celebrating

III

International Congress

Food Technology, Quality and Safety

CELEBRATING

FOOD PROCEEDINGS

ISBN 978-86-7994-050-6

III INTERNATIONAL CONGRESS “FOOD TECHNOLOGY, QUALITY AND SAFETY”, NOVI SAD 2016, SERBIA Publisher University of Novi Sad Institute of Food Technology Bulevar cara Lazara 1 21000 Novi Sad Main editor Dr Olivera Đuragić Editors Dr Tea Brlek Dr Jovana Kos Paper Review All abstracts and papers are reviewed by International Scientific Committee and competent researchers Technical editor Mr Tamara Sarafijanović Cover Boris Bartula, BIS, Novi Sad, Serbia Printed by “Futura” – Novi Sad, Serbia Number of copies 350 copies

Organization of Congress: INSTITUTE OF FOOD TECHNOLOGY, University of Novi Sad, Serbia

Congress is supported by: Ministry of Education, Science and Technological Development of the Republic of Serbia – Belgrade Provincial Secretariat for Higher Education and Scientific Research, Novi Sad, Serbia Ministry of Agriculture and Environmental Protection of the Republic of Serbia – Belgrade Provincial Secretariat of Agriculture, Water Economy and Forestry – Novi Sad, Serbia EFE – European Academy of Food Engineering Chamber of Commerce and Industry of Serbia, Serbia

GENERAL SPONSOR OF CONGRESS - LABENA FoodTech 2016 Congress President: dr Olivera Đuragić, Institute of Food Technology, University of Novi Sad FoodTech 2016 Organizing committee President: dr Pavle Jovanov, Institute of Food Technology, University of Novi Sad

III International Congress “Food Technology, Quality and Safety” – INTERNATIONAL SCIENTIFIC COMMITTEE Olivera Đuragić, Serbia, Institute of Food Technology, University of Novi Sad - president Maja Musse, Université Bretagne Loire (UBL), France Dragan Momĉilović, FDA, USA Zehra Ayhan, Faculty of Engineering, Sakarya UniversityTurkey Verica Dragović-Uzelac, Faculty of Food Technology and Biotechnology, Croatia Stefan Toepfl, German Institute of Food Technologies (DIL), Germany Jose Maria Lagaron, Institute of Agrochemistry and Food Technology, Spain Johanes Kahl, Exercise and Sports University of Copenhagen, Denmark Brijesh Tiwari, TEAGASC, Ireland Viktor Nedovic, Faculty of Agriculture, University of Belgrade, Serbia Nastasia Belc, National R&D Institute for Food Bioresources, Romania Yrjö Roos, University College Cork, Ireland Ţivko Nikolov, Department of Biological & Agricultural Engineering, Texas A&M University, USA Jana Hajslova, Institute of Chemical Technology, Prague, Czech Republic Vesna Tumbas, Faculty of Technology, University of Novi Sad, Serbia Bogdan Iegorov, Odessa National Academy of Food Technologies, Ukraine Ewa Rambialkowska, Faculty of Human Nutrition and Consumer Science, Warsawa, Poland Peter Raspor, University of Ljubljana, Slovenia

Radoslav Grujić, Faculty of Technology Zvornik, University of East Sarajevo, BIH Republic of Srpska Miomir Jovanović, Biotechnical Faculty Dean, University of Montenegro, Montenegro Ţeljko Knez, Faculty of Chemistry and Chemical Engineering, University of Maribor, Slovenia Mladen Brnĉić, Faculty of Food Technology and Biotechnology, Croatia Costas Biliaderis, Food Science and Technology, Aristotle University, Thessaloniki, Greece, Sonja Smole Mozina, Biotechnical Faculty, University of Ljubljana, Slovenia Boţidar Ţlender, University of Ljubljana, Department of Food Science and Technology, Ljubljana, Slovenia Harris Lazarides, Food Science and Technology, Aristotle University of Thessaloniki Aleksandra Martinović, Food Safety at Biotechnical Faculty, University of Montenegro, Montenegro Angel Angelov, Head of Center of Food Quality and Safety, University of Food Technologies, Bulgaria Diego Moreno-Fernández, Food Science & Technology Department, University of Murcia, Spain Dragana Miladinović, Instutut of Field and Vegetable Crops, Novi Sad Mladenka Pestorić, Institute of Food Technology, University of Novi Sad Milica Pojić, Institute of Food Technology, University of Novi Sad Olivera Šimurina, Institute of Food Technology, University of Novi Sad Pavle Jovanov, Institute of Food Technology, University of Novi Sad Marijana Sakaĉ, Institute of Food Technology, University of Novi Sad Predrag Ikonić, Institute of Food Technology, University of Novi Sad Aleksandra Mišan, Institute of Food Technology, University of Novi Sad Tatjana Tasić, Institute of Food Technology, University of Novi Sad Tea Brlek, Institute of Food Technology, University of Novi Sad Anamarija Mandić, Institute of Food Technology, University of Novi Sad Miroslav HadnaĊev, Institute of Food Technology, University of Novi Sad Tamara Dapĉević HadnaĊev, Institute of Food Technology, University of Novi Sad Bojana Filipĉev, Institute of Food Technology, University of Novi Sad Jelena Pejin, Faculty of Technology, University of Novi Sad, Serbia Branko Bugarski, Faculty of Technology and Metallurgy, University of Belgrade, Serbia Milica Radosavljević, Maize Research Institute “Zemun Polje”, Serbia Dragan Ţivanĉev, Institute of Food Technology, University of Novi Sad, Serbia Tanja Radusin, Institute of Food Technology, University of Novi Sad, Serbia Elizabet Janjić-Hajnal, Institute of Food Technology, University of Novi Sad, Serbia Ljubiša Šarić, Institute of Food Technology, University of Novi Sad, Serbia Jovana Kos, Institute of Food Technology, University of Novi Sad, Serbia Marija Bodroţa-Solarov, Institute of Food Technology, University of Novi Sad, Serbia Nebojša Ilić, Institute of Food Technology, University of Novi Sad, Serbia Ivan Milovanović, Institute of Food Technology, University of Novi Sad, Serbia Jelena Tomić, Institute of Food Technology, University of Novi Sad, Serbia Aleksandra Novaković, Institute of Food Technology, University of Novi Sad, Serbia Aleksandra Torbica, Institute of Food Technology, University of Novi Sad, Serbia

III International Congress “Food Technology, Quality and Safety” – ORGANIZING COMMITTEE Olivera Đuragić, Institute of Food Technology, University of Novi Sad Tea Brlek, Institute of Food Technology, University of Novi Sad Jovana Kos, Institute of Food Technology, University of Novi Sad Olivera Šimurina, Institute of Food Technology, University of Novi Sad Tamara Dapĉević HadnaĊev, Institute of Food Technology, University of Novi Sad Jelena Tomić, Institute of Food Technology, University of Novi Sad Jelena Krulj, Institute of Food Technology, University of Novi Sad Jovana Kojić, Institute of Food Technology, University of Novi Sad Ivan Milovanović, Institute of Food Technology, University of Novi Sad Marijana Sakaĉ, Institute of Food Technology, University of Novi Sad Dubravka Škrobot, Institute of Food Technology, University of Novi Sad Miona Belović, Institute of Food Technology, University of Novi Sad Alena Tomšik, Institute of Food Technology, University of Novi Sad Milica Pojić, Institute of Food Technology, University of Novi Sad Zdenka Marković, Institute of Food Technology, University of Novi Sad, Serbia Bojana Šarić, Institute of Food Technology, University of Novi Sad, Serbia Nataša Nedeljković, Institute of Food Technology, University of Novi Sad, Serbia

FoodTech 2016 Congress – HONORARY BOARD Prof. dr Milica Pavkov Hrvojević, dean, Faculty of Sciences, University of Novi Sad Prof. dr Sneţana Brkić, dean, Faculty of Medicine, University of Novi Sad Prof. dr Nedeljko Tica, dean, Faculty of Agriculture, University of Novi Sad Prof. dr Milica Petrović, dean, Faculty of Agriculture, University of Belgrade Prof. dr Miladin Kostić, rector, State University in Novi Pazar Prof. dr Miroslav Ćirković, director, Scientific Veterinary Institute Novi Sad Dr Petar Kljajić, director, Institute of Pesticides and Environmental Protection, Belgrade Dr Vesna ĐorĊević, director, Institute of Meat Hygiene and Technology, Belgrade Dr Jelena Begović, director, Institute of Molecular Genetics and Genetic Engineering, Belgrade Prof. dr Vladeta Stevović, dean, Faculty of Agronomy Ĉaĉak Prof. dr Jovan Stepanović, dean, Faculty of Technology in Leskovac Dr Vladimir Crnojević, director, BioSense Institute, Novi Sad Prof. dr Radomir Malbaša, dean, Faculty of Technology, University of Novi Sad

FoodTech 2016 Congress – EXECUTIVE COMMITTEE Olivera Đuragić, Institute of Food Technology, University of Novi Sad Pavle Jovanov, Institute of Food Technology, University of Novi Sad Jovanka Lević, Institute of Food Technology, University of Novi Sad Tea Brlek, Institute of Food Technology, University of Novi Sad SlaĊana Rakita, Institute of Food Technology, University of Novi Sad Jovana Kos, Institute of Food Technology, University of Novi Sad Đuro Vukmirović, Institute of Food Technology, University of Novi Sad

III International Congress “Food Technology, Quality and Safety”

SADRŽAJ BROCCOLI FROM FARM TO HEALTH – RESEARCH AND CHALLENGES Diego A. Moreno1*, Nieves Baenas1, Débora Villaño2, María Teresa López-Chillón2, Pilar Zafrilla2, Cristina García-Viguera1 ……………………………………………………………

1

MOLECULAR ANALYSIS OF NS Cucurbita moschata COLLECTION Dragana Miladinović1*, Aleksandra Dimitrijević1, Milka Brdar-Jokanović1, Ivana Imerovski1, Vladimir Sikora1, Ana Marjanović Jeromela1, Siniša Jocić1 ...................................................... 6 FIBER HEMP AS A VALUABLE SOURCE OF NUTRIENTS AND NUTRACEUTICALS Tijana Zeremski*1, Biljana Kiprovski1, Vladimir Sikora1, Jegor Miladinović1, Svetlana Balešević Tubić1 .........................................................................................................................

10

LACTOSE FERMENTATION IN MILK BY KOMBUCHA Katarina Kanurić*, Spasenija Milanović, Bojana Ikonić, Eva Lončar, Mirela Iličić, Vladimir Vukić, Dajana Vukić …………………………………………………………………………..

16

BIOAVAILABILITY AND BIOACTIVITY OF GLS/ITC OF BRASSICACEAE SPROUTS AS HEALTH PROMOTERS Nieves Baenas*, Cristina García-Viguera, Diego A. Moreno …………………………………

23

STABILITY OF WATER-IN-OIL EMULSIONS CONTAINING WATER OR ETHANOL GARLIC EXTRACT - INFLUENCE OF TYPE AND CONCENTRATION OF EMULSIFIER Jelena D. Ilić 1*, Branimir M. Bajac1, Branislava G. Nikolovski1 …………………………….

29

THE GARLIC EXTRACT W/O/W DOUBLE EMULSIONS – FROM EXTRACTION TO RELEASE CHARACTERISATION Jelena D. Ilić 1*, Ivana S. Lončarević1, Jovana S. Petrović1, Branislava G. Nikolovski1………

35

MOLECULAR DOCKING SYMULATION THROUGH THE ANGIOTENSIN CONVERTING ENZYME INHIBITION BY MILK DIPEPTIDES Vladimir Vukić*, Dajana Vukić, Spasenija Milanović, Mirela Iličić, Katarina Kanurić ...........

42

QUALITY ATTRIBUTES OF COOKIES ENRICHED WITH BETAINE Bojana Filipčev*, Jelena Krulj, Jovana Kojić, Olivera Šimurina, Marija Bodroža Solarov, Mladenka Pestorić ......................................................................................................................

46

CHEMICAL-MINERAL CONTET AND RHEOLOGICAL PROPERTIES OF SESAME AND SPELT FLOUR Jelena Filipović*1, Milenko Košutić1, Rada Jeftić-Mučibabić1, Vladimir Filipović2, Milica Nićetin2, Marija Radojković2 .......................................................................................................

52

APPLICATIONS OF POTENTIAL PROBIOTIC BACTERIA AND OMEGA-3 FATTY ACIDS IN YOGURT PRODUCTION AND IMPACT ON SENSORY QUALITY Milica Mirković1, Nemanja Mirković1*, Dušanka Paunović1, Jelena Miočinović1, Dušan Kekuš1, Viktor Nedović1, Zorica Radulović1.............................................................................

56

MYCOTOXINS SURVEY IN IMPORTED WHEAT COMMODITY DURING 2016 IN ALBANIA Afërdita Shtëmbari*1, Dritan Topi 2.............................................................................................

62

LACTIC ACID FERMENTATION OF CABBAGE JUICE BY PROBIOTIC STRAIN Darko Dimitrovski, Marija Cvetkovska Stojanovska, Jana Simonovska*, Donka DonevaSapceska ......................................................................................................................................

67

THE EFFECT OF OSMOTIC TREATMENT ON ANTIOXIDANT ACTIVITY OF CELERY ROOT Milica Nićetin*1, Lato Pezo2, Vladimir Filipović1, Biljana Lončar1, Jelena Filipović3, Tatjana Kuljanin1, Stanislava Gorjanović2...............................................................................................

74

RHEOLOGICAL CHARACTERISTICS OF DOUGH ENRICHED WITH CAROB AND SUGAR BEET FIBRES Dragana Šoronja-Simović1, Zita Šereš1, Nikola Maravić1*, Sonja Smole-Možina2, Lucija Luskar2, Ivana Nikolić1, Marijana Đorđević1..............................................................................

80

NEUROPROTECTIVE EFFECT OF ARISTOTELIA CHILENSIS (MAQUI BERRY) EXTRACT AND DAPSONE IN A STATUS EPILEPTICUS MODEL Luis Bautista Orozco1, Amadeo Gironés Vilaplana2, Eva González Trujano3, Cristina García Viguera4, Diego A. Moreno4,*, Araceli Díaz Ruíz 5, Camilo Ríos 5, Sandra Orozco Suárez1….

86

WATER ACTIVITY AND SOME CHEMICAL PARAMETERS IN DRY PORK PRODUCED WITH LESS AMOUNT OF SODIUM CHLORIDE Slobodan Lilić1*, Danijela Vranić1, Vladimir Korićanac1, Ivana Branković Lazić1, Jelena Jovanović1, Brankica Lakićević1..................................................................................................

92

THE EFFECT OF WHOLE GRAIN BUCKWHEAT FLOUR STORAGE ON THE CONTENT OF PHENOLIC COMPOUNDS Dubravka Škrobot1*, Aleksandra Mišan1, Marijana Sakač1, Anamarija Mandić1, Mladenka Pestorić1, Pavle Jovanov1............................................................................................................

96

EFFECT OF CITRIC ACID ADDITION ON ANTIOXIDANT PROPERTIES OF BLACKBERRY SYRUP Ana Kalušević1,2*, Dragana Paunović1, Ana Salević1, Jasmina Rajić1, Dušica Đinović1, Tanja Petrović1, Viktor Nedović1………………………………………………………………

101

INFLUENCE OF EXTRACTION CONDITIONS ON BIOACTIVE PROFILE OF RASPBERRY LEAVES Ana Salević1*, Ana Kalušević1, Viktor Nedović1........................................................................

106

AURICULARIA AURICULA-JUDAE (BULL.:FR.) WETTST. 1885 CYTOTOXICITY ON BREAST CANCER CELL LINE (MCF 7) Aleksandra Novaković1,3*, Maja Karaman2, Sonja Kaišarević2, Tanja Radusin1, Mirjana Beribaka3, Nebojša Ilić1………………………………………………………………………..

112

TWO LAYER CHITOSAN-BEESWAX COATING FOR APPLICATION ON ARTIFICIAL COLLAGEN CASINGS Nevena Hromiš*, Vera Lazić, Senka Popović, Danijela Šuput, Sandra Bulut, Natalija Džinić, Branislav Šojić, Vladimir Tomović ............................................................................................

116

POSSIBILITY TO MAINTAIN MODIFIED ATMOSPHERE IN POUCHES MADE FROM BIOPOLYMER MATERIALS Sandra Bulut*, Vera Lazić, Senka Popović, Danijela Šuput, Nevena Hromiš, Ljiljana Popović .......................................................................................................................................

122

PROPERTIES OF ALGINATE-BASED EDIBLE FILMS INCORPORATED WITH CAPSICUM OLEORESIN Mishela Temkov1*, Jana Simonovska1, Darko Dimitrovski1, Sandra Mojsova2, Vesna Rafajlovska1, Elena Velickova1 .......................................................................................

128

OXIDATIVE CHANGES IN OSMOTICALLY DEHYDRATED PORK MEAT PACKED UNDER MODIFIED ATMOSPHERE WITH AND WITHOUT STARCH EDIBLE COATING Danijela Šuput*, Vera Lazić, Branislav Šojić, Senka Popović, Nevena Hromiš, Sandra Bulut .................................................................................................................

135

THE INFLUENCE OF PROCESS PARAMETERS ON THE CHANGE OF PAPRIKA COLOUR (Capsicum annuum L.) DURING STORAGE Miona Belović1*, Aleksandra Novaković1, Žarko Kevrešan1, Jasna Mastilović1……………...

140

HIBRID PLA/SILICA COMPOSITES WITH IMPROVED THERMAL STABILITY Tanja Radusin1*, Ivan Ristić2, Branka Pilić2, Tamara Erceg2, Aleksandra Novaković1.............

146

STARCH BASED FOAMED PACKAGING MATERIALS Maciej Combrzyński, Leszek Mościcki*, Agnieszka Wójtowicz, Tomasz Oniszczuk, Marcin Mitrus ..........................................................................................................................................

151

CONSUMER PERCEPTION OF MEAT PRODUCTS IN SERBIA AND GERMANY Jasna Djinovic-Stojanovic1, Klaus Troeger2, Milan Ristic2,*, Nemanja Knezevic3, Milica Damnjanovic3 …………………………………………………………………………………..

157

CONSUMER ACCEPTANCE AND TEXTURE PROFILE ANALYSIS OF GRILLED GOAT CHEESES Zorana Miloradovic1, Jelena Miocinovic1*, Nemanja Kljajevic1, Sanja Tesevic**, Nikola Tomic1..........................................................................................................................................

163

SENSORY PROPERTIES OF BREAD FORTIFIED WITH GRAPE POMACE Monika Šporin1, Martina Avbelj2, Boris Kovač1,3, Sonja Smole Možina2..................................

169

EVALUATION OF FETA TYPE CHEESE QUALITY BY SENSORY AND INSTRUMENTAL METHODS Miona Belović1*, Nebojša Ilić1, Mladenka Pestorić1, Nurgin Memiši2, Aleksandra Novaković1, Rada Jevtić-Mučibabić1, Dubravka Škrobot1……………………………………..

175

INFLUENCE OF SENSORY AND TEXTURAL PROPERTIES ON CONSUMERS ACCEPTABILITY OF WHITE BRINED CHEESE Emilija Najdenovska, Elena Velichkova, Bozhidar Ristovski*, Mirjana Bocevska.....................

180

IMMOBILIZATION OF CANDIDA ANTARCTICA LIPASE B ONTO MODIFIED SILICA NANOPARTICLES AND ITS APPLICATION FOR THE SYNTHESIS OF LASCORBYL OLEATE Marija Ćorović1, Katarina Banjanac1, Nevena Prlainović2, Ana Milisavljević1, Milica Carević1*, Aleksandar Marinković3, Dejan Bezbradica1 ...........................................................

186

SYNTHESIS OF GALACTITOL GALACTOSIDE USING TRANSGALACTOSYLATION ACTIVITY OF β-GALACTOSIDASE FROM ASPERGILLUS ORYZAE Milica Carević*, Katarina Banjanac, Nevena Lukić, Aleksandra Jakovljević, Marija Ćorović, Ana Milisavljević, Dejan Bezbradica .........................................................................................

193

MOLECULAR IDENTIFICATION OF PLANT GROWTH PROMOTING BACILLUS SPECIES ISOLATED FROM THE SOIL IN VOJVODINA Dragana Bjelić1, Maja Ignjatov1, Jelena Marinković1, Tatjana Popović2, Zorica Nikolić1, Jelica Gvozdanović-Varga1, Žarko Ivanović2..............................................................................

200

RATING ENDANGERMENT OF HUMAN HEALTH FROM POLLUTION CAUSED BY MERCURY IN WATER AND FOOD Veljko Đukić1, Đorđe Okanović2, Biljana Đukić1......................................................................

206

ELECTROCHEMICAL BEHAVIOUR OF IMIDACLOPRID ON BISMUTH THIN FILM ELECTRODE Ana Đurović1, Zorica Stojanović1, Snežana Kravić1, Zvonimir Suturović1, Tanja Brezo1*, Spasenija Milanović1..................................................................................................................

212

SEED OIL FROM RED HOT PEPPER FORMULATED IN NANOEMULSIONS Jana Simonovska1*, Darija Cör2, Željko Knez2, Vesna Rafajlovska1........................................

218

MOLECULAR IDENTIFICATION OF PATHOGENETIC FUSARIUM SPECIES, THE CAUSAL AGENTS OF GARLIC (Allium sativum L.) CLOVES ROT IN SERBIA Маја Ignjatov1*, Dragana Bjelić1, Tatjana Popović2, Zorica Nikolić1, Dragana Milošević1, Jelica Gvozdanović- Varga1, Žarko Ivanović2.............................................................................

224

BIOLOGICAL POTENTIAL OF POLYSACCHARIDE EXTRACTS OBTAINED FROM COMMERCIALLY GROWN OYSTER MUSHROOM STRAIN Milena Pantića*, Dunja Duvnjaka, Danka Matijaševića, Aleksandra Sknepneka, Tomasz Słowińskib, Jadwiga Turlob, Miomir Nikšića ..............................................................................

229

BIOPROCESS PROSPECTS FOR DEVELOPING CO-PRODUCTS FROM MICROALGAE - CHLORELLA VULGARIS Sayali Kulkarni1 and Zivko Nikolov1*........................................................................................

234

MEAT INDUSTRY EFFLUENTS Zvonko Nježić1*, Đorđe Okanović1 ............................................................................................

241

GAS HOLDUP AND LIQUID VELOCITY IN A MEMBRANE AIRLIFT BIOREACTOR Predrag S. Kojić1, Nataša Lj. Luki 1*, Dragan Lj. Petrović1, Svetlana S. Popović1 ...................

246

MASS-TRANSFER IN ALCOHOL SOLUTIONS IN AN EXTERNAL-LOOP AIRLIFT BIOREACTOR Predrag S. Kojić1, Nataša Lj. Lukić1*, Svetlana S. Popović1, Dragan Lj. Petrović1 ..................

250

IMPROVEMENT OF BIOACTIVITY AND TEHNOLOGICAL PROPERTIES OF WHEY PROTEIN Tanja Krunić1*, Maja Bulatović2, Milka Borić2, Marica Rakin2 ...............................................

254

ALGINATE CAPSULES AS CARRIER FOR APPLICATION OF GLUCOSE OXIDASE IN WINE INDUSTRY Verica Petkova, Irina Mladenoska*............................................................................................

261

VALIDATION DATA FOR AFLATOXIN DETERMINATION IN MAIZE BY LCMS/MS Gorica Vuković1*, Vojislava Bursić2, Jovana Kos3, Radmilo Čolović1 Đuro Vukmirović3, Ferenc Bagi2 ...............................................................................................................................

267

PUBLIC HEALTH RISK OF HISTAMINE FROM CANNED SARDINES Jelena Babic1*, Sandra Jaksic1, Brankica Kartalovic1, Aleksandra Aleksic-Agelidis2, Miroslav Cirkovic1, Jelena Petrovic1 ..........................................................................................

274

INHIBITORY EFFECT OF ESSENTIAL OILS FROM PLANTS ON LISTERIA MONOCYTOGENES Dragana Kalaba1*, Vesna Kalaba 2, Milka Stijepic 3Jovana Glusac3 ........................................

279

THE CONTENT OF BIOACTIVE COMPOUNDS IN DRY APRICOTS FROM ORGANIC AND CONVENTIONAL PRODUCTION Ewelina Hallmann1*, Katarzyna Świąder1, Anna Rogowska1 , Ewa Rembiałkowska1................ 286

COMPARISON OF SELECTED PHYSICOCHEMICAL AND SENSORY PROPERTIES OF TRADITIONAL FERMENTED SAUSAGES PRODUCED IN VOJVODINA (NORTHERN SERBIA) Predrag Ikonić1*, Branislav Šojić2, Tatjana Tasić1, Marija Jokanović2, Vladimir Tomović2, 290 Snežana Škaljac2, Aleksandra Novaković1 ................................................................................ PRODUCTION QUALITY OF FALSE FLAX (Camelina sativa (L.) Crantz) 'STEPSKI' SEED, A NEW PROMISING OILSEED CROP FOR DIFFERENT USES Ana Marjanović Jeromela1*, Sreten Terzić1, Sandra Cvejić1, Milan Jocković1, Tijana Zeremski1, Dragana Miladinović1, Siniša Jocić1, Johann Vollmann2 .........................................

296

THE INFLUENCE OF THE FOOD SURFACE AND MATRIX ON NOROVIRUS DETECTION BY RT-qPCR Dragoslava Radin*, Ivana Zivkovic ............................................................................................ 300 CYPRODINIL AND FLUDIOXONIL FUNGICIDE RESIDUES AND DISSIPATION IN LETTUCE Sanja Lazić, Dragana Šunjka*, Milica Pantelić, Slavica Vuković, Nemanja Brzaković ............

306

SAFE USE OF NEONICOTINOIDS IN SOME VEGETABLES IN TERMS OF RESIDUES Sanja Lazić, Dragana Šunjka*, Slavica Vuković .......................................................................

311

EFFECT OF ACRYLAMIDE TREATMENT ON MORPHOMETRICAL PARAMETERS OF PANCREATIC BETA CELLS Milena Stošić 1, Jelena Marković 2, Milica Matavulj 2 ...............................................................

316

THE CONTENT EXPLORATION OF SOME FOOD ADDITIVES IN DIFFERENT SAMPLES OF INSTANT COFFEE 3in1 AND 2in1 Milena Miljković N.1*, Vojkan Miljković M.2, Violeta Rakić3, Marija Petrović1 .....................

321

THE POULTRY MARKET IN UKRAINE: PROBLEMS, OBJECTIVE AND SOLUTIONS Natalia Povarova, Liudmyla Melnyk*………………………………………………………….

325

QUALITY OF BEVERAGE OF HYDROLYZED MILK PERMEATE Dragana Ilić-Udovičić 1*, Spasenija Milanović 2, Mirela Iličić 2, Anamarija Mandić 3, Dajana 329 Vukić 2, Katarina Kanurić 2, Vladimir Vukić 2 ....................................................................... CADMIUM IN BEEF, PORK AND CHICKEN MEAT Biljana Pećanac, Jelena Aničić, Slobodan Dojčinović, Drago N. Nedić ..................................

335

IMPROVEMENT OF THE WHEAT STARCH SUSPENSIONS MICROFILTRATION PROCESS USING TWISTED TAPE AS TURBULENCE PROMOTER Bojana Ikonić1*, Olivera Šimurina2, Aleksandar Jokić1, Jelena Pavličević1, Zita Šereš1, Zoltan Zavargo1, Bojana Filipčev2 ............................................................................................. 341 DEVELOPMENT OF BAKERY PRODUCTION TECHNOLOGY FUNCTIONALITY LONG TERM IMPLEMENTATION Iryna Solonytska 1, Nataliya Tkathenko1, Vitalii Dobrovolskyi1 ................................................

