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SCIENTIFIC BULLETIN SERIES F. BIOTECHNOLOGIES Volume XVII, 2013

University of Agronomic Sciences and Veterinary Medicine of Bucharest Faculty of Biotechnologies

SCIENTIFIC BULLETIN SERIES F. BIOTECHNOLOGIES Volume XVII

2013

BucharesT

SCIENTIFIC COMMITTEE x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x

Veronika ABRAM - Biotechnical Faculty, University of Ljubljana, Slovenia Petru ALEXE - Faculty of Food Science and Engineering, University of “Dunarea de Jos Galati”, Romania Ioan ARDELEAN – Institute of Biology, Romanian Academy Narcisa BĂBEANU – Faculty of Biotechnologies, USAMV Bucharest, Romania Gabriela BAHRIM – Faculty of Food Science and Engineering, University of “Dunarea de Jos Galati”, Romania Gustavo V. BARBOSA-CANOVAS – Washington State University Pullman, State of Washington, USA Ingrid BAUMAN – Faculty of Food Technology and Biotechnology, Zagreb, Croatia Nastasia BELC – Faculty of Biotechnologies, USAMV Bucharest, Romania Daniela BORDA – Faculty of Food Science and Engineering, University of “Dunarea de Jos Galati”, Romania Calina Petruta CORNEA – Faculty of Biotechnology, USAMV Bucharest, Romania Paulo Jose do AMARAL SOBRAL – Depto de Eng. De Alimentos – FZEA USP, Pirassununga, Brazil Katherine FLYNN – European Association for Food Safety, Brussels, Belgium Helmut GLATTES – ISEKI Food Association, Austria Gustavo Fidel GUTIERREZ-LOPEZ – ENCB-IPN, National School of Biological Sciences, National Polytechnic Institute, Mexico Florentina ISRAEL – Faculty of Biotechnologies, USAMV Bucharest, Romania Stefana JURCOANE – Faculty of Biotechnologies, USAMV Bucharest, Romania Huub LELIEVELD – GHI Association Netherlands and EFFoST Executive Committee, Netherlands Florentina MATEI – Faculty of Biotechnologies, USAMV Bucharest, Romania Lynn McINTYRE – Food Microbiology, Harper Adams University College Edgmond, Newport, United Kingdom Amalia Carmen MITELUğ – Faculty of Biotechnologies, USAMV Bucharest, Romania Dumitru MILITARU – Institute Pasteur, Bucharest, Romania Anca NICOLAU – Faculty of Food Science and Engineering, University of “Dunarea de Jos Galati”, Romania Petru NICULIğĂ – Faculty of Biotechnologies, USAMV Bucharest, Romania Estela de Oliveira NUNES – Santa Catarina West University – UNOESC Biotechnological Nucleus, Brazil Nicolae PĂCALĂ – Faculty of Animal Husbandry and Biotechnology, USAMV Banat, Timisoara Romania Paola PITTIA – Dipartimento di Scienze, degli Alimenti University degli Studi di Teramo, Italy Mona Elena POPA – Faculty of Biotechnologies, USAMV Bucharest, Romania Cristina SILVA – ISEKI Food, Catholic University of Portugal Margarida VIEIRA – Directora do Dep. De Engenharia Alimentar, Instituto Superior de Engenharia, Universidade do Algarve, Portugal Medana ZAMFIR – Institute of Biology, Romanian Academy

EDITORIAL BOARD General Editor: Călina Petru‫܊‬a CORNEA Executive Editor: Mona Elena POPA Secretariat: Florentina MATEI, Florentina ISRAEL

PUBLISHERS: University of Agronomic Sciences and Veterinary Medicine of Bucharest, Romania – Faculty of Biotechnologies Address: 59 Mără‫܈‬ti Blvd., District 1, Zip code 011464, Bucharest, Romania, Phone: + 40 21 318 25 64, Fax: +40 21 318 28 88, E-mail: [emailprotected], Webpage: http://biotechnologyjournal.usamv.ro CERES Publishing House Address: 1 Pia‫܊‬a Presei Libere, District l, Zip code 013701, Bucharest, Romania Phone: + 40 21 317 90 23, E-mail: [emailprotected], Webpage: www.editura-ceres.ro

Copyright 2013 To be cited: Scientific Bulletin Series F “Biotechnologies“, Volume XVII, 2013 The publishers are not responsible for the content of the scientific papers and opinions published in the Volume. They represent the authors’ point of view. ISSN 2285-1364,

ISSN-L 2285-1364

International Database Indexing: CABI (www.cabi.org) from 2007 and DOAJ (www.doaj.org) from 2012.

SUMMARY

AGRICULTURAL BIOTECHNOLOGY In vitro cultivation of Laetiporus sulphureus and evaluation of its antimicrobial properties - Georgeta FIDLER, Gabriela POPA, Alina BUTU, Steliana RODINO, Calina Petruta CORNEA ……….………………………………...…………………… Induction of indirect organogenesis in vitro in Rhodiola rosea – an important medicinal plant - Krasimira TASHEVA, Georgina KOSTURKOVA ………….…… Camelina cultivation for biofuels production - Andra MORARU, Stefana JURCOANE, Delia DIMITRIU ………………………………………………………. In vitro study on the interaction between Bacillus thuringiensis and chemical pesticides used for corn crop protection - Mihaela Monica DINU, Ana-Cristina FĂTU, Sorin ùTEFAN, Ana Maria ANDREI ................................................................... Diversity analysis of tubered-bearing Ipomoea trifida (H.B.K.) G. DON. originated from Citatah West Java Indonesia based on chromosome traits - Tia SETIAWATI, Agung KARUNIAWAN ……………………………………….………………………. Influence of fertilization of soil with worm compost on the quality of peas - Tatiana BOCLACI, Larisa CREMENEAC ……………………………………….…….............. General aspects regarding waste management in sustainable development of agriculture - Larisa CREMENEAC, Tatiana BOCLACI ………..…………………… The selection of some tissue lines producers of anthocyanins in bilberry (Vaccinium mytillus L.) callus culture - Dorica BOTAU, Vanda BOLDA …….......... Antifungal activity of Astragalus onobrychis L. extracts - Ivan PAULIUC, Dorica BOTAU ………………………………………………………………………………… Antibacterial activity of Momordica charantia L. gemmotherapic extract - Ivan PAULIUC, Dorica BOTĂU …………………………………………………………… Estimating free radicals scavenging activity of some berries species - Gabriela LUğĂ, Florentina ISRAEL-ROMING, Daniela BĂLAN, Evelina GHERGHINA …… The influence of environmental conditions and planting date on sunflower oil content and fatty acids composition - Mihaela POPA, Narcisa BABEANU, Georgeta DICU, Andreea TEODORESCU, Nicolae BOAGHE ………………………………… Extraction, purification and Characterization of lectin from Phaseolus vulgaris L. cv. white seeds (White kidney bean) - Basheer A. AL-ALWANI, Mohammed A. JEBOR, Yasser H. JALIL …………………………………………………………….. Experimental results about potato callus induction - Andreea NISTOR, Mihaela CIOLOCA, Nicoleta CHIRU, Monica POPA …………………………………………...

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35 39 43 49 53 57 61

69 78

BIOTECHNOLOGY IN VETERINARY MEDICINE A stochastic epidemic model for dynamic of infectious diseases - Mioara VARGA ... Chlamydia psittaci in the parrots, pigeons and canaries in the city of Tirana - Gëzime SHEHU, Kristaq BERXHOLI, Zaçe MALAJ, Luljeta QAFMOLLA, Ymer ELEZI ……….

85 91

FOOD BIOTECHNOLOGY In vitro evaluation of antioxidative properties in Capsicum annuum, Vaccinium vitis-idaea and Melissa officinalis tinctures - Emanuel VAMANU, Adrian VAMANU, Elena DOPCEA …………………….…………………………………………….……. A comparative study on mountain area influence of milk samples from cow and sheep - Elena MARCU, Petru NICULITA, Ramona IANCU …………......................... Some important quality parameters of pork meat-biodegradable pack system monitoring at refrigeration storage - Vlad Ioan POPA, Elena TANASE, Mihaela GEICU-CRISTEA, Cristian-Andi NICOLAE, Raluca Augusta GABOR …….……... Evaluation of fructan contents in the taproots of plants Lactuca serriola L. and Sonchus oleraceus L. - Nadezhda PETKOVA, Panteley DENEV …………………….. Study on the interaction between the food matrix and the metal food cans - Marius Cristian BODA, Mona Elena POPA ………………………………………….……..… Studies on the aroma of Sauvignon wine - LuminiĠa VIùAN, Ricuta DOBRINOIU … Studies on the chromatic characteristics of red wines and color evolution during maturation - LuminiĠa VIùAN, Ricuta DOBRINOIU ……………………………….… Study of some changes that occur during meat fermentation - Olga DRĂGHICI, Delia CIUCUR, Mihaela-Daniela EPURE, Maria BICA ……………….……………….

99 105

110 117 123 127 132 137

INDUSTRIAL AND ENVIRONMENTAL BIOTECHNOLOGY In vitro evaluation of crude oil degradation potential of some Pleurotus ostreatus isolates - Gabriela POPA, Mihai Bogdan NICOLCIOIU, Calina Petruta CORNEA …... Biodegradable materials and its applications in medical tools development – A review - Elisabeta Elena TĂNASE, Maria RÂPĂ, Ovidiu POPA ……………………. Optimization of Trichoderma strain cultivation for biocontrol activity - Iuliana RAUT, Mariana CALIN, Gelu VASILESCU, Melania Liliana ARSENE, Luiza JECU, Tatiana SESAN ………………………………………………………………………….. Novel fungal collagenase from Aspergillus oryzae - Daniela BĂLAN, Florentina ISRAEL-ROMING, Petruta Călina CORNEA, Evelina GHERGHINA, Gabriela LUğĂ, Florentina MATEI, Mihai CURTAùU ………………………………………… Screening for high lipase producing microorganisms - Alexandra GHIORGHI‫܉‬Ă, Gheorghe CÂMPEANU, Vasilica MANEA, Mi‫܈‬u MOSCOVICI, Eugenia MOCANU, Corina IONESCU ……………………………………………………………………….. Selection of yeast strains with enhanced lipolytic activity - Alexandra GHIORGHI‫܉‬Ă, Gheorghe CÂMPEANU, Vasilica MANEA, Angela CĂ‫܇‬ĂRICĂ, Călina Petru‫܊‬a CORNEA ................................................................................................... Studies on diacerein biodegradability - Caterina TOMULESCU, Eugenia MOCANU, Misu MOSCOVICI, Gabriela SAVOIU, Maria PETRESCU, Nicoleta DOBRE ………. Growth and activity of cellulase-amylase enzyme Penicillium nalgiovense and Aspergillus tamarii molds isolated from cow rumen fluid - Yuli ANDRIANI, Sukaya SASTRAWIBAWA, Ratu SAFITRI, Abun ......................................................................

143 147

154

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164

171 177

181

FOOD SAFETY Essential oils utilization in food industry - A literature review - Adriana Laura MIHAI, Mona Elena POPA ...............................................................................................

187

Research on the correlation between physico-chemical, sensory analysis of smoothie type products and consumer preferences - Andreea STAN, Mona Elena POPA ………. Selective determination of nitrite in cured meat products using a nonconducting polymer film based sensor - Emilia OCNARU, Melania ARSENE, Gelu VASILESCU, Mihaela BADEA DONI ……………..…………………………………... Dioxins and furans contamination of food and their toxicological implications on the human body. Mini-review - Elena PRUTEANU, Petru NICULITA, Luminita CATANA, Mioara NEGOITA, Alina BALEA ……….....................………….....…… The microwaves effects on liquid foods - Ionica DELIU, Daniela GIOSANU, Constantin STANESCU ……………………………………………………………….. Phytochemicals, antioxidant and Į- amylase inhibitory activities of Smyrnium olusatrum l. Leaf, flower and fruit- Chokri MESSAOUD, Amina BENABDALLAH, Mohamed BOUSSAID ………………………………………………………………… Characterization and development of chromatografic method for simultaneous determination of artificial sweeteners in soft drink samples - Madalina JURCOVAN, Nicole-Livia ATUDOSIEI, Valentina LAZIN, Daniela MIHAILA .......................…… Decontaminating mycotoxins in agricultural produce following bioremediation approach- Shinawar Waseem ALI, Muhammad Saleem HAIDER, Muhammad RIAZ, Muhammad Hassan MUSHTAQ ………………………................................................. Effect of extract of Ginkgo biloba on vegetable oils - Olga DRĂGHICI, MariaViorela CODOI …………………………………………………………..………………

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198

202 208

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MISCELLANEOUS Interaction of Aspergillus niger hyphae and spores with colloidal silver nanoparticles Ovidiu IORDACHE, Calina Petruta CORNEA …………………….…………………... Fungal strains isolated from several cases of human dermatophytoses - Mariana CALIN, Iuliana RAUT, Olguta DRACEA, Luiza JECU, Veronica LAZAR …………. Screen-printed carbon electrodes modified with prussian blue and a nonconducting electropolymerized film for selective determination of H 2 O 2 in beverages- Florentina HUTANU, Emilia OCNARU, Melania-Liliana ARSENE, Mihaela BADEA-DONI ………………………………………………………………… Supporting students for a biotech career- Florentina MATEI, Mioara VARGA, Silvana DANAILA-GUIDEA, Mariana IORDACHE, Stefana JURCOANE, Camelia DIGUTA ……............................................................................................................

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AGRICULTURAL BIOTECHNOLOGY

Scientific Bulletin. Series F. Biotechnologies, Vol. XVII, 2013 ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

IN VITRO CULTIVATION OF LAETIPORUS SULPHUREUS AND EVALUATION OF ITS ANTIMICROBIAL PROPERTIES Georgeta FIDLER1,2, Gabriela POPA1, Alina BUTU2, Steliana RODINO2, Calina Petruta CORNEA1 1

University of Agronomical Sciences and Veterinary Medicine, Faculty of Biotechnologies, 59 Marasti, District 1, 011464, Bucharest, Romania, Phone: +40 (21) 318 22 66, Fax: +40 (21) 318 28 88, e-mail: [emailprotected] 2 National Institute of Research and Development for Biological Sciences, 296 Splaiul Independenԑei, District 6, 060031, C.P. 17-16, Bucharest – Romania, Phone: 021-220.77.80; 021-220.79.09 Fax: 021-220.76 95; e-mail: [emailprotected] Corresponding author e-mail: [emailprotected] Abstract Laetiporus sulphureus (Bull. Fr.) Murill., is a wood-rotting basidiomycete mushroom well known for it nutritional value. In this study, alcoholic and aqueous extracts obtained from a Romanian isolate of L. sulphureus cultivated on various culture media were investigated for the antimicrobial properties. PDA, malt extract as solid media and PD (I), malt extract (II), YPG (III) and Hwang (2008)(IV) -as liquid media were used for in vitro cultivation of L. sulphureus, in order to evaluate the optimal medium for an efficient biomass production of L. sulphureus. Between all media tested, best results regarding the growth of mycelia, were obtained when I, II, IV media were used. Only on IV (Hwang) culture medium was observed the typically orange pigment elaborated by fungus. Alcoholic and aqueous extracts of fruit bodies and submerged mycelium developed in liquid media were analyzed against strains of Candida albicans ATCC10321, Candida parapsilopsis CBS604, Escherichia coli, Bacillus cereus, Staphylococcus aureus, Enterococcus faecalis, Pseudomonas fluorescens and Pseudomonas aeruginosa. The results shown that the types of culture media for biomass production of L. sulphureus and their aqueous and alcoholic extracts tested against these pathogens have different effects on inhibitory activity. Between the two types of extracts tested aqueous extracts were inferior to alcohol extract in their inhibitory activity on all organisms except Candida sp. in interaction with L. sulphureus aqueous extract from biomass developed on IV medium. Keywords: antimicrobial properties, in vitro cultivation, Laetiporus sulphureus

Trametes versicolor, Ganoderma applanatum and G.lucidum, Laetiporus sulphureus has been found to be an excellent source of natural products with therapeutic properties. Those mushrooms provide a rich variety of secondary metabolites and polysaccharides that have been proved to posses significant antimicrobial activities (Siljegovic et al., 2011). The fruiting bodies of L. sulphureus contain N-methylated tyramine derivatives (Rapior et al., 2000), polysaccharides (Alquini and Carbonero, 2004), terpenoids, laetiporic acids and other compounds (Weber et al., 2004; Davoli et al., 2005). From L. sulphureus submerged mycelia cultures have been isolated various polysaccharides (Hwang et al., 2008; Hwang and Yun, 2010) with therapeutic evidences. For these reasons, the goals of our studies were to

INTRODUCTION Laetiporus sulphureus (Bull. Fr) Murril (Aphyllophorales, Polyporaceae) is a wood – rotting basidiomycete mushroom which grow on mature and old-growth trees in forests or in urban parks. It is know as a destructive pathogen of the trees that cause butt and trunk rots (Holsten et al., 2001; Sinclair and Lyon, 2005). L. sulphureus is characterized by an intense orange colour, fleshy basidiocarps and tubular hymenopores (Banik et al., 1998) and can be harvested as an edible fungus with reliable nutritional value. Because of its medical properties L. sulphureus has been used in some therapies as antitumor, antiviral, antimicrobial treatments (Wasser and Weis, 1999). Among several other mushrooms like

find the optimal medium for an efficient biomass production of L. sulphureus and to investigate the antimicrobial activities of alcoholic and aqueous extracts from fruit bodies and submerged mycelium developed in liquid media, against some pathogenic agents.

liquid media were analyzed against strains of Candida albicans ATCC10321, Candida parapsilopsis CBS604 (from the collection of MICROGEN, Bucharest), Escherichia coli, Bacillus cereus, Staphylococcus aureus, Enterococcus faecalis, Pseudomonas fluorescens and P. aeruginosa (from the collection of Faculty of Biotechnologies Bucharest). To determine the antimicrobial activities of tested extracts, 1 ml from each bacterial and yeasts suspensions was inoculated on Luria Broth or YPG media, respectively, in Petri plates. After removing the excess suspension using a micropipette, sterile filter paper discs soaked in fungal extracts were placed on the surface of the inoculated medium. 24 hours after incubation at 370C and 270C respectively, occurrence of inhibition halos was observed.

MATERIALS AND METHODS In vitro culture establishment. Samples of the fruit bodies of Laetiporus sulphureus, collected from Sinaia woods, were surface sterilizing and cutting out a piece of trama using a sterile scalpel. The pieces were placed in Petri dishes on 2% malt extract agar and PDA (potatodextrose-agar) media and incubated at 250C for a week. After the mycelium growing on the medium surface, mycelia agar discs (5 mm diameter) obtained from the active growth areas were placed in 100 ml Erlenmeyer flasks, each with 50 ml PD (potato-dextrose) (I), 2% malt extract (II) and YPG (yeast peptone glucose) (III) liquid media. We also used a medium (IV) prepared according Hwang et al (2008) (20g/l glucose, 2g/l peptone, 2g/l yeast extract, 0,46g/l KH2PO4, 1g/l K2HPO4, 0,5 g/l MgSO4), in order to determine the optimal growing medium for in vitro culture of L. sulphureus. After inoculation the samples were incubated at 250C in a rotary shaker at 148 rpm for 10 days. The biomass obtained from each liquid medium was filtrated and weighed.

RESULTS AND DISCUSSIONS The submerged cultivation of mushrooms is a promising method for obtaining pharmaceutical compounds and was applied for various edible and medicinal species (Petre et al., 2012). For L. sulphureus there are only few reports regarding the submerged cultivation and fungal pellet obtaining in order to obtain various metabolites (Sivulski et al., 2009; Hwang et al., 2008). For this reason, one of the aims of our experiments was to evaluate the effects of different media compositions on the submerged mycelium growth of L. sulphureus and to achieve maximum biomass production. For this purpose L. sulphureus fresh mushroom was firstly cultivated on two agar media (malt extract and PDA). The mycelium grown on the surface of these media was used for inoculation of four different liquid media: I-PD (potatodextrose), II- malt extract, III-YPG (yeast peptone glucose) and IV- Hwang (2008). After ten days of culture on these tested media the results shown that the best growth of the mycelia biomass (as pellets) was observed on Hwang medium followed by PD and malt extract media. The poorest growth was encountered on YPG medium. The wet weight of filtrated mycelia biomass harvested from Hwang medium (8.26 g/100ml) was significantly superior to that obtained on PDA

Extracts preparation. For extracts preparation the biomass developed on each liquid medium tested was used. A mixture of mycelium and medium from the in vitro culture was separated by filtration. The filtrated mycelia mass was grounded and used for the extracts preparation. For aqueous extract, 1 ml of distilled water per 1 g of mushroom material was added. In the case of alcohol extract, 1 ml 70% ethyl alcohol was added to sample (1 g wet weight). Also, ethyl alcohol (70%) was used as negative control. The aqueous and alcoholic solutions were then centrifuged at 5000 rpm for 10 minutes. The supernatant was kept at 4 0C and used to determine the antimicrobial activity of L. sulphureus extracts. Antimicrobial activity. Alcoholic and aqueous extracts of submerged mycelium developed in

(2.4 g/100 ml) and malt extract (1.30 g/100ml) media, respectively. The mycelia growth as pellets is of great interest from practical point of view, facilitating the extraction procedures

I-PD

of biological active compounds and which can be used to prepare functional food (Petre et al., 2012).

