Bartłomiej

Department of Chemistry, Wrocław University of Environmental and Life Sciences,


Poland

146. Protasiewicz Monika

University of Warmia and Mazury, Faculty of Food Science, Chair of Food Biochemistry,


Poland

147. Pudło Anna



Poland

148. Pusz Wojciech

Department of Plant Protection, Wrocław University of Environmental and Life Sciences

Grunwaldzki 24A, 53-363 Wrocław

Poland

149. Rakicka Magdalena



Poland

150. Regiec Piotr

Department Food Storage and Technology, Faculty of Food Science, Wrocław University of Environmental and Life Sciences


Poland

151 Reiter Tamara


A-8010 Graz Austria

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152. Resch Verena

Department of Chemistry, University of Graz A-8010 Graz Austria

153. Robak Małgorzata



Poland

154. Romaniuk Ewa



Poland

155. Ropa Anna



Poland

156. Rossignol Tristan


Domaine de Vilvert, 78352 Jouy-en-Josas,

France

157. Roszak Anna Zofia

Department of Genetics, Faculty of Biology, Institute of Experimental Biology, Adam Mickiewicz University Poznań


Poland

158. Rut Wioletta



Poland

159. Rymowicz Waldemar



Poland

160. Rywińska Anita



Poland

161. Salvesen Guy S.

Sanford-Burnham Medical Research Institute 10901 North Torrey Pines Road, La Jolla

USA

162. Santos-Buelga Celestino



163. Schwitalla Jan


Beutenbergstraße 11/a, 07745 Jena

Germany

164. Serafin Monika

Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology


Poland

165. Serek Paweł



Poland

166. Sieniawski Krzysztof

Stanisława Żurawskiego 17/19, 10-689 Olsztyn

Poland

167. Skrzypek Tomasz

Laboratory of Confocal and Electron Microscopy, Interdisciplinary Research Center, John Paul II Catholic University of Lublin

Doświadczalna 4 20-290 Lublin

Poland

168. Smuga Małgorzata



Poland

169. Snipas Scott

Sanford-Burnham Medical Research Institute 10901 North Torrey Pines Road, La Jolla

USA

170. Sordon Sandra



Poland

171. Sowiński Józef

Department of Crop Production, Wrocław University of Environmental and Life Sciences

Grunwaldzki 24a,50-363 Wrocław

172. Starowicz Piotr

Chair of Food Biochemistry, Faculty of Food Science, University of Warmia and Mazury in Olsztyn

Cieszyński 1, 10-726 Olsztyn-Kortowo

Poland

173. Stempniewicz Regina



Poland

174. Strąk Ewelina



Poland

175. Świeca Michał

Department of Biochemistry and Food Chemistry, University of Life Sciences


Poland

176. Świtalska Marta

Ludwik Hirszfeld Intitute of Immunology and Experimental Therapy, Polish Academy of Sciences, Department of Experimental Oncology,

Weigla 4, 53-114 Wrocław

Poland

177. Świzdor Alina



Poland

178 Sygit Maciej

SYGMA Sp. z o.o. Ruska 61, 50-079 Wrocław

Poland

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178. Szczech Magdalena

Research Institute of Horticulture Pomologiczna 18, 96-100 Skierniewice

Poland

179. Szczepańska Ewa



Poland

180. Szmigiel Beata



Poland

181. Szmyt Agnieszka



Poland

182. Sznurowska Magdalena



Poland

183. Szołtysik Marek



Poland

184. Szopa Jan

Department of Genetics, Plant Breeding and Seed Production, Wrocław University of Environmental and Life Sciences

pl. Grunwaldzki 24A, 53-363 Wrocław

Poland

185. Szwajgier Dominik

Department of Biotechnology, Human Nutrition and Science of Food Commodities,University of Life Sciences in Lublin

Skromna 8, 20-704 Lublin,

Poland

186. Szydełko-Rol-ska Ewelina



Poland

187. Taherzadeh Mohammad J.

Swedish Centre of Resource Recovery, University of Borås, Sweden

Allégatan 1, Borås Sweden

188. Terentjeva Margarita

Institute of Food and Environmental Hygiene, Faculty of Veterinary Medicine, Latvia University of Agriculture

K. Helmana iela 8LV-3004 Jelgava

Latvia

189. Tomaszewska- -Ciosk Ewa

Department Food Storage and Technology, Wrocław Uniwersity of Environmental and Life Sciences


Poland

190. Tronina Tomasz

Faculty of Food Science, Department of Chemistry, Wrocław University of Environmental and Life Sciences


Latvia

191. Turlej Eliza

NeoLek Laboratory of Experimental Anticancer Therapy, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Department of Experimental Oncology


Poland

192. Vegarud Gerd Elisabeth

Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences

1432 Ås, Norway

193. Walczak Paulina

Department of Chemistry, Wrocław Uniwersity of Environmental and Life Sciences


Norway

194. Waśko Adam

Department of Biotechnology, Human Nutrition and Science of Food Commodities, University of Life Sciences in Lublin


Poland

195. Wawrzeńczyk Czesław



Poland

196. Wietrzyk Joanna

Ludwik Hirszfeld Intitute of Immunology and Experimental Therapy, Polish Academy of Sciences, Department of Experimental Oncology


Poland

197. Wilczak Aleksandra



Poland

198. Wińska Katarzyna



Poland

199. Wińska Katarzyna



Poland

200. Winterbourn Christine

University of Otago Christchurch New Zealand

201. Witkowska Danuta



Poland

202. Wojtatowicz Maria



Poland

203. Worsztyno-wicz Paulina



Poland

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204. Woźniak Edyta



Poland

205. Wróbel- -Kwiatkowska Magdalena



Poland

206. Zambrowicz Aleksandra



Poland

207. Żarowska Barbara



Poland

208. Zawadzka Katarzyna



Poland

209. Żołnierczyk Anna K.

Department of Chemistry, The Faculty of Food Science, Wrocław University of Environmental and Life Sciences


Poland

210. Żymańczyk- -Duda Ewa



Poland

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noTeS

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noTeS

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General informaTionS

On BRIA2015 Conference

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VENuE

LANGuAGEBADGES AND REGISTRATION

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Documents

Biotechnology – Research and Industrial Applications · Ewa Huszcza Tomasz Boruczkowski Filip Boratyński Zbigniew Lazar Michał Piegza Monika Grzegorczyk Mateusz Kropiwnicki Joanna