347

CONTENTS OF TOTAL PHOSPHORUS IN DIFFERENT MEAT PRODUCTS Vladimir Kurćubić1*, Pavle Mašković1.......................................................................................

352

BIOFILM FORMING ABILITY OF FOOD-BORNE RELATED PATHOGENS Ivana Čabarkapa1*, Dubravka Milanov2, Radmilo Čolović1, Zorica Tomičić1, Ana Varga1, Olivera Đuragić1, Sanja Popović1, Marija Škrinjar3...................................................................

358

MOLECULAR AND MORPHOLOGICAL IDENTIFICATION OF ASPERGILLUS SPECIES ON CORN SEEDS Tijana Barošević1, Ferenc Bagi1, Dragana Budakov1, Sándor Kocsubé2, János Varga†2, Mila Grahovac1, Vera Stojšin1 ...........................................................................................................

365

PRESENCE OF MYCOTOXINS AND THEIR CO-OCCURRENCE IN WHEAT FROM THE AUTONOMOUS PROVINCE OF VOJVODINA, SERBIA Elizabet Janić Hajnal*, Jovana Kos, Aleksandra Torbica .........................................................

372

ASSESSMENT OF GOOD ENVIRONMENTAL PRACTICES IN ABATTOIRS Simo Čegar1, Ilija Đekić2, Nada Šmigić2, Dragan Antić3, Bojan Blagojević1*………………...

379

MUSHROOM EXTRACTS AS NOVEL BACTERIAL ANTI-ADHESION COMPOUNDS Meta Sterniša1, Jovana Vunduk2, Anita Klaus2, Miomir Nikšić2, Sonja Smole Možina1*...........

384

CARVACROL AS ANTIMICROBIAL AGENT TOWARD PROTOTHECA ZOPFII Ivana Čabarkapa1, Dubravka Milanov2, Ružica Tomičić3, Bojana Kokić1, Dragana Plavšić1, Ljiljana Kostadinović1, Ljiljana Suvajdžić4 ................................................................................

390

MONITORING OF NEONICOTINOID RESIDUES IN HONEY SAMPLES FROM AUTONOMOUS PROVINCE OF VOJVODINA Pavle Jovanov1*, Marijana Sakač1, Jovana Kos1, Bojana Šarić1, Aleksandar Ivezić2, Nataša 397 Nedeljković1, Milica Pojić1...................................................................................................... PROTEIN DIGESTIBILITY-CORRECTED AMINO ACID SCORE AND DIGESTIBLE INDISPENSABLE AMINO ACID SCORE IN RICE, RYE AND BARLEY Soňa Nitrayová*1, Matej Brestenský1, Jaroslav Heger1, Peter Patráš1……………………….. 402 THE RATIO OF NEUTRAL TO ACIDIC MUCINS IN RAT COLON AFTER SINGLE ORAL ACRYLAMIDE APPLICATION Ivana Koledin*1, Renata Kovač1,, Vesna Rajković1,, Milica Matavulj1........................................

408

CHANGES IN MYCOTOXINS OCCURRENCE IN MAIZE FROM REPUBLIC OF SERBIA Jovana Kos1*, Elizabet Janić Hajnal1, Ivan Milovanović1, Bojana Šarić1, Pavle Jovanov1, Jasmina Lazarević1, Ljubiša Šarić1 ............................................................................................. 412 TOXIGENIC POTENTIAL OF ASPERGILLUS FLAVUS CULTURES ISOLATED FROM WHEAT GRAINS Jelena Krulj*1, Aleksandra Bočarov Stančić2, Saša Krstović3, Jovana Kojić1 Jovana Đisalov1, Tea Brlek1, Marija Bodroža Solarov1…………………………………………………………..

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COLOR STABILITY OF VIRGIN OLIVE OIL AT MODERATE TEMPERATURES TESTING Tanja Lužaić1*, Vesna Vujasinović2, Seddiq Esalami1, Biljana Rabrenović3…………………..

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ANALYTICAL STUDY OF KALINJOT OLIVE CULTIVAR IN THE SOUTHERN REGIONS OF ALBANIA Stela Velo1 *, Dritan Topi2 ...........................................................................................................

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ENTERECOCCUS SPP. PRESENCE DURING FERMENTATION, DRYING AND STORAGE OF PETROVSKÁ KLOBÁSA Vesna Janković1, Brankica Lakićević1, Vesna Đorđević1, Tatjana Tasić2, Predrag Ikonić2, Natalija Džinić3................................................................................................

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ANETHUM GRAVEOLENS SEED OIL: ANTIBACTERIAL ACTIVITY AGAINST ESCHERICHIA COLI Ana Varga1*, Milica Aćimović2, Ivana Čabarkapa1, Bojana Filipčev1, Dragana Plavšić1, Zvonko Nježić1 ...........................................................................................................................

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EFFECT OF REPLACING BACKFAT WITH MICROCRYSTALLINE CELLULOSE GEL ON QUALITY OF FRANKFURTER Doncheva Toni1, Djordje Okanović2*, Tatjana Tasić2, Dragana Plavšić2, Nedjo Jelić3, Simo Duvnjak1 .....................................................................................................................................

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TOXIGENICITY OF FUNGI ISOLATED FROM SERBIAN CEREAL KERNELS Aleksandra Bočarov-Stančić1*, Saša Krstović2, Slavica Stanković3, Jelena Krulj4, Snežana Janković1, Marija Bodroža-Solarov4, Igor Jajić2 ……………………………………………

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FIVE YEARS STUDY OF SEASONAL VARIATIONS OF MILK COMPOSITION Tanja Keskic1*, Zorana Miloradovic2, Vladimir Bandzov1, Miladin Gavric1, Predrag Pudja2, Jelena Miocinovic2 .....................................................................................................................

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BIOCHEMICAL CHARACTERIZATION OF SATUREJA HORVATII S.L., LAMIACEAE Emilia Šefer1*, Nevena Grujić-Letić1..........................................................................................

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EXTRACTION AND CHEMICAL CHARACTERIZATION OF PURPLE WILLOW (SALIX PURPUREA L.) Emilia Šefer1*, Nevena Grujić-Letić1..........................................................................................

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BREAD WHEAT YIELDS AND YIELD TRENDS IN DIFFERENT MANAGEMENT SYSTEMS OF LONG-TERM TRIALS IN SLOVENIA Aleš Kolmanič*1, Branko Lukač 1, Lovro Sinkovič1, Vladimir Meglič1 ......................................

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EFFECT OF DIFFERENT MANAGEMENT SYSTEMS ON YIELD AND MULTIMINERAL COMPOSITION OF OAT GRAINS (Avena sativa L.) Lovro Sinkovič 1*, Aleš Kolmanič1, Marijan Nečemer2, Branko Lukač1, Vladimir Meglič1……

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IMPROVEMENT OF FORMULATION OF WHEAT GRAIN BATCH WITH IMPROVED QUALITY Alla Borta1 ……………………………………………………………………………………... 480 THE INCREASE OF TECHNOLOGICAL PERFORMANCE OF FLOUR FOR THE PRODUCTION OF BREAD Olivera Šimurina1*, Dragana Jovanović2, Mirjana Demin2, Bojana Filipčev1, Dragana Šoronja-Simović3, Bojana Ikonić3, Mladenka Pestorić1 ……………………………………….

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RELATIONSHIP OF STARCH DAMAGE TO WHEAT DOUGH RHEOLOGICAL BEHAVIOUR Slađana Rakita*1, Aleksandra Torbica1, Miroslav Hadnađev1, Tamara Dapčević Hadnađev1, Milica Pojić1 …………………………………………………………………………………

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GLUTOPEAK METHOD: ASSESSMENT OF ITS ABILITY TO DISCRIMINATE AMONG WHEAT FLOURS OF DIFFERENT QUALITY Miroslav Hadnađev1*, Tamara Dapčević Hadnađev1, Milica Pojić1 ………………………….

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THE EFFECT OF MIXING AND THERMAL PROCESSING ON THE CONTENT AND COMPOSITION OF FREE AND BOUND PHENOLIC ACIDS IN WHEAT FLOUR DOUGH Nada Nikolić1*, Jelena Mitrović1, Miodrag Lazić1, Ivana Karabegović1, Gordana Stojanović2, Jasna Mastilović3………………………………………………………………….

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MILLABILITY AND FERMENTABILITY OF VARIOUS ZP MAIZE GRAIN FOR WET-MILLING AND BIOETHANOL PRODUCTION Marija Milašinović-Šeremešić1*, Milica Radosavljević1, Valentina Semenčenko1, Dušanka Terzić1, Ljubica Dokić2, Ljiljana Mojović3…………………………………………………….

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VARIABILITY OF SEED OIL QUALITY IN SUNFLOWER GERMPLASM Sandra Cvejić1*, Siniša Jocić1, Milan Jocković1, Ana Marjanović Jeromela1, Dragana Miladinović1, Ivana Imerovski1, Aleksandra Dimitrijević1 ……………………………………

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EFFECT OF HULL ON OXIDATIVE STABILITY OF COLD PRESSED SUNFLOWER OIL OF OLEIC TYPE Ranko Romanić*1 ……………………………………………………………………………...

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EFFECT OF GROWTH STAGE ON NUTRITIVE VALUE AND MINERAL COMPOSITION OF DANDELION (TARAXACUM OFFICINALE WEB.) AT SPRING CUT Branko Lukač1*, Lovro Sinkovič1, Aleš Kolmanič1 Marijan Nečemer2, Vladimir Meglič1……… 520 MORPHOLOGICAL FEATURES, PRODUCTIVITY AND QUALITY OF POT MARIGOLD (Calendula officinalis L.) cv. “DOMAĆI ORANŽ“ Vladimir Filipović1*, Vladan Ugrenović2, Dragoja Radanović1, Tatjana Marković1, Vera Popović3, Milica Aćimović3, Vladimir Sikora3 …………………………………………………

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SHELF LIFE OF RAMSONS (Allium ursinum L.) UNDER DIFFERENT STORING CONDITIONS Alena Tomšik1*, Jasna Mastilović1, Žarko Kevrešan1, Senka Vidović2 ....................................

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VARIATION IN SWEETNESS OF THE CHESTNUT FRUITS (Castanea sativa Mill.) FROM MACEDONIA Marjan Slavkov1, Bozhidar Ristovski2*, Mirjana Bocevska2 ………………………………….

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COLOR PROFILE OF SOUR CHERRY VARIETIES FROM MACEDONIA Enise Sipahi1, Bozidar Ristovski1*, Jerneja Jakopic2, Ana Slatnar2, Franci Stampar2, Mirjana Bocevska1 ......................................................................................................................

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ANTHOCYANINS IN TRADITIONALLY PREPARED SOUR CHERRY LIQUEURS Bozidar Ristovski1*, Jerneja Jakopic2, Ana Slatnar2, Franci Stampar2, Mirjana Bocevska1

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ANTIFUNGAL ACTIVITY OF PARSLEY ESSENTIAL OIL Dragana Plavšić 1*, Gordana Dimić 2, Đorđe Psodorov1, Ljubiša Šarić1, Ivana Čabarkapa1, Dragan Psodorov3, Anamarija Mandić1 ……………………………………………………….

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INFLUENCE OF DIFFERENT DRYING METHODS ON PHYSICO-CHEMICAL PROPERTIES OF RASPBERRIES Anita Vakula1*, Milan Đurica2, Zdravko Šumić1, Aleksandra Tepić Horecki 1, Senka Vidović1, Branimir Pavlić1 ……………………………………………………………...

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SEPARATION OF PECTIN FROM SUGAR BEET JUICE BY BINARY SISTEM CALCIUM SULPHATE/ALUMINIUM SULPHATE Tatjana Kuljanin1*, Vladimir Filipović1, Milica Nićetin1, Biljana Lončar1, Ana Muzalevski1, Rada Jevtić-Mučibabić2 ………………………………………………………………………..

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THE POTENTIAL USE OF ABY-6 STARTER CULTURE IN FERMENTATION OF SOY BASED SUBSTRATES

Milka Borić1, Maja Bulatović1, Danica Zarić2, Tanja Krunić3*, Marica Rakin1 ……...

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CELERY ROOT OSMOTIC DEHYDRATION MASS TRANSFER KINETICS COMPARISON IN TWO OSMOTIC SOLUTIONS Vladimir Filipović1*, Milica Nićetin1, Biljana Lončar1, Violeta Knežević1, Jelena Filipović2, Lato Pezo3 ……………………………………………………………………………………...

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OSMOTIC DEHYDRATION OF FISH IN COMPLEX HYPERTONIC SOLUTION Biljana Lončar1*, Lato Pezo2, Vladimir Filipović1, Milica Nićetin1, Violeta Knežević1, Tatjana Kuljanin1, Lidija Jevrić1 ………………………………………………………………

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APPLICATION OF PCA METHOD FOR TEXTURAL PROPERTIES OF THREE SERBIAN TRADITIONAL DRY FERMENTED SAUSAGES Marija Jokanović1*, Bojana Ikonić1, Predrag Ikonić2, Vladimir Tomović1, Tatjana Tasić2, Snežana Škaljac1, Branislav Šojić1, Maja Ivić1, Natalija Džinić1 ……………………………...

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INDIGENOUS AND NON-INDIGENOUS PATHOGENS IN FISH MEAT: THE IMPORTANCE FOR HUMAN HEALTH Dragana Ljubojević1*, Dubravka Milanov1, Vladimir Radosavljević2, Miloš Pelić1, Nikolina Novakov3, Brankica Kartalović1, Miroslav Ćirković1.................................................................. ESSENTIAL OILS AND THEIR INHIBITORY EFFECT ON SALMONELLA ENTERITIDIS, ESCHERICHIA COLI, LISTERIA MONOCYTOGENES AND PSEUDOMONAS AERUGINOSA Vesna Kalaba1, Vesna Gojković2 Željka Marjanović Balaban 3, Dragana Kalaba4 ..................

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BETAINE CONTENT IN CEREAL AND PSEUDOCEREAL Jovana Kojić*1, Nebojša Ilić1, Jelena Krulj1, Anamarija Mandić1, Bojana Filipčev1, Tea Brlek1, Marija Bodroža Solarov1 .........................................................................................

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EFFECT OF THE OSMOTIC PRE-TREATMENT ON THE CONVECTIVE AIR DRYING KINETICS OF APRICOT Ivan Pavkov1*, Mirko Babić1, Milivoj Radojčin1, Zoran Stamenković1, Siniša Bikić2, Maša Bukurov2 ............................................................................................................................

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EFFECT OF SUGAR BEET MOLASSES ADDITION ON THE COMPOSITION OF SUGAR PRODUCTS Rada Jevtić-Mučibabić1, Bojana Filipčev1, Branislav Bogdanović2, Jovana Kojić1, Olivera Šimurina1, Jelena Filipović1, Jasmina Živković1 ........................................................... EFFECT OF RAINWATER ON HUNGARIAN WHEAT QUALITY Balázs P. Szabó1, Ernő Gyimes1, Antal Véha1.............................................................................

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BROCCOLI FROM FARM TO HEALTH – RESEARCH AND CHALLENGES 1

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Diego A. Moreno *, Nieves Baenas , Débora Villaño , María Teresa López-Chillón , Pilar Zafrilla , 1 Cristina García-Viguera 1

Phytochemistry Lab. Food Science and Technology Dept., CEBAS-CSIC, Campus universitario de Espinardo – 25, 30100 Murcia, Spain 2 Dpto. Ciencias de la Salud. Universidad Católica ‘San Antonio’ de Murcia (UCAM). Campus de los Jerónimos, 30107 Murcia, Spain

*Corresponding author: E-mail address: [email protected]

ABSTRACT Nowadays, society accepts the direct relationship between foods and health. In this sense, fruits and vegetables are the major components in balanced diets, particularly for their implications in the prevention of certain conditions and types of cancer and chronic diseases. Broccoli (Brassica oleracea L. var. italica) is a cruciferous food very rich in glucosinolates (GLS) and their bioactive hydrolysis products, the isothiocyanates (ITC), which may exert beneficial biological activities against different conditions through protection against inflammation and oxidative stress in the cells, slowing or preventing a number of cancers and other diseases, such as cardiovascular or neurodegenerative. Broccoli inflorescences and sprouts are the main dietary sources of glucoraphanin (GR), precursor glucosinolate of sulphoraphane (SFN), widely studied in different pathological conditions. Besides, U.S. Food and Drugs Administration (FDA) recently approved glucoraphanin as GRAS (generally accepted as safe) for use in foods and functional ingredients boosting the interest and potential of Brassica foods for health worldwide. Current research includes multidisciplinary work integrating the food chain: Evaluation of the pre- and post-harvest factors affecting the phytochemical composition of foods; Agro-waste recovery by elaboration of new ingredients for culinary applications and food industry; Enriching foods in bioactive phytochemicals using sustainable practices; Evaluating the in vivo bioavailability and metabolism of GLS/ITC; Incorporation of new foods in clinical interventions for adult health. This holistic approach from plant to food and health, will allow us to generate knowledge for the challenges of the society, and is necessary to let consumers know that Brassica vegetables likely provide excellent health effects. Keywords: Brassicaceae, glucoraphanin, functional food, inflammation, health

INTRODUCTION Nowadays it is well accepted in society a positive and direct relationship between food and health. In this sense, fruits and vegetables are a very important part of a balanced diet, particularly for their role in the prevention of certain diseases and types of cancer. People’s diet offer a greater and more diverse group of plant bioactives than do drugs, and they often do not realise that many drugs are derived from the compounds originally discovered in plant foods. Numerous epidemiological studies indicate that Brassica vegetables in general, and broccoli in particular, protect humans against cancer since they are rich sources of glucosinolates as well as possessing a high content of flavonoids, vitamins and mineral nutrients. Some criteria have been proposed to evaluate the possibilities of developing new “functional foods” to reduce the risk of specific cancers; largely in broccoli, which is associated with cancer protection. Processing conditions, domestic cooking, etc., affect the health-promoting properties of broccoli and these have been widely studied (Moreno et al., 2006). Therefore, it is important to ascertain the relevance of broccoli and cruciferous foods for human health, through different levels of research and development and innovative applications, from the knowledge of phytochemicals and health-promoting compounds in broccoli and their analysis, the influence of agricultural, processing and technological parameters on the composition and quality of the food and to scientifically validate its

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functionality through bioavailability, metabolism and bioactivity from in vitro to the clinical interventions and studies.

BROCCOLI: A WELLNESS FOOD RICH IN HEALTH-PROMOTING BIOACTIVES Broccoli (Brassica oleracea L. [Italica Group]) belongs to Brassicaceae plant family including a larg variety of species and cultivars, some of the most known are B. oleracea (i.e. cabbages, Brussels sprouts, broccoli), B. rapa (turnips), B. napus (radishes), Sinapis spp. (mustards), etc. In the recent years, these crops are increasingly consumed for organoleptic characteristics and possible health benefits as a good source of bioactive compounds.

Figure 1. Nutrients and phytochemicals in crucifers (broccoli) and associated health benefits

The sulphur-nitrogen containing compounds glucosinolates (GLS) are almost exclusively found in this family, being their beneficial health effect supposed to be induced by their hydrolysis (bioactive) products, the isothiocyanates (ITC). The phenolic compounds mainly studied are flavonols, anthocyanins and hydroxycinnamic acids, which principal bioactivity is their antioxidant capacity. The carotenoids, as well as vitamins (C, E, K) have also been considered as nutrients with biological activity. GLS have no known bioactivity themselves, but are hydrolysed to release ITC, highly reactive compounds that can activate the mammalian cellular transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and by so doing, improve host defense. The increasing detoxification enzymes to eliminate chemical carcinogens and additional mechanisms, many of them triggered by the activation of the transcription factor Nrf2, have been uncovered. The phytochemical wealth of Brassica foods is gathering attention from the scientific community for being potentially protective for the cardiovascular system and against certain types of cancer and neurological disorders, mainly because of their anti-inflammatory and antioxidant properties (Baenas et al. 2016). Therefore, there is a role for optimizing the content of GLS and the myrosinase-dependent action to form bioactive ITC from the GLS to increase the efficacy of Brassica foods for health and wellbeing, acting in the different steps of the food chain from the seeds to the plant and food for human health (Jeffery, 2014; Moreno et al., 2006).

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BROCCOLI AGRI-FOOD CHAIN RESEARCH Different factors could affect the bioactive compounds and phytonutrients of broccoli and could be classified into pre- and post-harvest aspects. The first group implies agronomic and environmental conditions or genetic and ontogenetic factors. Broccoli varieties for different production cycles (‘Naxos’, ‘Parthenon’, etc.) performed best in the areas of production under adverse conditions as found in Mediterranean SE Spain, where they overcome the semiarid climate with saline irrigation to maintain the all-year around exports (Dominguez-Perles et al. 2011). The application of elicitors during head formation could be beneficial for the enrichment in phytochemicals in broccoli, enhancing its nutritional value without using genetic approaches (Moreno et al., 2008). The available evidences indicate that both genetic background and crop management strategies are involved in the postharvest quality, bioactivity and bioavailability of the bioactive compounds in broccoli and cruciferous foods. Broccoli is mainly consumed cooked, and domestic handling and processing, including cooking processes, have been shown to considerably affect its health-promoting compounds (López-Berenguer et al. 2007), with great losses of vitamin C and significant losses of phenolic compounds and glucosinolates, depending on the cooking procedure, the presence of water and the time of cooking. Thus, genotype, fertilization, irrigation water, as well as age and harvesting time are the most important pre-harvesting factors, which may affect the bioactive composition of broccoli. The post-harvest factors such as storage, transport, and domestic cooking have been widely studied in our research group with the focus on the wide bioactive content of broccoli and how the pre- and post-harvest factors affect these healthpromoting phytochemicals (Baenas et al. 2016; Moreno et al., 2006, 2007).

INNOVATION WITH INGREDIENTS FROM BY-PRODUCTS The broccoli trade represents a major socio-economic activity in Murcia (SE Spain), and many by-products of the agri-food industry may be useful as source of nutrients and potentially functional ingredients, giving the opportunity to obtain added value products. Previous studies have been focused on edible florets, but we were interested also in adding value to broccoli by-products that represent a real problem in the production sites because of no intended use for this material has been envisaged. The broccoli-derived by-products are a good source of bioactive ingredients for food industry and reduction of environmental problems (Dominguez-Perles et al., 2010). Broccoli ingredient added to green tea resulted in a combination of phytochemicals with anti-tumoral activity (Dominguez-Perles et al. 2012), with potential for further developments in mechanistic models and the design of new functional foods and beverages.

HEALTH BENEFITS OF BROCCOLI CONSUMPTION To prevent and treat obesity is a world priority. Obesity is defined as the abnormal or excesive accumulation of fat which can negatively affect health and involves imbalaces in the glucose metabolism, high blood pressure, dyslipidaemia, and is characterised by a chronic proinflammatory state. The studies in human adults showed that the inflammation markers, interleukin-6 (IL-6) and C-reactive protein (PCRe), and the tumour necrosis factor-alpha (TNF-α) are elevated when there is a high visceral fat. Nowadays, more than 55% of the adult Spanish population is obese and almost 60% of the population of the EU-27, is overweight or obese. It is urgent and necessary to develop safe and effective strategies for the treatment of this pathology. The use of foods rich in bioactive and bioavailable compounds to act in the adipose tissue (adiposity, inflammation), may represent an useful tool for nutritional and clinical interventions to control and treat this chronic condition (AGL2013-46247-P Project). Broccoli (Brassica oleracea L. var. Italica), from the cruciferous family (Brassicaceae), is rich in glucosinolates , bioactive compounds, and their hydrolysis products, the isothiocyanates, 3


may exert a beneficial activity against different health conditions. The broccoli sprouts (Brassica oleracea var. italica) are germinated seeds of only a few days of age, rich in bioactive phytochemicals (vitamins, minerals, polyphenolic natural antioxidants and glucosinolates), with concentration levels up to 10 times those found in adult broccoli (PérezBalibrea et al., 2008). The broccoli sprouts are rich in glucoraphanin, glucosinolate precursor of the isothiocyanate sulforaphane (SFN), widely studied because of its health-promoting characteristics. The beneficial effects of the consumption of broccoli are linked to the ITC formed by the hydrolitic action of the plant myrosinase during mastication and the glucosidases present in the gastrointestinal tract (Jeffery, 2014; Moreno et al. 2006). However, there are no studies showing the beneficial effect of the GLS/ITC on obese population with associated chronic inflammation. The results of this research project will give us the possibility of incorporate the broccoli sprouts as a healthy food in the control and treatment of obesity and for the general population wellbeing.

CONCLUDING REMARKS The increasing awareness among scientist, food manufacturers as well as consumers of the beneficial effects of the phytochemicals present within Brassicaceae family (i.e. broccoli) has led to different methods to improve the quality and production of these vegetables. These investigations on increasing levels of Brassica phytochemicals may have a potential for human intervention studies to investigate the effects of a specific compound on human health. The recent works suggest that the potential for chemoprevention by dietary intervention will needs the establishment of dietary recommendations including 3 to 5 servings of cruciferous foods per week, besides the current recommended 3-to-5 a day portions of fruits and vegetables, based in scientific and epidemiological evidences of the influence of bioactive compounds from broccoli and cruciferous-based foods on the different stages of degenerative diseases and cancer.

ACKNOWLEDGEMENTS This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) through Research Project AGL2013-46247-P, and the Grant for Research Groups of Excellence from the Murcia Regional Agency for Science and Technology (Fundación Séneca), Project 19900/GERM/15. N. Baenas was funded by a FPU (Formación Profesorado Universitario) grant of the Spanish Ministry of Education.

REFERENCES Baenas N., Francisco M., Velasco P., Cartea M.E., García-Viguera C., Moreno D.A. (2016). Bioactive Compounds from Brassicaceae as Health Promoters. In: Luis R. da Silva and Branca Silva (Eds.), Natural Bioactive Compounds from Fruits and Vegetables as Health Promoters: Part 2. (pp. 27-47). Sharjah, United Arab Emirates: Bentham Science Publishers Ltd. ISBN: 978-1-68108-244-8. Domínguez-Perles R., Martínez-Ballesta M.C., Carvajal M., García-Viguera C., Moreno D.A. (2010). Broccoliderived by-products – A promising source of bioactive ingredients. Journal Food Science 75 (4), C383-92. Dominguez-Perles R., Martínez-Ballesta M.C., Riquelme F., Carvajal M., García-Viguera C., Moreno D.A. (2011). Novel varieties of broccoli for optimal bioactive components under saline stress. Journal Science Food Agriculture 91(9), 1638-1648. Domínguez-Perles R., Moreno D.A., García-Viguera C. (2012). Analysis of the tumoral cytotoxicity of Green tea-infusions enriched with broccoli. Food Chemistry 132 (3), 1197-1206. Jeffery E.H. (2014). Cancer prevention with Brassica vegetables. ISHS 2015. XXIX IHC – Proceedings of the VI Int. Symp. on Human Health Effects of Fruits and Vegetables (FAVHEALTH 2014). Acta Hortic. 1106, 143-146. DOI: 10.17660/ActaHortic.2015.1106.22 López-Berenguer C., Carvajal M., Moreno D.A., García-Viguera C. (2007). Effects of microwave cooking conditions on bioactive compounds present in broccoli inflorescences. Journal Agricultural Food Chemistry 55 (24), 10001-10007. Moreno D.A., Carvajal M., López-Berenguer C., García-Viguera C. (2006). Chemical and biological characterisation of nutraceutical compounds of broccoli. Journal Pharmaceutical and Biomedical Analysis 41 (5), 1508-1522.