II-Malt extract

III- YPG

IV- Hwang

Figure 1. Aspect of submerged mycelia of Laetiporus sulphureus in different liquid media

An interesting aspect of our results was the accumulation of orange pigment in mycelium in submerged conditions (in Hwang medium) (figure 2); in the other media the mycelium was uncoloured.

techniques, found that the pigment is a polyene of non-isoprenoid biosynthetic origin named laetiporic acid. The practical significance of this compound is not very clear but it could be used as food colorant. In the second investigation, alcoholic and aqueous extracts obtained from biomass developed on each liquid medium tested (I, II, III, IV) were analyzed for their ability to inhibit six strains of Gram positive and Gram negative bacteria: E. coli, B. cereus, S.aureus, E. faecalis, P. fluorescens and P. aeruginosa and two pathogenic yeasts: Candida albicans ATCC 10231 and Candida parapsilopsis CBS 604. The results obtained shown that the culture media used for submerged cultivation of L. sulphureus mycelium influenced not only the growth rate but also the biological activities of aqueous and alcoholic extracts against pathogens (Table 1).

Figure 2. Laetiporus sulphureus orange pigment elaborated in Hwang medium

Davoli et al (2005), investigating the orange pigment produced by fruit-bodies of L. sulphureus using modern spectroscopic

Table 1. Pathogen interactions with L. sulphureus extracts obtained from biomass developed on different liquid culture media (I, II, III, IV)

It was shown that the inhibitory action of aqueous extracts were inferior to alcohol extracts against all the pathogenic organisms tested. However, the aqueous extract from biomass developed on IV (Hwang) medium presented clear inhibition area against both Candida species (Figure 3). Alcoholic extracts of biomass provided from IV (Hwang) medium were optimal for inhibition of S. aureus and C. albicans. Clearly inhibition halo was observed

on S. aureus, E. coli E. faecalis, P. fluorescens and against both Candida species when the alcoholic extract from biomass provided to I (PD) medium was used (Figure 3). Alcoholic extract from biomass growth on III (YPG) medium had the best inhibitory activity against C. albicans and E. coli, but the alcoholic extracts from mycelium growth in malt extract medium inhibited only the growth of E. coli strain.

Figure 3. Inhibitory activity of L. sulphureus alcoholic extracts from biomass developed on I (PD) medium against Candida sp. (a, b -1). M=control (70% alcohol)

had the best inhibitory action for inhibition of S. aureus and C. albicans. Moreover, alcoholic extract from L. sulphureus biomass developed in PD medium inhibited Candida sp., S. aureus, E. coli E. faecalis and P.fluorescens. The results obtained are promising but optimisations of the growth conditions as well as the extraction procedures are necessary in order to recover larger quantities of biological active compounds.

CONCLUSIONS The goals of our studies were to find the optimal medium for an efficient biomass production of L. sulphureus and to investigate the antimicrobial activities of alcoholic and aqueous extracts from submerged mycelium developed in liquid media, against some pathogenic agents. The data from in vitro growth tests provides significantly evidences on biological differences between mycelia biomass developed on various media used. Between all media tested for maximum biomass production, the best growth of the fungal biomass, including as pellets, was observed on Hwang medium follow by PD and malt extract media. Only in Hwang culture medium was observed the typically orange pigment elaborated by fungus. Alcoholic and aqueous extracts from submerged mycelium cultivated in different liquid media tested against several microbial strains demonstrate the influence of culture conditions on inhibitory activity. It was shown that aqueous extract from submerged mycelia developed on Hwang media was inferior to alcohol extract in their inhibitory activity on all pathogenic organisms tested except Candida sp. Alcoholic extracts of biomass provided from IV (Hwang) medium

REFERENCES Alquini, G., Carbonero, E.R., 2004. Polysaccharides from the fruit bodies of the basidiomycete L. sulphureus. FEMS Microbiology Letters, 230 (1): 47-52. Banik, M.T., Burdsall, H.H. Jr., Volk, T.J., 1998. Identification of group within Laetiporus sulphureus in the Unite States based on RFLP analysis of the nuclear ribosomal DNA. Fol. Cryptolog Estonica Fasc., 33: 9-14. Davoli, P., Mucci, A., Schenetti, L., Weber, R.W.S., 2005. Laetiporic acids, a family of non carotenoid polyene pigments from fruit-bodies and liquid culture of L. sulphureus (Polyporales, Fungi). Phytoc. 66:817-823. Holsten, E., Hennon., P., Trummer, L., Schultz, M., 2001. Insects and diseases of Alaskan Forests. Tehnical Report R10TP-87, USDA Forest Service, Alaska. Hwang, H.S., Lee, S.H., Baek, Y.M., Kim, S.W., Jeong, Y.K., Yun, J.W., 2008. Production of extracellular polysaccharides by submerged mycelial culture of L. sulphureus var. miniatus and their insulinotropic properties. Appl. M. Biotech.78: 419-429. Hwang, H.S., Yun, J.W., 2010. Hypoglycemic effect of polysaccharides produced by submerged mycelial culture of

Sinclair, W.A., Lyon, H.H., 2005. Diseases of trees and shrubs, 2nd edn. Cornell University Press, Ithaca. Siwulski, M., Pleszczynska, M., Wiater, A., Wong, J.J., Szczodrak, J., 2009. The influence of different media on the Laetiporus sulphureus (Bull.: Fr.) Murr. mycelium growth. Herba Polonica, 55 (3):278-284 Wasser, S.P., Weis, A.L., 1999. Medicinal properties of substances accurring in higher Basidiomycete mushrooms: current perspective. Int. J. M. Mus.1:31-62. Weber, R.W.S., Mucci, A., Davoli, P., 2004. Laetiporic acid, a new polyene pigment from the wood-rotting basidiomycete L. sulphureus (Polyporales, Fungi). Terahedron Lett. 45: 10751078.

Laetiporus sulphureus on streptozotocin-induced diabetic rats. Biotechnol. Bioprocess Eng., 15: 173-181. Petre, M., Teodorescu, A., Patrulescu, F.., 2012. Biotechnology of submerged fermentation to produce nutritive mycelial biomass through controlled cultivation of edible and medicinal mushrooms, Biotechnologies, 16, 89-93 Rapior, S., Konska, G., Andory, C., Bessiere, J.M., 2000. Volatile composition of Laetiporus sulphureus. Crytogamie Mycol., 21: 67-72. Siljegovic, J.D., Stojkovic, D.S., Nicolic, M.M., Glamoclija, J.M., Sokovic, M.D., Ciric, A.M., 2011. Antimicrobial activity of aqueous extract of L. sulphureus (Bull.: Fr.) Mur. Proc. Nat. Sci.120: 297-303.

Scientific Bulletin. Series F. Biotechnologies, Vol. XVII, 2013 ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

INDUCTION OF INDIRECT ORGANOGENESIS IN VITRO IN RHODIOLA ROSEA– AN IMPORTANT MEDICINAL PLANT KrasimiraTASHEVA and Georgina KOSTURKOVA Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria, Corresponding author email: E-mail: [emailprotected] Abstract Rhodiola rosea L. is an endangered medicinal plant due to over harvesting in Bulgaria and in other European countries. The root and rhizomes are rich in pharmacologically and therapeutically active substances like flavonoids, phenolic acids that make the plant of commercial importance. The seed propagation in nature is very poor as far as only of 2 to 30 % of the seeds germinate depending on the ecologically geographic conditions. Thus, in vitro techniques are suitable for propagation of this species. In vitro propagation is possible by direct and indirect organogenesis. The latter is much more difficult in many species, including golden root. The present study aimed to understand the conditions, which provoke undifferentiated tissue initiation and development of shoots. The effects of different plant growth regulators in various concentrations oncallus induction and indirect plant regeneration were investigated. Regenerative callus was received on Murashige and Skoog medium containing 2,4–1 Dichlorophenoxyacetic acid (0.1 mgl )within 28 days. The adventitious buds were differentiated when the calli were –1 subcultured on Murashige and Skoog (1962) medium supplemented with 6-benzylaminopurine (1.0 mgl ) and indole–1 –1 3-acetic acid (0.1 mgl ) within 4 - 5weeks. Half-strength solid MS with indole-3-butyric acid (2.0mgl )and IAA (0.2 –1 mgl ) exhibited the best in vitro rooting. These results contribute to the understanding of processes of growth and development in vitro of Rhodiola rosea and possibilities for selected of valuable clones and establishment of propagation schemes. Keywords: calli, golden root, indirect organogenesis, regeneration.

Biotechnological techniques using cells, tissues, organs or whole organisms, growing and developing in in vitro conditions, are suitable for genetic manipulations (Khan ̖t al., 2009), and to obtain valuable compounds (Rao andRavishankar, 2002). During the last years, propagation of medicinal plants in in vitro conditions has gained increasing interest to the industry and has been more widely used for the necessities of the pharmaceutical industry. The way leading to production of great number of identical plants, what is a subject of clonal/micro-propagation, is one of the most preferable approaches from a commercial viewpoint. This approach could be used to create ex situ and in vitro collections, as well as for commercial propagation to obtain raw material for pharmacetical and cosmetic industries (Julsing et al., 2006). In vitropropagation allows obtaining of a large plant mass for a short period of time (Tripathi and Tripathi, 2003). Developed

INTRODUCTION More than 2000 plant species in Europe are used for production of medical and nutraceutical herb preparations. The wild species represent 70 % from this production and only 30 % from the species are cultivated in the field. However, a significant number of the wild species are rare, disappeared and/or under protection (Varbanova, 2002; Evstatieva et al., 2007). One of the reasons is the increasing interest of pharmaceutical industry towards these plants and respectively more intensive use of their natural resources which are not unlimited. This raises the question of searching for and development of alternative methods friendly to the environment. (Tasheva and Kosturkova, 2013). Such ones are the biotechnological methods which are important for breeding, propagation and conservation of valuable medicinal plants.

and increases resistance to infection diseases. Due to the low toxicity and the absence of side effects of its extract, golden root is widely used in food and cosmetic industry. In Bulgaria, there are no systematic and comprehensive studies of this endangered species and data on the application of modern biotechnological and phytochemical approaches. Creating integrated technologies for breeding, preservation and cultivation of this valuable species is a major premise for its cultivation in different mountain regions in Bulgaria. The few biotechnologically investigations concerning species from Rhodiola genus refer particularly to the process of indirect organogenesis. Callus induction followed by plant regeneration of Rh. coccinea, Rh. sachalinesis, Rh. rosea were studied by some authors (Kirichenko et al., 1994; Furmanova et al., 1995; Ishmuratova, 1998; Yin et al., 2004; Sha Hongetal., 2008; Liu Jianfeng et el., 2007; Liu Jian-feng et al., 2009). The present study aimed to understand the conditions which provoke undifferentiated tissue initiation and development of buds and shoots investigating the effects of different plant growth regulators in various concentrations oncallus induction and indirect plant regeneration.

micropropagation methods have been reported for many medicinal plants but this list is increasing (Tasheva and Kosturkova, 2013). Experimental approaches used for in vitro propagation of medicinal plants could be broadly divided/classified in three categories: (1) Isolation of meristems and stem tips and stimulation of their growth which depends on different factors. The effect of plant regulators and their combinations to micropropagation of medicinal plants is reported by Makunga et al., 2003; Debnath, 2009; Keng et al., 2009; Yusuf et al., 2011. (2) The second way includes induction of adventitious buds from leaves, stems and root segments or obtaining callus from these organs. The plant regeneration by organogenesis is well developed in some medicinal and aromatic plants, but it varies widely for different plant species, which requires an individual approach to study the conditions for regeneration, as well as determining the factors controlling growth and differentiation for each type of species (Patra et al. 1998). (3) The third way is somatic embryogenesis, which is the theoretically most effective to obtain plants (Martin, 2004; Paramageetham et al., 2004; Fiuk and Rybczynski, 2008, Robinson et al., 2009). To determinate, the relationship between different groups of phytoregulators is essential for these because success of tissue culture work depends on phytoregulators’ type and concentration. The process of differentiation of unorganized callus tissue, initiation of the buds and roots depends on the appropriate combination of auxins and cytokinins in the nutrient medium (Sagare et al., 2000). The influence of the cytokinins and auxins on in vitro cultures depends on the in vitro systems, plant species and in many cases even to the variety or/and ecotype. The natural and synthetic auxins and cytokinins induce typically physiological responses in plants. The specificity of the investigated object from one side and endogenous phytohormones from other side determine the effects of the exogenously applied phytoregulators. Rhodiola rosea is a medicinal plant under protection in Bulgaria and other European countries. The extract from root and rhizomes has a number of applications. It has adaptogenic, antitoxic and antihypoxic action,

MATERIALS AND METHODS Plant material. Explants isolated from in vitro propagated plants, obtained in our previous work were used in these experiments (Tasheva and Kosturkova, 2010a, b). Culture media. Nutrient media composition for callus induction was Murashige and Skoog (1962) medium containing zeatin 2.0 mg/l (Trans form), IAA 0.2 mg/l, casein hydrolisate 1000 mg/l, sucrose 3%, and agar-agar 0.6% (Tasheva and Kosturkova. 2010a, b). Nutrient medium composition for induction of organogenic callus designated as mediumDwas basic Murashige and Skoog (1962) nutrient medium enriched with 0.1 mg/l 2,4-D, 3% sucrose and 0.6 % agar-agar. Nutrient medium composition for bud formation was Murashige and Skoog (1962) nutrient medium containing BAP 1.0 mg/l, IAA

of calli may be due to accumulation of the auxins in the main cutting edges which stimulates cell proliferation especially in the presence of cytokinins (Martin 2000). The application of plant growth regulators tested concentrations significantly affects the growth and the development of callus and its differentiation.

0.1 mg/l, sucrose 3% and agar-agar 0.6% (designated as BA medium). Nutrient medium composition for shoot formation and plant was Murashige and Skoog (1962) medium containing zeatin 2.0 mg/l (mixed isomers), IAA 0.2 mg/l, casein hydrolisate 400 mg/l, sucrose 3%, and agaragar 0.6% (Tasheva and Kosturkova. 2010a, b). The pH of all culture media was adjusted to 5.7 – 5.8 prior and autoclaved at 1.1 kg.cm-2, 121°C for 20 min. Culture conditions. The calli explants were cultivated in test tubes or Petri dishes. ˃he frequency of regenerants induction was evaluatedperiodically with an interval of 28 days. Necrotic tissue was removed during each sub-cultivation. The average number and size of the buds, shoots and regenerants from one explant were scored. Obtained regenerants were separated from the callus and cultivated on media for multiplication followed by rooting. The cultures were cultivated (induced and maintained) in a growth room with artificial illumination (fluorescent lamps) under a 16 h photoperiod at temperature of 21 - 23°C and light intensity 20 ʅʺ m-2 s-1 for callus initiation and maintenance and 40 ʅʺ m-2 s-1 for organogenesis induction and bud/shoot formation. The regenerants which had formed roots in vitro were transferred to small pots containing soil, peat and perlite.

Figure 1 Regenerated plants and calli formation on MS medium containing 2.0 mg/l zeatin, 0.2 mg/l IAA and 1000 mg/l casein hydrolisate

RESULTS AND DISCUSSIONS Callus induction. Callus like structures or callus in the shoot basis was formed using the developed scheme for micropropagation (Tasheva and Kosturkova. 2010a, b). About 0.6 – 1.0 % from the plants formed non differentiated tissue. In most of the cases (about 85 – 90%) callus was solid, grainy, with greenbrown color (Figure 1 and Figure 2) and was considered appropriate for the present experiments. Auxins, cytokinin and auxin/cytokinin interactions are usually considered the most important factors for regulating growth and organized development in plant tissue and organ culture. Plants generally require these two classes of growth regulators. The formation

Figure 2. Formed and isolated callus tissue

Induction of organogenic callus and proliferation. Obtained callus when transferred to fresh nutrient medium D (containing MS salts and vitamins, 0.1 mg/l 2,4-D, 3% sucrose and 0.6% agar-agar) formed buds for 2 passages with 28 days duration of every passage (Figure 2 and Figure 3). Cultivation of the calli on this media for a one passage did not bring to revealing of organogenic capacity of calli.

plants to be multiplied on the same medium (Figure 5) or on a medium containing zeatin (cis/trans = 82/18% and IAA (for a short period of time) (Figure 6). Studied concentrations of growth regulators and their combination, stimulated cell division in the tissues, buds growth and appear of the first leaf. Received healthy plants are ready for in vitro cultivation. These preliminary results show that BAP supported indirect organogenesis process by stimulating the cells to reach the competent phase for cell division.

Figure 3. Isolated calli and cultivated on MS medium containing 0.1 mg/l 2,4-D

Figure 5. Isolated buds and shoot formation on MS medium containing BAP 1 mg/l and IAA 0.1 mg/l

Bud and shoot induction. Bud formation was stimulated when the calli were cultivated on BA medium (MS nutrient medium containing BAP 1.0 mg/l, IAA 0.1 mg/l, sucrose 3% and agar-agar 0.6% (Figure 4).Thus, in the present study it was found that the BAP and BA concentration with auxin (IAA and BA) induced the organogenesis.

A high concentration of cytokinin inhibits callus formation and increases buds induction. The combinationof cytokinin and auxin has a positive effect on cell division and differentiation.

Figure 6. Plant multiplication on MS medium containing BAP 1 mg/l and IAA 0.1 mg/l

Figure 4. Indirect organogenesis and bud formation on MS medium containing BAP 1.0 mg/l, IAA 0.1 mg/l

Callus is dedifferentiated and unorganized mass of parenchyma cells formed by the proliferation of parent tissue. Callus tissue is a good source of genetic variability and adventitious shoot formation. Callus induction is a prerequisite for

Multiplication of the obtained regenerants. The frequency of regenerated plants from callus cultures was very low not exceeding 5 – 6 %. However, it was possible obtained mini

induction and bud formation. The protoplasts formed callus on MS medium containing 2,4-D (1.0 mg/l), zeatin (0.5 mg/l), 0.5 M/l manitol and500 mg/l casein hydrolysate for 40-days period of cultivation. The callus formed adventitious buds on MS nutrient medium added with BA and NAA (1.0 mg/l and 0.1 mg/l, respectively). The buds grew and induced roots on ½ʺS for 30 days. In vitro rooting. The process of mini plants rooting is an important for their next step to adaptation in ex vitro conditions (Hazarika, 2003). The processes of rooting have specific requirements and rhyzogenesis was not obtained in all of the cited references. The rooting process on MS media without auxins has been studied in many plant species, such as Echinaceapurpurea (Korachetal., 2002), Carlinaacaulis (Trejgelletal., 2009). The frequency of the induced roots on medium without auxins may be due to the presence of endogenous auxins in regenerated buds/shoots (Minocha, 1987). Root induction of the growing in vitro shoots was achieved by adding auxins in nutrient media or on media without regulators which depends on species genotype (Rout et al., 1989). Different species had different potential to form roots and the optimal conditions are determined empirically. Moderate to high concentration of all auxins in the media inhibited root growth.

adventitious shoot formation and also for the other in vitro genetic improvement including induction of somaclonal variations and embryoids. Difficulties in working with callus cultures of different Rhodiola species were reported by other authors, too. Kirichenko et al. (1994) and Yin et al. (2004) studied the possibilities for callusogenesis, organogenesis and regeneration form leave explants of Rh. rosea. Furmanova et al. (1995) examined the effects of the different nutrient media at the regeneration and callus formation capability of Rh. rosea and study biologically active substances in the roots in wild species. Ishmuratova (1998) demonstrated that the ecotype is also a factor influencing the processes of effective callusogenesis and organogenesis. The latest capability is studies in seeds and rhizomes from three ecotypes Rhodiola growing in high Altai using MS nutrient medium containing different phytoregulators – BA, IAA, NAA, IBA and 2,4-D. The author specified that the choice of the optimal nutrient medium (MS containing BAP (0.2 mg/l) and IAA (0.1 mg/l) for explant development is depended on ecotype. Callus induction followed by plant regeneration of Rhodiola coccinea was studied byof ShaHongetal. (2008). The authors obtained three-type calli and noted that only one type of them was embryogenic. Similarly to our observations the role of BA and IAA was crucial. The embryogenic callus was obtained on MS nutrient medium containing BA (0.5 mg/l) and IAA (2.0 mg/l) and is suitable for regeneration. Callus induction (88.33 %) in different colors (yellow, green and red) was obtained of Rhodiola sachalinsis using leaf explants and cultivated on MS medium additionally with BA (2.0 mg/l) and NAA (0.5 mg/l) (Liu Jianfeng et el., 2007). They concluded that only green callus is able to regeneration on MS media containing BA (1.0 mg/l) and NAA (0.1 mg/l) with comparatively low percent (21.33 %) after long period of cultivation 50 days. The necessity for a long cultivation period was noted in our experiments, too. Regenerants were successfully rooted on½ MS. In the same species Rhodiola LiuJian-feng et al. (2009) obtained protoplast cultures from leaves of in vitro propagated plants, followed by callus

Figure 7. Root induction on MS medium containing BAP 1.0 mg/l and IAA 0.1 mg/l after 36 days cultivation.

The presence of auxin for the rhizogenesis process is necessary for many medicinal species. The concentration of IBA, for example, plays key role for stimulation of root formation for a number of plants as Centaurea rupestris (Perica, 2003), Wedelia chinensis

mg/lIAAand 0.4 mg/lGA3, reported in our previous papers (Tasheva and Kosturkova, 2010 a,b) (Figure 8 and 9). Adaptation ex vitro. Determination of the optimal conditions for adaptation of the invitro obtained plants in exvitro condition after the stage of rooting is a significant step for completed propagated scheme report. Adaptation of the regenerants in external conditions is the one critical stage of micripropagation. Adaptation of the plants obtained from this process of indirect regeneration was performed by the use of the scheme mentioned in our previous studies (Tasheva and Kosturkova, 2010a,b)

(Kameri et al., 2005), Emilia zeylanica (Robinson et al., 2006). Reducing the amount of salts in nutrient media for rhizogenesis had different effects to root formation and depend on the species. In many medicinal plants, rooting is more successful when the macro/micro salts (sometimes vitamins) are reduced twice or more Saussurea obvallata (Joshi and Dhar, 2003); Ensete ventricosum (Birmeta and Welander, 2004); Ecliptaalba (Baskaran and Jayabalan, 2005; Carlina acaulis (Trejgell et al., 2009), and sucrose is reduced to 2.0, 1.0 or 0.5%.

A model protocol for indirect organogenesis: 1. Callus induction on nutrient media containing zeatin 2.0 mg/l, IAA 0.2 mg/l and casein hydrolisate 1000 mg/l (MSZ1 medium). The callus was isolated and cutting on the explants with 0.5/1.0 cm in size. 2. Cultivation of the calli on MS media containing 0.1 mg/l 2,4-D (D medium) for 28 days, 22oC and 16/ 8photoperiod - 2 passages 3. Buds regeneration on MS media containing BAP 1.0 mg/l and IAA 0.1 mg/l for 28 days, 22oC and 16/ 8photoperiod 4. Propagation of plants regenerants on MS nutrient medium containing1.0 mg/l BAP and 0.1 mg/l IAA (BA medium) or on MS containing 1.0 mg/l zeatin and 0.1 mg/l IAA for 28 days, 22oC and 16/ 8photoperiod 5. Rooted of the regenerants on ½MS media containing 2.0 mg/lIBA, 0.2 mg/lIAAand 0.4 mg/lGA3(½ MS rooting medium) for 28 days, 22oC and 16/ 8photoperiod. 6. Adaptation in exvitro conditions (Tashevaand Kosturkova, 2010 a,b)

Figure 8. Roots formed on MS medium containing 2.0 mg/lIBA, 0.2 mg/lIAAand 0.4 mg/lGA3

CONCLUSIONS

Figure 9. Propagation on MS medium containing zeatin (cis-trans form).

As a result of our experiments the following scheme of consecutive culture media for indirect organogenesis and regeneration could be proposed: MSZ2 їDїDїBAї½ MS.

When regenerated shoots attained a height of 1.0-1.3 cm they were excised and transferred to MS medium for root induction. In our experiments rooting was observed on the same medium used for multiplication (Figure 7). Root formation needed about 28-36 days period of time, which is longer period in comparison with the use of half strength MS nutrient medium containing 2.0 mg/lIBA, 0.2

Plants produced in this study appeared normal without observed morphological or phenotypical abnormalities and successfully developed in pots. The present system could be used as an alternative one for multiplication of selected valuable clones of this important

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Scientific Bulletin. Series F. Biotechnologies, Vol. XVII, 2013 ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

CAMELINA CULTIVATION FOR BIOFUELS PRODUCTION Andra MORARU1, Stefana JURCOANE1, Delia DIMITRIU2 1

University of Agronomic Sciences and Veterinary Medicine of Bucharest - BIOTEHGEN, 59 Marasti Blvd., 011464, Bucharest, Romania 2 Manchester Metropolitan University, CATE, John Dalton Building, Chester Street, Manchester, UK Corresponding author email: [emailprotected]

Abstract Recently studies have been carried out regarding Camelina sativa benefits. This oilseed plant that belongs to Brassicaceae family presents a major interest due to the fact that it can be a renewable resource for sustainable biofuels production. The oil obtained by crushing the seeds is rich in polyunsaturated fatty acids and can be processed in order to obtain biokerosene for aviation. Camelina oil has the property to resist at minus 47-48°C, the requirement for aviation fuel, due to negative temperature in the area the aircraft fly. In our study, Camelina sativa was cultivated in several locations from Romania: in the North (Iasi and Satu-Mare county), in the West (Arad county) and in the South (Calarasi county). Two camelina varieties were tested: GP 202 and GP 204. The aim of this study is to assess the camelina yield potential and identify the optimum cultivation technology. The climatic conditions and the soils were monitored during the tested period. The production was influenced by the sowing date. The best results were obtained at Iasi (2.9 t/ha), Calarasi (1.2 t/ha) and Satu-Mare (1.2 t/ha). Keywords: aviation, biofuel, Camelina yield, Camelina sativa, cultivation technology.

and diseases (Putnam et al. 1993; Gugel and Falk, 2006; Martinelli and Galasso, 2010; Moser, 2010) It requires minimal or no tillage and no special equipment (Dobre et al, 2011). The oil obtained from Camelina sativa seeds is rich in polyunsaturated fatty acids (Abramovic and Abram, 2005) and can be used for various purposes: biodiesel (Fröhlich and Rice, 2005; Kruczyŷski,2013), biojet fuel (Moser, 2010; Shonnard et al., 2010, Jurcoane et al. 2011), heating oil, naphtha, liquefied petroleum gas (EPA, 2013), paint, varnish and cosmetics industry (Zubr,1997). Moreover, the by-product that remains after camelina seed crushing can be used in animal feeding in small amounts (Putnam et al. 1993, Jurcoane et al. 2011). Our study is aimed at identifying the best camelina cultivation technology using lowinputs and testing the potential of Camelina sativa varieties in several locations from Romania.

INTRODUCTION The issue of plant-based biofuel sustainability is very complex. The whole chain involved in biofuel production depends on multitude of factors, such as: GHG (greenhouse gases) emissions reduction, land use change, indirect land use change, land availability (food vs. fuel dilemma), agricultural practices, water consumption reduction, limiting negative effects on biodiversity, biofuels efficient use in the economic and energy sectors, logistics and social impact (Lee et.al, 2008; Moser, 2010; Shonnard et al., 2010; Amigun et al., 2011). According to EPA (Environmental Protection Agency). Camelina sativa can be successfully used for advanced biofuel production, meeting all the above mentioned criteria. Camelina sativa is an annual plant belonging to Brassicaceae family. It adapts to cold climate and can be cultivated in northern regions (Gugel and Falk, 2006;Moser, 2010). Its recent research has focused on introducing it in rotational crops with cereals (Moser, 2010), as well as in double cropping (Schillinger et al., 2012; Gesch and Archer, 2013). Its vegetation period is short (85-100 days) and, unlike rapeseed, it is more resistant to drought, pests

MATERIALS AND METHODS Camelina sativa cultivation and harvesting In Romania, during 2011-2012, Camelina sativa was cultivated in the North (Iasi and Satu-Mare county), in the South (Calarasi

rows was 25 cm. The following soil works were carried out: ploughing, disc harrowing, combinatory and rolling (before and after sowing). In March 2012, 150 kg/ha ammonium nitrate was applied. Harvesting was done on 27 June 2012 with a Fendt combine. In Arad County, the tests were carried out in Buteni village. In this location, 2 ha were sown with Camelina sativa GP 202 using a SUP 21 seeding machine. The seeding rate was 10 kg/ha. The distance between rows was 12.5 cm. Soil tillage was represented by ploughing, disc harrowing and rolling (before and after sowing). The harvesting took place on 11 July 2012 using a Claas Lexion combine. Camelina seeds conditioning After the harvest, the seeds were stored by farmers and were dried naturally (not by using special equipment). They were aired by farmers until they reached a humidity level of 8-9%. The entire yield obtained from all the demo trials was sent to Iasi for conditioning because this location was best equipped i.e. a suitable sieve with round holes of 1,5 mm. The separation process was made for each variety and impurities were removed. A second conditioning was necessary to be performed. The quantity left this process was also stored in Iasi. Analysis methods After harvesting and conditioning, IBNABalotesti (National Research Development Institute for Animal Biology and Nutrition) analysed the seed oil content. Regarding the soil samples the following analyses were conducted: soil pH using a pH meter, the total carbon and nitrogen content using LECO methodology and the available soil phosphorus content after Olsen's method.

county) and in the West (Arad county) in nonirrigated conditions. Two Camelina sativa varieties were tested: GP 202 and GP 204, German varieties. The camelina crop was sown in different periods at the depth of 1-2cm. The climatic conditions and the soil were monitored during testing period. Soil samples were taking during camelina vegetation at the depth of 20cm. In Iasi county (Tiganasi village location), 2 ha were sown with Camelina sativa GP 202 and 1 ha with GP 204 variety, using an Amazone sowing machine. The distance between rows was 25 cm, according with Imbrea et al. recommendations, 2011. The previous crop was represented by mustard. Before sowing, the following soil works were carried out: stubble ploughing after previous crop harvesting, rolling and leveling. Inputs applied for camelina crop were the highest from all locations. On the middle of August 2012, 200 kg/ha N: P: K 15:15:15 and 200 kg/ha N: P: K5:15:30 were applied. During flowering period and when the plants had almost all the capsules formed, 3 kg/ha of foliage fertilizer were applied. The seeding rate was 5 kg/ha for GP 202 and 5.5 kg/ha for GP 204. Only in this location phytosanitary treatments were applied (1 l/ha Folicur solo and 0.1 l/ha Calypso) and also a desiccant (3l/ha Reglone) was used a week before harvesting time. The harvest took place on 27 June 2012 using a Class Lexion combine, seeding conditioning being done in the same time with harvesting, using a Selector EF 3802 equipment that had very low diameter sieves. In Satu-Mare County, the trial was located in Acas village. 2 ha were sown with camelina using a SUP 21 seeding machine. Only Camelina sativa GP 202 was tested. The previous crop was maize. The soil tillage works made were: ploughing, disc harrowing, milling and rolling (before and after sowing). The seeding rate was 10 kg/ha and 200 kg/ha NPK 15-15-15 were applied in autumn. The harvesting was done on 10 July 2012 using a Claas Lexion combine. In Calarasi County the trial was located near Fundulea city (about1 km). 1 ha for each variety was tested. The seeding rate was 6 kg/ha for both varieties. The previous crop was represented by maize. The distance between

RESULTS AND DISCUSSIONS In 2011-2012, the climatic conditions recorded in Romania were not favourable for agriculture. In November in the tested locations a low amount of precipitation was recorded (< 2mm). For this reason, the camelina crop planted in autumn did not emerge in December. For the most areas from Romania, in May was recorded a prolonged period with high amount of precipitation followed by a period of drought and high temperatures in June. For instance, In Iasi county from 15 May 2012 to 25 May 2012

daily rainfall was recorded, the amount of precipitations reaching 78.4 mm (from the amount recorded during the whole month). The land from Iasi and Calarasi locations and Fundulea are preferred for agricultural activities. Regarding the soils from Satu Mare, Iasi and Calarasi (see Table 1), these had a slightly acid pH, and the soils from Arad a neutral pH. The C/N ratio showed that soil fertility had normal

values in Calarasi (C/N: between 10 and 15) and higher values for Iasi trial. The phosphorus concentration determined by Olsen method varied in the tested soils. None of these samples had a very low P concentration (< 4 ppm). The samples taken from Sate Mare, Arad, Calarasihad a low P concentration, which proves that for future tests P fertilization is mandatory.

Table 1. Soil tests results from different locations from Romania during camelina flowering period Location (county) Iasi Calarasi Satu-Mare Arad

pH 6.25 6.62 6.67 7.15

Total Nitrogen (%) 0.14 0.15 0.14 0.14

Total Carbon (%) 2.52 1.84 1.03 1.35

The camelina crop cultivated in autumn had a good resistance over the winter period and started to emerge in April (Table 2). In April 2012 the plants had 4-6 leaves. During April and May the plants grew rapidly. At the end of the May in Satu Mare, Iasi and Calarasi locations, and all the plants had their capsules formed (Figure 1). The weed infestation was low and no effects on camelina production. In Calarasi county the presence of few plants

C/N 18 12.27 7.36 9.64

P (ppm) 20.31 11.83 15.84 15.13

infested with Peronospora camelinae was noticed but no phytosanitary treatments were required. When the distance between rows was 25 cm, the plants were more robust and had numerous branches. Only in Iasi an insecticide and a fungicide with systemic action were applied to prevent Meligethes aeneus and Peronospora camelinae from damaging the plant.

Table 2. Sowing, emergence and harvesting period. Location (county) Iasi Calarasi Satu-Mare Arad

Sowing date 21 November 2011 9 November 2011 10 November 2011 4 March 2012

Emergence date 05 April 2012 20 March 2012 27 March 2012 11 April 2012

For the spring trial carried out in Arad county, the flowering occurred in the middle of May and continued at the beginning of June (see Figure 2). No fertilizers no phytosanitary treatments and no desiccant were used. Harvest reaping started in mid June and finished in July. The harvesting period was the same for both Camelina sativaGP 202 and GP 204 varieties. In Arad County the yield obtained was 0.76 t/ha but the seed oil percentage was higher (32.38%). We consider that the optimal period for harvesting was exceeded.

Harvesting date 27 June 2012 27 June 2012 10 July 2012 11 July 2012

Figure 1. Camelina sativa GP 202 variety, Calarasi County, 31 May 2012

In Iasi County, the harvesting was done at the right time and the losses were minimal. The most predominant weed was Chenopodium album but after treatment it was eliminated (see Figure 5). The seed oil percentage was higher for GP 202 variety (28.91%) and lower for the GP 204 variety (23.88%). The best obtained yield, 2.9 t/ha was obtained for GP 202 variety. For GP 204 variety, 2.2 t/ha were obtained.

Figure 2. Camelina sativa GP 202 variety, Arad County, 3 June 2012

In Satu-Mare, only a part of trial was infested with Peronospora camelinae (Figure 3, 4). No phytosanitary treatments were applied. In SatuMare with low inputs application, 1.2 t/hawere obtained. The seed oil percentage was the best from all locations (32.95%). Therefore, Camelina sativa is not a demanding plant but its cultivation technology requires further study.

Figure 5. Chenopodium album in camelina crop, Iasi County, 07 June 2012

In Calarasi, the harvesting was done using a combine with minimum air flow. Also, the harvesting was done too late. Especially in Calarasi county plants are harvested earlier than in other regions. No dessicant was applied. The first capsules containing high amount of seeds matured earlier and were the first to shake. It is necessary to identify the optimum harvesting period. The gross production for each variety was 1.8 t/ha for GP 202 and 1.2 t/ha for GP 204. The seeds had 25.23% oil content for GP 202 variety and 22.23% for the GP 204 variety.

Figure 3. Satu Mare County-Plants infested with Peronospora camelineae, 2 June 2012

CONCLUSIONS The technology of camelina cultivation is a complex one which needs attention by the farmers, although the plant itself is not a demanding one. The best obtained yield, 2.9 t/ha, was obtained in Iasi for Camelina sativa GP 202 variety. We consider that the harvest occurred too late in the South of Romania when the yields are harvested earlier than the rest of the country. It is necessary to identify the optimal harvesting date.

Figure 4. Satu Mare County – General aspect of Camelina sativa crop, 2 June 2012

Gugel R.K., Falk K.C., 2006, Agronomic and seed quality evaluation of Camelina sativa in western Canada, Can. J. Plant Sci., 86, 1047–1058. Imbrea F., Jurcoane S., Halmajan H.V., Duda M., Botos L., 2011, Camelina Sativa -A new source of vegetal oils, Rom.Biotechnol.Letts., 16, 3, 6263-6270. Jurcoane,S., Dobre P., Florea C., Petre S.M., Ropota M., 2011, A useful plant source for renewable jet fuels, human nutrition and animal feed,, Rom.Biotechnol.Letts, IX, 1-4, 33-34. KruczyŷskiW. S., 2013, Performance and emission of CI engine fuelled with Camelina sativa oil, Energy Conversion and Management, 65, 1–6. Lee H., Clark W., Devereaux C., 2008 Biofuels and Sustainable Development, CID Working Paper 174. Martinelli T., Galasso I., 2011 Phenological growth stages of Camelina sativa according to the extended BBCH scale, Annals of Applied Biology, 158,87–94. Moser B.R., 2010, Camelina (Camelina sativa L.) oil as a biofuels feedstock: Golden opportunity or false hope?, Lipid Technology, 22, 12, 270-273. Putnam D., Budin J., Field L., Breene W., 1993, Camelina: a promising low-input oilseed, 314-322. In: J. Janick and J.E. Simon (eds.), New crops. Wiley, New York. Schillinger W.F., Wysocki D.J., Chastain T.G., Guy S., Karow R., 2012, Camelina: Planting date and method effects on stand establishment and seed yield, Field Crops Research, 130, 138–144. Shonnard David R.,Williams Larry, Kalnes Tom N., 2010, Camelina –derived jet fuel and diesel: Sustainable advanced biofuels, Environmental Progress Sustainable Energy, 29, 3, 382-392 ; Publisher John Wiley& Sons. Zubr Josef, 1997, Oil-seed crop: Camelina sativa, Industrial Crops and Products, 6, 2, 16, 113–119. Environmental Protection Agency (EPA), Regulation of Fuels and Fuel additives: Identification of Additional Qualifying Renewable fuel Pathways under the Renewable Fuel Standard Program, Federal Register/ vol. 78, no. 43/Tuesday, March 5, 2013/ Rules and Regulations.

The seed oil percentage was higher for GP 202 variety and lower for the GP 204 variety. The crops that are sowing later in spring mature non-uniformly, leading to yield losses. We consider that both Camelina sativa varieties (GP 202 and GP 204) meet the growth and development conditions for Romania. ACKNOWLEDGEMENTS We would like to thank the Manchester Metropolitan University for fully supportingthesustainable camelina crop production development in Romania. The research was financed from the project no. 1970/2011, entitled “Sustainable aviation biofuel using camelina as a feedstock-Romania as a case-study”. REFERENCES Abramovic H., Abram V., 2005, Physico-chemical properties, composition and oxidative stability of Camelina sativa oil, Food Technol. Biotechnol., 43, 1, 63–70. Amigun B., Musango J.K., Stafford W., 2011, Biofuels and sustainability in Africa. Renewable and Sustainable Energy Reviews, 15, 2, 1360 – 1372. Dobre P., Jurcoane S., 2011, Camelina cropopportunities for a sustainable agriculture, Scientific Papers, UASVMB, LIV, series A, 420-424. Fröhlich A., Rice B., 2005, Evaluation of Camelina sativa oil as a feedstock for biodiesel production Industrial Crops and Products, 21, 1, 25–31. Gesch R.W., Archer D.W., 2013, Double-cropping with winter camelina in the northern Corn Belt to produce fuel and food, Industrial Crops and Products, 44, 718–725.