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III International Congress “Food Technology, Quality and Safety” Moreno D.A., López-Berenguer C., Carvajal M., García-Viguera C. (2007). Health Benefits of Broccoli. Influence of Pre- and Post-harvest Factors on Bioactive Compounds. Food 1(2) 297-312. Moreno D.A., López-Berenguer C., Martínez-Ballesta M.C., Carvajal M., García-Viguera C. (2008). Basis for the new challenges of growing broccoli for health in hydroponics. Journal Science Food Agriculture 88 (8), 1472-1481. Pérez-Balibrea S., Moreno D.A., García-Viguera C. (2008). Influence of light on health-promoting phytochemicals of broccoli sprouts. Journal Science Food Agriculture 88 (5), 904-910.

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MOLECULAR ANALYSIS OF NS Cucurbita moschata COLLECTION 1*

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Dragana Miladinović , Aleksandra Dimitrijević , Milka Brdar-Jokanović , Ivana Imerovski , Vladimir 1 1 1 Sikora , Ana Marjanović Jeromela , Siniša Jocić 1

Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia


ABSTRACT Although Cucurbita moschata Duch, also known as tropical pumpkin, is being adapted to a wide range of climatic and soil conditions, it is a scientifically neglected species. Consumers appreciate its fruit due to its sweet and mild taste, as well as high nutritive value. Knowledge of genetic relationships among genotypes is essential for the effective utilization of germplasm, especially for poorly characterized species such as C. moschata. C. moschata collection in Institute of Field and Vegetable Crops, Novi Sad, consists of 38 accessions, comprising commercial cultivars and hybrids collected over a long period from all over the world; material resulting from Institute’s comprehensive breeding programs; as well as old cultivars and locally adapted populations. In molecular diversity studies, initial characterization of 19 accessions has been done with 5 SSRs, previously used for Cucurbita molecular diversity studies. All tested SSRs were polymorphic. In total, 14 bands were amplified, with each primer having at least two alleles. Cluster analysis showed great variability of genotypes within the collection. There was no correlation between clustering and geographical origin of the tested genotypes. The obtained results, in combination with phenotypic observations, will be used for establishment of a core collection of genotypes to be used in breeding for improved fruit quality, thus enabling creation of commercial varieties with improved nutritional properties. Keywords: Cucurbita moschata, variability, SSR analysis

INTRODUCTION Cucurbita moschata (Duchesne ex Lam.) Duchesne ex Poir. is a traditional vegetable crop with broad nutritional and economic importance, which is grown for the seeds, immature and mature fruit, processed products, traditional medicine, decoration, and livestock feed (Wu et al., 2011). Although it is adapted to a wide range of climatic and soil conditions, it is a scientifically neglected species. Diversity in plant genetic resources provides an opportunity for plant breeders to develop new and improved cultivars with desirable characteristics, which include both farmerspreferred traits such as yield potential and breeders preferred traits such as pest and disease resistance (Govindaraj et al., 2015). That is why the knowledge of genetic relationships among genotypes is essential for the effective utilization of germplasm, especially for poorly characterized species, such as C. moschata (Gwanama et al., 2000). Most of the analyses of genetic variability involving in C. moschata have focused on the establishment of phylogenetic relationships with other Cucurbita species using genetic markers (Baranek et al., 2000; Tsivelikas et al., 2009; dos Santos et al., 2012). Molecular diversity of C. moschata germplasm was analyzed by RAPD markers (Youn and Chung, 1998; Gwanama et al., 2000), as well as by PCR SSCP and chloroplast sequence data (Barboza et al., 2012) and SRAP and AFLP markers (Ferriol, et al., 2003; Wu et al., 2011). Gong et al. (2008) developed SSR-based genetic linkage map, while Wu et al. (2014) developed and validated SSR markers for this species. Collecting and determining the degree of variability within the different accessions is necessary prior to their use in breeding programs. The aim of our study was to investigate DNA polymorphism in accessions of C. moschata by using selected SSR primers. 6


MATERIAL AND METHODS Nineteen accessions of C. moschata originating from the Cucurbita spp. collection of Institute of Field and Vegetable Crops (IFVCNS), Novi Sad, Serbia, were used in this study (Table 1). Table 1. Accessions of C. moschata used in the study Accession Mo 1 Mo 2 Mo 3 Mo 4 Mo 5 Mo 7 Mo 8 Mo 11 Mo 12 Mo 15 Mo 19 Mo 22 Mo 23 Mo 30 Mo 33 Mo 34 Mo 35 Mo 38 2/15

Origin Nepal Tajikistan USA India Mexico Suriname South Korea Burkina Faso Zambia Egypt Zimbabwe Maldives Bolivia Serbia France Unknown Unknown Serbia Unknown

DNA was extracted from leaves by the use modified CTAB protocol (Permingeat et al., 1998) and DNA polymorphism was evaluated in reactions with five SSR primers,. PCR was performed as described by Dimitrijević et al. (2010). Products of PCR amplification were run on 2% agarose gels and visualized with the BIO-Print system (Vilber Lourmat, Marne-LaVallée, France). Each fragment that was amplified was treated as binary unit character and scored “0” for absence and “1” for presence. An unweighted pair group arithmetic mean method (UPGMA) cluster analysis was performed, using the average linkage method.

RESULTS AND DISCUSSION Five SSR primers produced 14 fragments which were all polymorphic, indicating a high degree of diversity. The similar results were obtained by Wu et al. (2014) who randomly designed 57 pairs of primers based on the SSRs developed in pumpkin and used them in 12 pumpkin varieties. The results of detection showed that, of the 57 pairs of SSR primers, 35 pairs for PCR were able to produce bands with polymorphism and 14 pairs of primers produced bands without polymorphism in 12 varieties, and eight pairs of primers were able to produce no or blurred bands. Wu et al. (2012), who analyzed C. moschata accessions with AFLP primers, also found that nine pairs of EcoRI/MseI primers that were used in the study produced 500 fragments, of which 75.57% were polymorphic. In contrast to them, Young and Chung (1998) found only 16 polymorphic bands out of 86 bands produced by 11 RAPD primers that were used for analysis of South Korean C. moschata accessions. Cluster analysis, based on 14 polymorphic DNA fragments amplified by 5 primers, was used to reveal relationships among 19 C. moschata accessions from IFVCNS collection. All the accessions grouped in a single cluster, except for Mo3, Mo 11 and Mo 23 (Figure 1). The greatest similarity was found between Mo 33 and Mo 34 and Mo 19 and Mo 22. Accession Mo 30, originating from Serbia was more similar to accession Mo 5, originating from Mexico, than to Mo 38, another accession from Serbia. Furthermore, the accessions from the same 7


continents did not group together. This is in contrast to the results of other authors who found close correspondence between genetic relatedness and geographical distribution of C. moschata accessions (Hazra et al., 2007). Ferriol et al. (2004) who analyzed C. moschata accessions from the Canary Islands, Spanish peninsula, as well as Central and South America with SRAP and AFLP markers found that with both markers, the accessions clustered according to geographical origin. The similar results were obtained by Young and Chung (1998) with RAPD markers and Gwanama et al. (2000) with RAPD markers. Similar to our results, Barboza et al. (2012) could not find a clear separation according to origin in C. moschata accessions from Mesoamerica using the intergenic markers ITS1–ITS2, the chloroplast intergenic region tRNL–F and sequencing. The authors suggested that the differences between earlier works with other markers and their research may by the fact that genetic material from Mesoamerica, which is considered to be the center of origin and domestication of C. moschata, was used in the study. This indicates that variability within the IFVCNS C. moschata collection is comparable to that found in different C. moschata centers of diversity, and that the accessions within it could be directly used in applied breeding programs. Mo8 Mo2 Mo1 Mo7 Mo15 Mo35 Mo33 Mo34 Mo4 2/15 Mo30 Mo38 Mo5 Mo12 Mo22 Mo19 Mo23 Mo3 Mo11

Figure 1. Dendrogram showing relationship among different accessions of C. moschata based on SSR markers using UPGMA algorithm and average distances

CONCLUSIONS The knowledge of the relationships between genotypes is essential for the proper use of germplasm, especially for species that are not well characterized (Gwanama et al., 2001). SSR analysis revealed the great variability of tested C. moschata accessions from IFVCNS collection. No correlation between clustering and geographical origin of the tested genotypes was found. The obtained results, in combination with phenotypic observations, will be used for establishment of a core collection of genotypes to be used in breeding for improved fruit quality, as well as other agronomically important traits, thus enabling creation of commercial varieties with improved nutritional properties.

ACKNOWLEDGEMENTS This work was supported by Ministry of Education, Science and Technological Development, Republic of Serbia, project TR 31025. 8


REFERENCES Baranek, M., Stift, G., Vollmann, J., Lelley, T. (2000). Genetic diversity within and between the species Cucurbita pepo, C. moschata and C. maxima as revealed by RAPD markers. Cucurbit Genetics Cooperative Report, 23, 73-77. Barbozaa, N., Albertazzia, F.J., Sibaja-Corderob, J.A., Mora-Umañac, F., Astorgad, C., Ramíreza, P. (2012). Analysis of genetic diversity of Cucurbita moschata (D.) germplasm accessions from Mesoamerica revealed by PCR SSCP and chloroplast sequence data. Scientia Horticulturae, 134 (1), 60-71. Dimitrijević, A., Imerovski, I., Miladinović, D., Tančić, S., Dušanić, N., Jocić, S., Miklič, V. (2010). Use of SSR markers in identification of sunflower isogenic lines in late generations of backcrossing. Helia, 33 (53), 191-198. dos Santos, M.H., Rodrigues, R., Simões Azeredo Gonçalves, L., Pombo Sudré, C., Gonzaga Pereira, M. (2012). Agrobiodiversity in Cucurbita spp. landraces collected in Rio de Janeiro assessed by molecular markers. Crop Breeding and Applied Biotechnology 12, 96-103. Ferriol, M., Picó, B., Fernández de Córdova, P., Nuez, F. (2004). Molecular diversity of a germplasm collection of squash (Cucurbita moschata) determined by SRAP and AFLP markers. Crop Science, 44 (2), 653-664. Gong, L., Pachner, M., Kalai, K., Lelley, T. (2008). SSR-based genetic linkage map of Cucurbita moschata and its synteny with Cucurbita pepo. Genome, 51 (11), 878-887. Govindaraj, M., Vetriventhan, M., Srinivasan, M. (2015). Importance of genetic diversity assessment in crop plants and its recent advances: An overview of its analytical perspectives. Genetics Research International, http://dx.doi.org/10.1155/2015/431487. Gwanama, C., Botha, A., Labuschagne, M. (2001). Genetic effects and heterosis of flowering and fruit characteristics of tropical pumpkin. Plant Breeding, 120, 271-272. Gwanama, C., Labuschagne, M.T., Botha, A.M. (2000). Analysis of genetic variation in Cucurbita moschata by random amplified polymorphic DNA (RAPD) markers. Euphytica 113, 19-24. Hazra, P., Mandal, A.K., Dutta, A.K., Ram, H.H. (2007). Breeding pumpkin (Cucurbita moschata Duchh. Ex Poir.) for fruit yield and other characters. International Journal of Plant Breeding, 1 (1), 51-64. Permingeat, H.R., Romagnoli, M.V., Vallejos, R.H. (1998). A simple method for isolating high yield and quality DNA from cotton (Gossypium hirsutum L.) leaves. Plant Molecular Biology Reporter,16, 1-6. Tsivelikas, A.L.1, Koutita, O., Anastasiadou, A., Skaracis, G.N., Traka-Mavrona, E., Koutsika-Sotiriou, M. (2009). Description and analysis of genetic diversity among squash accessions. Brazilian Archives of Biology and Technology, 52 (2), 271-283. Wu, J., Chang, Z., Wu, Q., Zhan, H., Xie, S. (2011). Molecular diversity of Chinese Cucurbita moschata germplasm collections detected by AFLP markers. Scientia Horticulturae, 128, 7-13. Wu, T., Luo, S., Wang, R., Zhong, Y., Xu, X., Lin, Y., He, X., Sun, B., Huang, H. (2014). The first Illuminabased de novo transcriptome sequencing and analysis of pumpkin (Cucurbita moschata Duch.) and SSR marker development. Molecular Breeding, 34 (3), 437-1447. Youn, S.J., Chung, H.D. (1998). Genetic relationship among the local varieties of the Korean native squashes (Cucurbita moschata) using RAPD technique. Journal of Korean Society Horticulture Science, 35, 429437.

9


FIBER HEMP AS A VALUABLE SOURCE OF NUTRIENTS AND NUTRACEUTICALS 1

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Tijana Zeremski* , Biljana Kiprovski , Vladimir Sikora , Jegor Miladinović , Svetlana Balešević Tubić

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Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia

*Corresponding author:

E-mail address: [email protected]

ABSTRACT Hemp (Cannabis sativa L.) is considered one of the oldest crops known to man. It was used in the Old World for thousands of years as an important source of food, fiber, and medicine. Although primarily grown for hemp fiber used for production of durable plastics and specialty papers, fiber hemp plant has been attracting growing interest worldwide as a valuable source of nutrients and nutraceuticals. There is also a growing interest over the valorization of hemp secondary metabolites especially cannabinoids, which are believed to be beneficial in a number of physiopathological processes. There are 54 fiber hemp varieties registered in EU and 4 hemp varieties registered in Serbia. The aim of this study was to evaluate medical cannabinoids and nutrients (oil and protein) contents of all fiber hemp varieties registered in Serbia. Total oil content in seeds of investigated varieties ranged from 21.09 to 32.09%. Fatty acid analysis showed that hempseed oil is high in PUFA (polyunsaturated fatty acids) content and polyunsaturated/saturated (P/S) ratio with linoleic (C18:2 ω-6) and linolenic (C18:3 ω-3) acids as the major polyunsaturated fatty acids. The cannabidiol (CBD) content in four analyzed varieties ranged from 1.00 to 1.27% and delta 9tetrahydrocannabinol (THC) content was approximately 3 times lower than the legal limit in EU. The obtained results indicate that all analyzed hemp varieties are an excellent source of nutrition and a valuable resource for the functional foods, nutraceutical and pharmaceutical industry. Keywords: fiber hemp, hemp seed, oil, cannabinoids

INTRODUCTION Hemp (Cannabis sativa L.) is considered one of the oldest crops known to man. It was used as an important source of food, fiber and medicine for thousands of years in the Old World (Zias et al., 1993). Although primarily grown for hemp fiber used for production of durable plastics and specialty papers, fiber hemp plant has been attracting growing interest worldwide for oil production. It has been recognized as a new, underdeveloped industrial oilseed crop in the European Union, in contrast to conventional oil crops such rapeseed and sunflower (Zanetti et al., 2013). Hemp seeds are traditionally used in food and folk medicinal preparations (Jones, 1995) or employed as a feed for birds and fishes (Deferne and Pate, 1996). Recent characterization of oil (Latif and Anwar, 2009) and protein isolates (Tang et al., 2006, 2009) from hemp seeds have highlighted that not only the fiber, but also the seeds hold very interesting commercial potential in food (Callaway, 2004; Matthäus and Brühl, 2008), feed (Hessle et al., 2008) and cosmetic applications (Sapino et al., 2005). Hempseed, in addition to its nutritional value, has demonstrated positive health benefits, including the lowering of cholesterol and high blood pressure (Jones, 1995). While fiber and seed are the main products, there is a growing interest over the valorization of hemp secondary metabolites. Hemp vegetative and reproductive organs are rich in various unique bioactive secondary metabolites, namely cannabinoids, terpenoids and flavonoids (Hazekamp et al., 2010). 10


Cannabinoids are terpenophenolics, a group of compounds typical of the genus Cannabis. Commonly, there are cannabidiol (CBD), delta 9-tetrahydrocannabinol (THC), cannabichromene (CBC) and cannabigerol (CBG). Cannabinol (CBN) is a degradation product of THC and it does not occur naturally in plant (Peschel and Politi, 2015). Cannabinoids have modulating effects on the human endocannabinoid system and are believed to be beneficial in a number of physiopathological processes (Izzo et al., 2009). There are 54 fiber hemp varieties registered in EU (EC, 2016) and 4 hemp varieties registered in Serbia. The aim of this study was to evaluate all fiber hemp varieties registered in Serbia as a source of nutrients and medical cannabinoids.

MATERIAL AND METHODS Four registered varieties of fiber hemp plant: Helena, Marina, Novosadska and Fedora 17 were grown in 2015, at the Department for Alternative Crops of the Institute of Field and Vegetable Crops in Novi Sad, Serbia. Plants used for analysis of cannabinoids were harvested in flowering phase and only flowering tops were used. Hemp seeds (approx. moisture content 6.6%) were used for the analyses of oil content and composition, as well as total protein content. Hemp flowering tops were dried and then homogenized using laboratory mill. Absolute ethanol (20 ml) was added to 200 mg of dry and homogenized sample weighed into an erlenmayer with a stopper, after which the solution was sonicated for 15 min. Decarboxylation step was performed prior to GC analysis by transferring 1 ml of solution to a 2 ml GC vial, than putting it into a heating unit (150 °C) for 12 min where the solvent was evaporated and cannabionids were decarboxylated. The residue was dissolved in 1.5 ml of ethanol and shaken well prior to analysis. Analysis of cannabinoids was performed on Agilent 7890 gas chromatograph equipped with flame ionisation detector (FID). The separation was performed on a fused silica capillary column (HP-5, 30 m × 0.25 mm i.d., and 0.25 µm film thickness). Helium was used as carrier gas at a constant flow of 1 ml/min. The temperature program was as follows: initial temperature of 200 °C was held for 2 min, then increased to 240 °C at a rate of 10 °C/min, and hold for 10 min. The injector and detector temperatures were set at 250 and 280 °C, respectively. The injected sample volume was 1 μl and split ratio was 1:10. Individual analytical standards for cannabidiol (CBD), cannabigerol (CBG) and cannabinol (CBN) were used for calibration. Quantitation of THC was performed with CBN analytical standard in accordance to method given by Poortnman-van der Meer et al. (1999). Total oil content were determined by the extraction of oil from ground hempseed (8h, 70 °C), performed in a Soxhlet extractor using 5 g of seed and 200 ml petroleum-ether followed by solvent removal under vacuum at 60 °C. To determine hempseed fatty acid composition and content 4 g of seeds were pressed in a hydraulic press to yield approximately 0.5 ml of oil available for GC analysis. Then, oils were converted into fatty acid methyl esters by transesterification using TMSH (transesterification agent). To the reaction vial with 270 µl of TMSH, exactly 30 µl of oil was added, well shaken in the vortex, and kept at room temperature for an hour. Analysis of fatty acid methyl esters was performed on Agilent 7890 gas chromatograph equipped with flame ionization detector (FID) and split/splitless injector (split ratio of 1:50). The separation was performed on a fused silica capillary column (HP-INNOWAX, 30 m × 0.25 mm i.d., and 0.25 µm film thickness). Helium was used as carrier gas at a constant pressure of 53 kPa at 50 °C. The temperature program was as follows: initial temperature of 50 °C was held for 1 min, increased to 200 °C at a rate of 25 °C/min, then increased to 230 °C at a rate of 3 °C/min, and hold for 18 min. The injector and detector temperatures were 11


set at 250 and 280 °C, respectively. Injected sample volume was 1 μl. The results were processed using ChemStation software and expressed as the percentage of individual fatty acids in the oil sample. Determination of protein content was performed according to AOAC Official Method 972.43.

RESULTS AND DISCUSSION Cannabionids content in four analyzed varieties of fiber hemp flowering tops extracts is presented in Table 1 and typical chromatogram of fiber hemp flowering tops extracts is given in Figure 1. Fiber hemp flowering tops extract is characterized by low THC content and high cannabidiol (CBD) content. In most EU countries the current upper legal limit for cultivation is 0.2% THC (Sarmento et al., 2015) and in Serbia the limit is 0.3% THC. The THC content in all four analyzed hemp varieties was approximately 3 times lower than legal limit in EU. The CBD content in analyzed varieties ranged from 1.00 to 1.27% which is in good correlation with CBD content reported in fiber hemp varieties grown in France (Thouminot, 2015) and slightly lower than average CBD content in eight varieties experimentally grown in Serbia from 1999 to 2005 in order to evaluate the effects of agroclimatic conditions on CBD synthesis and accumulation (Sikora et al., 2011). A strong influences of soil temperature at 5 cm and growing degree days (GDD), which are the most significant agroclimatic parameters influencing cannabinoids synthesis (Mandolino et al., 2003; Sikora et al., 2011), could have contributed to lower CBD content in varieties grown in 2015 (Table 1). Table 1. Phytocannabinoids content (%) in fiber hemp plant Hemp variety CBD Helena 1.27 Marina 1.00 Novosadska 1.21 Fedora 17 1.00 *nd - not detected

THC 0.07 0.06 0.07 0.05

CBG nd* nd nd nd

CBD is the most prevalent cannabinoid in the fiber-type hemp and presents a large array of pharmacological properties, as recently reviewed by Burstein (2015). CBD itself has been shown in animal and in vitro studies to possess, among others, anti-anxiety, anti-nausea, anti-arthritic, anti-psychotic, anti-inflammatory, and immuno modulatory properties (Burstein, 2015). It has also shown potential as therapeutic agent in preclinical models of central nervous system diseases such as epilepsy, neurodegenerative diseases, schizophrenia, multiplesclerosis, affective disorders and the central modulation of feeding behavior (Hill et al., 2012).

Figure 1. GC-FID chromatogram of flowering tops extracts of fiber hemp variety Helena

Total oil content in seeds of investigated varieties ranged from 21.09 to 32.09%, and it was similar to findings of other authors (Callaway, 2004; Peschel and Politi, 2015). Obtained 12


results showed that hemp seed oil contained linoleic (C18:2ω6) and linolenic (C18:3ω3) acids as the major polyunsaturated fatty acids (Table 2), as it was confirmed in some previous studies (Callaway, 2004; Da Porto et al., 2012). ‘Novosadska’ had the highest linoleic and α-linolenic acids and ‘Fedora’ γ-linolenic acid contents (Table 2). These fatty acids are known as essential fatty acids because humans cannot produce them themselves, and must obtain them in their diet. Hempseed oil has been suggested to be perfectly balanced in regards to the ratio (3:1) of these two essential polyunsaturated fatty acids (linoleic and linolenic acids) for human nutrition. Also, the oil is mostly used in cosmetic industry, because of the presence of α-linolenic acid, which is an ideal ingredient for light body oils and lipid-enriched creams, known for high penetration into the skin (Rausch, 1995). Hempseed oil was characterized by high content of PUFA (polyunsaturated fatty acids) and polyunsaturated/saturated (P/S) ratio (Table 2). When compared to other vegetable oils, hempseeds has much higher PUFA content (approximately 10% higher than corn, soy, sunflower seeds and 60% than olive fruits). A high ratio of P/S is regarded favorably for the reduction of serum cholesterol and arteriosclerosis and prevention of heart diseases (Rudel et al., 1998). Similarly, the ratio of ω-6 to ω-3 fatty acids was 3.3-4.2, which is lower than in sunflower and olive seeds, but higher than in rape seeds or flax seeds (Callaway, 2004). Table 2. Oil content and fatty acid composition (%) of hempseed oils. Hemp variety Fatty acid Helena Marina Novosadska Palmitic C16:0 7.18 6.11 6.22 Stearic C18:0 2.69 3.03 2.80 Oleic C18:1 10.99 12.48 12.36 Linoleic C18:2ω6 53.55 53.21 54.09 α-Linolenic C18:3ω3 15.58 16.48 17.16 γ-Linolenic C18:3ω6 1.72 0.62 0.78 Arachidic C20:0 0.84 0.83 0.76 Eicosenic 20:1 0.38 0.39 0.37 Behenic 22:0 0.56 0.35 0.31 % PUFA* 75.78 75.20 75.94 n6/n3 ratio 3.55 3.27 3.20 total oil content - Soxhlet 32.09 31.88 29.38 * Polyunsaturated fatty acids

Figure 2. Protein content (%) in whole and dehulled hemp seeds

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Fedora 17 7.48 2.69 14.29 53.22 13.41 2.97 0.87 0.46 0.36 72.69 4.19 21.09


From a nutritional standpoint, numerous factors are known to influence the quality of dietary proteins, most notably the amino acid composition and the digestibility of the protein (FAO/WHO, 1990). The amino acid composition of a plant protein can be influenced by factors such as variety/genetics, agronomic conditions such as soil fertility, and post-harvest processing effects that alter the ratio of seed components (i.e., dehulling) (House et al., 2010). Due to the latter, we analyzed protein content in both, whole and dehulled seeds. As it is presented in Figure 2, protein content was similar in the investigated hempseeds. The highest protein content was determined in seeds of Novosadska variety (35.38%). The remaining seed cake or meal after the cold pressing/extrusion-based processing of hempseed has significant oil content (approx. 10%) and high (>30%) protein content (Callaway, 2004; Silversides et al., 2005), and has been marketed as a source of vegetable protein. In addition to the hemp seed meal, whole hemp seed and dehulled hemp seed (hemp nuts) are found in the human food marketplace.

CONCLUSIONS Due to high levels of PUFAs in the oil and high protein content in seeds, analyzed fiber hemp varieties are excellent source of nutrition and valuable resource for the functional food and nutraceutical industries. Analyzed varieties are rich in CBD which makes them promising source of cannabinoids for pharmaceutical industry.

REFERENCES Burstein, S. (2015). Cannabidiol (CBD) and its analogs: a review of their effects on inflammation. Bioorganic and Medicinal Chemistry, 23, 1377-1385. Callaway, J.C. (2004): Hempseed as a nutritional resource, Euphytica 140, 65- 72. Da Porto, C., Decorti, D., Tubaro, F. (2012): Fatty acid composition and oxidation stability of hemp (Cannabis sativa L.) seed oil extracted by supercritical carbon dioxide, Industrial Crops and Products 36(1), 401-404. Deferne, J.L., Pate, D.W. (1996). Hemp seed oil: a source of valuable essential fatty acids. Journal of the International Hemp Association, 3, 4-7. European Commission. (2016). Plant variety database. (http://ec.europa.eu/food/plant/plant_propagation_material/plant_variety_catalogues_databases/search/pu blic/index.cfm) Hazekamp, A., Verpoorte, R. (2006). Structure elucidation of the tetrahydrocannabinol complex with randomly methylated β-cyclodextrin. European Journal of Pharmaceutical Sciences, 29, 340-347. Hessle, A., Eriksson, M., Nadeau, E., Turner, T., Johansson, B. (2008). Cold-pressed hempseed cake as a protein feed for growing cattle. Acta Agriculturae Scandinavica A: Animal Science, 58, 136-145. Hill, A.J., Williams, C.M., Whalley, B.J., Stephens, G.J. (2012). Phytocannabinoids as novel therapeutic agents in CNS disorders. Pharmacology & Therapeutics, 133, 79-97. House, J.D., Neufeld, J., Leson, G. (2010). Evaluating the quality of protein from hemp seed (Cannabis sativa L.) products through the use of the protein digestibility-corrected amino acid score method. Journal of Agricultural and Food Chemistry, 58 (22), 11801-11807. Izzo, A.A., Borrelli, F., Capasso, R., Di Marzo, V., Mechoulam, R. (2009). Non-psychotropic plant cannabinoids: new therapeutic opportunities from an ancient herb. Trends in Pharmacological Sciences, 30, 515-527. Joint FAO/WHO expert consultation on protein quality evaluation (1990). Protein Quality Evaluation (Report). Food and Agriculture Organization of the United Nations, Rome. Jones, K. (1995). Nutritional and medicinal guide to hemp seed. Rainforest Botanical Laboratory, Gibsons, B.C., Canada. Latif, S., Anwar, F. (2009). Physicochemical studies of hemp (Cannabis sativa) seed oil using enzymeassisted cold-pressing. European Journal of Lipid Science and Technology, 111, 1042-1048. Mandolino G., Bagatta A., Carboni P., Ranalli P., Meijer, E. (2003). Quantitative and qualitative aspects of the inheritance of chemical phenotype in Cannabis. Journal of Industrial Hemp, 8, 51-72. Matthäus, B., Brühl, L. (2008). Virgin hemp seed oil: an interesting niche product. European Journal of Lipid Science and Technology, 110, 655-661. Peschel, W., Politi, M. (2015). 1H NMR and HPLC/DAD for Cannabis sativa L. chemotype distinction, extract profiling and specification. Talanta, 140, 150-165. Poortman-van der Meer, A.J., Huizer, H. (1999). A contribution to the improvement of accuracy in the quantitation of THC, Forensic Science International, 101, 1-8. Rausch, P. (1995). Verwendung von hanfsameno¨ l in der kosmetik. In Bioresource hemp (2nd Ed.). NovaInstitute, (pp. 556-561), Cologne, Germany.