Scientific Bulletin. Series F. Biotechnologies, Vol. XVII, 2013 ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

IN VITRO STUDY ON THE INTERACTION BETWEEN BACILLUS THURINGIENSIS AND CHEMICAL PESTICIDES USED FOR CORN CROP PROTECTION Mihaela Monica DINU, Ana-Cristina FĂTU, Sorin ùTEFAN, Ana Maria ANDREI Research - Development Institute for Plant Protection, 6 Ion Ionescu de la Brad Blvd., District 1, 013813, Bucharest, Romania Corresponding author email: [emailprotected] Abstract Interactions between the entomopathogenic bacteria Bacillus thuringiensis and chemical pesticides used for corn crop protection is one of the most important factors that influence the effectiveness of the entomopathogenic microorganism. Bacterial biopreparates based on B. thuringiensis could be used along with chemical pesticides. The effect of chemical ingredients on bacteria viability is mandatory and it should be conducted first. Interactions between the entomopathogenic bacteria B. thuringiensis and chemical pesticides occurs when chemicals and bacterial biopreparates are applied simultaneously or mixed together. Selectivity on some biological parameters of B. thuringiensis was tested by making a mixture of B. thuringiensis with chemical pesticides. Different concentrations of chemical pesticides were mixed with sporulated and vegetative bacterial cultures. The effects of chemical plant protection products on sporulation and vegetative growth of B. thuringiensis were monitored. The paper presents the results of experiments aimed at determining the influence of some chemical pesticides used for corn crop protection on the B. thuringiensis multiplication and sporulation. Keywords: Bacillus thuringiensis, chemical pesticides, corn crop.

the chemical control methods. That is why the bacterial entomopathogenic insecticides should be especially compatible with traditional pest management practices. Chemical plant protecttion products is one of the most important factors that influence the effectiveness of entomopathogic bacteria B. thuringiensis used in corn pest control. Mixtures of bacterial biopreparates based on B. thuringiensis with different chemicals are possible and can be used in practice. The effect of chemicals on active substance of bacterial bioproducts must be checked. The interaction between entomopathogenic bacteria and pesticides can occur in the following ways: (1) Corn pests and diseases form a rich complex of species that cause damages in our country. Simultaneous control of diseases and pests could be made using plant protection chemicals. It is also very important to maintain a natural biological balance, to protect the environment and the useful insects. The systems of integrated protection in agriculture use chemical pesticides to control (a) Diseases: seedling damping-off (Pythium sp.), foot rot (Fusarium spp.), head smut (Sorosporium

INTRODUCTION Bacillus thuringiensis is an endospore-forming Gram-positive bacterium of economic importance due to its entomopathogenic capability and has been used as a safe microbial insecticide for over 50 years for caterpillars’ pest control. The insecticidal action of B. thuringiensis is attributed to protein crystals produced by the bacterium. B. thuringiensis based insecticides are popular with organic farmers because they are considered 'natural insecticides'. They differ from most conventional insecticides because they are toxic to only a small range of related insects (Hellmich, 2012). This is because specific pH levels, enzymes, and midgut receptors are required to activate and bind a given Cry toxin to midgut cells, which leads to pore formation in the insect's intestine and death (Federici 2002). Modern technology involves B. thuringiensis gene responsible for the production of the insecticidal protein incorporation into the maize genome for the corn borer control. Although B. thuringiensis based insecticides are an important tool for maize growers, they cannot completely replace

holci-sorghi), corn smut (Ustilago maydis), (b) Pests: maize leaf weevil (Tanymecus dilatecollis), European corn borer (Ostrinia nubilalis), wireworms (Agriotes sp.), locusts and (c) Annual and perennial monocotyledonous and dicotyledonous weeds (Amaranthus sp., Chenopodium sp., Sinapis sp., Capsella sp., Thlaspi sp., Cirsium sp., Hibiscus sp., Xanthium sp., Abutilon sp., Raphanus sp., Solanum sp., Polygonum convolvulus, Setaria sp., Echinochloa sp., Digitaria sp., Convolvulus arvense, Calystegia sepium). (2) Application of chemical pesticides in the mixture or simultaneously with bacterial bioproducts, depending on the evolution of insect pest, in order to make treatments more profitable. (3) Application of some pesticides containing bacterial preparation in order to obtain adequate efficacy.

MATERIALS AND METHODS T4 strain of B.thuringiensis var. thuringiensis, from Research-Development Institute for Plant Protection collection of micro-organisms, was used for this experiment. The following working method was used in order to identify the selectivity of plant protection products against entomopathogenic bacteria. Bacterial culture was grown in corn extract agar media which was mixed with each of the chemicals in the following three concentrations: the recommended concentration for use (c.u.), ½ (1/2c.u.) and ¼ (1/4c.u.) of recommended concentration for use. Test mixtures were sown in Petri dishes, which were incubated at 280C for 72 hours. Different chemicals from fungicides, insecticides and herbicides groups were tested (Table 1).

Table 1. Plant protection chemicals tested in combination with bacterial culture FUNGICIDES Chemical group Dithiocarbamates and thiuram derivatives Triazoles and imidazoles INSECTICIDES Synthetic pyrethroids

Various

Product (s.a.) Target organism ROYAL FLO 42 S Pythium spp. (thiram 480g/l) Fusarium spp. Pythium spp. VITAVAX 200 FF (carboxina 200g/l+thiram 200 g/l Fusarium spp. SIGNAL (cypermethrin 300 g/l) ACTARA 25 WG (thiamethoxam 25%) GAUCHO 600 FS (imidacloprid 600 g/l) COSMOS 250 FS (fipronil 250 g/l) CRUISER 350 FS (thiamethoxam 350 g/l)

Dose (conc.) 3,0 l / t seeds 2,5 l / t seeds

Agriotes spp.

2,0 l/t seeds

Tanymecus dilaticolis

0,100 kg/ha

Tanymecus dilaticolis Agriotes spp.

6,0-8,0 l pc/t seeds

Agriotes spp.

5,0 l/t seeds

Tanymecus dilaticolis Agriotes spp.

1,2 μl/one seed

HERBICIDES

Aminofosfats

Picoline derivatives

Sulfonylureas

Isoxazoles Mixtures

DOMINATOR (glyphosate 360 g/l) ROUNDUP (Glyphosate isopropylamine salt 360 g/l) CERLIT (fluroxypyr 250 g/l) MISTRAL 4 SC (nicosulfuron 40g/l)

Annual and perennial weeds

4,0 l/ha

Monocotyledonous and dicotyledonous weeds, annual and perennial (+Sorgum halepense from rhizomes) Convolvulus arvensis Calystegia sepium

4 l/ha (mixed with 100-150 l water/ha)

Sorghum halepense

1,5 l/ha

TITUS 25 DF (rimsulfuron)

Monocotyledonous weeds, annual and perennial – including S.halepense from 60 g/ha seeds and rhizomes- and some annual dicotyledonous weeds

CALLISTO 480 SC Annual weeds (mesotrine 480 g/l) CALLAM Annual and perennial dicotyledonous (tritosulfuron 12,5% + dicamba weeds 60%)

1,0 – 2,0 l/ha

0,350 l/ha 0,4 + 1,0 l/ha

Figure 1. Filter paper discs with sterilized distilled water on B. thuringiensis bacterial lawn (corn extract agar media)

Figure 2. Effect of mesotrine on halo formation and inhibition of the growth of Bacillus thuringiensis on corn extract agar media

Figure 3. Effect of glyphosate isopropylamine salt on halo formation and inhibition of the growth of Bacillus thuringiensis on corn extract agar media

In order to make a better observation on selectivity of plant protection products against

B. thuringiensis strain, a common working method was used for some of the variants.

bacterial colonies was close or equal to those of the control variant. Microscopic analysis showed no changes in vegetative or sporulated bacterial cells. The following issues were observed for the groups of chemicals: (a) fungicides have generally manifested stronger inhibitory effect on bacterial spores; (b) insecticides manifested, in general, high degree of selectivity against B. thuringiensis and (c) herbicides manifested the lowest degree of selectivity. There was an almost complete inhibition of bacterial growth, both in vegetative and sporulated culture when mixed concentration of DOMINATOR and ROUNDUP herbicides dose corresponded to recommended concentration for use. Partial inhibitory effect was maintained when the concentration was reduced by two or four times. Recent studies aimed at rice crops protection, revealed the compatibility between insecticides (thiamethoxam, labdacyhalothrin, malathion and fipronil) and B. thuringiensis strains (B. thuringiensis subsp. dendrolimus, B. thuringiensis var. kurstaki, B. thuringiensis var. thuringiensis and B. thuringiensis subsp. entomocidus) interaction. However, at a 101 concentration, ten times higher than the recommended concentration, some insecticides presented inhibitory effect in the bacterial development. The insecticide malathion inhibited the development of six out of seven evaluated B. thuringiensis strains at the higher concentration (Pinto et al., 2012). Batista-Filho et al. (2001) and Almeida et al. (2003) reported compatibility between B. thuringiensis bacterial growth and thiamethoxam insecticide. Silva et al. (2008) reported resistant B. thuringiensis var. kurstaki colonies expressing inhibition in the presence of some herbicides. Field toxicity studies have shown that when chemical insecticides manifest in vitro toxicity against B. thuringiensis, this does not suggest necessarily high field toxicity (Alves et al., 1998). It is recommended, though, chemical insecticides be used in the advised doses when using B. thuringiensis-based products.

Bacteria was inoculated in Petri dishes on corn extract agar media, followed by the placement of 4 paper discs with the evaluated insecticide in 4 points of the dish. In the control treatment, four filter paper discs with sterilized distilled water replaced the insecticide (Figure 1). The data was analyzed on the 4th and 7th day after the treatment application. Each Petri dish was analyzed for the absence or presence of the bacterial growth inhibition halo (Figures 2, 3) The tested chemicals are commonly used in maize crop protection. Observations followed B. thuringiensis colony diameter treated with pesticides variants, compared with untreated control variants. RESULTS AND DISCUSSIONS Results on the effect of chemicals on spore germination and vegetative multiplication of B. thuringiensis are presented in Tables 2, 3 and 4. Bacterial growth on agar media was noted as follows: + + + = very good growth, + + = good growth, + = weak growth. An overall analysis of the data presented reveals that the tested products fall into one of these 3 categories: (a) products with high selectivity towards B. thuringiensis (b) products with average (good) selectivity and (c) products with low selectivity (bacteriostatic). This analysis refers to how bacterial growth and spore germination of B. thuringiensis have been influenced by contact with plant protection chemicals Bacteriostatic properties of some chemicals influenced spore germination and vegetative multiplication in varying proportions. This effect was revealed through the partial inhibition of vegetative and sporal multiplication at high concentrations of the chemical. Bacterial cultures mixed with recommended concentration for use of chemicals belonging to this category and inoculated on agar media, developed colonies with a diameter from two to eight times lower than the bacterial colonies of untreated control variant. By decreasing the concentration of the chemical in the mixture experiments, development of bacterial cultures was registered normal parameters. The size of

Table 2. Influence of some fungicides on Bacillus thuringiensis Experimental mixture tested Bacterial lawn /Fungicide concentration Variants I (a) I (b) II (a) II (b)

Chemical substance + ROYAL FLO 42 S VITAVAX 200 FF

Bacillus thuringiensis vegetative sporulated vegetative sporulated

c.u. +++ +++ +++ +++

½ c.u. +++ +++ +++ +++

¼ c.u. +++ +++ +++ +++

Table 3. Influence of some insecticides on Bacillus thuringiensis Experimental mixture tested Bacterial lawn /Insecticide concentration Variants I (a) I (b) II (a) II (b) III (a) III (b)

Chemical substance + SIGNAL ACTARA 25 WG GAUCHO 600 FS

Bacillus thuringiensis vegetative sporulated vegetative sporulated vegetative sporulated

c.u. +++ +++ ++ ++ +++ +++

½ c.u. +++ +++ +++ ++ +++ +++

¼ c.u. +++ +++ +++ +++ +++ +++

Table 4. Influence of some herbicides on Bacillus thuringiensis Experimental mixture tested Bacterial lawn / Herbicide concentration Variants I (a) I (b) II (a) II (b) III (a) III (b) IV (a) IV (b) V (a) V (b)

Chemical substance + DOMINATOR ROUNDUP TITUS 25 DF CALLISTO 480 SC CALLAM

Bacillus thuringiensis vegetative sporulated vegetative sporulated vegetative sporulated vegetative sporulated vegetative sporulated

c.u. +++ +++ +++ +++ +++ +++

½ c.u. + + + + +++ +++ +++ +++ +++ +++

¼ c.u. ++ ++ ++ ++ +++ +++ +++ +++ +++ +++

Based on the data presented, the tested chemicals fit within these degrees of selectivity in relation to B. thuringiensis.

CONCLUSIONS The overall effect of the chemicals on B. thuringiensis efficiency is difficult to assess in field conditions. On one hand, we consider the average concentration of pesticides with which bacteria come into contact is in a lesser amount than the one tested in the laboratory. Occasionally, those which can be applied directly to the soil could exceed the normal dose. On the other hand, growth of B. thuringiensis on agar media, with optimal conditions, makes it more tolerant for chemicals compared to bacteria released into nature where it has to face less favorable conditions, competition with antagonists etc. Therefore, experimental variants which showed good selectivity of chemicals against bacteria B. thuringiensis in controlled laboratory conditions should be tested under field conditions too.

Table 5. Selectivity of chemicals against B. thuringiensis High selectivity ROYAL FLO 42 S Fungicides VITAVAX 200 FF SIGNAL GAUCHO Insecticides 600 FS CRUISER MISTRAL 4 SC TITUS 25 DF Herbicides CALLISTO 480 SC CALLAM.

Good selectivity

Low selectivity

ACTARA 25 WG ESTERON 60 ATRANEX 80 WP DOMINATOR ROMANEX ROUNDUP 500 SC ALANEX 48 EC

Federici B.A., 2002. Case study: Bt crops-a novel mode of insect control. Genetically Modified Crops: Assessing Safety, ed. K. T. Atherton, Taylor & Francis, 164-200. Hellmich R. L., Hellmich K. A., 2012. Use and Impact of Bt Maize. Nature Education Knowledge 3 (10) :4 Pinto L., Dörr N., Ribeiro A., De Salles S., De Oliveira J., Menezes V, Fiuza L., 2012. Bacillus thuringiensis monogenic strains: screening and interactions with insecticides used against rice pests. Braz. J. Microbiol. vol. 43 (2). Silva E.R.L., Alves L.F.A., Santos J., Potrich M., Sene L., 2008. Técnicas para avaliação in vitro do efeito de herbicidas sobre Bacillus thuringiensis Berliner var. kurstaki. Arq. Inst. Bio. 75 (1), 59-67.

REFERENCES Alves S.B., 1998. Controle microbiano de insetos. 2. ed. São Paulo: FEALQ, 326p. Almeida J.E.M., Batista-Filho A., Lamas C., Leite L.G., Trama M., Sano A.H., 2003. Avaliação da compatibilidade de defensivos agrícolas na conservação de microrganismos entomopatogênicos no manejo de pragas do cafeeiro. Arq. Inst. Biol. 70 (1), 79-84. Batista-Filho A., Almeida J.E.M., Lamas C., 2001. Effect of thiametoxam on entomopathogenic microrganisms. Neotrop. Entomol. 30 (3), 437-447.

Scientific Bulletin. Series F. Biotechnologies, Vol. XVII, 2013 ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

DIVERSITY ANALYSIS OF TUBERED-BEARING Ipomoea trifida (H.B.K.) G. DON. ORIGINATED FROM CITATAH WEST JAVA INDONESIA BASED ONCHROMOSOME TRAITS Tia SETIAWATI1, Agung KARUNIAWAN2 1

Department of Biologi Universitas Padjadjaran, Jl. Raya Bandung-Sumedang Km. 21 Jatinangor, 45363, Sumedang, Indonesia 2 Department of Agronomy Universitas Padjadjaran, Jl. Raya Bandung-Sumedang Km. 21 Jatinangor, 45363, Sumedang, Indonesia Corresponding author email: [emailprotected]

Abstract Ipomoea trifida is considered as a wild crop relative to sweet potato Ipomoea batatas. A set of 10 accessions was selected for tubered-bearing I. trifida originated from Citatah West Java was evaluated on their diversity based on chromose traits. A field trial and laboratory analysis were conducted at Universitas Padjadjaran Bandung Indonesia. Relationship between species identified by cluster analysis and Principal Component Analysis (PCA). The results showed that the observation on the 10 accession of tubered-bearing I. trifida using nine chromosome traits produced dissimilarities distance (Euclidean coefficient) ranging from 1.75 to 6.22. Dendogram generated at a dissimilarity distance of 5.23 showed the formation of three main clusters. Principal Component Analysis (PCA) produced first two principal component (PC1 and PC2), which has been able to explain 89.64% of the total variation. It is concluded that thare are highy diveristy between 10 accession of tubered-bearing I .trifida based on chromosomes traits. Keywords: Chromosome, Ipomoea trifida, Principal Component Analysis.

of the yields, levels of dry matter, starch, increased levels of protein (Kobayashi & Miyazaki, 1976) and resistance to certain pests and diseases as black rot root diseases (Shiotani and Kawase, 1989; Komaki, 2001) and scab disease (Hartana, 1994). Wild relativesI. trifida originated from Citatah West Java has morphotype variations, so to determine the level of genetic diversity and relationship among accessions need to be done the clustering by morphological or cytogenetic studies (chromosome traits). Morphological characterization done through observation of the phenotypic appearance, while cytogenetic studies done through observation of chromosome number or ploidy level and form of chromosomes. These characters can be used as a chromosome differentiating factor for identifying genetic variation in plants that will be useful for breeders in developing and improving the quality of crops. Most wild relatives of sweet potato are found of tetraploid or diploid (Renwarin etal., 1994) and have not been characterized, so many potential sources of genetic diversity is unknown. So far it is not known the level of morphotype variation,

INTRODUCTION Wild relative of sweet potato Ipomoea trifida is considered as having a potential source of genes to support plant breeding programs of domesticated sweet potato (Ipomoea batatas (L.) Lam.). Hambali (1988) reported that the highest genetic and phenotypic diversity of wild relatives of sweet potato in Indonesia is in the Citatah-West Java. The results of field observations in Citatah by a Padjadjaran University Team in 2010 have collected 168 accessions of wild relatives of sweet potato that have not been identified. Based on morphological variation in flowers and leaves, supposedly 168 accessions comprising I. trifida and I. triloba (Agung Karuniawan, personal communication, 2010). Wild relatives of sweet potato were found in Citatah known by local residents as “huhuian” and “boled areuy”. Naturally wild relatives of sweet potato were found in Citatah grow as weeds in agricultural land of sweet potatoes, and other calcareous slope areas. Wild relatives of sweet potatoI. trifida has been used as a source of genes in sweet potato breeding to improve the character

genetic diversity and relationship among accessions of wild relatives of sweet potato originated from Citatah West Java. This information is necessary to support the management and utilization of germplasm. Information of the level of genetic diversity on germplasm material is needed by breeders to identify potential progenitor and will be useful also to prevent the use of closely related progenitor closely in crossing.

haploid complement chromosome lenght (HCL), the value of intrachromosomal index (A1) and interchromosomal index (A2). Analysis of genetic diversity performed using cluster analysis and Principal Component Analysis (PCA) with the software of XLstat 2009. RESULTS AND DISCUSSIONS Cluster analysis on ten accession of tuberedbearing I. trifida based on chromosome traits. Dissimilarities distance (Euclidean coeffisien) between ten accession of tubered-bearingI. trifida based on nine chromosome traits ranged from 1.755 to 6.224 (Figure 1).

MATERIALS AND METHODS Materials Research materials consisted of ten accessions of tubered-bearing I. trifida collection of the Faculty of Agriculture, Universitas Padjadjaran. Root tips used as materials for preparations for observation of chromosomes. The chemicals required include a solution of 0.002 M 0.8-hydroksiquinolin, fixative solution (ethanol: glacial acetic acid = 3:1), a solution of 4N HCl, solution of 45% acetic acid, and 2% orcein. Methods Chromosome preparations Root tips that meristimatis obtained from stem cuttings grown on medium (soil: compost: manure = 1:1:1). Chromosome preparations made using the squash method of Darnaedi (1990). The root was cut 1 cm from the root tip and soaked in a solution of0.002 M 8hydroksiquinolin for 3-5 hours at 18-200C. Subsequently the roots were fixed in a mixture solution of ethanol: glacial acetic acid (3:1) for 48 hours and transferred to a solution of 4N HCl for 10 minutes. Subsequently the root was immersed in 45% acetic acid solution for 10 minutes. Staining of preparations carried out using 2% orcein for 10 minutes on top of a glass object, then closed, heated and pressed. Observations and data analysis Observation of chromosome using light microscopy. Chromosomes on prometafase or early metaphase stage photographed and made the micrography. Chromosome captured images magnified and printed with a computer program, then print out of the chromosome picture was used for observation of chromosome number, chromosome size (the length of the long arm (q), the length of the short arm (p) and total length (q + p)), centromere index (CI), shape of chromosomes,

Figure 1. Dendogram of clustering on ten accession of tubered-bearing I. trifida

Dendogram generated at a dissimilarities distance of 5.228 showed the formation of three main clusters, namely cluster I consists of 4 accessions (accession 118, 19, 15, and 81); Cluster II consists of 3 accessions (accession 180, 149, and 99); and cluster III consists of 3 accessions (accession 40, 1 and 13). Highest dissimilarities distance of 6.224 possessed by the accession of 118, 19, 15, and 81 are joined in cluster I. Thus it can be assumed the four accession have the mostdistant relationship to other accession based on the chromosome traits were observed. The lowest dissimilarities distance of 1.755 is owned by the accession of 99 and 149 showed that the two accessions have a high similarity of chromosome traits so that the realtionship between the two accessions are very close. The high degree of similarity between the two accessions probably

traits of the short arm (p), centromere index (IS), the number of metacentric chromosomes (m), the number of submetacentric chromosome (sm) and intrakchromosomal asymmetry index (A1) on PC2 contributes for 36.393% of the variation that arises among the tested accessions. Therefore, until the second principal component (PC2) was able to explain 89.640% of the total variation (Table 1.). To see the pattern of distribution of ten accessions tubered-bearing I. trifida can be seen in biplot graphic (Figure 2.). Accessions that are in the same quadrant indicates that the accessions have a very close relationship, however if they are in a different quadrant with an angle > 900 then the accessionshave a distant relationship.