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III International Congress “Food Technology, Quality and Safety” Rudel, L.L., Kelly, K., Sawyer, J.K., Shah, R., Wilso, M.D. (1998). Dietary monounsaturated fatty acids promote aorti atherosclerosis in LDL receptor-null ApoB100-overexpressing transgenic mice. Arteriosclerosis, Thrombosis and Vascular Biology, 18, 1818-1827. Sapino, S., Carlotti, M.E., Peira, E., Gallarate, M. (2005). Hemp-seed and olive oils: their stability against oxidation and use in O/W emulsions. Journal of Cosmetic Science, 56, 227-251. Sarmento, L., Carus, M., Grotenhermen, F., Kruse, D., Brenneisen, R., Grassi, G., Knapsack, C. (2015). Scientifically Sound Guidelines for THC in Food in Europe, Nova-Institute, Hürth, Germany, Sikora, V., Berenji, J., Latković, D. (2011). Influence of agroclimatic conditions on content of main cannabinoids in industrial hemp (Cannabis sativa L.). Genetika, 43 (3), 449-456. Silversides, F.G., Lefrançois, M.R. (2005). The effect of feeding hemp seed meal to laying hens. British Poultry Science, 46, 231-235. Tang, C.H., Ten, Z., Wang, X.S., Yang, X.Q. (2006). Physicochemical and functional properties of hemp (Cannabis sativa L.) protein isolate. Journal of Agricultural and Food Chemistry, 54, 8945-8950. Tang, C.H., Wang, X.S., Yang, X.Q. (2009). Enzymatic hydrolysis of hemp (Cannabis sativa L.) protein isolate by various proteases and antioxidant properties of the resulting hydrolysates. Food Chemistry, 114, 14841490. Thouminot, C. (2015). Hemp breeding in France: overview and prospects (in French). Oilseeds & fats Crops and Lipids, 22(6), D603. Yu, L.L., Zhou, K.K., Parry, J. (2005). Antioxidant properties of cold-pressed black caraway, carrot, cranberry, and hemp seed oils. Food chemistry, 91 (4), 723-729. Zaneti, F., Monti, A., Berti, M.T. (2013). Challenges and opportunities for new industrial oil seed crops in EU27: A review. Industrial Crops and Products, 50, 580-595. Zias, J., Stark H., Sellgman J., Levy R., Werker E., Breuer A., Mechoulam R. (1993). Early medical use of cannabis. Nature, 363 (6426), 215.

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LACTOSE FERMENTATION IN MILK BY KOMBUCHA Katarina Kanurić*, Spasenija Milanović, Bojana Ikonić, Eva Lončar, Mirela Iličić, Vladimir Vukić, Dajana Vukić Faculty of Technology, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia *Corresponding author: E-mail address: [email protected]

ABSTRACT The effect of kombucha starter culture on the kinetics of lactose transformation as well as on the rheological properties in the different phases of milk fermentation (pH values of 5.4, 5.1, 4.8 and 4.6) at 42 °C were investigated. The results of rheological properties were compared with those obtained for the samples prepared with yoghurt and probiotic starters. Lactose fermentation in milk by kombucha starter lasted 8 h. Change of lactose concentration is consisting of two retaining stages and very steep decline in - between. The analysis of the rate curve showed that the reaction passes through maximum after 4 h. After reaching the maximum, the reaction rate decreases rapidly. The best model to present the curve of fermentation change of lactose is sigmoidal Boltzmann's function. Kombucha starter exhibited a similar influence on the casein networking pattern as the yoghurt and probiotic starter cultures. The viscosity values measured at pH 5.4 indicate similarity of the gelation processes taking place in the samples involving kombucha and probiotics. The sample produced with kombucha had the highest value of complex modulus, which resulted in better rheological characteristics of the final product. Keywords: kombucha, milk, lactose fermentation, rheological properties

INTRODUCTION Kombucha is a traditional fermented beverage with a history of several thousand years in the East and remains quite popular in the West because of a large number of claims regarding its therapeutic potential against a host of maladies. It is known as symbiotic association of yeasts and acetic acid bacteria whose metabolic activities on sweetened tea produce a pleasant slightly sweet, carbonated acidic refreshing beverage consumed worldwide. The microbial composition of kombucha cannot be given because it depends on the culture origin. Novel research (Marsh et al., 2014) has showed significant presence of lactic acid bacteria in kombucha. The major bacterial genera in 5 kombucha samples from various localities were Gluconacetobacter (over 85% in most samples) and Lactobacillus (up to 30%). Only trace populations of Acetobacter were detected (lower than 2%). Radulović et al. (2010) have also isolated and identified lactic acid bacteria from kombucha tea. The kombucha fermentation process also leads to the forming of cellulose pellicles floating at the surface of the growth medium, due to the activity of some strains such as Acetobacter xylinum (Johnsy et al., 2005; Kallel et al., 2012). Fermented milks have been an important component of nutrition and diet. Yoghurt is among the most popular fermented milk products. It is medium-acid fermented milk, characterized by a smooth, viscous gel with a characteristic flavor. Yoghurt is a protein network formed by casein micelles entrapping serum and fat globules. The distribution of the other components has an important effect on the rheological behavior of yoghurt. It is a typical example of weak viscoelastic gel. Dynamic rheological measurements of yoghurt are of interest as they are directly related to its viscous and solid properties. The first attempt to mathematically describe the process of the gel viscoelastic properties evolution in the course of gelation was introduced by Scott-Blair and Burnett (1958) in 1958, and later (Scott-Blair and Oosthuizen, 1961) empirical regression model was refined. The regression model describes the experimental results beyond the gel point, showing an initial 16


rapid increase of the shear modulus, process inflection and its subsequent approaching to the equilibrium level at t → ∞ (Smykov, 2015). Lactose is present in the milk of all mammals with only a few minor exceptions and it is the main carbohydrate in dairy products. It is a unique disaccharide, composed of glucose and galactose. During yoghurt fermentation, lactose present in the milk is converted to lactic acid which decreases the pH of the product. As a consequence, casein micelles are destabilized when the pH reaches about 5.3 and irreversibly coagulate to form a gel (Duboc and Mollet, 2001). Several studies showed the technological and nutritional potential of kombucha application in the development of new fermented dairy products (Malbaša et al., 2009; Iličić et al., 2012; Milanović et al., 2012; Vukić et al., 2014). Therefore, the aim of this paper was to investigate the effect of kombucha starter culture on the kinetics of the lactose transformation as well as on the rheological properties in the different phases of milk fermentation.

MATERIAL AND METHODS Materials Milk Homogenized and pasteurized cow’s milk from AD Imlek, Division Novi Sad Dairy, was used for the production of kombucha fermented milk products. The composition of milk was as follows: fat content – 2.0 g/100g, total solids – 10.59 g/100g, total proteins – 3.30 g/100g and lactose – 4.39 g/100g, ash 0.69 g/100g. Kombucha inoculum Kombucha inoculum was prepared according to procedure published by Milanović et al., (2002). Kombucha is cultivated on a black tea (Camellia sinensis – oxidized, 1.5 g/L) with sucrose concentration of 70 g/L. The tea was cooled to room temperature, after which inoculum from a previous fermentation was added in concentration of 10 % (v/v). Incubation was performed at 25 °C for 7 days. Inoculum in concentration of 10 % (v/v) was used for milk fermentation. Samples production During the fermentation process involving kombucha, yoghurt or probiotic starter culture, samples were taken at the pH values of 5.4, 5.1, 4.8 and 4.6. The particular samples were designated by the capitals K (kombucha), P (probiotic) and Y (yoghurt), and the corresponding pH value. The kombucha inoculum was added to the milk in an amount of 10% (Vukić et al., 2014). The probiotic starter culture was Probio-Tek® from CHR Hansen (Denmark) containing: Lactobacillus acidophilus LA-5®, Bifidobacterium lactis BB-12®, and Streptococcus thermophilus. The yoghurt starter culture Yo-Flex® from CHR Hansen contained the thermophilic cultures: Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. The commercial starters (probiotic and yoghurt) were added according to the manufacturer's specification – 0.005 g/100g. All the samples were produced in triplicate at 42 °C.

Methods pH Values The pH values were determined using a pH-meter (pH Spear, Eutech Instruments Oakton, UK). Sugar content The content of lactose and galactose at were detected in all samples using specific enzymatic tests K-LACGAR 12/05 (lactose and D-galactose) purchased by Megazyme, 17


Ireland. Products of the reactions were monitored using spectrophotometer (T80+UV/VIS Spectrometer PG Instruments Ltd, UK). HPLC analysis of lactic acid Lactic acid content was determined according to the modified method of Jayablan et al. (2007). Four grams of samples were transferred into a 25 mL volumetric flask, 5 mL of bidestiled water was added and filled up with acetonitrile (Mallinckrodt Baker, Inc., Netherlands). The obtained solution was mixed for 5 min and then was filtered through membrane syringe filter (with diameter pore of 0.45 μm). Agilent 1100 system (USA) with C– 8 column and UV–DAD detector was used for determination. The mobile phase was a mixture of 20 mM potassium dihydrogen phosphate, pH 2.4 and methanol (97:3). The flow rate and column temperature was maintained as 1.0 mL/min and 28 °C, respectively. Detection was carried out at 220 nm. Lactic acid (Supelco analytical, USA) was used as a standard. Each sample was prepared and injected in triplicate. Lactose kinetics The change of lactose concentration during fermentation of milk with kombucha was fitted to the sigmoidal Boltzmann's model. Rheological properties The viscosity of the samples was measured at 5 °C using a viscometer HAAKE RheoStress 600HP (Karlsruhe, Germany) with a cone and-plate-sensor PP60Ti (gap 1 mm). Stress sweeps were increased from 0.1 to 100 Pa to determine the linear viscoelastic range of the samples at a constant frequency of 1Hz. The frequency sweeps from 1 to 10 Hz were performed at a constant shear stress of 1Pa to obtain dynamic complex parameters: the storage modulus (G’), the loss modulus (G’’) and the phase angle (tanδ), which represents the ratio of G’’ and G’. The onset of gelation was determined by measuring the modulus during the sample production. Replicate measurements for each sample were done independently. Statistical analysis Statistical analysis of results was carried out with the computer program "Statistica 9”.

RESULTS AND DISCUSSION The pH value was measured as indicator of the milk fermentation by kombucha, at 42 °C (Figure 1).

Figure 1. Change of pH during milk fermentation by kombucha at 42 °C

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Fermentation curve is characterized by a slight increase during the first two hours (from the starting pH 6.07 to pH 6.2). The next two hours, the pH value gradually decreased. Further decrease in pH value took place until the end of the fermentation. Decrease of pH during the milk fermentation is a result of lactose transformation. Lactose molecules bind to the enzyme's active sites where they are hydrolyzed into glucose and galactose, which are then metabolized through a number of consecutive and parallel reactions in the cell. Seto et al. (1997) have reported that Acetobacter strains, which are abundant in kombucha medium, consumed preferentially glucose as carbon source. In line with this, glucose is preferred to galactose as a carbon source by kombucha microflora. Lactose consumption and the accumulation of galactose and lactic acid during fermentation of milk by kombucha were examined and presented in Figure 2. During the fermentation, the lactose is first converted into glucose and galactose which are further converted into other products such as pyruvic acid, lactic acid, carbon dioxide, water, ethanol and others, through a series of consecutive and parallel reactions (Тamime and Robinson, 2004; Surono and Hosono, 2011). Reaction mixture changes its qualitative and quantitative composition during the milk fermentation. At the beginning of the process, lactose content was 4.39 g/100g and decreases continuously during the fermentation. The best model to present the change of lactose (as well as sucrose, researched by Lončar et al., 2014), is sigmoidal Boltzmann's function. Change of lactose concentration at 42 °C is consisting of two retaining stages and very steep decline in–between (Figure 2). The time interval when the change of constituents was most evident is between the 4th and the 6th hours.

Figure 2. Kinetics of lactose fermentation

The analysis of the lactose consumption rate (Figure 3) showed that the lactose was consumed at maximum rate after 4 hours. After reaching the maximum, the reaction rate decreases rapidly, probably because of the suppression of metabolic activity of the present microorganisms due to the production of organic acids. Similar results were obtained by Chen and Liu (2000) who investigated prolonged fermentation of sucrose by kombucha. The effect of kombucha starter culture on the rheological properties was examined by the storage modulus G’ and loss modulus G’’ analyses. The results were compared with those obtained for the samples prepared with yoghurt and probiotic starters. As the gel forms, the values of the storage modulus G’ and loss modulus G’’ show an increase, but their ratio changes depending on the gel formation phase (Horne, 1999; Xu et al., 2008; Jacob et al., 2011). 19


Figure 3. Reaction rate of lactose fermentation

(Lines show mathematical models, symbols present experimental values)

Figure 4. Complex modulus of the samples produced by starters: a) kombucha; b) yoghurt; c) probiotic

It is evident from Figure 4 that the networking of casein micelles caused an increase in the storage modulus (G’) and loss modulus (G’’) during the fermentation, i/e. in stronger bonds and more rigid casein network at increased complex modulus. Samples produced with kombucha had the highest values of the complex modulus, which indicates better rheological 20


characteristics of the final product. It is evident that the major changes in the values of viscosity and modulus during the milk fermentation using kombucha inoculum take place in the first phase of casein micelles networking, which is in accordance with observations concerning the samples produced with the other two starters. As the fermentation proceeds further, the pH value decreases and the viscosity changes are less pronounced due to the approaching of the casein isoelectric point. Different types of starter culture induced different gelation rates and final gel viscosity of the sample.

CONCLUSIONS Milk fermentation by kombucha at 42 °C showed specific biochemical behaviors. The best model to present the change of lactose during fermentation is sigmoidal Boltzmann's function. Change of lactose concentration is consisting of two retaining stages and very steep decline in–between. The major differences in the measured rheological properties of the samples were observed in the first phase of casein gelation. The samples produced with kombucha had the highest value of complex modulus, which resulted in the better rheological characteristics of the final product.

ACKNOWLEDGEMENTS Authors want to thank Ministry of Education, Science and Technological Development of Republic of Serbia for the financial support of research presented in this article, Project No. 46009.

REFERENCES Chen, C., Liu, B. Y. (2000). Changes in major components of tea fungus metabolites during prolonged fermentation. Journal of Applied Microbiology, 89, 834-839. Duboc, P., Mollet, B. (2001). Applications of exopolysaccharides in the dairy industry. International Dairy Journal, 11, 759-768. Horne, D. S. (1999). Formation and structure of acidified milk gels. International Dairy Journal, 9 (3-6), 261268. Iličić, M., Kanurić, K., Milanović, S., Lončar, E., Đurić, M., Malbaša, R. (2012). Lactose fermentation by Kombucha – a process to obtain new milk–based beverages. Romanian Biotechnological Letters, 17(1), 7013–7021. Jacob, M., Nöbel, S., Jaros, D., Rohm, H. (2011). Physical properties of acid milk gels: Acidification rate significantly interacts with cross–linking and heat treatment of milk. Food Hydrocolloids, 25, 928-934. Jayabalan, R., Marimuthu, S., Swaminthan, K. (2007). Changes in content of organic acids and tea polyphenols during kombucha tea fermentation. Food Chemistry, 102, 392-398. Johnsy, G., Ramana, K.V., Sabapathy, S.N., Bawa, A.S. (2005). Physico-mechanical properties of chemically treated bacterial (Acetobacter xylinum) cellulose membrane. World Journal of Microbiology and Biotechnology, 21, 1323-1327. Kallel, L., Desseaux, V., Hamdi, M., Stocker, P., Ajandouz, E. H. (2012). Insights into the fermentation biochemistry of Kombucha teas and potential impacts of Kombucha drinking on starch digestion. Food Research International, 49, 226-232. Lončar E., Kanurić, K., Malbaša, R., Đurić, M., Milanović, S. (2014). Kinetics of saccharose fermantation by Kombucha. Chemical Industry & Chemical Engineering Quarterly, 20 (3), 345-352. Malbaša, R., Milanović, S., Lončar, E., Đurić, M., Carić, M., Iličić, M., Kolarov, Lj. (2009). Milk–based beverages obtained by Kombucha application. Food Chemistry, 112, 178-184. Marsh, A.J., Sullivan, O., Hill, C., Ross, R.P., Cotter, P.D. (2014). Sequence-based analysis of the bacterial and fungal compositions of multiple kombucha tea fungus samples. Food Microbiology, 38, 171-178. Milanović, S., Carić, M., Lončar, E., Panić, M., Malbaša, R., Dobrić, D. (2002). Primena koncentrata čajne gljive u proizvodnji fermentisanih mlečnih napitaka. Food Industry – Milk and Dairy Products, 13 (1-2), 813. Milanović, S., Kanurić, K., Vukić, V., Hrnjez, D., Iličić, M., Ranogajec, M., Milanović, M. (2012). Physicochemical and textural properties of kombucha fermented dairy products. African Journal of Biotechnology, 11 (9), 2320-2327.

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III International Congress “Food Technology, Quality and Safety” Radulović, Z., Paunović, D., Iličić, M., Mirković, N., Petrušić, M., Obradović, D. (2010). Promena mikroflore čajne gljeve tokom skladištenja fermentisanih mlečnih napitaka. Food Industry – Milk and Dairy Products, 21 (1-2), 99-102. Scott-Blair, G.W., Burnett, J. (1958). Physical changes in milk caused by the action of rennet: III. Effects of separation, homogenization and pasteurization and of varying calcium content on the rigidity moduli and viscosities of curd. Journal of Dairy Research, 25, 297-303. Scott-Blair, G.W., Oosthuizen, J.C. (1961). A viscometric study of the breakdown of casein in milk by rennin and rennet. Journal of Dairy Research, 28, 165-173. Seto, A., Kojima, Y., Tonouchi, N., Tsuchida, T., Yoshinaga, F. (1997). Screening of bacterial celluloseproducing Acetobacter strains suitable for sucrose as a carbon source. Bioscience, Biotechnology, and Biochemistry, 61, 735-736. Smykov, J.T. (2015). Kinetics of Milk Gelation. Part II. Mathematical Modeling. In E. Karpushkin (Ed.), Rheology, Principles, Applications and Environmental Impacts, Chapter 4 (pp. 83-103), New York: Nova Publishers. Surono, S., Hosono, A. (2011). Fermented Milks – Types and Standards of Identity. In J. W. Fuquay (Ed.), Encyclopedia of Dairy Sciences, 2nd Edition (pp. 470-476), London: Elsevier Academic Press. Тamime, A.Y., Robinson, R.K. (2004). Yoghurt, Science and Technology, Woodhead Publishing Ltd, Cambridge, UK. Vukić, V., Hrnjez, D., Kanurić, K., Milanović, S., Iličić, M., Torbica, A., Tomić, J. (2014). The effect of kombucha starter culture on the gelation process, microstructure and rheological properties during milk fermentation. Journal of Texture Studies, 45, 261-273. Xu, Z.-M., Emmanouelidou, D. G., Raphaelides, S. N., Antoniou, K. D. (2008). Eﬀects of heating temperature and fat content on the structure development of set yogurt. Journal of Food Engineering, 85, 590-597.

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BIOAVAILABILITY AND BIOACTIVITY OF GLS/ITC OF BRASSICACEAE SPROUTS AS HEALTH PROMOTERS Nieves Baenas*, Cristina García-Viguera, Diego A. Moreno Phytochemistry Lab. Dept. of Food Science and Technology, CEBAS-CSIC, Campus de Espinardo, 30100, Espinardo, Murcia, Spain. *Corresponding author: E-mail address: [email protected]

ABSTRACT Cruciferous sprouts are fresh plant foods very interesting because their higher contents of nutrients and phytochemicals compared to the adult plants. Different strategies could be carried out in order to produce sprouts rich in bioactive compounds, such as appropriate conditions and days of germination, along with treatments with elicitors. In this work, 8-day-old broccoli (Brassica oleracea var. italica) and radish (Raphanus sativus cv. Rambo) sprouts were selected due to their high content in glucosinolates, in order to evaluate the absorption and metabolism of their bioactive compounds, isothiocyanates and indoles, as well as validate certain biological activities. The absorption and metabolism of sulforaphane from broccoli sprouts were studied in vitro using human intestinal and hepatic cancer cells, showing antiproliferative activity and, therefore, assisting the anticancer mechanisms in the cells. On the other hand, sulforaphene from radish sprouts was demonstrated to be metabolized through the mercapturic acid pathway, showing also a beneficial effect in the glucose metabolism of the organism model Drosophila melanogaster. Additionally, the antinociceptive (analgesic) effect of broccoli sprouts was evaluated in rodent models, suggesting their potential use in treatment of pain without the adverse effect of analgesic drugs. The selection of suitable cruciferous varieties for sprouts production with high contents of phytochemicals will maximize their promoting properties for their possible use in preclinical and clinical trials, even without increasing the overall vegetable consumption. Keywords: Sulforaphane, sulforaphene, anticancer, energy metabolism, antinociception.

INTRODUCTION The vegetables from the family Brassicaceae have been widely approved for its beneficial effects on human health through epidemiological studies (Jahangir et al., 2009), as good sources of a variety of nutrients and phytochemicals that may work synergistically against certain types of cancer, cardiovascular diseases, neurodegeneration and diabetes (DinkovaKostova and Kostov, 2012). Cruciferous sprouts are usually collected and commercialized at 7 or 8 days of age after germination, considering this young physiological state the optimum for consumption in terms of biomass and size, which allow manipulation, as well as for their health-promoting compounds content, since sprouts have significantly greater concentrations of phytochemicals than mature plants (10-100 times) (Cevallos-Casals and CisnerosZevallos, 2010; Hanlon and Barnes, 2011). Glucosinolates (GLS) are hydrolyzed to the biologically active isothiocyanates (ITC) and indoles among other compounds in presence of the enzyme myrosinase when there is a tissue disrupted in the plant (Grubb and Abel, 2006) and also by the action of the gut microflora upon human ingestion (Angelino and Jeffery, 2014). The exposure of Brassicaceae plants to exogenous treatments with elicitors, such as the phytohormones, salicylic acid (SA), jasmonic acid (JA) and methyl jasmonate (MeJA), resulted in an increase in GLS (Ku et al., 2014), through induction of the defense mechanisms in the plant and expression of genes involved in the production of secondary metabolites (Ferrari, 2010). The beneficial effects of crucifers on human health, like antibacterial, antifungal, antitumor, anti-mutagenic, anti-inflammatory, neuroprotective and antioxidative activities, which have 23


been demonstrated through many in vivo and in vitro studies, are based on the induction of the Keap1/Nrf2/ARE pathway, which regulates the transcription of many antioxidant and antiinflammatory genes that preserve cellular homeostasis and induce detoxification enzymes that process and eliminate carcinogens and toxins before they can cause damage (Stefanson and Bakovic, 2014). In this work, the possible health-promoting effects of broccoli and radish 8-day-old sprouts, through investigation of the absorption and metabolism of their bioactive compounds, and their effects in cancer proliferation, glucose metabolism and antinociception have been investigated in order to foster their application as healthy and fresh vegetables.

MATERIAL AND METHODS Broccoli (Brassica oleracea var. italica) and red radish (Raphanus sativus cv. Rambo) sprouts were allowed to grow for 8 days and treated with the elicitor MeJA (250 µM) for 4 days before collection. Sprout samples were collected, weighed (fresh mass), flash frozen in liquid nitrogen, stored at −80 ºC and lyophilized prior to analyses and their use in the in vitro and in vivo experiments. Caco-2 and HT-29 cells, as well as HepG2 cells, which feature characteristics of intestinal epithelial cells and hepatocytes, respectively, were selected for studying the antiproliferative effect of broccoli sprouts extract and their pure compounds sulforaphane (SFN) and glucoraphanin (GRA), as well as their absorption and metabolism by these cell lines. The in vivo model Drosophila melanogaster was used in order to study absorption and metabolism of bioactive compounds of radish sprouts, analyzed by UHPLC-MS-QqQ (Dominguez-Perles, et al., 2014) and their effect in the regulation of the glucose metabolism and expression of the spargel gene. Broccoli sprouts aqueous extract was administered at 50, 100, 250 and 500 mg/kg via i.p., and at 500, 1000 and 2000 mg/kg orally, to Swiss albino mice and female Wistar rats, submitted to the writhing (Collier et al., 1968) and formalin tests (Wheeler-Aceto et al., 1990), respectively. Gastric damage (Robert et al., 1979), as possible adverse effect observed in the effect of anti-inflammatory, was also explored in the effect of significant dosage (100 mg/kg BSE i.p.).

RESULTS AND DISCUSSION After the evaluation of the GLS content in broccoli and radish sprouts during the germination time (4, 8, and 12 day-old-sprouts), the day 8 of germination was selected as appropriate size for commercialization and consumption, even though the content in phytochemicals decrease during germination due to a dilution effect of tissue expansion (Figure 1).

Figure 1. Total GLS content in broccoli and radish sprouts during germination

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The exogenous treatment of sprouts with MeJA (250 µM) showed an increase in total GLS of 84 % and 23 %, in broccoli (712 mg/100g F.W.) and radish sprouts (452 mg/100g F.W.), respectively (data not shown). Once the previous results were obtained, the evaluation of the absorption and metabolism of GLS/ITC present in 8-day-old broccoli sprouts (containing 1µM of SFN), as well as the pure compounds SFN (1 µM) and GRA (50 µM), was approached by the Caco-2 and HepG2 cell lines. In our work, cells were capable of metabolize SFN, accumulated predominantly as glutathione conjugates (SFN-GSH) but also as SFN-cysteine (SFN-CYS), which were determined in cells lysates and in the culture medium in nanomolar concentrations (0.5–90 nM) (data not shown). In addition, broccoli sprouts and SFN showed dose-time dependent antiproliferative activity in Caco-2, HT-29 and HepG2 cancer cell lines, while GRA did not achieve the half-maximal inhibitory concentration (IC50). A lower IC50 was observed after broccoli sprouts treatment (~2 µM) compared to SFN (~50 µM), suggesting that other compounds present in the food matrix may act biologically in synergy with SFN (Figure 2).

Figure 2. Cell growth Inhibition (%) after application of broccoli sprouts extract, sulforaphane and glucoraphanin solutions. IC50 shows the half-maximal inhibitory concentration

Moreover, to study the absorption and metabolism of bioactive compounds present in radish sprouts cv. Rambo we used Drosophila melanogaster, as a model organism. Flies were subjected to a diet supplemented with radish sprouts (10.6 g/L) for 10 days, containing 50 µM of sulforaphene (SFE). Flies homogenizes after this treatment showed nanomolar concentrations, per gram on a fresh weight basis, of SFE (1.11 nmol/g F.W. in flies), as the predominant ITC, and SFN and I3C (0.998) was prepared by dissolving of 0.0125% (w/v) of PTSA in external water phase without emulsifier and measuring absorbance of diluted dye solutions using a UV-VIS spectrophotometer (HALO DB-20S, Dynamica, U.K.) at 374 nm. For yield determination, PTSA (0.3% w/v) was dissolved in the internal water phase and double W/O/W emulsions were prepared as previously described. After emulsion preparation and during storage time, samples of W/O/W emulsions were centrifuged for 30 min at 4500 rpm (at 20 °C) using a centrifuge (Univerzal 320R, Hettich, Germany) for emulsion phase separation. An aliquot of each centrifugated sample was filtrated through 0.2 and 0.1 µm syringe filter (Macherey-Nagel, Düren, Germany) to remove the residual oil after centrifugation and their absorbance was measured at 374 nm. As a blank solution, samples prepared in the same way from the W/O/W emulsions without dye in the internal water phase were used. The concentration of PTSA in the external water phases of W/O/W emulsions was determinated after preparation and during storage time from standard calibration curve as an average result of three individual samples. The entrapment yield (Y) was calculated using Eq. (3) (Surh et al., 2007): (3) where, Ci is concentration of PTSA encapsulated into internal water phase of W/O/W emulsions, Ce is concentration of PTSA measured in the external water phase after preparation and during storage time, φwo is the volume fraction of internal water phase in W/O emulsions and φwow is the volume fraction of W/O droplets in the W/O/W emulsion.

RESULTS AND DISCUSSION In this study the role of the active components that should be protected from environmental influences and encapsulated at double emulsion carriers were water garlic and ethanol garlic extract. The mentioned active ingredients were primarily used for the simple W/O emulsion preparation, which than were used for preparation of W/O/W emulsions by two different techniques: homogenization and stirred cell membrane emulsification. Preparation of simple W/O emulsions with encapsulated water garlic extract implies the usage of prepared extract as an internal water phase of double emulsions, stabilized with 4% of PGPR, with added salt to increase the stability of internal droplets (Landfester, 2006). However, previous study had shown that the preparation of W/O emulsions containing the ethanol garlic extract requires their mixing with water phase (containing added salt (NaCl)) and usage a suitable selected emulsifier (4% of PGPR) (Ilić et al., 2015). In that way prepared W/O emulsions were used for preparation of double W/O/W emulsions. Differences in individual formulations were reflected as differences in external water phase composition, i.e. addition of different osmotic regulator in W 2 for equalization of the osmotic pressures between water compartments which are presented in Table 2. Table 1. Droplet size and span number of different formulations of the W/O/W emulsions with encapsulated water or ethanol garlic extract in internal water phase, prepared with different osmotic regulators in W 2 and two different technique of emulsion preparation Preparation procedure Homogenization Stirred cell ME

Osmotic regulator in W2 no regulator glucose NaCl glucose

Water garlic extract W/O/W D4,3 [µm] span [-] 54.76 ± 0.07 1.14 ± 0.001 58.86 ± 2.62 1.14 ± 0.020 65.93 ± 2.30 1.02 ± 0.021 54.66 ± 0.93 0.62 ± 0.018

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Ethanol garlic extract W/O/W D4,3 [µm] span [-] 58.81 ± 4.52 1.23 ± 0.017 39.80 ± 0.57 1.13 ± 0.031 54.88 ± 0.09 1.13 ± 0.007 57.77 ± 0.16 0.73 ± 0.003


Obtained droplet size and span value of double emulsions with encapsulated garlic extracts prepared using homogenizer and stirred cell ME are given in Table 1. It can be observed that droplet size of emulsions with water garlic extract as an internal water phase depends of the external water composition, where the smallest droplets were obtained in formulation with no matched osmotic pressure (54.76µm ± 0.07). However, the droplet size distribution (span value) was similar in all formulation, because it was a parameter that mostly depends on preparation procedure (Vladisavljević et al., 2004). The main goal of using a stirred cell ME was to obtain emulsions with narrow droplet size distribution. By ME preparation procedure at optimal conditions (N=850 rpm and ∆Ptm=20 kPa), a significant reduction of droplet size was not achieved (D4,3=54.66µm ± 0.93) in comparison with homogenization, but the droplet size distribution was significantly improved with a decrease of the span value up to 0.62 ± 0.02. A similar observation can be noticed when ethanol garlic extract was implemented into double W/O/W emulsion. Usage of ME led to formation of oil droplets with span value of 0.73 ± 0.003 in comparison with span values of about 1.2 obtained by homogenization.

Figure 2. The yield of PTSA after preparation and during storage time of W/O/W emulsions with encapsulated water garlic extract prepared with different osmotic regulators in W 2 (glucose, NaCl and without regulator) by homogenization (solid symbols) and stirred cell ME at N=850rpm and ∆Ptm=20kPa (open symbols)

Figure 3.The yield of PTSA after preparation and during storage time of W/O/W emulsions with encapsulated ethanol garlic extract prepared with different osmotic regulators in W 2 (glucose, NaCl and without regulator) by homogenization (solid symbol) and stirred cell ME at N=850rpm and ∆Ptm=20kPa (open symbol)

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Due to the complex composition of garlic extracts incorporated in the W/O/W emulsions, PTSA dissolved in the internal water phase was used for estimation of encapsulated material yield of the double emulsions. In Fig. 2, Y of double W/O/W emulsions was given with encapsulated water garlic extract immediately after the preparation and during time. Also, the influence of used emulsification technique for emulsion preparation on the Y was presented. The yield of W/O/W emulsions prepared by homogenization (Fig. 2) was high (Y≥99%) in formulation with matched osmotic pressures between water compartments with glucose, while after two month of storage, 93.41% ± 0.9 of material still was retained in the internal water phase. Usage of salt as an osmotic pressure regulator gave smaller initial yield (91.89% ± 0.9), but release kinetic of encapsulated material was similar. Formulations without balanced osmotic pressures showed significantly lower initial encapsulation efficiency (76.6% ± 1.9) and higher release during time causing release of about 50% of encapsulated material. When ethanol garlic extract was implemented in the internal water phase, the yield of W/O/W emulsions, given on Fig. 3, was 99.08% ± 1.3, 97.98% ± 1.6 and 99.35% ± 0.8 for emulsions without matched osmotic pressures, glucose as osmotic additive and NaCl as osmotic regulator, respectively. Release kinetics of encapsulated material was similar for all investigated formulation and seemingly independent of osmotic pressures regulation. Unexpectedly, after two month of storage encapsulation yield of emulsions prepared by homogenization with no matched osmotic pressures was 83.59% ± 1.3, while Y of formulation with added osmotic regulators was 77.77% ± 2.6 and 86.7% ± 0.9 for glucose and NaCl, respectively. The method for preparation strongly influenced on Y weather the double emulsions were used as carriers of water or ethanol garlic extract (Fig. 2 and Fig. 3). Initial yield of encapsulated material was similar for both techniques, but release was significantly slower for emulsions prepared by ME, probably as a consequence of uniformity of droplet size distribution and less coalescence of oil droplets. For emulsions with water garlic extract, after 60 days of storage, 97.55% ± 0.9 was still retained in the internal water phase, while for emulsion with encapsulated ethanol extract retained fraction was 97.05% ± 0.2.

CONCLUSIONS The initial encapsulation yield and release of the marker dye from W/O/W emulsions with encapsulated water garlic extract depends on the addition and type of osmotic pressure regulator in the external water phase. When the ethanol garlic extract was implemented in the internal water phase, the yield of W/O/W emulsions was similar for all investigated formulation and seemingly independent of osmotic pressures regulation. Method for preparation influenced the encapsulation yield and the droplet size distribution weather the double emulsions were used as carriers of water or ethanol garlic extracts. Encapsulation yield during time was significantly higher for emulsions prepared by ME in comparison to homogenization.

ACKNOWLEDGEMENTS This work was supported by the Ministry of Education and Science of the Republic of Serbia within the Integrated and Interdisciplinary Research Project No. 46010, 2011-2016.

REFERENCES Adachi, S., Imaoka, H., Ashida, H., Maeda, H., Matsuno, R. (2004). Preparation of microcapsules of W/O/W emulsions containing a polysaccharide in the outer aqueous phase by spray-drying. European Journal of Lipid Science and Technology, 106, 225–231. Dickinson, E. (2011). Double Emulsions Stabilized by Food Biopolymers. Food Biophysics, 6, 1–11.

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III International Congress “Food Technology, Quality and Safety” Dragosavac, M. M., Holdich, R. G., Vladisavljević, G. T., Sovilj, M. N. (2012). Stirred cell membrane emulsification for multiple emulsions containing unrefined pumpkin seed oil with uniform droplet size. Journal of Membrane Science, 392-393, 122–129. Dragosavac, M. M., Sovilj, M. N., Kosvintsev, S. R., Holdich, R. G., Vladisavljević, G. T. (2008). Controlled production of oil-in-water emulsions containing unrefined pumpkin seed oil using stirred cell membrane emulsification. Journal of Membrane Science, 322, 178–188. Herzi, S., Essafi, W., Bellagha, S., Leal-Calderon, F. (2012). Influence of diffusive transport on the structural evolution of W/O/W emulsions. Langmuir, 28, 17597–17608. Ilić, J. D., Lončarević, I. S., Petrović, J. S., Nikolovski, B. G. (2015). Preparation of the W/O/W emulsions containing garlic extract incorporated into internal water phase using stirred cell membrane emulsification. Journal of Hygienic Engineering and Design. Journal of Hygienic Engineering and Design, 12, 139–145. Jiménez-Colmenero, F. (2013). Potential applications of multiple emulsions in the development of healthy and functional foods. Food Research International, 52(1), 64–74. Landfester, K. (2006). Synthesis of Colloidal Particles in Miniemulsions. Annual Review of Materials Research, 36(1), 231–279. Lanzotti, V. (2006). The analysis of onion and garlic. Journal of Chromatography A, 1112(1-2), 3–22. Nikolovski, B., Ilić, J., Sovilj, M. (2016). How to formulate a stable and monodisperse water-in-oil nanoemulsion containing pumpkin seed oil: The use of multiobjective optimization. Brazilian Journal of Chemical Engineering, in press. Muschiolik, G. (2007). Multiple emulsions for food use. Current Opinion in Colloid and Interface Science, 12, 213–220. O` Regan, J., Mulvihill, D. M. (2009). Water soluble inner aqueous phase markers as indicators of the encapsulation properties of water-in-oil-in-water emulsions stabilized with sodium caseinate. Food Hydrocolloids, 23(8), 2339–2345. Pays, K., Giermanska-Kahn, J., Pouligny, B., Bibette, J., Leal-Calderon, F. (2002). Double emulsions: How does release occur? Journal of Controlled Release, 79, 193–205. Perez-Moral, N., Watt, S., Wilde, P. (2014). Food Hydrocolloids Comparative study of the stability of multiple emulsions containing a gelled or aqueous internal phase. Food Hydrocolloids, 42, 1–8. Surh, J., Vladisavljević, G. T., Mun, S., McClements, J. D. (2007). Preparation and Characterization of Water / Oil and Water / Oil / Water Emulsions Containing Biopolymer-Gelled Water Droplets. Journal of Agricultural and Food Chemistry, 55(3), 175–184. Tang, S. Y., Shridharan, P., Sivakumar, M. (2013). Impact of process parameters in the generation of novel aspirin nanoemulsions - Comparative studies between ultrasound cavitation and microfluidizer. Ultrasonics Sonochemistry, 20(1), 485–497. Ushikubo, F. Y., Cunha, R. L. (2014). Stability mechanisms of liquid water-in-oil emulsions. Food Hydrocolloids, 34, 145–153. Vladisavljević, G. T., Lambrich, U., Nakajima, M., Schubert, H. (2004). Production of O/W emulsions using SPG membranes, ceramic α-aluminium oxide membranes, microfluidizer and a silicon microchannel plate - A comparative study. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 232, 199– 207. Vladisavljević, G. T., Williams, R. A. (2005). Recent developments in manufacturing emulsions and particulate products using membranes. Advances in Colloid and Interface Science, 113(1), 1–20. Vladisavljević, G. T., Mitrović, M. V. (2000). Membrane emulsification - state of the art. Chemical Industry, 12(54), 523–529. Zhao, G., Hu, C., Sun, R., Ni, S., Xia, Q. (2015). Effect of Emulsification Process on Multiple Lipid Particles Encapsulating Both Coenzyme Q10 and Tea Polyphenols. Journal of Food Process Engineering, 38(2), 144–154.

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MOLECULAR DOCKING SYMULATION THROUGH THE ANGIOTENSIN CONVERTING ENZYME INHIBITION BY MILK DIPEPTIDES Vladimir Vukić*, Dajana Vukić, Spasenija Milanović, Mirela Iličić, Katarina Kanurić 1

Faculty of Technology Novi Sad, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia

*Corresponding author: Vladimir Vukić E-mail address: [email protected]

ABSTRACT Molecular docking is a method which predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex. Insight into ligand binding to the enzyme, active site residues and their interactions, provide valuable information about the catalytic mechanism, possible ways for enzyme inhibition or improvement of catalytic reaction through rational design of the enzyme. In recent years, a great attention has been paid to discovery and synthesis of novel bioactive peptides with angiotensin-I-converting enzyme (ACE) inhibition. Therefore, potential of the ACE inhibition by milk dipeptides will be presented through molecular docking simulation. Threedimensional coordinates for the molecular structure of Angiotensin-I converting enzyme was retrieved from Protein Data Bank data base. The geometry optimization of all compounds was carried out using the MMFF94 force field with the Gasteiger-Marsili charges using the Powell conjugated gradient algorithm method. Docking simulations were performed using the program Surflex-Dock with flexible H atoms. According to our results, milk dipeptides inhibit ACE through zinc independent mechanism. Among dipeptides, the highest ACE inhibitory activity has the peptides with TRP at their C-terminus. This could be due to deep TRP position into the hydrophobic pocket at the end of the catalytic channel, where it makes hydrophobic interactions with nonpolar residues. Furthermore, their polar groups make polar interactions with catalytic GLU384 and other polar residues, which inhibit its catalytic function. Dipeptides with low ACE inhibitory activity not enter the hydrophobic pocket, and therefore do not make strong interactions with the enzyme. Keywords: Molecular docking, ACE, Milk dipeptides

INTRODUCTION In order to understand a protein’s function it is important to be able to predict and understand the interactions a protein undergoes with other biomolecules. Molecular docking is a method which predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex. This method has two major parts: an algorithm creating potential protein–ligand complex geometry (pose generation) and a function predicting the binding affinity of the ligand to the protein based on the complex geometry (scoring). As a result a large number of potential ligand poses are obtained, as well as docking score (binding affinity). Insight into ligand binding to the enzyme, active site residues and their interactions, provide valuable information about the catalytic mechanism, possible ways for enzyme inhibition or improvement of catalytic reaction through rational design of the enzyme. These interactions often give important hints on the protein’s function (Stockwell, 2004). Moreover, small molecules can inhibit or activate protein functions and therefore, play a dominant role in pharmaceutical research. C-domain of mammalian somatic angiotensin-I converting enzyme (sACE) is mainly responsible for Ang II formation (Fuchs et al., 2008; van Esch et al., 2005), which makes it an important drug target in the treatment of cardiovascular diseases. Two histidine residues positioned in the functional motif HEXXH and a third glutamate residue (distantly positioned) bind the zinc atom, which acts as a cofactor (Coates, 2003). Residue Glu384 acts as the catalytic base and protonates the amine product. In recent years, a great attention has been paid to discovery and synthesis of novel bioactive peptides with angiotensin-I-converting enzyme (ACE) inhibition. Milk proteins have a leading role as a source of bioactive peptides 42


and ACE inhibitors (Iwaniak and Dziuba, 2009; Iwaniak et al., 2014). Therefore, the aim of this research is to investigate ACE inhibition by milk dipeptides through molecular docking simulation.

MATERIAL AND METHODS An ACE inhibitory milk dipeptides database, containing the amino acid sequences and the activities expressed as the concentration of the peptide (μM) necessary to inhibit the activity by 50% (IC50), was constructed with data taken from the literature and the Biopep database (Tab. 1) (Minkiewicz et al., 2008). Table 1. Milk dipeptides used for the molecular docking analysis with their ACE inhibitory activity Dipeptide IC50 (µM) Dipeptide IC50 (µM) YL 50 IR 695 AP 230 KR 380 FP 315 KW 1.63 FY 8 LW 50 IG 1200 RL 2439 MY 193 VM 120 VG 1100 VR 52 VY 4 VW 0.5 YP 120 YA 460 GA 2000 YG 1523 GL 2500 YW 4.15

The 3D structures of all dipeptides are constructed using Chemsketch, a chemically intelligent drawing interface freeware (O'Boyle, 2010). Energy minimization was performed using an MMFF94 force field, with the Gasteiger-Marsili charges using the Powell conjugated gradient algorithm method with convergence criterion of 0.01 kcal/mol Å, using the maximum iterations set to 1000. The three-dimensional structure of Angiotensin-I converting enzyme (ACE, EC 3.4.15.1) were retrieved from the Protein Data Bank data base (PDB ID: 4APH:A). Atomic charges were assigned using the MMFF94 method. Protein structure was prepared for molecular docking simulations using the following parameters: polar hydrogen atoms were added, protonation types and termini treatment was enabled. Docking simulations were performed using the program Surflex-Dock with flexible H atoms [Jain, 2003]. Obtained results were visualized using the PyMol program (The PyMOL Molecular Graphics System). Using our molecular docking protocol, Ang I was successfully re-docked with the crystal structure of human sACE with an RMSD of 1.894 Å, which suggests that the appropriate methods were used in the present study.

RESULTS AND DISCUSSION Among the investigated dipeptides, the highest ACE inhibitory activities (Table 1) have those with tryptophan at their C-terminus. Docking simulation of the VW dipeptide revealed that Trp is positioned within the hydrophobic pocket at the end of the catalytic channel (Fig 1A). As the simulation revealed, its high inhibitory activity is the result of the Trp hydrophobic interactions between with Phe527 (3.80Å), Phe457 (4.32Å) and Tyr523 (3.50Å). Carboxil group at the C-terminus makes H bonds with Gln281 (2.90Å), Tyr520 (2.95Å) and His513 (2.98Å). Furthermore, dipeptide VW makes ionic interactions with Lys511 (2.83Å). Free amino group makes ionic interactions with catalitic residue Glu384 (2.76Å), as well as H bond with His383 (2.80Å). Amino acid Trp in dipeptide KW binds similarly compared with Trp in dipeptide VW (Fig. 1B). Deep into the hydrophobic pocket it makes hydrophobic 43


interactions with Val380 (4.33Å) and Val379 (3.08Å). Furthermore, it makes ionic interactions with Glu384 (3.12Å), as well as H bonds with Tyr520 (2.01Å) and His353 (2.18Å). Amino acid Lis is stretched along the active site and makes polar interaction with His410 (3.17Å) at its other end. Dipeptides YW and VY bind similarly with previously described peptides, (results not shown).

Figure 1. Docking results of ACE (PDB ID: 4APH:A) (shown in brown and white) with selected milk dipeptides (shown in green): A) VW, B) KW, C) IG, D) GL, E) IR. H-bonds and hydrophobic interactions formed between residues and molecule are shown as dotted lines with yellow colour. The nonpolar hydrogen atoms are removed for clarity

In the obtained docking simulation, dipeptides with the lowest ACE inhibitory activity were not positioned into the hydrophobic pocket and did not make hydrophobic interactions with the active site of the enzyme. These dipeptides mostly contain glycine are the shortest among the investigated dipeptides. Dipeptide IG is the weakest inhibitor among the dipeptides (Fig. 1C). It doesn’t make hydrophobic interactions with the enzyme, which explains its weak inhibitory activity. It makes H bonds with His353 (3.07Å), Tyr520 (2.94Å) and Gln281 (2.93Å), as well as ionic interactions with catalytic Glu384 (3.02Å) and Lys511 (3.07Å), which makes it a specific inhibitor for ACE. Dipeptide GL also makes electrostatic interactions with Glu384 (2.97Å) and doesn’t make hydrophobic interactions with the enzyme (Fig. 1D). Also, it makes H bond with His383 (3.67Å). Although dipeptide IR contains hydrophobic amino acid Ile, it has short hydrophobic chain. Therefore, it makes weak hydrophobic interactions with residues at the end of catalytic channel, which results in low ACE inhibitory activity. Molecular docking simulation of IR into the enzyme active pocket revealed its binding into the hydrophobic pocket by Ile amino acid (Val380 (3.61Å) and Val379 (3.60Å)). Furthermore, through Arg amino acid it makes electrostatic interactions with Lys511 (2.81Å), Tyr520 (3.18Å) and catalytic Glu384 (2.79Å) (Fig. 1E). Results of scoring function revealed ligands binding affinities to the enzyme (Tab. 2). Peptides with high inhibition have high total scores. Although total score of dipeptide KW is slightly higher than VW, its IC50 value is lower compared to VW. This could be the result of polar interaction contribution to the scoring function that should be taken with caution when the inhibition mechanism is mostly depended on hydrophobic interactions. Dipeptides with the lowest total score have the lowest IC50 values. These results indicate that total score 44


values correlates with experimental IC50 values and could be reliable parameter for prediction of inhibitory activity. Results obtained it this research could be used in further investigations and should provide a framework for design of new ACE inhibitory peptides as drug candidates. Table 2. Molecular docking results of selected dipeptides obtained by SurflexDock scoring function Dipeptide Total score IC50 (µM) VW 13.34 0.5 KW 13.71 1.63 YW 12.44 4.15 VY 11.78 4 IG 8.73 1200 GL 8.31 2500

CONCLUSIONS Docking simulation of the VW dipeptide revealed that Trp is positioned within the hydrophobic pocket at the end of the catalytic channel, which is responsible for its high inhibitory activity. Amino acid Trp in dipeptide KW binds similarly compared with Trp in dipeptide VW. In the obtained docking simulation, dipeptides with the lowest ACE inhibitory activity were not positioned into the hydrophobic pocket and did not make hydrophobic interactions with the active site of the enzyme. These dipeptides mostly contain glycine, which doesn’t make hydrophobic interactions with the enzyme. Total score values correlates with experimental IC50 values. Obtained results could be used in further design of ACE inhibitory peptides as drug candidates.

ACKNOWLEDGEMENTS Financial support from the Ministry of Education, Science and Technological development of the Republic of Serbia (Grant III 46009) is highly appreciated.

REFERENCES Coates, D. (2003). The angiotensin converting enzyme (ACE). The International Journal of Biochemistry & Cell Biology, 35, 769–773. Fuchs, S., Xiao, H.D., Hubert, C., Michaud, A., Campbell, D.J., Adams, J.W., Capecchi, M.R., Corvol, P., Bernstein, K.E. (2008). Angiotensin-converting enzyme C-terminal catalytic domain is the main site of angiotensin I cleavage in vivo. Hypertension, 51, 267–274. Iwaniak, A., Dziuba, J. (2009). Animal and plant origin proteins as the precursors of peptides with ACE inhibitory activity. Proteins evaluation by means of in silico methods. Food Technology and Biotechnology 47 (4), 441–9. Iwaniak, A., Minkiewicz, P., Darewicz, M. (2014). Food-Originating ACE Inhibitors, Including Antihypertensive Peptides, as Preventive Food Components in Blood Pressure Reduction. Comprehensive Reviews in Food Science and Food Safety, 13 (2), 114-134. Jain, A.N. (2003). Surflex: fully automatic flexible molecular docking using a molecular similarity-based search engine. Journal of Medicinal Chemistry, 46, 499–511. Minkiewicz, P., Dziuba, J., Iwaniak, A., Dziuba, M., Darewicz, M. (2008). BIOPEP database and other programs for processing bioactive peptide sequences. Journal of AOAC International, 91, 965-980. O'Boyle, N.M., Banck, M., James, C.A., Morley, C., Vandermeersch, T., Hutchison, G.R. (2011). Open Babel: an open chemical toolbox. Journal of Cheminformatics, 3:33. Stockwell, B.R. (2004). Exploring biology with small organic molecules. Nature, 432, 846–854. The PyMOL Molecular Graphics System (2015) v. 1.8 Schrödinger, LLC. (www.pymol.org) van Esch, J.H., Tom, B., Dive, V., Batenburg, W.W., Georgiadis, D., Yiotakis, A., van Gool, J.M., de Bruijn, R.J., de Vries, R., Danser, A.H. (2005). Selective angiotensin-converting enzyme C-domain inhibition is sufficient to prevent angiotensin I-induced vasoconstriction. Hypertension, 45, 120–125.

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QUALITY ATTRIBUTES OF COOKIES ENRICHED WITH BETAINE Bojana Filipčev*, Jelena Krulj, Jovana Kojić, Olivera Šimurina, Marija Bodroža Solarov, Mladenka Pestorić Institute of Food Technology, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia *Corresponding author: E-mail address: [email protected]

ABSTRACT Although not essential, betaine is an important nutrient which strongly affects general human health by performing several significant physiological roles in human organism. Primarily, betaine acts as an osmolyte and protects the cells from osmotic stress and allows waste product concentration in urine. Secondarily, betaine is a methyl donor in transmethylation reactions. The later physiological function has been related to numerous health benefits betaine has been claimed for: reduced risk for cardiovascular, hepatic and cerebral diseases. Even though it is thought that betaine deficiency is not common in general population of western nations, recent surveys inferred that betaine intake is likely to be on a low side. Elderly population (aged >50 years) or other vulnerable populations (diabetic and homocystinuria patients), may be at a higher risk of inadequate intake. Food enrichment with betaine may be one of the feasible ways to improve the betaine status in general population. In this study, it was investigated the effect of fortifying cookies with increasing betaine levels (5003000 mg/100 g flour) on the cookie quality traits. Most cookie quality attributes (spread, height gain, hardness, fracturability) were not significantly affected by rising betaine levels. It was observed that increasing betaine doses contributed to more prominent vividness and yellow color intensity of upper cookie surface. Sensory evaluation revealed that there were no differences in the perception of texture within the enriched cookies but somewhat better ratings were received for cookies with lower fortification levels (500-1000 mg/100 g flour) regarding taste, total quality and acceptability. This is most probably due to perception of aftertaste (slight burning sensation) at higher levels of enrichment. The enriched cookies were 7-40 times higher in betaine in comparison to the control sample. The estimated betaine loss during baking ranged from 17-29%. Keywords: betaine, cookies, quality, enrichment,dietary intake

INTRODUCTION Nowadays, consumers are drawing more attention on the health aspects of foods in addition to classical, quality and nutritional issues. This attitude has contributed to the growth of functional food market. Functional foods are foods intended to be consumed as a part of a normal diet that claim to improve wellbeing or health. Examples of functional foods include foods that contain specific minerals, vitamins, fatty acids or dietary fiber, and foods with added biologically active substances. In the study of van Kleef et al. (2005), it was shown that consumers tend to prefer functional food concepts that primarily communicate disease-related health benefits in carriers with a healthy image or health positioning history and that consumer evaluations primarily differ to the extent that health claims are personally relevant in addressing an experienced disease state. Consequently, the concepts of “functional foods” and “health claims” are complementary and mutually associated. Development of food formulations eligible to bear a health claim poses a considerable challenge to food technologists. Betaine is a natural compound first derived from sugar beet. Later, it was found in many organisms (bacteria, plants, invertebrates, mammals). In mammals, betaine performs dual roles: a) it preserves osmotic equilibrium and maintains the structure of tissues in the kidney; b) it is a methyl donor which is important for homocysteine metabolism (Craig, 2004). The later role has been associated with the ability of betaine to reduce levels of circulating 46


homocysteine in human organism which captured substantial attention of nutritionists. Plasma homocysteine is a biomarker for elevated vascular disease risk (Bates et al., 2010 in Ross 2014). A betaine-rich diet has been implied to efficiently lower the cardiovascular risk by reducing plasma homocysteine (Olthof et al., 2003). Food rich in betaine includes sugar beet, beet molasses, wheat bran and germ, spinach, bread and shrimps (Craig, 2004). Human may also obtain betaine from foods rich in choline (Craig, 2004). However, studies aimed to estimate the dietary intake of betaine showed large national, international and gender variations among populations investigated and reported mainly low daily intakes (>150 mg/day). Health effects may be expected at betaine daily intakes of 1500 mg and higher (Commission Regulation, 2012). Food enrichment with betaine has been proposed as a more practical and sustainable way to increase betaine intake than modification of dietary patterns that require planning of diets based solely on food naturally rich in betaine (Atkinson et al., 2009). This work was aimed to investigate the effects of betaine incorporation into classical tea cookie formulation. High enrichment levels were considered in order to reach betaine content in cookie high enough to enable a health claim to be made on the product. According to EU legislation, health claim related to contribution of betaine to normal homocysteine metabolism, can be made on foods containing at least 500 mg betaine per serving providing that the daily intake of betaine is 1500 mg (Commission Regulation, 2012). Besides the analysis of general quality attributes such as physical, textural, sensorial and color properties, the content of betaine and betaine loss during baking were also investigated.