that the two accessions is the same material. Afuape et al. (2011) state that the genotypes that showed high similarity can be expected as duplicate genotypes so besides morphological characterization, molecular characterization is needed to confirm whether these genotypes are the same material with a different name or whether these genotypes came from the same parent. Principal component analysis on ten accessions of tubered-bearing I. trifida based on chromosome traits. Principal component analysis performed on ten accessions of tubered-bearing I. trifida to see relationship and chromosome traits affecting variation appeared between accessions. Based on Eigen value > 1, then there are two pricipal components that have been able to explain 89.64% of the variation total of accessions tested (Table 1.). Table 1. Principal component analysis (PCA) on ten accession of tubered-bearing I. trifida based on nine chromosome traits No Traits 1 Lenght of the long arm (q) 2 Lenght of the short arm (p) Total lenght of chromosome 3 (TL) 4 Centomere Index (CI) Number of metacentric 5 chromosome (m) Number of submetacentric 6 chromosome (sm) haploid complement 7 chromosome lenght (HCL) 8 Intrachromosomal index (A1) 9 Interchromosomal index (A2) Eigen Proportion (%) Cumulative (%)

Principal Component (PC) 1 2 -0.892 0.414 -0.014 0.988 -0.660

0.735

0.863

0.484

0.903

0.332

-0.903

-0.332

-0.660

0.733

-0.851 -0.155 4.792 53.247 53.247

-0.500 0.589 3.275 36.393 89.640

Figure 2. Biplot of PC1 and PC2 of ten accession tubered-bearing I. trifida base on chromosome traits

Ten accessions spread in four quadrants of biplot. Accession 15, 19, and 81 are in the same quadrant, indicating that the three accessionshave a close relationship. The three accessions also have close relationship with accession 118 that was in different quadrants as forming an acute angle ( 0.5 (Zubair, 2004). The first Principal Componen (PC1) contributes the proportion of variation by 53.247%in ten accessions of tubered-bearing I. trifida provided by almost all the tested chromosome traits except the traits of the short arm (p) and interchromosomal asymmetry index (A2). The

Ten accessions of tubered-bearing I. trifida originated from Citatah West Java has a broad genetic diversity.Tests on the ten accession of tubered-bearing I. trifida using nine chromosome traits produced dissimilarities distance (Euclidean coeffisien) ranged from 1.755 to 6.224. Dendogram generated at a dissimilarities distance of 5.228 showed the

The International Society for Tropical Root Crops. p. 469-473. The International Society for Tropical Root Crops in Collaboration with Department of Agriculture of Thailand, Bangkok, Thailand. Hartana, A. 1994.Ipomoea trifida, sumber keragaman genetik dalam pemuliaan ubi jalar (Ipomoea batatas). Laporan Akhir. Bogor. Kobayashi, M., Miyazaki, T., 1976. Sweetpotato breeding using wild related species. Proc. IV Symp. Int.Soc. Trop. Root Crop. Taiwan:AVRDC. Komaki, K., 2001. Phylogeny ofIpomoea species closely related to sweet potato and their breeding use. Bull. Natl. Inst. Crop Sci, 1, 1-56. Renwarin, J. , Hartana, A., Hambali, G.G., Rumawas., F., 1994. Ubijalar tetraploid dan prospeknya sebagai sumber genetik dalam program pemuliaan ubi jalar pentaploid. Zuriat. 5, 2, 8-15. Shiotani, I., Kawase., T., 1989. Genomic structure of the sweet potato and hexaploidsIpomoea trifida (H.B.K.) Don. Japan. J.Breed, 39, 57-66. Zubair, M. 2004. Genetic Diversity and Gene Action in Mungbean. Thesis. Faculty of Crop and Food Sciences. University of Arid Agriculture, Rawalpindi. Pakistan.

formation of three main clusters.Principal Component Analysis (PCA) produced two first principal component (PC1 and PC2), which has been able to explain 89.64% of the total variation. ACKNOWLEDGEMENTS The research was supported by Universitas Padjadjaran Bandung Indonesia under the scheme of‘Riset Andalan’ 2010-2012 REFERENCES Afuape, S., Okocha, P., Njoku, 2011. Multivariate assessment of the agromorphological variability and yield components among sweetpotato (Ipomoea batatas (L.) Lam) landraces. African Journal of Plant Science, 5, 2, 123-132. Darnaedi, D., 1990. Training Teknik Sitologi. Herbarium Bogoriensis. Balitbang Botani. Puslitbang Biologi LIPI, Bogor. Hambali, G.G., 1988. Tuberization in diploid Ipomoea trifida from Citatah, West Java, Indonesia. In: Howeler, R.H. (Eds.). Proceedings of The Eighth Symposium of

Scientific Bulletin. Series F. Biotechnologies, Vol. XVII, 2013 ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

INFLUENCE OF FERTILIZATION OF SOILWITH WORM COMPOST ON THE QUALITY OF PEAS Tatiana BOCLACI, Larisa CREMENEAC Scientific and Practical Institute of Biotechnologies in Animal Husbandry and Veterinary Medicine, 15 Scolara Street, 6225, Village Maximovca, district Anenii Noi, Republic of Moldova Corresponding author email: [emailprotected] Abstract Worm compost is one of the final products obtained as a result bioconversion technology of organic waste by worm cultivation. The product obtained is a natural organic fertilizer ecological, composed of grains of various sizes, color dark brown, odorless, hygroscopic with long acting. In this article are presented the results of research obtained in the field experiment during the period of three years. The experiment was organized in field conditions of Technological-Experimental Station 'Maximovca'.As research material was used worm compost-organic fertilizer and peas variety 'Renata'. In experiment were included 3 groups, with a surface such (two-experimental, one-control). Experimental on the batches before sowing, was incorporated organic fertilizer dose of 4t/ha and 3t/ha from dejections of cattle, as a result of use of technology bioconversion of organic waste by worm culture. The control group was grown peas with natural background. The research was conducted in order to assess the influence worm compost on the quality of peas. In the outcome research found that worm compost influences the beneficial the development at the phases phenology of the agricultural crops. Early development of agricultural crops at different phases phenology conditional increasing the quality of production the harvest, resistance in the adverse climatic conditions and at different maladies, allowing obtaining output ecological agricultural. Analysis of research results obtained revealed thatworm compost has a positive impact on the quality of output of pea by increasing total nitrogen, crude protein and the decrease content of nitro compounds. The nitrogen content as total and crude protein in the samples of pea grown with fund worm compost, increased by properly with 42,38%-126,50% si 42,37%-127,50% and nitro compounds diminished by 5,76%-65,11%. Thus, into results of the investigations it was found improve the quality of peas intended grown with fund worm compost compared with the cultivated fund natural. Keywords: fertilizer organic, peas, quality, nitro compounds, protein gross.

use of which help increase of soil fertility, harvest and improving the quality of agricultural production. In worm compost considerable are concentrated qualities of enzymes, vitamins, stimulators of growth, non-pathogenic microflora. Worm compost rests role in the development of ecological agriculture (Cremeneac, 2003). As a result of the investigations it was found that in the worm compost is well balanced content of macro-and microelements, which allows dose reduction of incorporation into the soil, which is 8-12 times lower than dose of ordinary compost. It was found that a tonne of worm compost contain 270-300 kg of humus. It allows to significantly decreasing period for completing the amount of humus in the soil, thus restored soil fertility and resistance to wind and alluvial erosion.

INTRODUCTION To obtain ecological production both as animal feed and for human alimentation, it is necessary to use certain technologies to improve the environment. Branches of the national economy are important producers of organic waste that pollutes components of nature (soil, water, air). In science and practice of world were performed research directed at reducing negative influence of organic waste on the environment, paying special attention to their performance methods of bioconversion. An effective method is considered bioconversion of organic waste by worm cultivation that as the new direction of agrobiological science and practice worth fundamental research. The purpose of this biotechnology is to obtain worm compost-ecological organic fertilizer, the

Worm compost can be used in cultivation of all agricultural crops (greenhouse and open field). Influence on their development at different phenological stages, harvest and quality of agricultural production. According to research found that using worm compost is welcome in the greenhouse practice of vegetable growing, where of rule uses considerable amounts of mineral fertilizers, chemicals for pest and disease destroying crops. This hampers to obtain ecological agricultural production, thus reducing demand for agricultural products on the market (Cosolapova et al., 1996). As a result of previous studies found that worm compost had a positive effect on the quality of forage crops (maize alfalfa and fodder beet) grown with worm compost fund. Quantity of nitro-compounds in maize alfalfa and fodder beet grown with worm compost fund reduced corresponding from 2,10 to 2,66, 3,47 to 3,76, 1,10 to 1,14 times compared with plants grown with mineral fertilizer (Boclaci et al., 2012). So, incorporation of worm compost in the soil improves soil fertility, influence early development of agricultural crops in different phenological stages, increasing yields and improved quality of obtained production.

On control lot was grown peas with natural fund. The research was conducted over three years, including the first, second and third year of action of worm compost. During the experiment was determined by using the usual methods, total nitrogen content and crude protein (Petuhova et al., 1989), and nitro-compounds content-electro-colorimetric method (Razumova et al., 1986). All analyzes were conducted using samples of peas in their natural state. In order to assess the influence of soil fertilization with worm compost on quality of peas in different phenological stages, research has been carried out over three years. Each year was conducted investigations over phases: early flowering, total flowering, pod formation and total ripening. During the experiment has been observed that in all phenological phases of the experimental plants I and II growing by worm compost fund, total nitrogen content, crude protein and nitrocompounds was higher than in the control group cultivated naturally fund. As a result of studies found that incorporation of worm compost in the soil, at a dose of 4t/ha-3t/ha, the quality of peas has improved in all phenological stages. During the experiment the dependence of phenological stages of development samples of peas were taken in order to assess its quality.

MATERIALS AND METHODS To appreciate the influence of worm compost on quality of peas experiment was organized under the field of Technological-Experimental Station 'Maximovca'. As research material was used organic fertilizer and peas variety 'Renata'. In experiment (Table 1) were included 3 groups, with a surface such (two-experimental and one-control).

RESULTS AND DISCUSSIONS Analyzing the results of research conducted in the first year of action of organic fertilizer (Table 2) revealed a significant increase of total nitrogen and crude protein content in plants of experimental groups. This increase constituted duly 126,50% 127,50% in plants of the experimental group I and 65,66%, 65,58% in plants of the experimental group II. Regarding the content of nitro compounds in the first year of action of worm compost in peas samples collected from experimental group I, the nitrate content decreased by 53,69%, and nitrite 53,70% compared with control group. In peas samples collected from the experimental group II nitrate and nitrite content decreased, corresponding to 53,74% and 52,67%.

Table 1. The scheme of the experiment No 1 2 3

Type of culture Peas Peas Peas

Lots I-experimental II-experimental III-control

Condition of experiment Worm compost, 4t/ha Worm compost, 3t/ha Natural fund

On experimental lots before sowing, was incorporated organic fertilizer dose of 4t/ha (experiment I) and 3t/ha (experiment II) from cattle manure as a result of use bioconversion technology of organic waste by worm growing.

Table 2. Assessing the influence of worm compost on quality of peas from the experiment

(experimental lot II). The content of nitrocompounds decreased the exception of the nitrate content in peas samples collected from experimental group I and nitrite content in plants of the experimental group II. Compared with control lot in the second year of action content of crude protein and of total nitrogen increased corresponding by 51,11,% and 51,09% (experimental lot I) and 45,40% (experimental lot II). Comparing the amount of nitro-compounds in plants of experimental groups with those of their control group was found reduction with 41,51,%-50,85,% exception being nitrite content of peas samples collected from the experimental lot II. So the results obtained and in the second year of action worm compost quality of peas was directly related to the influence of worm compost.

Variant of experiment Indices No 1 2 3 4

Total nitrogen,% Crude protein,% Nitrites, mg/kg Nitrate, mg/kg

Control

Experiment I4t/ha

Experiment II -3t/ha

1,66

3,76

2,75

10,40

23,66

17,22

289,80

134,20

138,20

4,86

2,25

2,30

This increase constituted duly 126,50% 127,50% in plants of the experimental group I and 65,66%, 65,58% in plants of the experimental group II. Regarding the content of nitro compounds in the first year of action of worm compost in peas samples collected from experimental group I, the nitrate content decreased by 53,69%, and nitrite 53,70% compared with control group. In peas samples collected from the experimental group II nitrate and nitrite content decreased, corresponding to 53,74% and 52,67%. So the results obtained show beneficial influence of worm compost on quality of peas in the first year of action of fertilizer, increasing total nitrogen and protein contentand diminished content of nitrates and nitrites. The same regularity was followed in the second year of action of fertilizer on peas cultivation with fund of worm compost (Table 3).

Table 4. Assessing the influence of worm compost on quality of peas from the experiment

No Indices

Total 3,02 nitrogen,% 2 Crude protein,% 18,88 3 Nitrites, mg/kg 190,00 4 Nitrate, mg/kg 1

1 2 3 4

Total nitrogen,% Crude protein,% Nitrites, mg/kg Nitrate, mg/kg

Variant of experiment Experiment Experiment III Control 4t/ha -3t/ha 3,15

4,76

4,88

19,69

29,75

28,63

265,00

155,00

130,24

3,12

5,00

4,34

4,30

27,13 66,30 -

26,88 72,30 -

The results presented in Table 4 show that the values of total nitrogen and crude protein in peas, in the third year of action of worm compost, compared to their plants collected in second year both the control lot and the experimental decreased as corresponding with 4,13%, 4,11% (control group), 8,82%, 8,81% (experimental lot I) and 6,11% (experimental lot II). Comparing the results obtained in the third year with those obtained in the first year of action of worm compost was found that the value of these indices has increased accordingly but it has exceeded that of the first year of action of worm composting with 81,93%; 81,54% (control group), 15,43%, 14,67% (experimental lot I) and 56,36%, 56,10% (experimental lot II). In peas collected from the control lot, experimental I and II experimental, in the third year of action of worm compost nitrate value decreased, corresponding to 34,44%, 50,60%

Table 3. Assessing the influence of worm compost on quality of peas from the experiment Indices No

Variant of experiment Experiment Experiment I Control II -3t/ha -4t/ha

Total nitrogen and crude protein values?? both in samples of plant collected from the control group and in the-in the experimental groups increased corresponding to 89,80%, 89,33% (control group), with 26,60%, 25 70% (experimental lot I) and 66,55%, 66,26%

and 47,61% in comparison with the results in the first year and corresponding to 28,30%, 57,23% and 44,48% compared with the results obtained in the second year of action of worm compost. In comparison to the control group in samples of peas, the experimental lot I, total nitrogen content and crude protein increased, corresponding to 43,71% and 43,70%, and the ones collected from experimental lot II-with 42,38% and 42,37%. The amount of nitrates in samples of peas from the experimental group decreased by 65,11% I and experimental group II-with 61,95% compared to that in samples collected from the control lot. In samples of peas from all groups, in the third year of action of worm compost nitrite were not detected. So, researches that were made in the third year of action of worm compost found the influence of fertilizer on quality of peas.

total nitrogen and crude protein content in peas plants in experimental groups increased by 42,38% 126,50% and 42,37%-127,50% compared to that in the control lot plants; the value of nitro-compounds diminished on experimental plants with 5,76%-65,11% compared to the control group of plants. REFERENCES Boclaci, T., Cremeneac, L., 2012, Some peculiarities of forage crops with fund of viermicompost and mineral fertilizer, Iasi, p.152-156 Cremeneac L., 2003. Bioconversion of organic wastes in Moldova. Summary information, Typography INEI,Chisinau, 32 p. Kosolapova A., Smyshlyaev E., Kosolapov I., 1996, Worm's culture and its capabilities. Ryazan, 72 p. Petuhova, E., Besarabova, P., Antonova, O., 1989, Zootechnical feed analysis, Moscow VO 'Agropromizdat', 238 p Razumov, V., 1986, Reference book laboratory-chemist for feed analysis, Moscow, Rosselhozizdat, 1986, 300 p.

CONCLUSIONS Fertilization of soil with worm compost in doses of 3t/ha and 4t/ha improved quality of peas over three years:

Scientific Bulletin. Series F. Biotechnologies, Vol. XVII, 2013 ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

GENERAL ASPECTS REGARDING WASTE MANAGEMENT IN SUSTAINABLE DEVELOPMENT OF AGRICULTURE Larisa CREMENEAC, Tatiana BOCLACI Scientific and Practical Institute of Biotechnologies in Animal Husbandry and Veterinary Medicine, 15 bcolarĉ Street, 6225, Village Maximovca, District Anenii Noi, Republic of Moldova, Phone: 373-22-359-351, fax. 373-22-359-350, Email: [emailprotected], [emailprotected] Corresponding author: [emailprotected] Abstract. The paper summarizes the world and own experience using worm culture (earthworms) for utilization of various organic wastes, use of products worm cultivation (worm compost, biomass, worms and alarm pheromone) in a number of industries. Recognized features of physiological development and maintenance of worm culture, the stages and the basic conditions of industrial cultivation technology, the product features and their use. The bioconversion process of organic wastes is implemented in the Experimental Section of the Scientific and Practical Institute of Biotechnologies in Animal Husbandry and Veterinary Medicine. The studies conducted by several states, including Moldova, concluded that worm compost has a positive influence on crop productivity, reducing growth period, their resistance to unfavourable climatic conditions and common plant diseases. Worm cultivation opens up new perspectives and opportunities for introducing technology of obtaining the protein not only for feed purposes, but also in the food industry for the production ecological products. The results of research showed that the products obtained from biomass worms and alarm pheromone, can serve as raw material for the preparation of medicinal products used in medicine and in veterinary medicine for the control and prevention of various diseases. Technology of bioconversion of organic waste using worm culture is designed for landowners, farmers, ranchers, and agricultural enthusiasts, and others as an alternative method for the sustainable development of agriculture. The research conducted and analysis of results about the development of worm cultivation found that: worm cultivation is a branch that has the ability to solve stringent ecological problems existing in some sectors of the national economy, contributing to the development of sustainable agriculture and otained ecological agricultural productions, products obtained as a result of worm cultivation can be widely used in crop, livestock, human and veterinary medicine. Keywords: bioconversion, organic wastes, organic fertilizer, worm compost, worm culture.

Special attention in the last decades of the twentieth century was directed to use worm culture biotransformed as organic waste. Was found that worm culture (earthworms), as biotransformed, is able to process organic waste of different origin, transforming them into valuable organic fertilizer - worm compost (biohumus, biocompost). An important support in the elaboration bioconversion technology of organic waste has served long observations made on the lives of earthworms in nature. It was found that search of food earthworms swallow soil particles with organic waste, which enter the digestive tract of worm culture is enriched with micro- and macro turning into worm compost, characterized by major agrochemical properties. "Domestication" of

INTRODUCTION Organic waste management, the diverse origin of ancient times had spreading range of practical people. Livestock waste, plant growing and food can be used as raw material in organic waste bioconversion technology by worm cultivation to obtain valuable organic fertilizer and biomass with a high content of protein (Condîreva et al., 1994; Cremeneac, 2003). Biotechnology used, along with organic waste management and production aspect, manifest an environmental aspect, which allows solving the problems of environment protection and sustainable development of agriculture.