MATERIAL AND METHODS Sample preparation The cookies were produced according to the formula (based on flour): wheat flour type 400 (100 g), trans-free vegetable fat (32.5 g), sugar (31.1 g), water (1.1 g), sodium bicarbonate 0.35-g and ammonium bicarbonate 0.20 g. Betaine was added at following doses (on flour basis): 0.5, 1.0, 2.0 and 3.0 g. All the ingredients were blended together in a bowl using a kitchen mixer for 7 min. After a homogenized dough was formed, it was left to rest for 10 min. Finally, dough was sheeted using a dough laminator to a thickness of 4.5 mm. Cookies were shaped with a hand cutter (dimensions 35 x 37 mm) and baked at 170 C for 8 min. Objective and sensory evaluation of cookies was carried out 24 h after baking. Methods Betaine content in the cookies was determined on HPLC equipped with a Kinetex column and ELSD detector. Method of extraction and detailed analytical parameters are given in Filipčev et al. (2015). Cookie dimensions were determined using a digital calliper. Spread was determined as a ratio of widht and height. Cookie texture was determined on a texture analyser TA-XTplus (Stable Micro Systems, England) using the 3-point bending rig. Color properties were measured using a Minolta Chromameter (CR-400, Japan) at standard observation angle of 2 and illuminant D65. The results were expressed according to CIE Lab color system - L* lightness (black 0/white 100); a* (green-/red+); b* (blue-/yellow+). Measurements were made on top surface at five points in “X” pattern. Sensory analysis was carried out with a 6-member panel. The panel consisted from experienced and trained panelists recruited from the staff of the Institute of Food Technology (Novi Sad, Serbia). The panelists scored the biscuits for flavor, aftertaste, texture, and overall quality using a 5-point scale where 5 represented the highest intensity and 1 represented the lowest intensity of the attribute. Flavor described the aromatic notes associated with those typical of cookies. Aftertaste was related to non-typical aromatic notes or sensations (here specifically it was described as a slight burning sensation in the mouth). Texture was evaluated as fracturability in the mouth, i.e. force necessary to break a cookie into crumbs. 47


Overall quality included the general evaluation of cookie quality including its appearance, color, texture, flavor and aftertaste. Score 5 indicated very good quality and 1 very poor quality. One-way ANOVA was used to analyse the effect of betaine addition on the cookie quality.Tukey’s test was used to differentiate means at a significance p40 S. aureus 20 ±0.0 20±0.0 20±0.0 20±0.0 L. monocytogenes 10±0.0 >40 40±0.0 >40 P. hauseri 40±0.0 >40 2.5±0.0 2.5±0.0 P. aeruginosa 10±0.0 40±0.0 10±0.0 10±0.0 E. coli 40±0.0 >40 >40 >40 E. coli O157:H7 40±0.0 >40 >40 >40 S. Enteritidis 20±0.0 20±0.0 20±0.0 20±0.0 S. Typhimurium 40±0.0 >40 >40 >40 Y. enterocolitica 40±0.0 40±0.0 10±0.0 10±0.0 *

Data are expressed as mean ± standard deviation (n = 3).

HA extract was the most active against Gram-negative bacteria Proteus hauseri with bactericidal concentration of 2.5 mg mL–1 followed by P. aeruginosa and Y. enterocolitica. 231


with microbicidal concentration of 10 mg mL–1. Among Gram-negative bacteria, HW extract was most effective against S. Enteritidis (microbicidal concentration of 20 mg mL–1) and P.aeruginosa with minimal inhibitory concentration of 10 mg mL–1. Based on obtained MBC data it can be concluded that HA polysaccharide extracts had stronger antibacterial activity than HW against tested Gram-negative bacteria. Biological activity of polysaccharide extracts might be mostly linked to different chemical composition of the extracts (Matijašević et al., 2016). According to chemical characterization of extracts, HW samples have higher content of proteins and phenolic compounds than HA extracts. Based on FTIR spectra, those extracts contain also more glucans than HA extracts. The low content of proteins and glucans in HA extract is probably due to denaturation, precipitation and damaging by sodium hydroxide solvent. This is probably reason of more pronounced biological activity of HW polysaccharide extracts. The mechanism of action of extracts as complexes of different compounds still remains unknown.

CONCLUSION HW and HA extracts obtained from P. ostreatus P80 have been recognized as a promising source of biologically active compounds such as glucans, peptides and phenols. Variations in their activities might be attributed to the differences in solubility in water, size of the molecules, branching rate and form. Tested microbial strains are recognized as agents of food poisoning, so mushroom extracts are recognized as potential food preservatives with antimicrobial and antioxidant activity.

ACKNOWLEDGEMENTS This work was supported by grant from the Polish National Science Centre DEC2013/09/B/NZ7/03978. The authors would like to acknowledge Ministry of Education, Science and Technological Development of the Republic of Serbia (Projects No. III 46010 and III 46001).

REFERENCES Alarcon, J., Aguila, S. (2006). Lovastatin production by Pleurotus ostreatus: effects of the C:N ratio. Zeitschrift für Naturforschung C, A Journal of Biosciences, 61, (1-2), 95-8. Barros, L., Cruz, T., Baptista, P., Estevinho, L.M., Ferreira, I.C. (2008). Wild and commercial mushrooms as source of nutrients and nutraceuticals. Food and Chemical Toxicology, 46, 2742–2747. Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein-dye binding. Analytical Biochemistry, 72, 248–254. Duvnjak, D., Pantić, M., Pavlović, V., Nedović, V., Lević, S., Matijašević, D., Sknepnek, Nikšić, M. (2016). Advances in batch culture fermented Coriolus versicolormedicinal mushroom for the production of antibacterial compounds. Innovative Food Science and Emerging Technologies, 34, 1-8. Khatun, S., Islam, A., Cakilcioglu, U., Guler, P., Chatterjee N. C. (2015). Nutritional qualities and antioxidant activity of three edible oyster mushrooms (Pleurotus spp.). Njas-Wageningen Journal Of Life Sciences.72-73, 1-5. Kim, H.J., Kim, J.S., Park, R.H. (2009). The different antioxidant and anticancer activities depending on the color of oyster mushrooms, Journal of Medicinal Plants Research, 3(12),1016–1020. Klaus, A., Kozarski, M., Niksic, M., Jakovljevic, D., Todorovic, N., Van Griensven, L.J.L.D. (2011). Antioxidative activities and chemical characterization of polysaccharides extracted from the basidiomycete Schizophyllum commune. LWT - Food Science and Technology. 44, 2005-2011. Kozarski, M., Klaus, A., Niksic, M., Jakovljevic, D., Helsper, J.P.F.G., Van Griensven, L.J.L.D. (2011). Antioxidative and immunomodulating activities of polysaccharide extracts of the medicinal mushrooms Agaricus bisporus, Agaricus brasiliensis, Ganoderma lucidum and Phellinus linteus. Food Chemistry. 129, 1667–1675. Manzi, P., Pizzoferrato, L. (2000). Beta-glucans in edible mushrooms. Food Chemistry. 68, 315–318. Matijašević, D., Pantić, M., Rašković, B., Pavlović, V., Duvnjak, D., Sknepnek, A., Nikšić M. (2016).The Antibacterial Activity of Coriolus versicolor Methanol Extract and Its Effect on Ultrastructural Changes of Staphylococcus aureus and Salmonella Enteritidis. Frontiers in Microbiology. 7:1226 Mau, J.L., Tsai, S.Y., Tseng, Y.H., Huang, S.J. (2005). Antioxidant properties of hot water extracts from Ganoderma tsugae Murrill. LWT – Food Science and Technology, 38(6), 589-597

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III International Congress “Food Technology, Quality and Safety” Ramirez-Anguiano, A.C., Santoyo, S., Reglero, G., Soler Rivas, C. (2007). Radical scavenging activities, endogenous oxidative enzymes, and total phenols in edible mushrooms commonly consumed in Europe. Journal of the Science of Food and Agriculture, 87, 2272-2278 Silva, S., Martins, S., Karmali, A., Rosa, E. (2012). Production, purification and characterization of polysaccharides from Pleurotus ostreatus with antitumour activity. Journal of the Science of Food and Agriculture. 92(9), 1826-1832. Stamets, P. (2000). Growing Gourmet and Medicinal Mushrooms Paperback . Ten Speed Press, 3 th edition, 283-327. Tseng, Y.H., Yang, J.H., Mau, J.L. (2008). Antioxidant properties of polysaccharides from Ganoderma tsugae. Food Chemistry, 107(2), 732-738.

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BIOPROCESS PROSPECTS FOR DEVELOPING CO-PRODUCTS FROM MICROALGAE - CHLORELLA VULGARIS 1

1

Sayali Kulkarni and Zivko Nikolov * Biological and Agricultural Engineering, Texas A&M University College Station, Texas, United States *Corresponding author: E-mail address: [email protected]

ABSTRACT Recent algal bioproducts development efforts have taken on a more comprehensive approach that include high-value food proteins, phycobiliproteins, carotenoids, vitamins, fatty acids and antibacterial metabolites. This comprehensive list of potential products with a broad range of physico-chemical properties and end applications requires a holistic development approach as their isolation and fractionation is rather complex resulting in high-energy and manufacturing cost. Algal proteins have high nutritive value and desirable functional properties, which make them good candidates for application in foods. In this study, we explored several bioprocessing options for extraction of highquality protein from Chlorella vulgaris. To break the rigid cell wall of C. vulgaris for maximal release of algal proteins, several physical and mechanical lysis methods such as sonication, high-pressure homogenization, and bead-beating were compared. Homogenization was best suited for protein extraction releasing 3 times more protein than sonication and ball milling. Protein fractionation by isoelectric precipitation and membrane filtration was compared. Acidification of protein extracts from pH 7.0 to 4.0 was necessary for complete ribulose-1, 5-bisphosphate carboxylase/oxygenase precipitation. Protein-rich concentrate generated by 300 kDa MWCO ultrafiltration had essential amino acid scores comparable or better than FAO/WHO recommended scores. Keywords: microalgae, protein extraction, membrane filtration, cell lysis, chlorophyll pigments

INTRODUCTION The versatile uses of microalgae food, feed and energy have been identified decades ago (de la Noue & de Pauw, 1988). One of the major interests is to make use of microalgae for the production of biofuels (Chisti, 2007). Significant efforts have been made in upstream technologies to increase biomass (de la Noue & de Pauw, 1988) productivity and oil accumulation in biomass by carrying out media optimization and molecular manipulation of metabolic pathways (Mercer & Armenta, 2011). However, multiple high-value co-product extraction is essential to make microalgae an economically viable option for competing with traditional hydrocarbon feedstocks. From various life cycle and techno economic analysis, it has been found out that biofuels-from-algae platform can’t be sustainable unless other high value products such as important proteins can be extracted from algae (Guedes et al., 2011; Safi et al., 2014) Microalgae are a rich source of protein, containing 20-60 % protein depending on the species (Becker, 2007). Chlorella vulgaris is a microalgae that can accumulate high amount of lipids, carbohydrates as well as proteins (Liang et al., 2009). C. vulgaris contains 51-58% protein (dw basis) and comparable amino acid profile to WHO/FAO standards. The major high value protein in algal extracts is ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO). It has a potential to be an ingredient in food and feed. Purified RuBisCO is an odorless, tasteless, white powder with a nutritive value as good as other food proteins (Barbeau & Kinsella, 1988). The rigidity of C. vulgaris cell wall (Gerken et al., 2013) is one of the main obstacles for extracting internally stored bio-products. For releasing and fractionating valuable compounds, biomass must be subjected to an efficient cell wall disruption treatment. Various cell lysis methods have been tested out on C. vulgaris. Some of them are 234


acid & alkaline treatment, autoclaving, bead milling, and electroporation, french press, manual grinding microwaves, osmotic shock and ultrasonication (Safi et al., 2014). Previous research has shown that protein fractions obtained by homogenization and tangential flow filtration from C. vulgaris have great amino acid profile excellent emulsifying properties (Ursu et al., 2014). However, complete cell disruption is required for protein solubilization and enhanced emulsification properties. Keeping this in mind, the first specific objective was to compare three mechanical methods of cell lysis: high-pressure homogenization, sonication and bead milling. Different extraction conditions also effect the amount of protein released (Ursu et al., 2014). Hence, after the selection of the best method for cell lysis, the next objective of this research was to compare protein extraction at neutral (7.0) and basic (12.0) pH. C. vulgaris was homogenized at neutral pH. Finally we aimed to fractionate the main high value product RuBisCO by step-wise acid precipitation at pH 6, 5 and 4. The pellet and supernatant fractions were analyzed for total soluble protein and RuBisCO. The last step involved obtaining a RuBisCO-rich fraction of protein by membrane filtration using a 300 kDa membrane. In order to evaluate the quality of the fraction, amino acid analysis was carried out.

MATERIALS AND METHODS Chlorella vulgaris biomass was obtained from Global Algae Innovations in the form of a wet paste (24 % solids). Biomass was stored at -80 0C. Mechanical methods of cell lysis Three different methods of cell lysis checked: homogenization, bead-beater and sonication. Frozen wet biomass was mixed with RO water (pH 6.75) with biomass to buffer ratio of 1:5, and mixed end-over-end for 15 minutes. Homogenization of biomass was carried out using Emulsiflex c3 (Avestin, Canada) high-pressure homogenizer for 3 passes at 17000 psi. Effectiveness of bead-beating for cell lysis was tested by mixing biomass with 0.5 mm beads in BeadBeater (Bartlesville, OK) for a total of 16 minutes. Sonication was carried out for 30 min with 30 s on/off intervals at 4 °C using a sonicator (Sonifier 250, Branson, USA) at 30% output control and 30% duty cycle with a microprobe (1/8′ microtip A3-561 Branson, USA) After cell lysis, lysate was centrifuged at 10000 rpm for 20 min at 4 °C. Effect of pH on protein extraction Frozen biomass was mixed with RO water at a biomass to buffer ratio of 1:5 and divided into two parts, one at pH 6.75 and at the other at pH 11.8 (pH adjustment was done using 1M NaOH). Homogenization was carried out for 5 passes at both pH 6.75 and pH 11.8. Three replicates were carried out. Total soluble protein (TSP) was measured after every pass. JMP pro 11 was used for statistical analysis. RuBisCO fraction using acid precipitation Clarified cell-free extracts (CFE) obtained after homogenization were subjected to step-wise acid precipitation. CFE at pH 6.75 was brought to the pH of 6 using 0.2 N HCl, mixed end over end for 15 minutes and centrifuged at 10000 rpm for 20 mins at °C. pH of the supernatant from this stage was adjusted to 5 and centrifuged under same conditions. The pH of the supernatant at pH 5 was then adjusted to pH 4, which was followed by centrifugation to obtain a pellet and supernatant at pH 4. The pellets and supernatants at every step were analyzed for TSP and presence of RuBisCO by SDS-PAGE and Western blot. Membrane filtration for fractionation of RuBisCO-rich protein concentrate In order to fractionate a RuBisCO rich protein concentrate, membrane filtration was carried out on homogenized CFE. Membrane used in the first stage was 300 kDa MWCO hollow fiber module (Spectrum Laboratories, Houston, TX). Concentration of permeate from the first 235


step was carried out using a 3 kDa MWCO hollow fiber membrane (GE Healthcare, Marlborough, MA). The detailed description of the entire process in summarized in Figure 1. TSP in every stream was measured by the Bradford assay. Fraction A and fraction B were obtained by freeze-drying the respective concentrates. Amino acid analysis was conducted to analyze the nutritive value of every fraction. Total carbohydrates in fraction A and B were measured.

Figure 1. Membrane filtration process used to generate different protein fractions

Protein, total carbohydrates and amino acid analysis Total soluble protein (TSP) was determined by Bradrdford assay (Bradford, 1976) using Commassie plus assay kit (ThermoScientific). Absorption at 595nm was measured using VERSA max microplate reader (Molecular Devices). NuPAGE Novex Bis-Tris pre-cast gradient gels (4–12%) from Invitrogen™ (1.5 mm X 10 wells), were used for SDS-PAGE electrophoresis. Reducing buffer was prepared using NuPAGE LDS sample buffer (4X) and 2-mercaptoethanol (9:1). Samples were reduced by using a 1:3 ratio of reducing-buffer sample, followed by heating at 70 °C for 10 min. NuPAGE MES SDS Running Buffer (20X) stock solution was used to prepare 1X running buffer in RO water. NuPAGE antioxidant was added to ensure reduced samples during electrophoresis. Gels were run for 35 min at constant voltage (200 V). For SDS analysis, the gels were stained in Coomassie™ G-250 stain (ThermoFisher Scientific) followed by destaining in RO Water. For Western blot analysis, the gel was transferred to nitrocellulose membranes using iBlot® 7-Minute Blotting System (Life Technologies Corporation). After protein transfer, membrane was blocked with 2.5 % fat-free milk in TBS buffer (pH 7.5) containing 0.05% Tween. RuBisCo was detected using anti-RuBisCO antibody produced in chicken and anti-chicken antibody produced in rabbit (Sigma Aldrich, St Louis MO). Blots were visualized with nitro-blue tetrazolium (NBT) and 5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt (BCIP-Sigma FAST). Amino acid distribution was determined by hydrolysis of samples, followed by derivatization and separation of amino acids using HPLC (performed by Protein Chemistry Lab, Texas A&M University). Total carbohydrates estimation was carried out using phenol-sulfuric acid method (Dubois et al., 1956). Standards ranging from 12.5-100 ug/ml were prepared by mixing glucose in RO water. 1 ml of standards and samples were mixed with 1 ml of 5% phenol

236


solution. 5 ml of concentrated sulfuric acid was added to the mixture. After mixing well, samples kept at room temperature for 25 min and absorbance was measured at 490 nm.

RESULTS AND DISCUSSIONS Comparison of method of cell lysis We compared three different mechanical methods of cell lysis: high-pressure homogenization, sonication and bead milling. The efficiency was assessed by the amount of TSP extracted from frozen wet biomass. As seen in Figure 2 homogenization works the best in order to break the tough cell wall of chlorella compared to other physical methods of cell lysis. Protein amount released via homogenization is 3 times greater than sonication and 1.5 times greater than bead milling.

Figure 2. Comparison of protein extraction from C. vulgaris using different cell lysis methods

Effect of pH on protein extraction In order to check the effect of pH on protein extractability, homogenization of wet biomass was carried out at neutral and basic conditions.

Figure 3. Protein released at neutral and basic pH

As seen in Figure 3, TSP released at pH 11.75 was slightly higher than that at pH 6.8, but wasn’t significantly different. The large deviation in TSP could be result of variability in protein content of different batches of biomass used. Since change in pH didn’t significantly impact protein release, it was decided to carry out homogenization at neutral pH. RuBisCO fractionation using acid precipitation The major high value protein in algal extracts is RuBisCO. C. vulgaris was homogenized at neutral pH. In order to fractionate RuBisCO, step-wise acid precipitation was carried out at pH 6, 5 and 4. The pellet and supernatant fractions were analyzed for total soluble protein (Figure 4a) and RuBisCO (Figure 4b). As seen in the Figure 4a, pH 5 supernatant and pH 4 237


pellet have significantly lesser protein than the clarified cell extract, and based on western blot analysis still contains significant amount of RuBisCO. Pellet fraction at pH 4 can be carried forward to the next purification step. Acid precipitation of algal protein at pH 5 and below pH 5, reduces DNA (by 9%) as well as chlorophyll (70%) content in algal cell lysates (Munjal et al., 2015). Membrane filtration for fractionation of RuBisCO-rich protein concentrate Membrane filtration fraction labeled as Concentrate 300kDa AD (after dialysis) in Figure 5, contained significant amount of RuBisCO and other algal proteins (lanes 3 and 5). Freezedried protein concentrate (Fraction A in Figure 1), consisted of 45% protein (estimated by amino acid analysis; 36 % if estimated by Bradford assay), and another 10-14 % consisted of carbohydrates. Fraction B, had 43% protein by weight and 18-20% carbohydrates. (a)

(b)

TSP (mg)

500

Step-wise acid precipitation

0 Clarified cell lysate

pH 6

pH 5

pH 4

Pellet

Figure 4a. TSP distribution during step-wise acid precipitation; Figure 4b: Western blot for distribution of RuBisCO. Lane 1: cell free lysate; lane 2: supernatant at pH 6; lane 3: pellet at pH 6; lane 4: pellet at pH 5; lane 5: supernatant at pH 5; lane 6: supernatant at pH 4; lane 7: pellet at pH 4

Figure 5. SDS-PAGE gel for membrane filtration fractions. Lane 1: molecular weight marker; lane 2: cell-free extract (CFE); lane 3: 300 kDa concentrate before diafiltration diluted; lane 4: 300 kDa permeate before diafiltration ; lane 5: 300 kDa concentrate after diafiltration; lane 6: 300 kDa permeate after diafiltration; lane 7: 3 kDa concentrate after diafiltration; lane 8: 3 kDa permeate after diafiltration. 1X, 10X, 30X indicate the dilution of samples for loading on to SDS-PAGE gel

238


Comparing the amino acid profile of Fraction A and B, it was found that Fraction A has higher percentage (data not shown) of essential amino acids (methionine, phenylalanine, isoleucine, leucine, lysine & valine) than Fraction B. Fraction B had higher amount of glutamic acid/glutamate. All other amino acids are similarly distributed in both fractions. Essential amino acid distribution of RuBisCO isolated from alfalfa leaves and other protein sources is listed in Table 1 (Hood et al., 1981). This RuBisCO isolate contained 98% protein and 1-1.8 % carbohydrates. Fraction A has comparable amino acid profile to RuBisCO isolate, other protein sources meet the FAO/WHO standards. However, it had only 45% protein by weight. It should be noted that removal of impurities from fraction A in order to increase weight percentage of total protein is necessary, in order to compare its nutritive value (per gram) to other pure protein isolates. Table 1. Essential amino acid chemical scoring for Fraction A Amino acid

a

a

RuBisCO (g/ 16g N)

6.4 5 7.4 6.6 8.8

8 5.3 6.7 6.6 10

6.5 5.3 6.7 4.9 9.4

Fraction A (g/ 100 g protein) 6.5 5.5 6.5 5.2 9.6

10.1

11.2

12.8

10.4

Whole egg (g/ 16g N)

5.5 4.0 5.0 4.0 7.0 6.0

Lysine Threonine Valine Isoleucine Leucine Tyrosine and Phenylalanine

e

Casein (g/ 16g N)

FAO/WHO (g/ 16g N)

b

c

d

Scoring (%) >100 >100 >100 >100 >100 >100

FAO/WHO reference (1973) b (Harper, 1977) c (Gordon & Kalan, 1974) d Hood (1981) e This study. Values might be slightly lower than reported considering that tryptophan and cysteine haven’t been included. f Fraction A compared to FAO/WHO reference

CONCLUSIONS High-pressure homogenization was the best method for lysis of C. vulgaris cells and complete protein extraction. Protein extraction is higher at basic pH, but the difference is not significant after 5 passes through the high-pressure homogenizer. Acid precipitation (pH 5) can remove other proteins, and partially purify RuBisCO. Membrane fractionated, RuBisCOenriched fraction has amino acid profile comparable to FAO/WHO standards, and can potentially be further developed and formulated for a variety of food applications.

REFERENCES Becker, E. 2007. Micro-algae as a source of protein. Biotechnology advances, 25(2), 207-210. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry, 72(1), 248-254. Chisti, Y. 2007. Biodiesel from microalgae. Biotechnology advances, 25(3), 294-306. de la Noue, J., de Pauw, N. 1988. The potential of microalgal biotechnology: a review of production and uses of microalgae. Biotechnology advances, 6(4), 725-770. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P., Smith, F. 1956. Colorimetric method for determination of sugars and related substances. Analytical chemistry, 28(3), 350-356. Gerken, H.G., Donohoe, B., Knoshaug, E.P. 2013. Enzymatic cell wall degradation of Chlorella vulgaris and other microalgae for biofuels production. Planta, 237(1), 239-253. Gordon, W., Kalan, E. 1974. Proteins of milk. Fundamentals of dairy chemistry, 87-124. Guedes, A., Amaro, H.M., Malcata, F.X. 2011. Microalgae as sources of high added‐value compounds—a brief review of recent work. Biotechnology Progress, 27(3), 597-613. Harper, A. 1977. Human amino acid and nitrogen requirements as the basis for evaluation of nutritional quality of proteins. Hood, L., Cheng, S., Koch, U., Brunner, J. 1981. Alfalfa proteins: Isolation and partial characterization of the major component—Fraction I protein. Journal of Food Science, 46(6), 1843-1850.

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f

III International Congress “Food Technology, Quality and Safety” Liang, Y., Sarkany, N., Cui, Y. 2009. Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions. Biotechnology letters, 31(7), 1043-1049. Mercer, P., Armenta, R.E. 2011. Developments in oil extraction from microalgae. European journal of lipid science and technology, 113(5), 539-547. Munjal, N., Kulkarni, S., Quinones, K., Tran, M., Mayfield, S.P., Nikolov, Z.L. 2015. Evaluation of pretreatment methods for primary recovery and capture of an antibody fragment (αCD22scFv) from Chlamydomonas reinhardtii lysates. Algal Research, 12, 455-462. Safi, C., Zebib, B., Merah, O., Pontalier, P.-Y., Vaca-Garcia, C. 2014. Morphology, composition, production, processing and applications of Chlorella vulgaris: A review. Renewable and Sustainable Energy Reviews, 35, 265-278. Ursu, A., Marcati, A., Sayd, T., Sante-Lhoutellier, V., Djelveh, G., Michaud, P. 2014. Extraction, fractionation and functional properties of proteins from the microalgae Chlorella vulgaris. Bioresource technology, 157, 134-139.

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MEAT INDUSTRY EFFLUENTS 1

Zvonko Nježić *, Đorđe Okanović

1

1

Institute of Food Technology, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia

*Corresponding author: E-mail address:[email protected]

ABSTRACT Sustainability of the system of the process management, safety, quality and environment protection in the chain of large-scale production of foods depends on the availability of the methods that enable the realization of measurements of the corresponding indicators in production, processing and distribution, through the use of investigative methods. Utilization of these methods should be sustainable with respect to the speed with which the results are obtained, cost of the performed investigations and the significance of the obtained results. Important aspect of the sustainability of massive food production chain is the effect of the processes on the environment. In the meat industry, the by-products make up to 70 percent of the slaughtered animal weight, and their utilization and non-harmfull disposal are regarded as a priority with respect to the ecological risk and the influence on the environment. Meat industry with its location in the suburban towns and villages is often a source of unpleasant odor. Rodent and insect colonies on those sites are a source of infection and a great danger to human health. Keywords: Meat industry, Effluents, Environment protection

INTRODUCTION The environment is endangered with creation and accumulation of wastes, so more attention is given to its protection and upgrading. With the endeavor of manufacturing even larger quantities of goods aimed at fulfilling human needs for higher standard of living and creating optimal conditions for maintaining a state of health, the contemporary civilization generates great quantities of wastes that adversely influence the environment, degrading it to such a degree that it becomes harmful to human and animal health (Okanović et al., 2008). A part of slaughterhouse by-products is very often discarded as animal wastes, which, as such, contaminate the environment, and they can cause contagious diseases in humans and animals. If animal by-products are not adequately disposed of, they undergo a rapid degradation. This degradation process is accompanied by creation of decomposition products, mainly gases (ammonia, hydrogen sulphide, mercaptans, etc.), as well as of other decomposition products, such as fatty acids, aromatic substances and others. As such, they not only pollute the atmosphere with harmful gases and gases that have unpleasant odors, but they also contaminate the soil, food and water as well. This objection also imposes the performance of investigations aimed at defining how these producs should be utilized in a safe and ecologically justified manner. The contaminants include numerous inorganic and organic compounds that reach the organism through the polluted air, water, and nutrition. Initial quantities are low, but with time they accumulate in individual tissues and organs, causing sicknesses, degenerations or death of the organism. It seems that the aspiration for better productivity creates an opposite effect as well. This is absolutely true in agricultural and animal production, which has undergone various changes. Higher consumption of the protein rich foods steers the animal production towards breeding of even higher number of animals. That tendency is made possible with industrial preparation of animal feeds and with higher automatization and mechanization of the animal growth processes (Okanović et al., 2007). Dead animals and inedible slaughterhouse by-products, in the form of wastes from the production and processing, if not safely disposed, can become a serious barrier to further improvement of manufacturing, in this case of foods, and as such they represent significant contaminants of the environment (Ristić et al., 2008). Animal wastes and inedible decaying by-products contaminate not only the air with unpleasant and toxic gases, but also soil, foods 241


and water resources as well. Ristić (2003) states that the predominant part of blood ends up in sewage, i.e., in effluents, and that only minor part of blood is collected and processed. This leads to surface waters becoming physically polluted, and at the same time, blood in such environments represents substrate for microorganisms’ growth, many of which are pathogenic for humans and animal world. Biological oxygen demand (BOD) of blood, according Baras et al. (1991), amounts to some 100,000 mgO2/l. Pollution of surface water with blood in the year of 1989 in former Yugoslavia amounted to about 57 x 109 kg which is the equivalent of about one million inhabitants. Pollution of the environment with animal wastes shows other adverse effects as well. It is well known that locations where organic waste substances are heaped up and decay regularly provide ideal living conditions for insects and rodents. They enable spreading of infections and contribute to the degradation of visual impression of the environment which they inhabit. Improper manipulation of dead animals and farm and slaughterhouse by-products also cause the contamination of soil, surface- and underground water, foods and various utilities, thus making them inconvenient or less valuable for utilization (Ristić 2007). WASTE WATERS FROM THE MEAT PROCESSING INDUSTRY Food industry consumes great quantities of technical water, so the extraction and separation of starch and proteins, which are in the case of wheat flour separated in the form of gluten in one of some twenty patented ways, necessitates such quantities of water, which, on average, are up to 8 times higher than the quantities of the starting raw material (Nježić et al., 2006). Sugar industry needs up to 17 times higher quantity of water when compared to sugar beet, the water has to be recycled, thus decreasing the needs of incoming water quantity. Slaughterhouse industry is a great consumer of bacteriologically and physicochemically correct water; it is believed that for each slaughtered cow or swine some 500 to 1,000 liters of water are used. With regard to water used in slaughter house industry, we have to make distinction between technological water and water for sanitary purposes that must have drinking water quality (Baras et al., 2002). 

Process water should fulfill claims of the WHO for drinking water,



Sewage systems and waste disposal systems should be designed and constructed in a manner whereby the risks of contaminations of the process water are avoided.

Water that comes in immediate contact with manufacturing streams has to be exclusively of sanitary quality. Ice and steam also ought not to represent a threat to proper health and safety of food. The largest part of water consumed in slaughterhouses appears in the technological waste effluents. For effluent disposal, all areas have to possess the corresponding numbers of gutters or water drains. Gutters must have efficient system of prevention for returning of unpleasant odors from the sewage. Waters contain high quantities of organics, chlorine and residues of substances used for cleaning and sanitation (BOD 5=200-1500 mg O2/l; COD=800-1700 mg O2/l) (IvančevTumbas.,et al., 2004). Therefore, waste waters from slaughterhouses should, prior to being disposed into some natural receiver or local sewage system, pass through pretreatment and/or biological treatment. Pretreatment of waste waters includes fat removal, sedimentation of the suspended particles and chlorination. Sewage systems for atmospheric and sanitary waste waters are united with the technological waste eater systems only after the equipment for their pretreatment (BOD 5 0.05) difference were confirmed in 527


the height and the impact of different fertilizers applied. Plants with a maximum biomass were obtained in the treatment with commercial organic fertilizer “Italpollina”; on average they weighed 336.2 g, which was higher by 121.0 g compared to the average plant weight obtained in the variants fertilized with organic mineral fertilizer “Fertizel” (215.2 g). On the other hand, the plants treated with this fertilizer had the highest number of flowers per plant (52.0), but also the lowest average flower weight (1.7 g) in relation to the average flower weights of plants fertilized with “Italpollina” (2. 0 g) or with “LAB1” (1.8 g). However, the maximum flowers mass per plant in the first year had the plants treated with “Italpollina” (93.6 g). The dry flower yields calculated per hectare, as the most relevant indicator of pot marigold flower productivity, confirmed that the best yields are obtained on plots treated with “Italpollina” (1924.4 kg ha-1) which significantly differed (p> 0.05) from the yields treated with “Fertizel” (1524.0 kg ha-1) and from the control plots (1678.8 kg ha-1). The best chemical quality of pot marigold flowers – i.e. the high total carotenoids content has also been obtained from plots treated with “Italpollina” (1360 mg kg -1) but this time, even slightly better with “LAB1” (1376 mg kg-1). Table 3. Statistical indicators of morphological, productive and qualitative properties of pot marigold resulting from the application of different nutrition amounts in the year 2014 Treatments “Fertizel” “Italpollina” 4:4:4 “LAB1” Control Average Cv (%) LSD 5%

The average weight of the dry flower (g)

The dry flowers mass / plant (g)

The dry flowers yield (kg -1 ha )

Total carotenoids (mg/kg)

1.3

55.9

1750.1

1678

51.7

1.4

72.4

1858.7

1887

40.8 32.3 41.9 10.2 5.3

1.3 1.1 1.3 0.6 0.7

53.0 35.5 54.2 6.3 6.3

1899.9 1631.2 1785.0 123.4 562.3

1756 1532 1713 202.2 512.4

Plant height (cm)

The mass of the whole plant (g)

Number of flowers / plant

33.8

200.1

43.0

42.1

231.4

40.7 30.2 36.7 4.8 4.8

190.1 172.8 198.6 24.6 24.2

In the second year, between the plant heights reached with the implementation of "Italpollina" and "LAB1" on one side, and the plant height reached with the implementation of "Fertizel" and the control (plots without fertilization control), highly significant variation in the plant height was recorded (Table 3). As in the first year, in the second year of investigation the maximum average mass of the plants was recorded in the plots fertilized with “Italpollina” (231.4 g), except that due to a heavy rainfall in the 2014, the plant mass in this variant, compared to those obtained in the 2013, was lower for about 100 g. Number of flowers per plant prove to be in positive correlation with the mass of the whole plant. Application of all fertilizers increased the average number of flowers and the average mass of dry flowers compared to the control treatment. The calculated average mass of dry flowers per plant proves to be the highest in the second year in the variant fertilized with “Italpollina”. The yield of flowers obtained in the second year and expressed per hectare was the highest on plots fertilized with the compost “LAB1” (1899.9 kg ha-1), statistically differing (p>0.05) from the control (1631.2 kg ha-1). The coefficient of variation (Cv) for the obtained dry flower yields under different fertilization treatments accounted for 123.4 %. In the second year, the content of carotenoids in the flowers increased and it ranged from the lowest value 1532 mg kg -1 in the control till the highest one 1887 mg kg-1 in variant with “Italpollina”. As in accordance with Kumar and Singh (2011) who reported that the application of fertilizers with higher nitrogen content significantly increased the height of pot marigold plants, in our study the largest plant height was achieved with “Italpollina” (52.2 cm). Similarly to results of the “LAB1” (51.4 cm), both values statistically differ (p> 0.05) from the heights obtained with other two variants, the “Fertizel” (44.7 cm) and the control (43.5 cm). The greatest influence on formation of the whole plant mass was achieved again by “Italpollina”; on these plots the average weight of the whole plant was 405.2 g, the value being higher by 7.3% compared to those obtained with “LAB1” by 22.4 % with “Fertizel” or by 25.2% compared to the values in 528


control plots. Differences in the weights of whole plants prove to be statistically significant (p> 0.05). The values for number of flowers, the average dry flower weights and the dry weight of flowers per plant were the highest in flowers collected from the plots fertilized by “Italpollina”, as well as the yields of dry flowers (2631.0 kg ha-1). On the contrary, the total carotenoid content was the lowest in the aforementioned fertilization variant (2058 mg kg -1), the highest being in flowers harvested from plots treated with “Fertizel” (2451 mg kg-1). Table 4. Statistical indicators of morphological, productive and qualitative properties of pot marigold resulting from the application of different nutrition amounts in the year 2015 Treatments “Fertizel” “Italpollina” 4:4:4 “LAB1” Control Average Cv (%) LSD 5%

The mass of the whole plant (g)

Number of flowers / plant

314.5

60.2

52.2

405.2

51.4 43.5 47.9 13.3 3.9

375.5 303.1 349.6 35.8 23.8

Plant height (cm)

44.7

The average weight of the dry flower (g)

The dry flowers mass / plant (g)

The dry flowers yield (kg -1 ha )

Total carotenoids (mg/kg)

2165.0

2451

74.2

3.3

244.9

2631.0

2058

56.8 42.8 58.5 34.3 6.4

2.7 1.9 2.6 1.5 0.4

153.4 81.3 157.5 168.7 9.5

2346.0 1804.0 2236.5 367.8 367.3

2226 2120 2214 243.7 598.1

2.5

150.5

Generally, the highest values of all investigated parameters were recorded in the third experimental year. This point to the fact that the environmental conditions greatly influenced morphological, productive and qualitative traits of cultivated pot marigold flower, which is in agreement with findings reported in other few investigations with cultivated pot marigold (Novak et al., 1999; Martin and Deo, 2000; Chaparzadeh et al., 2004). The impact of the external environment can certainly be very significant for variations of agrobiological properties of Calendula officinalis and for the content of carotenoids in its different genotypes (Adamović, 2010). Compared to the previous findings (Kishimoto et al., 2005, Cox & Eaton, 2011, Elhindi, 2012, Khalid & da Silva, 2012, Lim, 2013, Hasan et al., 2013) our investigation showed significant differences in yields and qualitative traits of dry pot marigold flower. In studies of Atiyeh et al. (2002), where different vermicomposts were investigated on cultivated tagetes (the plant which is increasingly used in organic farming, so as the pot marigold), due to its fungicidal properties, the obtained results showed a significant differences achieved with tagetes grown in substrate mixed with vermicompost in comparison to those grown without it.

CONCLUSION According to our experience in the three-year experimental production of pot marigold flowers conducted in Serbia with cultivar ˝Domaći oranž˝ and based on the results obtained with implementation of different fertilizers, the following can be concluded: 1. Application of both the commercial organic fertilizer “Italpollina” (NPK 4: 4: 4) and the

organic compost “LAB1” significantly improved morphological, productive and qualitative traits of pot marigold flower. 2. In the three-year research period, the highest average values for the plant height, the whole plant mass, the number of flowers per plant, the average flower weight, the average mass of flowers per plant and the yield of flowers per hectare were achieved on plots fertilized by “Italpollina” (NPK 4: 4: 4), while the positive impacts on the same parameters was also achieved by the compost “LAB1”. The remaining two treatments (organic fertilizer “Fertizel” and the control) achieved significantly worse results. 3. In the three agro-ecologically different experimental seasons, the best productivity of pot marigold flowers was recorded in the third year (2236.5 kg ha-1) which was for 451.5 kg ha-1 higher compared to those obtained in the second and for 497.9 kg ha-1 in the first one. 529


4. A similar relationship has been made with the total carotenoids content in pot marigold

flowers. The highest average value 2214 mg kg-1 was achieved in the third experimental season, while in the second one it was 1713 mg kg-1 and in the first one only 1297 mg kg1 . A significant increase in the amount of carotenoids was recorded in all fertilized plots compared to the control confirming that it improves healing and nutritional properties of pot marigold plants.

ACKNOWLEDGEMENTS We are grateful to the Ministry of Education, Science and Technology of Republic of Serbia for support in this investigation (Project III46006).

REFERENCES Adamović. S.D. (2010). Variranje agrobioloških osobina i sadržaja karotenoida različitih genotipova nevena u zavisnosti od vremena berbe. Bilten za alternativne biljne vrste, 42, 40-45. Andersen. F.A. (2001): Final report on the safety assessment of Calendula officinalis extract and Calendula officinalis. Int. J. Toxicol., 20 (2), 13-20. Atiyeh. R.M.. Arancon. N.Q.. Edwards. C.A.. Metzger. J.D. (2002). The influence of earthworm-processed pig manure on the growth and productivity of marigolds. Bioresource Technology, 81, 103-108. Chaparzadeh. N.. D'Amico. M. L.. Khavari-Nejad. R. A.. Izzo. R.. Navari-Izzo. F. (2004). Antioxidative responses of Calendula officinalis under salinity conditions. Plant Physiology and Biochemistry, 42 (9), 695-701. Cox. D.. Eaton. T. (2011). Organic fertilizer use leads to different growth response. nutrient use. and nitrogen leaching by marigold ‘First Lady’. Floral Notes, 24 (1), 5-8. Elhindi. K. (2012). Evaluation of composted green waste fertigation through surface and subsurface drip irrigation systems on pot marigold plants (Calendula officinalis L.) grown on sandy soil. Australian Journal Crop Science, 6 (8), 1249-1259. European Pharmacopoeia 7th edition (Ph. Eur. 7.0.) (2011). Council of Europe. Strasbourg. Filipović. V.. Ugrenović. V. (2015). Pot Marigold – One of the More Demanded Medicinal Plants. International Scientific Meeting „Sustainable agriculture and rural development in terms of the Republic of Serbia strategic goals realization within the Danube region - Regional specifics. Proceedings. 296-313. Hasan. A.E.. Bhiah. K.M.. Al-Zurfy. M.T. (2014). The impact of peat moss and sheep manure compost extracts on marigold (Calendula officinalis L.) growth and flowering. Journal of Organic Systems, 9 (2), 56-62. Khalid. K.A.. da Silva. J.T. (2012). Biology of Calendula officinalis Linn.: focus on pharmacology. biological activities and agronomic practices. Medicinal and aromatic plant science and biotechnology, 6 (1), 12-27. Kishimoto. S.. Maoka. T.. Sumitomo. K.. Ohmiya. A. (2005). Analysis of Carotenoid Composition in Petals of Calendula (Calendula officinalis L.). Biosci. Biotechnol. Biochem., 69 (11), 2122-2128. Krinsky. N. (2001). Carotenoids as antioxidants. Nutrition, 17, 815-817. Legha. M.R.. Prasad. K.V.. Singh. S.K.. Kaur. C.. Arora. A.. Kumar. S. (2012). Induction of carotenoid pigments in callus cultures of Calendula officinalis L. in response to nitrogen and sucrose levels. In Vitro Cellular & Developmental Biology-Plant, 48 (1), 99-106. Lim. T.K. (2013). Calendula officinalis. In: Lim. T.K. (Ed.). Edible Medicinal And Non-Medicinal Plants: Volume 7. Flowers. Springer Science & Business Media, 213-244. Martin. R.J.. Deo. B. (2000). Effect of plant population on calendula (Calendula officinalis L.) flower production. New Zealand Journal of Crop and Horticultural Science, 28, 37-44. Novak. J.. Zitterl-Eglseer. K.. Franz. Ch. (1999). Genotype x environment interaction in Calendula officinalis L. Acta Horticulturae, 502, 67-70. Re. T.A.. Mooney. D.. Antignac. E.. Dufour. E.. Bark. I.. Srinivasan. V.. Nohynek. G. (2009). Application of the threshold of toxicological concern approach for the safety evaluation of calendula flower (Calendula officinalis) petals and extracts used in cosmetic and personal care products. Food Chem. Toxicol., 47, 1246-1254. Sarić. M.. Kastori. R.. Petrović. M.. Stanković. Ž.. Krstić. B.. Petrović. N. (1990). Praktikum iz fiziologije biljaka. Naučna knjiga. Beograd. Sausserde. R.. Kampuss. K. (2014). Composition of carotenoids in calendula (Calendula officinalis L.) flowers. In 9th Baltic Conference on Food Science and Technology" Food for Consumer Well-Being" FOODBALT 2014. Jelgava. Latvia. 8-9 May. 2014. Latvia University of Agriculture. Faculty of Food Technology, 13-18. STATISTICA (Data Analysis Software System). v.10.0 (2010). Stat-Soft. Inc. USA (www. statsoft.com). Su. Q.. Rowley. K.G.. Itsipoulos. C.. O’Dea. K. (2002). Identification and quantitation of major carotenoids in selected components of the Mediterranean diet: green leafy vegetables. figs and olive oil. European Journal of Clinical Nutrition, 56, 1149-1154. Tucakov. J. (2014). Lečenje biljem – novo. izmenjeno i dopunjeno izdanje. Vulkan izdavaštvo d.o.o.. Beograd. Yeum. K.J.. Russell. R.M. (2002). Carotenoid bioavailability and bioconversation. Annual Review of Nutrition, 22, 483-504.

530


SHELF LIFE OF RAMSONS (Allium ursinum L.) UNDER DIFFERENT STORING CONDITIONS 1

1

1

Alena Tomšik *, Jasna Mastilović , Žarko Kevrešan , Senka Vidović

2

1

Institute of Food Technology, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia 2 Faculty of Technology, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia

*Corresponding author: Alena Tomšik E-mail address: [email protected]

ABSTRACT Allium species have been used in the traditional medicine for many centuries as edible and medicinal plant. In past few years interest for Allium ursinum L., (wild garlic) as wild aromatic plant is significantly growing thanks to its high content of specific compounds with health benefits. Broad spectrum of biological activities obtained from A. ursinum such as antioxidant, cytostatic, antimicrobial, and antidiabetic were reported with the highest content of biologically active compounds in fresh leafs before flowering. Due to short period of availability of fresh wild garlic leafs during spring season, its shelf life was evaluated in dependence of post harvest storing conditions. Leaf stalks of wild garlic bunches (20 leafs per bunch) were immersed in water in order to preserve the water regime and stored at different temperatures (0, 5, 10 and 20° C) during two weeks. Weight loss, colour change and sensory properties (wilting, yellowness, and decay) were monitored sensory for examined samples. In addition the changes in respiration rates during the storage period for wild garlic leafs stored at different temperatures were compared. The storing of samples at 20 °C resulted in significant colour change in comparison to other temperatures in 4 days, while the samples stored at 10 °C changed significantly the colour in 7 days. Due to preservation of water regime there was no weight loss registered for samples stored at 0, 5 and 10 °C, while the samples stored at 20 °C suffered from weight losses. For samples stored at 0 and 5 °C respiration rate was low and constant during the storage period, while the samples stored at 10, and especially 20 °C had higher initial respiration rate that dropped rapidly. Due to shrivelling of leafs and colour changes the samples stored at 20 °C lost their market value in less than 7 days, samples stored at 10 °C in less than 14 days, while the samples stored at 5 and 0 °C were characterized with quality acceptable in longer period. Keywords: Alium ursinum, postharvest, temperature storage, colour change, sensory evaluation

INTRODUCTION Allium ursinum, known as wild garlic is a perennial herbaceous species with wide-spread distribution. It can be found on natural stands from the Mediterranean region to Scandinavia during early and mid springtime. Due to its preference for high air humidity it can be also found on shallow, and fairly dry places (Sobolewska, Podolak, & Makowska-Was, 2013). Wild garlic often forms dense populations known as beech forests. Allium ursinum has a distinct garlic-like scent for which the presence of sulfur containing compounds that are the most characteristic constituents in Allium species is responsible. Sulfur-containing compounds of wild garlic are also responsible for its traditional use for culinary and medicinal purposes. The potential health benefits of A. ursinum have been attributed mainly to the sulfur containing compounds. Several biological activities of A. ursinum plants and extracts, such as antioxidant, cytostatic, antimicrobial, and antidiabetic were reported (Sobolewska et al., 2013). Apart from sulfur-containing substances A. ursinum has been also reported as a good source of phenolic compounds (Gîtin, Dinicǎ, Neagu, & Dumitrascu, 2014; Oszmiański, Kolniak-Ostek, & Wojdyło, 2013). Broad spectrum of biological activities obtained from A. ursinum and its extracts, as well as the presence of chemical compounds with high terapeutical potential, contribute to the growing interest for exploitation of this plant (Tomšik et al., 2015).

531


According to Schmitt et al. (2005) the relative quantitative profile of the investigated sulfur compounds mainly sulfoxides (alliin, methiin, isoalliin and propiin) differs during the vegetation period. The period of active growth of A. ursinum lasts approximately 3-3.5 months starting in early spring, between late February and early March (Sobolewska et al., 2006). A. ursinum is usually marketed at green markets and more recently in supermarkets. For the marketing purposes the leafs are tied in a bunches and immersed in shallow water in order to preserve the water regime of the leafs. The bunches are exposed to the customers usually at ambient temperature or, in the best case in cold departments of the supermarkets (10-15°C). Wilted, decayed or yellowish bunches are not rare sight at the market. Due to short leaf shelf life under currently applied conditions, significant quantities of picked and to the market delivered A. ursinum, ends up as waste, with a plethora of valuable nutrients and bioactive compounds lost. The main aim of this research is to evaluate A. ursinum shelf life and its limitations under currently applied way of preparation for the market but with the application of different temperature regime in the distribution chain. Due to short period of availability of fresh wild garlic leafs during spring season optimisation of post-harvest storing conditions, might result in the extension of the period of availability of fresh A. ursinum at the market and reduction of its loses.

MATERIAL AND METHODS A. ursinum was collected in the forests of Fruška Gora mountain (Vojvodina, Serbia) in April 2016. Right after picking, leafs were transported to the postharvest technology pilot plant. Leafs were tied into bunches (20 leafs/bunch). The bottom parts of the stalks were cut (1-2 cm) in order to ensure equal surface for contact with water for all stalks. Bunches were immersed in water and kept in conditioned storage chambers at 0, 5, 10 and 20 °C with RH>90% for 14 days, i.e. until the deterioration of a critical sensory property under the level acceptable for the market. Weight loss was calculated based on the measurement of five bunches from each storage conditions after removal of excess of immersion water, every 2 days. Color changes were registered using MINOLTA Chroma Meter CR-400 (Minolta Co., Ltd., Osaka, Japan). The results were expressed as a* (- intensity of green) and b* (+ intensity of yellow tones). Measurements were performed on 10 leafs from 3 bunches per treatment. Sensory properties were evaluated by 3 trained assessors on the scale from 5 (without deterioration) to 1 (extremely deteriorated). Value 3.5 was considered to be the lower limit of the acceptability of the produce at the market. Respiration rate was measured every day for 4h. For purpose of respiration rate measurement approximately 100 g of leafs was closed in sealed container and after 4h at examined temperature regime the changes in headspace gas were registered using Oxybaby 6i (Witt-Gasetechnik GmbH & Co. KG, Witten, Germany). Obtained results were used to calculate daily respiration rates (ml CO2 produced/g of leafs X 24h). Based on daily respiration rates, cumulative respiration rates for 5 days were calculated.

RESULTS AND DISCUSSION Weight loss, sensory properties, colour changes and respiration rates are compared for the product stored at 0, 5, 10 and 20 °C. The results are presented on Figures 1, 2, 3 and 4 respectively. A. ursinum stored in bunches immersed in water does not suffer the weight loss if it is kept at the temperature of 10 °C or less (Figure 1). At higher temperature (20 °C), the weight losses 532


occur first slowly and after certain period much rapidly until the 5th day, and afterwards the weight loss stops.

Figure 1. Weight loss of A. ursinum immersed in water in a bunches in dependence of storage temperature

A. ursinum in bunches immersed in water becomes unacceptable for the market after more than 5 days of storage at 20 °C due to very intensive yellowing and wilting of the leafs and appearance of signs of rottening of the leafs (Figure 2).

Leaf Rotening 5

Mark

4

0 2

3

4 7

2

9 12

1

14 0 0

5

10

20

Temp of storage (°C)

Figure 2. Deteoration of sensory properties of A. ursinum immersed in water in a bunches in dependence of storage temperature

Oppositely A. ursinum stored at 10 °C suffered from more expressed rottening due to which it lost marketability, while yellowing and wilting was registered but it did not exceed the limits of marketability. A. ursinum stored at 0 and at 5 °C did not lose marketability in respect to observed aspects of deterioration during 14 days of storage. Minor variation in evaluation at these temperatures were probably the consequence of variability of starting properties of stored bunches. 533


The color changes of A. ursinum bunches varied also in dependence of storage temperature (Figure 3). Bunches stored at 20 °C were characterized with rapid increase of yellow tone intensity (Figure 3a) while the intensity of green tone (Figure 3b) first increased more rapidly, than at lover temperature -a* was higher indicating occurrence of the process of synthesis of green pigments. After 4 days, intensity of green tone rapidly decreased at 20 °C pointing out decomposition of green pigments. At lower temperatures intensity of green and yellow colour slowly but increased steadily during the whole storage period. 45 35

b*

25 15 5 -5

Figure 3. Changes of color properties of A. ursinum immersed in water in a bunches in dependence of storage temperature

Figure 4. Respiration of A. ursinum immersed in water in a bunches in dependence of storage temperature

Respiration rates (Figure 4) had the same trend but largely different intensities at different temperatures. At 20 °C and 10 °C respiration rate was much higher. Similar respiration rates and different sensory properties and colour changes resulting in quite better preservance of A. ursinum stored at 10 °C in comparison to 20 °C point out that the deteoration of quality at 534


20 °C is not only the consequence of respiration but also of a number of other processes being more expressed at this higher temperature. Respiration rates at 0 and 5 °C were similar and much lower, presenting thus preconditions for better preservation of A. ursinum at these lower temperatures.