The field chosen for worm cultivation was located in an area accessible for use the organic waste, less inclined, with drainage into the ditch, for the water from sectors because worm culture not support high humidity. Sectors for worms cultivation were oriented in the direction of predominantly wind in order not be situated in the wind and to receive the sufficient amount of warmth, with south exposition. Formed sectors for worm cultivation in household were oriented from north to south. The next step was to prepare the sectors and departments. for worm cultivation According to the technology of bioconversion the organic waste for worm cultivation in households is practiced by pairs sectors 1m wide and 50m long. The distance between the two sectors can be 1m, and of the pair - 4,0m -4,5m Taking into account these requirements STE "Maximovca" were used 5 sectors for worm cultivation, in which initially were placed about 125 tons of organic waste (manure of cattle) that were previously undergone fermentation during 6 months. During six months in sectors were added as supplementary food for still about 25 tonnes. So, in total have undergone processing about 150 tons of organic waste. Each sector was divided into 25 sections, with dimensions 1,0m × 2,0m. Bioconversion technology of the organic waste by worm cultivation in this household was held at the open air. In the first year of work the main objective was the accumulation of increased population the earthworm Red Hybrids of Californiatherefore the process has been used only two sectors that have been placed organic waste and worm culture. In each sector for worm cultivation were placed about 25 tons of organic waste subjected to bioconversion to increase population of earthworm, which can then be used to manage organic waste and obtain worm compost. Each sector for worm cultivation were divided into sections, where was placed each a ton of organic waste. Nutritive substrate thickness in the sectors for worm cultivation was 25-35cm - summer and 35-45cm - winter. Under one month nutritive substrate from sectors was sprayed for a week - every day, then once a week. After the period of

earthworm served as the basis for the management of organic waste and use of worm culture in the process of bioconversion of organic waste in the branches of the economy in general and especially in agriculture,of food industry, of household waste and of sludge from wastewater as a result of treatment. Based on the research conducted and results obtained (Gorodniy, 1990; Melnik, 1994; Cremeneac, 2003) was elaborated a complex program of management and bioconversion of organic waste by worm cultivationthat includes following directions: bioconversion of organic waste by worm cultivation to obtain valuable organic fertilizer, biological transformation of organic waste and getting worm compost, obtaining biologically active preparations of tissues worm culture, prospects and effectiveness in agriculture of worm compost (as fertilizer) and biomass earthworm as the protein supplement in animal feed rations) to obtain otained ecological agricultural production (Cremeneac et al., 2012). So in the article are described aspects of organic waste management for use in environmental quality of life and nutrition of worm culture and possibilities of using products from sustainable development of agriculture in addressing issues of environmental protection in society. MATERIAL AND METOD Research in the domain management of organic waste and appreciation of worm compost have been carried out in the Technological-Experimental Station "Maximovca", where he was held the household for worm cultivation and the experiment in field conditions in order to evaluate the influence of worm compost on quality and yield of agricultural crops. In organizing of household for worm cultivation was taken into account the objectives and tasks that must be solved. It was found that the dominant household for worm cultivation will be: processing of organic waste, increasing the population of earthworm, production of worm compost for resort needs.

active acidity - using pH meter, total nitrogen - using the Kjeldahl method; nitro-compounds – using the electro colorimetric method, magnesium, calcium, phosphorus and potassium - using the usual methods (Petuhova et al.,1989). Determining the harvest productivity was made by weighing. Control counting of earthworm population was carried out by special method, which consists in using a form with size 10cm × 10cm, with a length of 20 cm (corresponding nutritive substrate thickness of sectors). With this form are taken three samples of nutritive substrate from each section. By counting is determined the number of earthworms of all ages in all three samples, and then the necessary calculations were made taking into account the surface section and then sectors. Subsequently, in the second and subsequent years, the population of earthworm obtained was used as organic waste biotransformed in the 5 sectors. So, the objectives and the proposed duties for organic waste management in sustainable agriculture have been met. Statistical processing of the results was performed according to the method developed by E.Mercurieva

spraying, the substrate prepared for worm cultivation has undergone biochemical analysis to determine quality indices. Initially, and then permanently in order to determine the quality of the organic waste placed in areas for worm cultivation was make analyzes: biochemical, determination of active acidity (pH), ammonia nitrogen content, total nitrogen, organic matter, magnesium, phosphorus, potassium and calcium and microbiological taking into consideration pathogenic and non-pathogenic microflora present, using the usual methods (Razumova, 1986). Also to determine the quality of nutritional substrate for worm culture is used "50-the earthworm test" that provide control of the organic waste by using 50 earthworms. In a container were placed 23kg of organic waste that is located 50 earthworms. If after 24 hours the earthworms does not leaving the substrate and were active when it is considered that organic waste can be used as a nutritional substrate for worm culture. Complete cycle of processing of organic waste vary from 5 to 8 months, depending on climatic conditions earthworms and population density located to substrate . As a result of use of bioconversion technology of organic waste by worm cultivation final products were obtained: worm compost - valuable organic fertilizer, organic mass of earthworms and ecologycal production. For assessment purposes the influence of viermicompostului on quality and yield of agricultural crops in conditions of field was organized experiment in which research materials have served viermicompostul and agricultural crops: peas, fodder beet and corn. For each agricultural crop were used a control group (with natural background) and two experimental groups (with background of worm compost). Worm compost was incorporated into the soil at a dose of 4t/ha (experimental group I) and 3t/ha (experimental group II). To determine the quality of nutrient substrate, organic fertilizer produced (of worm compost) and cultivated agricultural crops with fund of worm compost were used following methods: the determination of

RESULTS AND DISCUSSIONS As a result of biochemical analyzes of nutritive substrate placed for worm cultivation sectors, it was found that it coincided with necessary parameters for bioconversion of organic waste by worm cultivation, being used as a nutritive and life medium for worm culture (Table 1). Through the quality control of nutritive substrate according to test "50 of earthworm" also was confirmed quality of nutritive substrate because after 24 hours of placement of earthworms in substrate, all 50 earthworms used in testing, remained alive and active. In April in each section were placed each 2,000 mature earthworms. Placement of earthworms was made early morning, since under the influence of sunlight they quickly hide in the substrate.

Table 1. Chemical composition of the nutrient substrate

No 1 2 3 4 5 6 7 8

Indices Active acidity (pH), units Ammoniacal nitrogen, mg / kg Total nitrogen, % Organic substanc, % Magnesium, % Phosphorus(P2O5), % Potassium (K2O), % Calcium, %

cocoons to one mature earthworm, 1,2 juvenile earthworms to a one cocoon and 2,6 juvenile earthworms to one mature earthworm. After three months, this correlation has changed constitute: 2,6 cocoons to one mature earthworm, 3,4 a juvenile earthworms to one cocoon and 8,7 juvenile earthworms to one mature earthworm. To the end of the sixth correlations cocoons: mature earthworms and juvenile earthworms: earthworms mature decreased and correlation juvenile earthworms: cocoons remained at level of the first month. These changes at the end of the sixth were influenced by increased earthworm population in sectors, which resulted decline in reproduction. According to the results obtained after six months of earthworms population in a sector was about 2004750 individuals of all ages. Such in both sectors was obtained for worm cultivation a population of about 4 million earthworms, which was later used for biotransformed as the organic waste 5 sectors ready for bioconversion of waste by worm cultivation. After being determined quality of nutritional substrate for worm cultivation in the sectors in these worms were placed culture reasons in a section 32000 earthworms or earthworm 800000 in a sector. Complete duration of processing of nutritional substrate is 6 months. During the experimental period nutritive substrate was sprayed with water (according to necessity). For worm cultivation sectors were covered with straw in order to reduce evaporation - summer and protection from the cold - winter. During the experimental period requirements have been bioconversion technology of organic waste by worm cultivation (humidity - 70-80 %, content of ammoniacal nitrogen 1,0 to 20mg / kg, active acidity (pH) - 6,8 to 7,6 units and content of cellulose - 30 %). After 30 days from the beginning of the experiment, it was found that worm culture completely processed the nutritive substrate, so started adding new amounts of nutritive substrate, as additional food. This process was conducted (according to necessity) every 1014 days. At the end of the experimental period worm culture was separated from the substrate and placed in other sectors, prepared

Index value, M ±m 7,57 ± 0,08 5,56 ± 0,57 0,83 ± 0,63 30,35 ± 0,60 1,17 ± 0,52 0,65 ± 0,32 0,68 ± 0,01 1,55 ± 0,35

During the first week, daily, investigations were performed to establish the presence of abnormalities in behavior and development of worm culture. As a result of investigations it was found that at the end of the first week, mature individuals began to use in nutrition the substrate in which they were placed. This showed that the substrate is benific for use as living environment for worm culture. During of investigations, over 6 months, were made observations on the process of reproduction of worms culture by conducting monthly control count of individuals of all ages present in sections. Results regarding the developer population earthworm are presented in Table 2. Table 2. Dynamics of population development of earthworm of California Red Hybrid

No. 1 2 3 4

Population of earthworm Mature earthworms Juvenile earthworms Cocoons Total: (mature earthworms + juvenile earthworms)

During of investigations, month One Three Six month month month 2000

2000

10077

5125

17490

70113

4320

5200

20740

7125

19490

80190

Analysis of the results has found that over six months, the number of mature earthworms in a section increased 5 times compared to their number in the initial stage. During the investigation were found significant changes in correlation: Cocoons: mature earthworms, earthworms juvenile: cocoons and juvenile earthworm:mature earthworms. It was found that in the first month these indices were 2,2

in advance. As a result of bioconversion was obtained about 90 tons of worm compost, valuable organic fertilizer. In Table 3 are exposed quality indices ofworm compost obtained as a result of bioconversion of organic waste by worm cultivation.

According to the research found that one tonne of compost worm contained an amount of 270-300kg of humus. This allows to significantly decreased period for completing the amount of humus in the soil, thus restored soil fertility and soil resistance to wind and alluvial erosion. In order to determine the particularities of the development process of plants and agricultural productivity cultivated with fund of worm compost in field conditions of Technological-Experimental Station "Maximovca" was organized experiment. At the initial stage of the experiment it was found that all cultures of the experiment cultivated with fund of worm compost sprang with 5-7 days earlier than those in control groups. This demonstrates that the beneficial influence viermicompostul germination and springing of agricultural crops. Comparing the development of plants in all variants was found that in lots of worm compost fund of agricultural crops have grown more intense, early flowering of peas and formation earlier of maize cobs took place 5-6 days earlier than in control groups. So, as a result of studies found that incorporation of worm compost in the soil, at a dose of 4t/ha, resulting in faster development of crops, reduce the period of flowering and ripening of crops. At the end of the experiment there was an essential difference in the productivity of crops, depending on the fund that was grown and crop type (Table 4). Analyzing obtained found that harvested pea obtained varied from lot to lot. The experimental groups I and II harvested grains peas exceeded that of the control group, respectively 47,50 % and 35,30 %.

Table 3. Quality indices of worm compost obtained from cattle manure

No 1 2 3 4 5 6 7 8 9

Indices

Index value, M ±m

Active acidity (pH), units Organic substance, % Total nitrogen, % Potassium (K2O), % Magnesium, % Phosphorus (P2O5), % Calcium, % Humus, % Nonpathogenic bacterial flora, colonies

32,92 ± 0,02 - 39,96 ± 0,03 1,09 ± 0,01 - 3,00 ± 0,04 1,92 ± 0,02 - 2,80 ± 0,05 1,18 ± 0,03 - 2,50 ± 0,04 1,37 ± 0,08 - 2,50 ± 0,06 1,62 ± 0,02 - 3,80 ± 0,05 29,66 ± 1,40 - 35,91 ± 1,90

7,81 ± 0,03 - 8,08 ± 0,02

2x1012 -3x1012

Comparing the values of worm compost obtained with the the nutritive substrate was found that the amount of active acidity, total nitrogen, potassium, magnesium, phosphorus and calcium have exceeded that of a the nutritive substrate, ie 3,17 % - 6,74 % , 31,32 % - 261,14 % 182,40 % - 311,8 %, 110,77 % - 284,62 % and 4,52 % - 145,20 %. Organic matter content decreased by 8,46 % - 31,66 % respectively. As a result of the investigations it was found that worm compost contain 100 times more nonpathogenic microflora (2x1012 colonies) than traditional compost. In worm compost are concentrated considerable quantities of enzymes, vitamins and stimulators of growth. It was also found that in the worm compost is well balanced content of macro-and microelements, which allows decreasing the dose of incorporation in soil of 8-12 times compared to ordinary compost. When using worm compost as organic fertilizer savings are considerable taking into account that at one hectare using 3-6 tons of worm compost compared to 40-70t/ha of traditional compost. Efficacy action of worm compost is kept over 3-4 years. So, according to the results obtained it was found that after the biochemical composition and usage, worm compost is upper traditional compost.

Table 4. Indices of agricultural crop productivity No. 1 2 3

Agricultural crop Peas Maize Fodder beet

Lots and quantity of yield, kg ExperiExperimental I mental II 10,640 15,700 15,400 57,000 77,000 69,000 630,000 1115,000 907,000 Control

Harvest of maize in the experimental group was 35,08 % and 21,05 % more increased than the control group of plants. On the experimental groups where fodder beet was cultivated with worm compost fund

different phenological phases contributing to increased harvest obtained per unit of area. Incorporation of compost worm in the soil, at a dose of 4t/ha and 3t/ha, expected decrease springing and ripening period and increases of agricultural crops harvest by 21,05 %76,98 %.

has collected 76,98 % and 43,96 % more productive than the control group. So, the results of investigations involving different crops and incorporation of worm compost in the soil reduce phenological stages of plant development increases yield of agricultural crops. Thus, as a result of organic waste management and their use as living environment and nutrition for the worm culture was possible solve some problems of the environment protection and obtaining increased quantities of agricultural production.

REFERENCES Condîreva M., Cremeneac L., Bahcivanji M., 1994. Biological processing technology of manure using the method worm’s cultivation. Recommendations. Agroinformreclama,Chisinau, 24 p. Cremeneac L., 2003. Bioconversion of organic wastes in Moldova. Summary information, Typography INEI, Chisinau, 32 p. Cremeneac L., Boclacl T., Chiruneԑ Z., 2012. Recommendations. Bioconversion technology of organic waste and use products obtained. Printing "Print-Caro", Chisinau, 79 p. Gorodniy N., 1996. Bioconversion of agro-ecosystems management, UkrISTEI, Kiev, 232 p. Melnick J., 1994. Vermiculture: the production and use, UkrISTEI, Kiev, 125 p. Petuhova E., Besarabova P., Antonova O., 1989. Zootechnical feed analysis, Moscow VO "Agropromizdat", 238 p Razumov V., 1986. Reference book laboratory-chemist for feed analysis, Moscow, Rosselhozizdat, 1986, 300 p.

CONCLUSIONS Bioconversion of organic waste by worms cultivation solves some important issues in the sustainable development of agriculture: the complete processing of organic waste, protect the environment, obtaining organic ecological fertilizer, long-acting, increasing crop yield and improving the quality of agricultural production. Qualitative indexes values of worm compost have exceeded those essential the nutritive substrate. Worm compost used for growing crops, beneficial influence their developers in

Scientific Bulletin. Series F. Biotechnologies, Vol. XVII, 2013 ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

THE SELECTION OF SOME TISSUE LINES PRODUCERS OFANTHOCYANINS IN BILBERRY (Vaccinium mytillus L.) CALLUSCULTURE Dorica BOTAU, Vanda BOLDA Faculty of Horticulture and Forestry, University of Agricultural Sciences and Veterinary Medicine of Banat, 119 Calea Aradului, 300645, Timisoara, Romania Corresponding author email: [emailprotected]

Abstract Our research aimed the obtaining a proliferative tissue at 7 bilberry (Vaccinium mytillus L.) local population, to select some anthocyanins producing tissue lines by in vitro culture. The tissue lines selected from ArieƔeni, Semenic, Retezat, Valea SebeƔului, Buces-Vulcan, Vadul Dobrii and Cornereva local population, with good results of callus growth capacity in subculture, produced anthocyanins (646 mgC3GE/100g). These results demonstrated the possibility to elaborate a production system of these metabolites in controlled conditions. Keywords: bilberry (Vaccinium mytillus L.) local population, callus, anthocyanins.

INTRODUCTION

MATERIALS AND METHODS

Among the plants that provide raw material for obtaining a wide range of natural medications (phytotherapeutical products), food supplements, natural colorants and preservatives, there is also included the species Vaccinium myrtillus L. In our country, but also worldwide, a sustaining preference for the consumption of phytotherapeutical preparations can be recognized, to the disadvantage of medicines prepared by chemical synthesis. This is due to the fact that herbal therapeutic products are better tolerated by the human body (on the background of a better compatibility at a metabolic level). The curative medicinal properties of bilberry are known since antiquity and they were exploited under various forms, from fruit and leaf teas, poultices, tinctures, powders, to complex drugs obtained nowadays (Maillefert, 2010). The bilberry plants have therapeutic importance both in human medicine and in veterinary medicine. Among the many effects of the active principles of bilberries there we can find: hypoglycemic, hypotensive, antiseptic, cholagogue, vasoprotectives of capillaries (eye, brain, peripheral), antidiarrheal, antioxidant (Martz et. al, 2010).

We used 7 local populations of bilberry collected from the central-west part of Transilvania: ArieƔeni, Semenic, Retezat, Valea SebeƔului, Buces-Vulcan, Vadul Dobrii and Cornereva. For callus inducing three types of explants were used: meristem, leaf and stem, grown in vitro on three culture media (Lloyd and McCown Woody plant medium-WPM, Anderson’s Rhododendron medium-AND and Mourashige-Skoog medium-MS), under the influence of ANA (naphtalen-acetic acid) two hormonal variants (V1, V2, table 1). Undifferentiated tissues obtained were subcultured on media supplemented with the same hormonal balances. Callus was sub-cultured in three culture cycle, on WPM medium supplemented with 1,5 mg/l ANA, Table 1. Hormonal balances tested for callus induction on culture media (WPM, AND, MS) Variant 1 2

Hormons mg/ml ANA BAP 1,5 1 1,5 1,5

AS 40

The total antioxidant capacity of the selected tissue lines from the ArieƔeni, Retezat and

Valea Sebesului populations has been studied, achieving good results concerning the ability of callus growth in subculture and obtaining plant biomass, with rich anthocyanin pigments (those calluses were visually selected that showed red cells, signaling the presence of anthocyanins). To determine the total antioxidant capacity we use extracts obtained from callus and foliar tissue of bilberry mother plant, using a rapid method, elaborated by Benzie and Strain (1996), modified by Varga et al. (2000); Szollosi and Varga (2002). The total anthocyanin content was expressed in cianidin 3-glucoside equivalents (C3GE) /100g fresh vegetal material-FW.

Figure 2. Graphical representation of bilberry callus percentage for different genotypes and hormonal balances on bilberry meristem explants.

However, meristem explants produced less callus (fig. 2). The genotype is another factor which influences callus production at bilberry. Only at the populations from Arieseni, Cornereva, Retezat and Valea Sebesului, callus was obtained in greater quantities comparing to the other studied genotypes, over 90% callus from leaf and stem explants (fig.1, 2, 3, 4, 5 and 6).

RESULTS AND DISCUSSIONS 1. Experimental results regarding the callus induction at bilberry local population Abundant and proliferative callus was obtained from stem and foliar explants, grown on WPM media, supplemented with 1.5 mg/l ANA + 1.5 mg/l BAP + 40 mg/l AS, demonstrating that the culture media, explant type and association of citokinine with auxine, in equal proportions and in the presence of adenine sulphate-AS (40 mg/l) are key factors in inducing callus at spontaneous bilberry.

Figure 3. Graphical representation of bilberry callus percentage for different genotypes and culture mediums on bilberry leaf explants.

Under WPM medium influence the leaf explant produced callus at all bilberry local population, with values between 61,67% and 88,33% (fig.3).

Figure 1. Graphical representation of bilberry callus percentage for different genotypes and culture medium on bilberry meristem explants.

AND medium determine the lowest variability between populations in terms of callus production. The best values registered WPM medium when it used meristem explants (fig.1).

Figure 4. Graphical representation of bilberry callus percentage for different genoyupes and hormonal balances on bilberry leaf explants.

Table 2. The effect of the genotype, callus type and AS concentration on the growth of the bilberry callus in subculture on the solid medium WPM (1,5 mg/l ANA+1,5 mg/l BAP) Genotype Arieseni Concentration Callus type (AS) Foliar Stem 40 AS y2,21 b x2,54 b 60 AS y2,50 a x2,78 a 80 AS x2,26 b x2,38 b Genotype Vadu Dobrii Concentration Callus type (AS) Foliar Stem 40 AS x2,13 a x2,25 b 60 AS y2,10 a x2,68 a 80 AS y1,99 a x2,65 a Genotype Cornereva Concentration Callus type (AS) Foliar Stem 40 AS y1,71 a x2,07 b 60 AS y1,60 a x2,35 a 80 AS y1,69 a x2,22 ab Genotype BuceƔ Vulcan Concentration Callus type (AS) Foliar Stem 40 AS x1,74 a x1,62 b 60 AS y1,61 ab x1,90 a 80 AS y1,50 b x2,04 a Genotype Retezat Concentration Callus type (AS) Foliar Stem 40 AS y1,92 b x2,35 b 60 AS y2,33 a x2,70 a 80 AS y2,20 ab x2,51 ab Genotype Valea SebeƔului Concentration Callus type (AS) Foliar Stem 40 AS y2,35 a x2,62 a 60 AS y2,56 a x2,83 a 80 AS x2,34 a x2,34 b DL5%=0,23 g DL1%=0,31 g DL0,1%=0,40 g

Figure 5. Graphical representation of bilberry callus percentage for different genotypes and culture media on bilberry stem explants.

The stem explant produced callus in the largest amount on WPM medium, with values between 63,33% at Vadul Dobrii population and 95% at Arieseni population.

Figure 6. Graphical representation of bilberry callus percentage for different genotypes and hormonal balances on bilberry stem explants.

The two populations Buces-Vulcan and Vadul Dobrii present lower results in callus growth. The best medium which provided a good callus growth was WPM and it was used in callus subculture. The callus growth in subculture is significantly influenced by the genotype and the presence of AS. Our results are in concordance with those obtained by Litwinczuk and Wadas (2008). The growth bilberry callus in subculture on WPM medium was influenced by genotype, callus type and AS concentration (table 2). It can observe that a significantly callus growth in subculture was registered at Retezat, Valea Sebesului and Arieseni populations (2,70-2,83) in presence of 60 mg/l AS. The origin of callus is a factor influencing in a certain extent the callus growth in subculture. The stem callus has a good growth compared with foliar callus at bilberry local population.

2. Results regarding the evaluation of the total anthocyanin content (on solid WPM media) The callus produced by Retezat, Valea Sebesului and Arieseni populations in subculture synthesised anthocyanins that was analysed (fig.7).

CONCLUSIONS Our results demonstrated the strong influence of genotype, culture medium and hormonal balance on callus induction and anthocyanin biosynthesis capacity of bilberry tissue cultivated in vitro. An abundant callus can be induced from leaf end stem explants at wild bilberry, on WPM medium, in presence of ANA and BAP in equal amount (1,5 mg/l) and 40 mg/l AS. In callus subculture, the genotype and amount of AS are important factors that allow selection of tissue lines with anthocyanin biosynthesis capacity.

Figure 7. The variance analysis for the total anthocyanin content (mg C3GE /100 g fresh weight) of the bilberry tissue lines obtained on solid WPM medium for the Arieseni (V1), Retezat (V2) si Valea Sebesului (V3) populations

REFERENCES Benzie IFF, Strain JJ, 1996. The ferric reducing ability of plasma (FRAP) as a measure of 'antioxidant power': the FRAP assay. Anal Biochem 239:70-76. Maillefert O., 2010. Vaccinium myrtillus L. : une plante médicinale à anthocyanosides. Universite Joseph Fourier. Faculte de Pharmacie de Grenoble. Litwinczuk W., Wadas M., 2008. Auxin-dependent development and habituation of highbush blueberry (Vaccinium × covilleanum But. Et Pl.) 'Herbert' in vitro shoot cultures. Scientia Horticulturae, 119, pp.41-48. Maillefert O., 2010. Vaccinium myrtillus L. : une plante médicinale à anthocyanosides. Universite Joseph Fourier. Faculte de Pharmacie de Grenoble0 .Martz F, Jaakola L, Julkunen-Tiitto R, Stark S., 2010. Phenolic composition and antioxidant capacity of bilberry (Vaccinium myrtillus) leaves in Northern Europe following foliar development and along environmental gradient. J Chem Ecol 36:1017-1028. Orlikowska T., 1986. Micropropagation of highbush blueberry, Fruit Science Reports, Vol.XIII, No. 3. Varga ISz, Szollosi R, Bagyánszki M., 2000. Estimation of total antioxidant power in medicinal plants (adaptation of FRAP). Curr Topics Biophys 24:219–225. Szollosi R, Varga ISz., 2002. Total antioxidant power in some species of Labiatae (adaptation of FRAP method). Acta Biol Szeged 46:125–127.

Figure 8. Callus with anthocyanins at the bilberry tissue lines from Retezat population

We selected tissue lines with higher anthocyanin content than control samples, for all three populations, due to the contribution of AS we suppose. The best antioxidant capacity was registered at Retezat bilberry population in presence of 60 mg/l AS (V2, 646 mg/ 100 g fresh weight).

Scientific Bulletin. Series F. Biotechnologies, Vol. XVII, 2013 ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

ANTIFUNGAL ACTIVITY OF ASTRAGALUS ONOBRYCHIS L. EXTRACTS Ivan PAULIUC, Dorica BOTĂU Faculty of Horticulture and Forestry, University of Agricultural Sciences and Veterinary Medicine of Banat, Timisoara, CaleaAradului Nr. 119, 300645 Phone: +40256/277.009; 0256/277.122; Fax: + 40256/200.296, Email: [emailprotected] Corresponding author email: [emailprotected] Abstract Ethanol and water extracts from aerial parts of Astragalus onobrychis were tested against 12 species of Candida, using the broth microdilution method, in order to determine the MIC values. The study showed a more potent activity on the ethanol extract with a MIC value of 2,57 mg/mL and a weaker activity on the water extract of 3,32 mg/mL on most of the Candida species studied. The results indicate that Astragalus onobrychis is a promising candidate for developing antifungal products. Keywords: Astragalus onobrychis, antifungal activity, broth micodilution, Candida.

species worldwide, which includes the plants commonly known as legumes (Wojciechowski, 2004). Astragalus is mainly distributed in cool to warm, arid and semiarid regions of the northern hemisphere, South America, and tropical East Africa; it is especially diverse in the south-western and Sino-Himalayan regions of Asia (ca. 1500–2000 spp.), in western North America (ca. 400–450 spp.), and the Andes of South America (ca. 100 spp.). Both the geographic center of diversity and the presumed origin of Astragalus in Eurasia – specifically in the steppes and mountains of south-western to south-central Asia and the Himalayan plateau. According to Ekuci and Ekim (2004) there are 142 species in Europe of which 50 are endemic. Several species of this genus are used in folk medicine due to their hepatoprotective, antioxidative biological activities and their antiviral properties. In Turkish folk medicine, the roots of Astragalus species are used for the treatment of leukemia and for the healing of wounds (Yesilada, 2005). Furthermore, A. mongholicus Bunge and A. membranaceus Bunge are among the most popular Chinese medicines and are used for a variety of diseases, including as an adjunctive in cancer chemotherapy. Some Astragalus products, such as gum tragacanth, are widely in use in the preparation of pharmaceuticals and as thickening agents in certain foods (Paul,

INTRODUCTION Candida albicans, the most commonly isolated opportunistic human fungal pathogen, can cause skin and mucosal infections as well as life-threatening systemic infections (Wang et al., 2010). Candida infections, or candidiasis, are difficult to treat and create very serious challenge in medicine. Therefore, screening and testing various plants for potential antifungal activities is very important in order to develop antibiotics. Although, C. albicans is the most commonly isolated yeast, other species are found with increasing frequency, including C. parapsilosis (Safdar, 2004). C. parapsilosis particularly affects critically ill neonates and surgical intensive care unit (ICU) patients, likely because of its association with parenteral nutrition and central lines (Kuhn et al., 2004). In recent decades, many studies have been carried out on different plant species to discover compounds of possible interest for antifungal applications. Among these studies, several have focused on the biological and phytochemical properties of different species of Astragalus, the largest genus of the family Fabaceae and, with over 2500 species, probably the largest genus of flowering plants (Teyeb, 2012). Fabaceae is the third largest family of angiosperm plant with approximately 730 genera and over 19400

The Candida strains used are: C. albicans, C. glabrata, C guilliermondii, C. inconspicua, C. krusei, C. lusitaniae, C. norvegica, C. parapsilosis, C. pulcherrima, C. zeylanoides, C. orthopsilosis, C. metapsilosis. Media YEPD agar and broth (1% (w/v) yeast extract, 2% peptone, 2% glucose/dextrose, 2% agar in distilled water), RPMI 1640 medium (Sigma –Aldrich , St. Louis, MO, USA; with L-glutamine , without sodium bicarbonate powered, buffered whit 0,165 mol 1-1 4-morpholinepropanesulfonic acid at pH=7,0). Inoculum preparation It was prepared a number of test tubes with YEPD agar medium in which the yeast strains were inoculated. The tubes were then incubated for 3 days at 37oC. Fungal Enumeration Fungal populations were determined by plate counting. A modified version of the method described by Montville (2008) was used. In this procedure, five samples of different dilutions were individually surface plated onto one plate, in the form of “lanes” and then incubated at 370C for 24 h. Plates with colonies ranging between 30 and 100 were considered for colony counting to determine the fungal populations.The densities for each strain is shown in (Table 1).

2007). Antibacterial activity has also been reported for some Astragalus species, such as A. gymnolobusFisch and A. brachystachys DC. In addition, bioactive saponins have been extracted from A. suberi L. (Jassbi, 2002). Goÿevac et al. (2008) investigated the antioxidant activity of methanol extract from the aerial part of A. glycyphyllos L. Another study showed that an ethanol extract of A. membranaceus roots may inhibit the growth of Trypanosomacruzi (Schinella, 2002). Astragalus onobrychis has a wide distribution. It can be found in Europe, Asia and northern Africa. It is the first Astragalus species to be described by Linnaeus in his famous book, Species plantarum from 1753 (Ranjbar, 2007). To the author’s knowledge, there are no previous studies of antifungal properties of A. onobrychis on Candida species. MATERIALS AND METHODS Plant material Fresh, young plants were collected in the spring, from the Botanic Garden of the University of Szeged. Preparation of extracts The plants were cleaned, washed and air dried under shade with occasional shifting and then powdered with a mechanical grinder and stored in an airtight container. Aqueous solutions were prepared with 10 g of plant powder and 100 ml distilled water. The alcoholic solution was made with 100 ml of 95% ethanol. The solution was put in an orbital shaker for 24 hours and kept in a dark room at all times. The aqueous extract was filtered with a vacuum pump and concentrated using the lyophilization process. The alcoholic extract was concentrated using a rotavap. The dried substance was measured on a digital scale. The solution for testing was made by diluting the dried substance and making solutions to a known concentration. The final concentration of the solutions, are as follows: Astragalus onobrychis ethanol extract: 41,2, 20,6, 10,3, 5,15 and 2,57 mg/mL. Astragalus onobrychis water extract: 26,6, 13,3, 6,65, 3,32 and 1,66 mg/mL. Microorganisms

Table 1. The densities measured for each strain Fungal strains C. inconspicua

CFU/ml 7,5 x 108

C. krusei C. glabrata

8,2 x 108 7,6 x 109

C. pucherrima

9,6 x 107

C. methapsilosis

9,8 x 109

C. guilliermondi C. albicans

9,2 x 108 8,2 x 108

C. norvegica

8,8 x 108

C. parapsilosis

7,8 x 109

C. kefyr C. lusitaniae

9,4 x 107 9,8 x 108

C. zeylanoides

6,4 x 108

Antifungal assay: In each well was put 1 ml of RPMI and inoculated with yeast. 100 ȝl of the stock solution of plant extract was prepared at the concentration of 41,2 mg/mL for the ethanol

Table 2: Ethanol extract

extract and 26,6 mg/mL for the aqueous extract and was added into the first wells. Then, 100 ȝl of their serial dilutions was transferred into four consecutive wells. The last well containing 100 ȝl of nutrient broth without the compound and 100 ȝl of the inoculum from each strip was used as negative control. The final volume in each well was 200 ȝl. The plates were covered with a sterile plate sealer. Contents of each well were mixed on plate shaker at 300 rpm for 20 s and then incubated at 370C for 48 h. Microbial growth in each medium was determined at 24 hours and at 48 hours by reading the respective absorbance (A) at 620 nm using an ASYS Jupiter plate reader (Biochrom Ltd., Cambridge UK) and confirmed by plating 5 ȝl samples from clear wells on nutrient agar medium. The MIC was defined as the lowest concentration of the compounds to show no growth of microorganisms. The tests were performed in triplicate. From the samples which showed activity, it was taken out 50 μl of solution and put in a Petri plate with YEPD agar medium and incubated at 370C. Statistical analysis Data were averages of three results ± Standard Deviations (SD) by using Microsoft Excel.

Species

MIC value mg/ml

Control

C. norvegica C. inconspicua C. zeylanoides C. pulcherrima C. guillermondi C. albicans C. krusei C. lusitaniae C. glabrata C. parapsilosis C. metapsilosis C. orthopsilosis

2,57 2,57 41,2 2,57 41,2 2,57 41,2 41,2 2,57 2,57 2,57 2,57

62,5 70,1 54,3 55,6 64,3 41,5 45,3 52,8 67,3 62,4 54,3 54,1

The results are in accordance with the one obtained by Sulaiman (2012) on A. atropilosulus subsp. abyssinicus from Saudi Arabia on Candida sp. He tested leaf extracts and obtained a MIC value of 13 mg/mL with an ethanol extract and 30 mg/mL with an aqueous extract. Also, Mikaeili (2012) obtained a MIC value of 160 mg/mL on Candida albicans, with an aqueous extract from Astragalus verus. Table 3: Aqueous extract Species MIC value mg/ml

Control

C. norvegica

3,32

54,2

C. inconspicua

26,6

65,2

RESULTS AND DISCUSSIONS

C. zeylanoides

26,6

43,2

The results obtained, show that A. onobrychis presents antifungal activity and inhibited the growth of most tested strains. As it can be observed from the tables below, a more intense inhibitory effect was noted in case of the ethanol extract and a weaker activity in the case of aqueous extract. As it can be seen from (Table 2), which shows the ethanol extract values, most of the 12 Candida species tested, had a MIC value of 2,57 mg/mL and a few were more resistant. (Table 3) presents the aqueous extract values, which are higher, that means the aqueous extract presents a weaker activity. In the case of C. lusitaniae, C. glabrata and C. metapsilosis, the MIC values could not be determined. No significant change was found at the 48 hours.

C. pulcherrima

26,6

44,7

C. guillermondi

3,32

46,2

C. albicans

26,6

57,3

C. krusei

26,6

53,2

C. lusitaniae

63,2

C. glabrata

61,6

C. parapsilosis

26,6

54,2

C. metapsilosis

67,1

C. orthopsilosis

26,6

61,3

In the case of isolated constituents, tested separately, the MIC values are much lower, for example 31,25 μg/mL for soyasaponin I, isolated from A. trimestris, and tested on C. albicans, which shows that the isolated compounds have a much more intense antimicrobial properties (El-Hawiet, 2010). Most Astragalus species exhibit a wide range of antimicrobial properties on very diverse microorganisms, like the ones tested by

Kuhn Duncan M., Pranab K. Mukherjee, Thomas A. Clark, Claude Pujol, Jyotsna Chandra, Rana A. Hajjeh, David W. Warnock, David R. Soll, and Mahmoud A. Candida parapsilosis, Ghannoum, 2004. Characterization in an Outbreak Setting, Emerging Infectious Diseases, Vol. 10, No. 6. Mikaeili Ali, Isaac Karimi, TayebehShamspur, BabakGholamine, MasoudModaresi, Ali Khanlari, 2012. Anti-candidal activity of Astragalus verus in the in vitro and in vivo guinea pig models of cutaneous and systemic candidiasis. Brazilian Journal of Pharmacognosy 22(5): 1035-1043. Montville T, Matthews K, 2008. Food Microbiology an Introduction. ASM Press, Washington, DC, USA, 2nd edition. Paul G., 2007. Actualités en phytothérapie. Phytothérapie 2: 100-106. RanjbarMassoud, Mohammad-Reza Rahiminejad and Ali-AsgharMaassoumi, 2007. Notes on the ShortStemmed Species of Astragalus Sect. Onobrychoidei (Fabaceae) in Iran. Willdenowia Bd. 37, H. 1, pp. 305312. doi:10.3372/wi34.34116. Safdar A., Perlin D.S., Armstrong D., 2002. Hematogenous infections due to Candida parapsilosis: changing trends in fungemic patients at a comprehensive cancer center during the last four decades. DiagnMicrobiol Infect Dis.; 44:11–6. Schinella GR, Tournier HA, Prieto JM, Ríos JL, Buschiazzo H, Zaidenberg A., 2002. Inhibition of Trypanosomacruzi growth by medical plant extracts. Fitoterapia 73: 569-575. Sulaiman Alrumman A., Mahmoud F. M. Moustafa, Saad A. Alamri, 2012. Anti-bacterial and anti-fungal investigation of Astragalus atropilosulus subsp. Abyssinicus. African Journal of Microbiology Research Vol. 6(34), pp. 6365-6369. DOI: 10.5897/AJMR12.778. Teyeb H., NahlaZanina, Mohamed Neffati, WahibaDouki, Mohamed FadhelNajjar. 2012. Cytotoxic and antibacterial activities of leaf extracts of Astragalus gombiformisPomel (Fabaceae) growing wild in Tunisia, Turk J. Biol. 36, 53-58. Wang Yu-Chao, Chung-Yu Lan, Wen-Ping Hsieh, Luis A Murillo, Nina Agabian, Bor-Sen Chen, 2010. Global screening of potential Candida albicans biofilm-related transcription factors via network comparison. BMC Bioinformatics, 11:53. Wojciechowski M.F., Lavin M., Sandesrson M.J., 2004. A phylogeny of Legumes (Leguminosae) based on analysis of the plastid MATK gene resolves many well-supported subclades within the family. Am. J. Bot.; 91:1846-1862. Yesilada E, Bedir E, Çalıú ø., Takaishi Y., Ohmoto Y., 2005. Effects of triterpenesaponins from Astragalus species on in vitro cytokine release. J, Ethnopharmacol 96 (1-2): 71-77.

Balachandar (2012). However, not all species do present antimicrobial properties, and one interesting example are the 13 Astragalus species from eastern Anatolia in Turkey, 4 of which are endemic, tested by Adigüzel (2009) on 40 different species of microorganisms. He found that at any concentration, “none of the extracts tested has inhibitory activity against any of the microorganisms tested” (Adigüzel, 2009). CONCLUSIONS The extracts prepared from aerial parts of A. onobrychis, with different solvents, exhibit a different antifungal effect. The ethanol extract presents a stronger antifungal activity than the aqueous extract. A. onobrychis extracts, inhibited almost all of the Candida species. ACKNOWLEDGEMENTS The authors are thankful to the Department of Microbiology from the University of Szeged for providing the Candida strains and to the Botanic Garden of the University of Szeged for providing the Astragalus onobrychis plant. REFERENCES AdigüzelAhmet, MünevverSökmen, HakanÖzkan, GülerayA÷ar, MedineGüllüce, Fikrettinùahin, 2009. In vitro Antimicrobial and Antioxidant Activities of Methanol and Hexane Extract of Astragalus Species Growing in the Eastern Anatolia Region of Turkey. Turk J Biol, 33, 65-71. doi:10.3906/biy-0805-1. Balachandar S, Jagadeeswari M, Dhanabalan R, Meenachi M, 2012. Antimicrobial activity of Astragalus membranaceus against diarrheal bacterial pathogens. Int J Pharm, 2(2): 416-418, ISSN 22491848. Ekuci M., Ekim T., 2004. Revision of the Section Hololeuce Bunge of the Genus Astragalus L. (Leguminosae) in Turkey. Turkish Journal of Botany, 28: 307–347. El-HawietAmr M., Soad M. Toaima, Aya M. Asaad, Mohamed M. Radwan, Nadia A. El-Sebakhy, 2010. Chemical constituents from Astragalus annularisForssk and A. trimestris L., Fabaceae. Brazilian Journal of Pharmacognosy 20(6): 860-865. ISSN 0102-695X. Goÿevac D, Zduniü G, Šavikin K., V. Vajs, N. Menkoviü, 2008. Antioxidant activity of nine Fabaceae species growing in Serbia and Montenegro. Fitoterapia 79: 185-187. Jassbi AR, Zamanizadehnajari S, Azar PA, Tahara S., 2002. Antibacterial diterpenoids from Astragalus brachystachys. Z Naturforsch 57 (11-12): 1016-21.

Scientific Bulletin. Series F. Biotechnologies, Vol. XVII, 2013 ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

ANTIBACTERIAL ACTIVITY OF MOMORDICA CHARANTIA L. GEMMOTHERAPIC EXTRACT Ivan PAULIUC, Dorica BOTĂU Faculty of Horticulture and Forestry, University of Agricultural Sciences and Veterinary Medicine of Banat, Timisoara, CaleaAradului Nr. 119, 300645Phone: +40256/277.009; 0256/277.122; Fax: + 40256/200.296,Email: [emailprotected] Email: [emailprotected] Corresponding author email: [emailprotected] Abstract The antibacterial activity of a gemmotherapic extract of Momordica charantia L., was investigated against 5 species of bacteria, Bacillus subtilis, Streptococcus faecalis, Serratiamarcescens, Pseudomonas aeruginosa and B. cereus v. mycoides, using the well diffusion assay. The extract was prepared from young buds of M. charantia, in according to the gemmotherapic principles. The result revealed that the extract had a significant inhibitory activity against Bacillus subtilis and Pseudomonas aeruginosa but moderate on the other species at a concentration of 50mg/mL. The results are indicating that the gemmotherapic extracts can be a viable alternative to the modern extraction techniques. Keywords: Momordica charantia, antibacterial activity, gemmotherapic extracts, well diffusion assay.

thatprotect them from infection (Lou et al., 2010). In this study, we tested a gemmotherapic extract from young buds of M. charantia based on the principle that gemmotherapic extracts have a more intense inhibitory activity compared with the traditional extracts (Braca, 2008). M. charantia is a climber belonging to family Cucurbitaceae, is commonly known as bitter gourd or bitter melon. The plant is native to Asia and it was recently introduced in culture in Romania. It is cultivated in the western part of Romania since the year 2006 (Crisan, 2007). The antimicrobial activity of M. charantia is well documented, the plant possessing high inhibitory effects on many species of bacteria and fungi (Abalaka et al., 2011), antiinflammatory activities (Koboriet al., 2008), (Lii et al., 2009) and antioxidant (Wu and Ng, 2008).

INTRODUCTION Gemmotherapyor plant stem cell therapy uses a wide variety of embryonic plant parts, collected in the spring at a critical stage in the plants growth when much of the plants energy is directed to the growing areas. Gemmotherapy is an important subsection of phytotherapy. Gemmotherapic extracts are known for their higher content in active compounds (Rozencwajg, 2008). They are prepared according to gemmotherapic principles from the French Pharmacopoeia, (as cited in Nicoletti, 2012), which consists in maceration of plant buds with equal thirds of water, alcohol and glycerin. In recent years, there have been a revival of natural, plant based antimicrobial agents. This trend is the consequence of the limited effectiveness of synthetic products to fight against newer, drug resistant bacteria. For this purpose, the antimicrobial properties of many plant compounds from a wide variety of plant species have been assessed (Karuppusamy, 2009). Further, about 80% of the drugs used in modern medicine are the products of plant origin (Patwardhan et al., 2004). Also, food preservatives derived from plants and herbs are of growing interest, since plant compounds often possess antimicrobial properties

MATERIALS AND METHODS Plant material collection Plants of M. charantia were cultivated in a garden at the University of Agricultural Sciences in Timisoara. Early buds were

collected from very young plants and put immediately in alcohol of 96% concentration. Preparation of gemmotherapic extracts The solutions were made with equal thirds of alcohol, glycerol and distilled water. The fresh buds were collected, cleaned, washed with distilled water and then put in the solution for extraction. The process of extraction took place for a week in a dark place at 100C, using an orbital shaker. The extract was then filtered, concentrated by using a rotavap and weighted. The dry material was diluted for the tests and filtered through a sterile membrane filter. Two concentrations were tested: one of 50 mg/mL and the second, of 1:10 dilution factor solution. Microorganisms The bacteria used in this experiment are two Gram-negative bacteria species: Pseudomonas aeruginosa and Serratiamarcescens and three Gram-positive bacteria: Bacillus subtilis, Bacillus cereus var. mycoides and Streptococcus faecalis. The bacterial cultures were grown in Mueller Hinton Agar (Merck) and Mueller Hinton Broth (Merck). Bacteria counting The bacteria were counted using a Burker chamber. The values are shown in (Table 1).