CONCLUSIONS The common practice of marketing of wild garlic (A.ursinum) leaves in bunches immersed in water at the green markets characterized with temperatures up to 20 °C in the period of its availability shorten significantly its shelf life. Under these conditions the shelf life of wild garlic is up to 4 days and afterwards it loses its marketability due to leaves wilting and yellowing. Introduction of practice to keep wild garlic bunches in the marketing process in the cold chain would result in significant extension of its shelf life, which might be extended to the period over two weeks. Under the conditions of cold chain with maintenance of temperature under 5oC wild garlic leaves have low respiration rate, sensory properties remain unchanged, green color of leaves becomes even more intensive and weight losses are minimal.

ACKNOWLEDGEMENTS The authors are grateful to Ministry of Education and Science of Republic of Serbia for financial support (Project number III 46001).

REFERENCES Gîtin, L., Dinicǎ, R., Neagu, C., & Dumitrascu, L. (2014). Sulfur compounds identification and quantification from Allium spp. fresh leaves. Journal of Food and Drug Analysis, (May), 2–7. http://doi.org/10.1016/j.jfda.2014.04.002 Oszmiański, J., Kolniak-Ostek, J., & Wojdyło, a. (2013). Characterization and content of flavonol derivatives of Allium ursinum L. plant. Journal of Agricultural and Food Chemistry, 61(1), 176–84. http://doi.org/10.1021/jf304268e Schmitt, B., Schulz, H., Storsberg, J., & Keusgen, M. (2005). Chemical characterization of Allium ursinum L. depending on harvesting time. Journal of Agricultural and Food Chemistry, 53(18), 7288–7294. http://doi.org/10.1021/jf0504768 Sobolewska, D., Janeczko, Z., Kisiel, W., Podolak, I., Galanty, A., & Trojanowska, D. (2006). Steroidal glycosides from the underground parts of Allium ursinum L. and their cytostatic and antimicrobial activity. Acta Poloniae Pharmaceutica - Drug Research, 63(3), 219–223. Sobolewska, D., Podolak, I., & Makowska-Was, J. (2013). Allium ursinum: botanical, phytochemical and pharmacological overview. Phytochemistry Reviews, 1–17. http://doi.org/10.1007/s11101-013-9334-0 Tomšik, A., Pavlić, B., Vladić, J., Ramić, M., Brindza, J., & Vidović, S. (2015). Optimization of ultrasoundassisted extraction of bioactive compounds from wild garlic (Allium ursinum L.). Ultrasonics Sonochemistry, 29, 502–511. http://doi.org/10.1016/j.ultsonch.2015.11.005

535


VARIATION IN SWEETNESS OF THE CHESTNUT FRUITS (Castanea sativa Mill.) FROM MACEDONIA 1

2

Marjan Slavkov , Bozhidar Ristovski *, Mirjana Bocevska

2

1

The Customs Administration of R. Macedonia, Central Laboratory, Arhimedova 5, 1000 Skopje, Macedonia 2 Faculty of Technology and Metallurgy, Ss. Cyril and Methodius University, Ruđer Bošković 16, 1000 Skopje, Macedonia *Corresponding author: E-mail address: [email protected]

ABSTRACT Sweet chestnut (Castanea sativa Mill.) in native populations in R. Macedonia is widespread in some regions, mostly located in west part. The chestnut fruits are organic food product very popular during winter days. Usually are consumed roasted or boiled. They are rich in nutrients, more or less sweet, and with attractive flavour. In this investigation the fruits of twelve native, uncultivated populations of chestnuts from three regions of R. Macedonia (two located at north, and one at southwest of the country-known as rich in chestnut), were collected and analysed. The shape and morphological characteristics (height, width, thickness, weight) of the chestnuts were analysed and their nutritional composition (moisture content, protein, fat, free sugars and minerals) was determined. The major component in the composition of the chestnut samples was water, followed by free sugars (12.2-17.5%), proteins, fats and ash. The composition of the sugars was identified by liquid chromatography, and sucrose has been determined as the dominant sugar, while fructose and glucose were detected only in traces in few of the analysed samples. It was found that chestnut varieties of northern regions are with higher content of sugars than those of southwestern region. The Pearson correlation between mass of chestnuts and sucrose content in dry mass was r=-0.7281. Although this correlation is moderate in some way, it points at long held myth developed by growers that smaller nuts are sweeter. Keywords: chestnut, morphological and nutritional characteristics, sweetness

INTRODUCTION Chestnut trees spread all over the world produce wood and chestnuts that have considerable economical value. European kind or sweet chestnut (Castanea sativa Mill.) is also widespread in some areas of R. Macedonia. On the north-west well known are regions of Skopje, Tetovo and Gostivar, on south-west regions of Debar and Struga, in central part regions of Kichevo and M.Brod and on south the regions of Bitola and Strumica. Mainly chestnut trees grown spontaneously in forest with some other broad-leaf trees or as chestnut stands. Sweet chestnuts are seeds, annually produced inside spiny dehiscent fruit that easily fall down from the trees (Demiate et al., 2001). From a compositional standpoint the chestnuts are one of the world’s most popular nuts which offer unique qualities compared to other nuts. Fresh chestnuts contain vitamin C and are much lower in fat than other common nuts but are good source of essential fatty acids (Senter et al., 1994). They contain as much starch as potato, so in some regions of the world the chestnut tree could earn the nickname “bread tree” (Lizotte and Fulbright, 2014). The lipids are precursor source for the formation of flavor compounds when chestnuts are heat processed before consumption (España et al., 2011) and free sugars contribute to chestnut sweetness (Pereira-Lorenzo et al., 2006). The consumers choose chestnuts according to their appearance, expecting it to have good taste and sweetness. In this investigation the morphological characteristics and nutrient composition of chestnut native species grown in three different regions of R. Macedonia were studied, aiming to find if some relations exist among them. 536


MATERIAL AND METHODS The chestnut seeds were collected under the naturally grown trees from three regions of R. Macedonia (Figure 1), in October 2014. The seeds of selected trees differ in appearance and flavour characteristics and behaviour to processing (roasting, boiling), according to experience of local population. Totally 12 different samples were collected, 5 samples from the region of Tetovo, 4 from region of Struga and 3 samples from Skopje region, and were adequate denoted. The samples from Tetovo as T1-T4, the samples from Struga region as V1-V4, and from Skopje as S1-S3. The seeds were evaluated in terms of their appearance, morphological characteristics and chemical composition.

Figure 1. Chestnut areas where chestnuts were collected (T-region of Tetovo, S-region of Skopje, V-region of Vevcani/Struga)

The characterisation (appearance, and morphological characteristics) was performed according to the UPOV (1989) on 20 seeds of each chesnut sample. Three characteristic dimensions of chesnuts were measured: L-length; D-diameter; T-thickness (Figure 2) by slide calipers.

Figure 2. The measured dimensions of chestnut seed: L-length; D-diameter; T-thickness

The mass was measured with analytical balance to accuracy of 0.001 g. Moisture content was determined by dehydration in an electric oven at 102 °C until constant weight (AOAC, 1965). Dehydrated seeds were ground and used for determination of protein, fats and ash by official methods (AOAC, 1965). The free sugars content was established by the method of Chenlo et al. (2010). The sugars were extracted using the mixture of methanol:water (80:20), 537


and analyzed by HPLC (HP 1000, Agilent Technologies, USA). The all chemical analyses were performed in duplicate. The statistical evaluation was performed by one-way analysis of variance (ANOVA test) with Minitab 17 (Minitab Inc. USA) followed by Duncan’s multiple range test with a significance level of p> Zn 2+

2+

> Cd

Al > Cu > Pb > Cd

2+

2+

2+

~ Ni

> Ca

 Ca2+

(1)

2+

(2)

It is evident that Cu2+ ions (equation 1) are more efficient than Ca2+ ions because of marked surface complexation ability of Cu2+ ions (Kuljanin, 2008; Lević et al., 2007). The selectivity

565


order displayed in equation 2 (Kinniburgh et al., 1999) was in accordance with the amount of positive charge on these ions. The studies in work (Lević et al., 2007), have shown that the salts CaCl2, CuSO4 and AlCl3 are more efficient in the separation of pectin from sugar beet juice than conventional method using CaO. Also, in the previous studies, a similar effect has been proved using pure compounds, CaSO4 and Al2 (SO4)3 (Kuljanin et al., 2014). In these works, the optimal amounts of Ca and Al salts were determined by zeta potential measurement method (Riddick et al., 1975). Because of possible adverse effects of CuSO4 in the processing of food, the use of Al2(SO4)3, AlCl3, CaSO4 or CaCl2 is recommended for purification the sugar beet juice. Aluminum and calcium salts are favourable not only for economic reasons but also for the preservation of the environment (Kuljanin et al., 2014). The aim of this work was to investigate the effects of binary system with equal shares of Al2(SO4)3 and CaSO4 (1 : 1; w/w) on the efficiency of pectin separation. The optimal coagulation and sedimentation are controlled by measuring the zeta potential of the pectin solution.

MATERIAL AND METHODS Pectin preparations have been obtained from sugar beets slices during the industrial processing of sugar beet (factory Žabalj). The crystal hydrate form of salts (CaSO 4 x 2H2O and Al2(SO4)3 x 18H2O) are used for obtaining aqueous solutions. These salts are manufactured at the factory "Zorka Pharma", Šabac (purity of salts was 99.0 % w/w). Studied solutions were prepared with de-ionized water and pH values of each solution was adjusted to the 7 (pH Meter MA 5740, “Iskra”, Kranj). The pectin preparations were extracted in acidic conditions (pH 1.0, for preparation P1 and pH 3.5, for preparation P2) in accordance with standardized laboratory methods of AOAC (2000). Procedure was repeated three times and basic composition of preparations were determined (Table 2). Extraction procedure has been described in (Kuljanin, 2008). Pectin solutions were obtained by dissolving 1 g of preparation in 250 cm3 of distilled water and leaving it overnight to swell. Binary solutions were prepared by dissolving 0.5 g of pure salt CaSO4 and 0.5 g of pure salt Al2(SO4)3 in 200 cm3 distilled water (proportion of salts, 1 : 1; w/w). From these binary solutions, adequate volume (cm3), using pipette, was added to 50 cm3 of pectin preparation solutions to obtain the corresponding concentration (ranging from 50 to 500 mg/dm 3) (Table 1). Table 1. Plan of experiments with model-solutions of pectin preparation Precipitant: CaSO4/ Al2(SO4)3 (1 : 1 w/w) 3 Flasks filled with 50 cm of pectin solution (0.1% w/w) pH 7 Concentration of CaSO4/Al2(SO4)3 3 (mg/dm ) Volume of CaSO4 3 solution (cm ) Volume of Al2(SO4)3 3 solution (cm )

50

100

150

200

250

300

350

400

450

500

0.31

0.63

0.94

1.25

1.56

1.88

2.18

2.50

2.81

3.13

0.29

0.60

0.90

1.19

1.48

1.78

2.07

2.37

2.68

3.00

Description of solutions mixing and procedure of zeta potential measurement is presented in the articles of Kuljanin et al. (2014); Kuljanin (2008). Zeta potential of pectin solutions was determined by the method of electrophoresis using a Zeta Meter ZM-77 (Riddick, 1975). An average value of 20 measurements of particles was used to derive the zeta potential in the tested binary solutions. 566


RESULTS AND DISCUSSION From Table 2, it can be seen that the preparations P1 and P2 have different compositions and degrees of esterification which is in accordance with the extraction conditions. Table 2. Basic physico-chemical composition of pectin preparation Preparation Solid content, g/100g 5 Equivalent of free COOH groups, X · 10 5 Equivalent of esterified COOH groups, Y · 10 Content of galacturonic acid, Gal.A, % Degree of esterification, DE

P1 82.25 10.60 27.55 66.31 72.21

P2 80.35 24.58 16.05 72.24 39.50

The equivalent of free -COOH groups are responsible for the amount of negative charge on pectin surface while the degree of esterification is inversely proportional to the number of binding sites for Ca2+ and Al3+ ions. Based on that, the pectin solution P2 showed the better binding characteristics for Ca2+ and Al3+ ions. By adding a mixture with the same ratios of Al2(SO4)3 and CaSO4, reduces the absolute value of zeta potential of pectin macromolecules. Change of the sign a zeta potential was observed within the whole tested concentrations of binary solution (Figure 1). This indicates that in addition to the simple charge neutralization and ion exchange, a mechanism of specific adsorption occurs (Kuljanin et al., 2014).

Figure 1. The influence of binary solution CaSO4/Al2(SO4)3 (1 : 1; w/w) to the change zeta potential of the pectin preparations P1 and P2

According to the Schulce-Hardy rule, a smaller amount of Al2(SO4)3 would be required for neutralization of the surface of pectin compared to CaSO4, since Al3+ ion carries larger charge. But it is also known that Ca2+ ions have a large dehydration effect of hydrophilic macromolecules such as pectin. From the literature is well known favourable impact of the SO42- anions on the coagulation of colloidal particles. In addition to CuSO4, the pure salts CaSO4 and Al2(SO4)3 proved to be most effective in the separation of pectin from sugar beet juice (Kuljanin, 2008; Kuljanin et al., 2014). In our tests, under the same experimental 567


conditions, the amount of mixture CaSO4/Al2(SO4)3 necessary to bring to zero zeta potential values, amounts 465 mg/dm-3 (705 mg/gp), for pectin P1 and 420 mg/dm3 (585 mg/gp), for pectin P2. These quantities are slightly larger than the required amount of pure compounds CaSO4 and Al2(SO4)3 (Kuljanin et al., 2014). On the other hand, these quantities are significantly less compared with conventional method beet juice purification where approximately 9 g CaO per g of pectin is used.

CONCLUSIONS Although the pure salts CuSO4, CaSO4 and Al2(SO4)3 proved to be most effective in the separation of pectin from sugar beet juice, for the environment protection and for economic reasons, examined binary system CaSO4/Al2(SO4)3 (1 : 1 w/w) can be used as a good replacement for the traditional coagulant CaO. However, reliable comparison between the proposed compounds and traditional coagulant CaO, require additional testing under industrial conditions of sugar beet juice processing. Also, additional researches in real industrial conditions are required.

ACKNOWLEDGEMENTS The Ministry of Education and Science of the Republic of Serbia financially supported this work. (Project TR -31055).

REFERENCES AOAC (2000). Methods of Analysis of Official Analytical Chemists, Washington, 44, 3-4. Dronnet V.M., Renard C.M.G., Axelos M.A.V., Thibault J.F. (1996). Characterisation and selectivity of divalent metal ions binding by citrus and sugar-beet pectins. Carbohydrate Polymers 30, 253-263. Haapala H., Goltsova N., Pitulko V., Lodenius M. (1996). The effects of simultaneous large acid and alkaline airborne pollutants on forest soil. Environmental Pollution 94, 159–168. Kinniburgh D.G., Riemsdijk W.H., Koopal L.K. Borkovec M., Benedetti M.F., Avena M.J. (1999). Ion binding to natural organic matter: competition, heterogeneity, stoichiometry and thermodynamic consistency, Colloids and Surfaces, A: Physicochem. Eng. Aspects P 151, 147-166. Kohn R. (1987). Binding of divalent cations to oligomeric fragments of pectin. Carbohydrate Research 160, 343-353. Kuljanin T. (2008). Sugar beet juice clarification by means of alternative coagulants and flocculants, PhD Thesis, Faculty of Technology, University of Novi Sad, Serbia. Kuljanin T., Jevrić L., Ćurčić B., Nićetin M., Filipović V., Grbić J. (2014). Aluminium and calcium ions binding to pectin in sugar beet juice: Model of electrical double layer, Hem. Ind. 68 (1) 89-97. Lević Lj., Tekić M., Djurić M., Kuljanin T. (2007). CaCl2, CuSO4 and AlCl3 & NaHCO3 as possible pectin precipitants in sugar juice clarification, Int. J. Food Sci. Tech. 42, 609-614. Riddick M.T. (1975). Zeta-Meter Manual (third ed.), New York.

568


THE POTENTIAL USE OF ABY-6 STARTER CULTURE IN FERMENTATION OF SOY BASED SUBSTRATES 1

1

2

3*

Milka Borić , Maja Bulatović , Danica Zarić , Tanja Krunić , Marica Rakin

1

1

Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11000 Belgrade, Serbia 2 IHIS Techno Experts d.o.o. Research Development centre, Batajnički drum 23, 11000 Belgrade, Serbia 3 Innovation center Faculty of Technology and Metallurgy, University of Belgrade, 11000 Belgrade, Karnegijeva 4, Serbia


ABSTRACT The aim of this study was selection of optimal fermentation parameters (inoculum level and temperature) for production of fermented soy milk based beverage, using starter culture ABY-6 specially designed for yogurt production. Samples inoculated with five different inoculum levels (0.05; 0 0.1; 0.2; 0.3; 0.4; 0.5%) were fermented at 39, 42 and 45 C. After 6h of fermentation, quality of fermented soy milk based beverages was evaluated by determining the viable cell count, pH value, titratable acidity, proteolytic activity and α-amino acid content. Based on the results all samples reached pH values in range 4.70-5.15 and titratable acidity in range 0 -1 15.6-24.4 SH. Highest viable cell count (9,08 log (CFU ml )) was obtained in sample inoculated with 0 0.5% (w/v) of starter culture, fermented at 42 C. After 6h of fermentation, in samples with highest viable cell count, proteolytic activity (538.46 µgLe/ml) and free α-amino acid content (249.54 mg/ml) were determined. The present study is the first report on use of commercial ABY-6 culture in soy milk fermentation. Commercial ABY-6 culture could be used for soy milk fermentation in order to formulate probiotic soybased beverage. Keywords: soymilk, fermentation, probiotic, proteolytic activity

INTRODUCTION Soy milk is a traditional oriental drink. It is a water extract of soybean that can be a good substitute for cow's milk. It contains a lot of mineral matters, vitamins, and the advantage is the lack of cholesterol, lactose and saturated fatty acids. Elevated cholesterol levels and consumption of saturated fatty acids are today associated with high blood pressure and atherosclerosis (Rosenthal et al., 2003). In addition, a large part of the population, especially in Asia and Africa do not tolerate lactose in cow's milk as they are lacking in their ßgalactosidase enzyme that breaks down lactose, or reduced activity of this enzyme. For such a person consuming cow's milk can cause digestive problems, so soy milk serves as a good substitute. Numerous studies show the positive effect of soy on health. It is believed that the soybeans effect on the reduction of cholesterol in the blood, that alleviates menopausal symptoms and reduce the risk of cancer, osteoporosis and other related diseases (Rivas et al., 2002; De Lewis and al., 2006). However, the presence of indigestible oligosaccharides (raffinose and stachyose) and specific beany flavor limited consumption of soy milk in Western countries. Since the fermentation can improve the taste of soy milk, reduce the proportion of raffinose and stachyose, fermented soy products are gaining in importance and they are considered as healthier than pure, raw soymilk for a number of reasons. Fermented soymilk have higher antioxidant activity (Wang et al., 2006), more desirable taste, contain digestible protein and have a lower content of oligosaccharides raffinose and stachyose that can cause digestive 569


disorders (Cruz et al., 1981). Fermentation is generally carried out with lactic acid bacteria, but also with some types of mold. It also explores the possibility of fermentation of soy milk with probiotic bacterias (Scalabrini 1998, Božanić 2006, Wang et al., 2006). Due to improper and unbalanced diet, the presence of various chemicals in food, and antibiotics therapy, intestinal microflora may be disturbed and harmful microorganisms may become dominant. Such disturbances in the intestinal microflora equilibrium may lead to the weakening of the entire physiological system and to a very hazardous and pathological symptoms of the disease. It is therefore recommended consumption of foods containing probiotic bacteria such as yogurt, acidophilus milk and similar products (Tamime, 2005). Since probiotic bacteria entered mainly through fermented milk products, persons who do not consume milk because of lactose intolerance, allergies to cow's milk protein (casein), or because of the belief, it was partially denied. Also, multiplies the value of food and health products (Hou et al., 2000).

MATERIAL AND METHODS Soymilk (Joya, Mona Naturprodukte GmbH, Austria) was purchased from local supermarket (Efedra bioshop, Serbia). Nutritional values of 100 ml of soymilk: Protein 3.9 g; fat 2.3 g (of which saturates 0.3 g); carbohydrate 3.7 g; fibre 0.3 g; salt 0.1 g. Microorganisms and media For production of fermented soymilk beverage was used commercial lyophilized dairy starter culture, known as Lactoferm ABY-6 (Biochem, Italia). Starter culture is mixture of four types of bacteria in the following ratio: - Streptococcus salivarius subsp. thermophilus 80% - Lactobacillus acidophilus 13% - Bifidobacterium bifidum 6% - Lactobacillus delbrueckii subsp. bulgaricus 1% The culture that consists of 10 g lyophilized starter powder is the one currently used in dairy industry. The culture was maintained according to the manufacturer's instructions at -18 °C until use (no longer than 20 mounts). For each experiment, 0.05; 0.1; 0.2; 0.3; 0.4; 0.5% (w/v) of starter culture was gently added in tempered soymilk. Experimental procedure Soymilk samples were tempered at 39, 42 and 45 °C and inoculated with 0.05; 0.1; 0.2; 0.3; 0.4; 0.5% (w/v) each. Flasks containing 100 ml of soymilk were incubated at 39, 42 and 45 °C in a water bath. During the incubation, samples (2 ml) were taken every 1 hour for determination of pH value. The fermentations were carried out for 6 h until pH=4.8±0.2 was reached. After 6 h fermentations were stopped by quick cooling. Chemical analysis The titratable acidity was determined by the Soxhlet-Henkel method (Varga, 2006), and the pH value was measured using a pH meter (Inolab, WTW 82362, Wellheim, Germany). Proteolytic activity was determined by OPA method (Church et al, 1983), with some modifications. Fermented samples were mixed with 2% and 12% trichloroacetic acid (TCA), respectively, in 1:3 ratio and incubated at 4 °C for 30 minutes. Samples were centrifuged for 15 min at 5000 rpm. After centrifugation, 50.0 μL of supernatant were mixed with 1.0 mL OPA reagent, and after 5 min apsorbances were mesuared at 340 nm against blank containing 50 µL TCA (2% or 12%) in a place of the sample. Proteolytic activitiy of lactic acid bacteria was calculated according to standard curve for L-leucin. 570


g (Le/mL)  434,7826 x A  4,552

Primary amino acids can be determined by ninhydrin reaction (Lie, 1973). The result is a blue dye that shows an absorbance maximum at about 570 nm. The amino acid leucine is used as a standard substance for this assay. Ninhydrin Color Reagent: 10 g Na2HPO4x12 H2O, 0.5 g ninhydrin, 0.3 g fructose are dissolved in a total of 100 mL distilled water. Stock may be stored refrigerated up to 2 weeks in an amber bottle. Leucine Standard Stock: Exactly 187.5 mg leucine is dissolved in distilled water and brought to exactly 100 mL. Standard stock may be stored at 0 - 4°C to avoid growth of molds. Leucine Standard Solution: 1 mL leucine standard stock is diluted into 100 mL (fi nal vol.) with distilled water. The standard contains 2 mg amino-nitrogen/L. Use freshly made for each daily assay run. Dilution solution: 2 g of potassium iodate (KIO3) is dissolved in 600 mL distilled water and 400 mL 96% ethanol added. Stable long term at 4 °C. One mL of fermented samples was diluted to 100 mL with distilled water and 2 mL transferred to test tubes. Ninhydrin color reagent (1 mL) is added and the loosely covered tubes heated in a boiling water bath for 16 min. The tubes are transferred to a cold water bath to bring to 20°C (within 20 minutes) and then 5 mL of dilution reagent is added, mixed and the absorbance recorded (within 30 minutes) at 570 nm against a blank containing 2mL of water in place of the sample

Microbiological analysis One millilitre of fermented sample was diluted with 9 mL of sodium chloride (0.85%, w/v), and mixed uniformly. Subsequent serial dilutions were prepared and viable cell count was determined using pour plate technique. MRS agar and anaerobic incubation at 37 °C for 48 h were used for the enumeration of viable cell count of probiotic bacteria. The experiments were performed in triplicate. Mean values were analysed using one-way ANOVA. The Tukey test was performed for means comparison (Origin Pro 8 (1991–2007), Origin Lab Co., Northampton, USA). Differences were considered significant at P < 0.05.

RESULTS AND DISCUSSION Fermentation of soymilk by commercial cultures designed for yoghurt production could be new way of including soymilk in human consumption. Fermentations were carried out at 39, 42 and 45 °C. Production performances were evaluated by determining pH, titratable acidity (SH), viable cell count (log (CFU/mL)) and proteolytic activity and free amino acids content. Changes in pH and titratable acidity (SH) during fermentation are specific for every temperature and inoculum level used in this research. pH values for different inoculum concentration (0.05, 0.1, 0.2, 0.3, 0.4 and 0.5% (w/v)) at 39, 42 amd 45 °C, are shown in Fig 1, a), b) and c), respectively. A gradual decrease of pH was observed in all samples during 6 h fermentation. Values of pH were ranged from 4.70 to 5.15 in all samples after fermentation. Based on the results, pH decreases faster in samples with increasing inoculum level and temperature during the fermentation. As shown in Fig 1 (d), titratable acidity (SH) indicated an increase during the fermentation which points to acid production by lactic acid bacteria. Titratable acidity of samples ranged from 15.2 to 24.4 °SH after fermentation. Highest 571


titratable acidity of 24.4 °SH was observed in sample with higher inoculum level and at 42 °C, which is the optimal temperature for ABY-6 culture. Stijepic et al. (2013), showed similar values of titratable acidity in fermentation of soymilk with probiotic lactic bacteria and inulin.

0,05% 0,1% 0,2% 0,3% 0,4% 0,5%

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26 24 22 20 18

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time (h) c)

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45°C

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Figure 2. Effect of inoculum concentration on a) pH at 39 °C, b) pH at 42 °C, c) pH at 45 °C, d) SH°

In our previous investigations with ABY-6 culture and whey as a substrate, fermentation time was shorter (4h), pH values were lower, titratable acidity showed similar values, but viable probiotic cell count showed lower values, from 4.88 to 5.10 log(CFU/mL) (Bulatović et al., 2014). The fermentation time with BTC culture (Bifidobacterium spp,Lactobacillus casei, and Streptococcus thermophilus) was 6-7 hours, but the viable probiotic cells count (Lactobacillus casei and bifidobacteria) did not increased importantly during fermentation time (Božanić et al. 2008). The fermentation time with ABT-5 culture (Lactobacillus acidophillus, Bifidobacterium spp., and Streptococcus thermophilus) was 7-8 hours and viable cell count met the requirement for probiotic minimum (~6 log(CFU/mL)) (Božanić et al, 2011). Therefore, to shorten the fermentation time and to improve growth of viable probiotic cell count, soymilk was fermented in this research by ABY-6 culture. The changes in viable cell count of probiotic bacteria in beverages with different inoculum level (0.05, 0.1, 0.2, 0.3, 0.4 and 0.5% (w/v)) fermented with ABY-6 for 6 h at 39, 42 and 45 °C are show in Fig. 2 a), b) and c), respectively. As indicated in Fig. 2(a-c) viable cell count of probiotic bacteria (L. acidophilus and B. bifidum) ranged from 6.77 to 9.08 log(CFU/mL) in samples with 0.05% (w/v) inoculum at 45 °C and 0.5% (w/v) inoculum at 42 °C, respectively, after 6 h of fermentation. It suggests that inoculum level has significant (P

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