The nutrient agar plates were seeded with 0.1 ml of standardized inoculums of each of the five test organisms. The inoculum was spread evenly over plate with a sterile glass spreader. Using a sterile cork-borer of 5 mm diameter, three holes were made into the Petri dishes seeded with bacterial culture. The bottoms of the holes were sealed with agar to avoid seepage. 50ʅl of extracts were introduced in the wells, using a micro liter syringe. Concentrations of 5 and 50 mg/ml extracts were reconstituted in distilled water and transferred into the wells. The plates were kept for 30 min at room temperature to allow diffusion of the extract, and then were incubated at temperature of 370C for 24 hours. After the incubation period, the zones of inhibition were measured using a digital caliper. In this study, the measurement is taken including the 5 mm diameter of the hole. Studies were performed in triplicates and the mean value was calculated. A solution of only alcohol, glycerol and water in equal ratios wasused as a negative reference. Statistical analysis Data were averages of three results ± Standard Deviations (SD) by using Microsoft Excel.

Table 1: The Burker chamber count data

RESULTS AND DISCUSSIONS

Bacteria species

Number of cells/ml

Pseudomonas aeruginosa

1.6x108

Serratia marcescens

1.7x108

Bacillus subtilis

8x107

Bacillus cereus var. mycoides

15x105

Streptococcus faecalis

2.2x108

In the (Table 2) are presented the mean zone of inhibition measured after 24 hours and in the (Figure 1) is represented the graph with the measurements. The 1:10 dilution factor solution presents little or no visible inhibitory effect, most likely because the concentration here is too low as shown in the (Figure 2). All values were expressed as means ± standard error means. From the measurements we obtained, it can be observed that B.subtilis and P.aeruginosa presented the highest sensitivity, the lowest being the S.marcescens. All bacteria species tested are susceptible to the extract, at 50 mg/mL concentration and intermediate at 5 mg/mL. The diameter of the zones of inhibition approximately doubles at a tenfold concentration. These results are similar with those reported by Supraja (2013), in a test with a 50 mg/mL concentration of methanol extract of M.

Well diffusion assay and antibacterial activity The antibacterial activity was determined using the hole in plate assay procedure (Perez et al., 1990). All bacterial cultures were maintained on nutrient agar slants at temperature of 40C and sub cultured onto nutrient agar broth for 24 hours prior to testing. The pure cultures of the microorganisms were inoculated onto Muller-Hilton nutrient broth incubated at temperature of 370C for 24 hours. 25 ml of nutrient agar was poured into the 100 mm plate, with an even depth of 4 mm on a level surface shaken and allowed to cool.

charantia on B.subtilis, with a ZOI (zone of inhibition) of 16 mm diameter. In the present study, we obtained a 20 mm diameter at the same concentration.

previous similar reports regarding this aspect (Somchit, 2010; Rahman, 2011).

Table 2: Antimicrobial activity of Momordica charantia by well diffusion method after 24 hours. Measurement taken including the 5 mm diameter of the hole Bacteria/Zone of inhibition in mm* Pseudomonas aeruginosa Serratia marcescens Bacillus subtilis Bacillus cereus var. mycoides Streptococcus faecalis

50mg/ml

5mg/ml

Control

17.4±0.2

11.6±0.3

8.2±0.4

11.4±0.5

10.2±0.4

9.0±0.2

20.8±0.2

12.4±0.4

7.8±0.3

15.4±0.4

7.6±0.2

5.8±0.4

13.6±0.2

11.4±0.3

6.2±0.2

Pseudomonas aeruginosa

Serratia marcescens

Bacillus subtilis

B. cereus var. mycoides

Figure 2. The zones of inhibition for the extracts of M. charantia at 50 mg/ml (left), 1:10 dilution (right) and reference (bottom), for Bacillus cereus var. mycoides, Bacillus subtilis, Pseudomonas aeruginosa and Serratia marcescens.

The use of antibiotics has reduced the incidence of infectious diseases but their extensive uses in therapy, has led to the appearance of drugresistant bacteria (Normanno et al., 2007), which is a major public health issue worldwide. For this purpose, numerous plant extracts were screened for antimicrobial properties that could protect people from microbial infections (Serra et al., 2008; Lou et al., 2010). The plant extracts can also be used in combination with traditional antibiotics.In the literature, there are reports regarding the use of plant crude extracts (Aqil et al., 2005, 2006)in combination with fewer amounts of antibiotics for anti-bacterial activities, especially for antibiotic-resistant bacteria, compared to antibiotics alone (Schmidt et al., 2008). CONCLUSIONS

Figure 1. Antimicrobial activity of Momordica charantia solutions and the reference, zone of inhibition in mm In another study made by Leelaprakash (2011), on a methanol leaf extract, he obtained a ZOI of 22 mm at

B.subtilis but at 100 mg/mL concentration and an 18 mm at P.aeruginosa at the same 100 mg/mL concentration. Although similar tests were performed with slightly different types of cork borers with diameter ranging from 5 to 8 mm, different extract volumes and different concentrations, the results from the present study, confirms the findings of Roopashree et al. (2008) and Costa et al. (2011) on crude alcoholic and water extracts of M. charantia. The data in this study indicate that Grampositive bacteria were more susceptible to inhibition as compared to Gram-negative bacteria. This finding confirms numerous

Based on the present study, it can be concluded that there is a great potential in using the gemmotherapic principles for plant extracts in the development of more potent and efficient antimicrobial agents.

vitro antimicrobial and antioxidant activity of Momordica charantia leaves. Pharmacophore, Vol. 2 (4), 244-252 ISSN 2229 – 5402. Lii C.K., H.W. Chen, W.T. Yun, and K.L. Liu, 2009. Suppressive effects of wild bitter gourd (Momordica charantia Linn. var. abbreviata ser.) fruit extracts on inflammatory responses in RAW 264.7 macrophages. J. Ethnopharmacol. 122: 227-233. Lou Z., H. Wang, W. Lv, C. Ma, Z. Wang, and S. Chen., 2010. Assessment of antibacterial activity of fractions from burdock leaf against food-related bacteria. Food Control. 21:1272-1278. Nicoletti M, Toniolo C., 2012. HPTLC Fingerprint Analysis of Plant Staminal Cells Products. J Chromat Separation Techniq 3:148. doi:10.4172/21577064.1000148 Normanno G., M. Corrente, G. La Salandra, A. Dambrosio, N.C. Quaglia, A. Parisi, G. Greco, A.L. Bellacicco, S. Virgilio, and G.V. Celano, 2007. Methicillin-resistant Staphylococcus aureus (MRSA) in foods of animal origin product in Italy. Intern. J. Food Microbiol. 117: 219-222. Patwardhan B., Vaidya A.D.B., Chorghade M., 2004. Ayurveda and natural products drug discovery. Curr. Sci., 86(6): 789-799. Perez C., Paul M., Bazerque P., 1990. An Antibiotic assay by the agar well diffusion method. Acta. Bio. Med. Exp. 15: 113-115. Rahman MS, Salehin MF, Jamal MAFM, Parvin A, Alam MK., 2011. Antibacterial activity of Argemonemexicana L. against water borne microbes. Research Journal of Medicinal Plant; 5(5): 621-626. Roopashree T.S., Raman Dang, Shobha Rani R.H., Narendra C., 2008. Antibacterial activity of antipsoriatic herbs: Cassia tora, Momordica charantia and Calendula officinalis. International Journal of Applied Research in Natural Products, Vol. 1(3), pp. 20-28. Rozencwajg, J., 2008. Dynamic Gemmotherapy, Integrative Embryonic Phytotherapy, Second Edition, 143 – 160. Schmidt B., D.M. Ribnicky, A. Poulev, S. Logendra, W.T. Cefalu, and I. Raskin, 2008. A natural history of botanical therapeutics. Metabolism 57: S3-S9. Wu, S.J. and L.T. Ng., 2008. Antioxidant and free radical scavenging activities of wild bitter melon (Momordica charantia Linn. var. abbreviata ser.) in Taiwan. LWTFood Sci. Technol. 41: 323-330. Somchit MN, Rashid RA, Abdullah A, Zuraini A, Zakaria ZA, Sulaiman MR, Arifah AK, Mutalib AR., 2010. In vitro antimicrobial activity of leaves of Acalyphaindica Linn. (Euphorbiaceae). African Journal of Microbiology Research; 4(20): 2133-2136 Supraja P., R. Usha, 2013. Antibacterial and phytochemical screening from leaf and fruit extracts of Momordica charantia. Int J Pharm Bio Sci; 4(1): (B) 787 – 793. ISSN 0975-6299.

Also, the gemmotherapic extracts obtained from M. charantiayoung shoots, using the classic gemmotherapic principles, exhibit an intensive antimicrobial activity. Further investigation is needed in order to study the synergy of fractions from Momordica charantia. ACKNOWLEDGEMENTS The authors are thankful to the Department of Microbiology from the University of Szeged for the support in carrying out this research. REFERENCES Abalaka M. E., Onaolapo J.A., Inabo H.I. and Olonitola, O.S., 2001. In vitro Antibacterial Assessment of Crude Extracts of the Leaves and Stem of Momordica charantia on Enteropathogenic and Pyogenic Bacterial Isolates. Fermentation Technology and Bioengineering, 2:43-48. Aqil F., M.S. Khan, M. Owais, and I. Ahmad, 2005. Effect of certain bioactive plant extracts on clinical isolates of betalactamase producing methicillin resistant Staphylococcus aureus. J. Basic Microbiol. 45: 106-114. Aqil F., I. Ahmad, and M. Owais, 2006. Evaluation of anti-methicillin-resistant Staphylococcus aureus (MRSA) activity and synergy of some bioactive plant extracts. Biotechnol. J.1: 1093-1102. Braca A, Siciliano T., 2008. Chemical composition and antimicrobial activity of Momordica charantia seed essential oil. Fitoterapia. 79:123–5. Costa José Galberto M., Eidla M. M. Nascimento, Adriana R. Campos and Fabiola F. G. Rodrigues, 2011. Antibacterial activity of Momordica charantia (Curcubitaceae) extracts and fractions. Journal of Basic and Clinical Pharmacy, Vol-002 Issue-001: 45-51. CrisanSimona, Hălmăgean L., 2007. Technological solutions with impact on the yield of Momordica charantia L. fruits (Cucurbitaceae) in Arad agroecological area. Buletin USAMV-CN, 64/2007. ISSN 1454-2382. Karuppusamy S., 2009. A review on trends in production of secondary metabolites from higher plants by in vitro tissue, organ and cell cultures. J. Med. Plants Res., 3: 1222-1239. Kobori, M., H. Nakayama, K. Fukushima, M. OhnishiKameyama, H. Ono, T. Fukushima, Y. Akimoto, S. Masumoto, C. Yukizaki, Y. Hoshi, T. Deguchi, and M. Yoshida, 2008. Bitter gourd suppresses lipopolysaccharide-induced inflammatory responses. J. Agric. Food Chem. 56: 4004-4011. Leelaprakash G., J. Caroline Rose, Gowtham B.M., Pradeep Krishna Javvaji, Shivram Prasad. A, 2011. In

Scientific Bulletin. Series F. Biotechnologies, Vol. XVII, 2013 ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

ESTIMATING FREE RADICALS SCAVENGING ACTIVITY OF SOME BERRIES SPECIES Gabriela LUğĂ, Florentina ISRAEL-ROMING, Daniela BĂLAN, Evelina GHERGHINA University of Agronomical Sciences and Veterinary Medicine Bucharest, Faculty of Biotechnologies, 59, Marasti Blvd., 011464, Bucharest, Romania, www.usamv.ro Corresponding author Florentina ISRAEL-ROMING: [emailprotected] Abstract Berries fruits contain phytochemical components with antioxidant activity, such as polyphenols, ascorbic acid, that have possible protective effects on human health. Free radicals can induce changes in different cell biomolecules such as lipids, proteins, and nucleic acids. This oxidative stress is involved in pathogenesis of many human diseases: cancer, cardiovascular diseases, osteoporosis, neurodegenerative processes. The objective of these researches was to estimate and compare the free radicals scavenging activity of total extracts of certain berries fruits species: raspberry (Rubus idaeus), strawberry (Fragaria ananassa), sea-buckthorn (Hippophaea rhamnoides). The evaluation involved determination of total phenols using spectrophotometrical method and of ascorbic acid content by HPLC method. Total antioxidant capacity was determined using the stable free radical diphenylpicrylhydrazyl (DPPH) method and calculating the parameter IC50 (the concentration of sample which is required to scavenge 50% of DPPH free radicals). As expected, sea-buckthorn fruits manifested the highest radical scavenging activity expressed as EC50 value (512.76 ȝg/ml). A linear correlation was obtained for total phenols content (Pearson’s correlation coefficient -0.945) and for ascorbic acid (-0.8607). Keywords: ascorbic acid, berries, scavenging activity, total phenols

health, therefore a preference for antioxidants from natural rather than from synthetic sources have imposed. Considering that, an increasing interest for investigating the antioxidants provided by berries fruits was observed in recent years. The objective of these researches was to estimate and compare the free radicals scavenging activity (antioxidant activity) of total extracts of certain berries fruits species: raspberry (Rubus idaeus), strawberry (Fragaria ananassa), sea-buckthorn (Hippophaea rhamnoides). The evaluation involved determination of total phenols using spectrophotometrical method and of ascorbic acid content by HPLC method. The total antioxidant capacity was determined using the stable free radical diphenylpicrylhydrazyl (DPPH) method and calculating the parameter EC50 (the concentration of sample which is required to scavenge 50% of DPPH free radicals).

INTRODUCTION Berries fruits are an important source of active natural biocompounds known to be responsible for free radicals scavenging activity. These fruits contain phytochemical components with antioxidant activity, such as polyphenols, ascorbic acid, that could be involved in preventing the occurrence of oxidative-stress related diseases, caused by the attack of free radicals on key biocomponents like lipids, proteins or nucleic acids (Mayne S.T., 2003; Pisoschi A., 2011). This oxidative stress is involved in pathogenesis of many human diseases: cancer, cardiovascular diseases, osteoporosis, neurodegenerative processes. Vitamin C is an electron donor, and this property accounts for all its known functions. As an electron donor, vitamin C is a potent water-soluble antioxidant in humans. Antioxidant effects of vitamin C have been demonstrated in many experiments in vitro (Kelly F.J., 1998; Padayatty S., et al, 2002). The food industries used some synthetic antioxidants for the protection against the oxidizing agents, but recent researches emphasized their possible toxicity for human

MATERIALS AND METHODS Biological materials. The analyzis were performed on three wild berries fruits species: three samples of raspberry (Rubus idaeus), five

samples of strawberry (Fragaria ananassa), and two samples of sea-buckthorn (Hippophaea rhamnoides) provided by the local market. The determinations were made in triplicate, using fresh fruits. The extractions were conducted according to the protocol used for each determination. Total phenolic content was performed according to the modified Folin-Ciocalteu assay (Singleton et al., 1999). The method consists in chemical reduction of Folin-Ciocalteu reagent (which is a mixture of tungsten and molybdenum oxides) and measuring the intensity of the obtained blue colour at 750 nm. The measurements were achieved with a UV/Visible ThermoSpectronic Helios spectrophotometer. Total phenols values were expressed in terms of gallic acid equivalent (GAE), which is a common reference compound. The ascorbic acid contentwas determined by HPLC-RP with UV detection. The mobile phase consisted in 0.1% phosphoric acid. Samples were centrifuged at 4,000 rpm for 10 minutes; 1 ml supernatant was ten times diluted with elution solvent. Before injection, samples were filtered using 0.22 μm PTFE filters. Data acquisition and processing were realized with EMPOWER software. Ascorbic acid detection was performed at 210 nm. Calibration curve was achieved using a 0.1 μg/ml standard solution of L-ascorbic acid, in five concentration levels with three injections for each level. The free radical scavenging activity (total antioxidant capacity) was determined using the stable free radical diphenylpicrylhydrazyl (DPPH) method according to Blois, M.S. (1958) procedure adaptated by Brand-Williams W. (1995) for complexes matrices. Briefly, a 100 PM solution of DPPH in methanol was prepared and 2 ml of this solution was mixed with 1 ml of different concentrations of berries fruits extract in 80% aqueous methanol. After 30 min incubation in dark at room temperature, absorbance was measured at 515 nm. The percentage of the radical scavenging activity (RSA) was calculated as follows: % RSA = (1-[Asample/Acontrol t=0])/100 DPPH solution in 80% methanol was used as a control. Gluthation at various concentrations (25 to 200 Pg/ml) was used as a standard. The EC50 parameter for each sample, defined as the concentration of sample which is required

to scavenge 50% of DPPH free radicals, was calculated from the non linear regression curve of Log concentration of the sample extracts (Pg/ml) against the percentage of the radical scavenging activity. The statistical analysis was performed using the one-way Analysis of Variance (ANOVA). Pearson’s correlation coefficient (r) was used to calculate the relationship between the DPPH and total polyphenol contents and ascorbic acid content of the three berries fruits species. RESULTS AND DISCUSSIONS The phenolics constitute a very divers and widespread group of biochemical compounds occurred in natural vegetal sources. Biological effects of polyphenols are attributed to their antioxidant effects, so that their determination is of considerable interest. The biochemical analysis performed in this study indicated high values of the total phenols content in the tested berries (table 1), well known for their rich content in bioactive compounds (Battino M. et al, 2009; Ribera A.E. et al., 2010). The results emphasized seabuckthorn as the richest phenolics source among the tested fruits. The total phenols content in sea-buckthorn fruits was 669.63 mg GAE/100g, which is 1.93 times higher than in strawberry and 1.63 times higher compared to raspberry. Other authors found that the total content of phenols in sea-buckthorn depends on the cultivar and varies from 828.7 to 1099.6 mg/100g (Novruzov E., 2005; Seglina D. et al., 2008). Table 1. The total phenols content in the tested berries Samples Strawberry Raspberry Sea-buckthorn

Total phenols (mg GAE/100g fresh weight) 345.34 r 3.93 410.78 r 6.39 669.63 r 8.18

Regarding the level of the ascorbic acid content (table 2), also sea-buckthorn fruits reached the highest amount (1154.24 mg/100g), which was according with the expectations. The results of ascorbic acid content in sea-buckthorn agree with those reported by Gutzeit D. et al. (2008) and Christaki E. (2012).

Table 2. The ascorbic acid content in the tested berries Samples Strawberry Raspberry Sea-buckthorn

take into account that, in contrast with the standard, the berries extracts are complex mixtures of numerous compounds with different properties.

Ascorbic acid (mg/100g fresh weight) 64.21 r 2.18 52.02 r 1.50 1154.24 r 4.15

Table 3. EC50 values of DPPH scavenging activities of the studied berries

For estimating the antioxidant capacity of tested berries fruits was used the DPPH method because is adequate for screening antioxidant compounds formed by small molecules (such as phenols or ascorbic acid), considering that the reaction intensity can be measured using a spectrophotometrical method (Nickavar B. et al, 2009). The EC50 values for every tested fruits were calculated for further comparison. For this purpose the extracts of selected berries were screened for their possible radical scavenging activity (RSA). Extracts in different concentrations of selected berries exhibited a high antioxidant activity, expressed as percentage of DPPH reduction (figure 1). The measurements indicated the highest antioxidant activity for sea-buckthorn fruits, confirming the expectations due to their rich content in total phenols and ascorbic acid. The EC50 values were calculated for all the tested berries fruits (table 3), showing that seabuckthorn fruits manifested the highest scavenging activity (512.76 ȝg/ml).

Samples Strawberry Raspberry Sea-buckthorn Standard (gluthatione)

EC50 (ȝg/ml) 727.63 620.05 512.76 79.66

The rich content in phenols and ascorbic acid of the berries may cause the antioxidant properties of these fruits. The correlation between total phenols content, as well as ascorbic acid content, and the antioxidant activity of certain fruits and vegetables extracts was studied by different authors (Pellegrini N. et al, 2003; Franco D. et al, 2008; Dvorakova M. et al, 2008), which reported an increasing antioxidant activity correlated with the concentration of these active biocompounds. In the present paper a correlation study was performed between the radical scavenging activity (expressed as EC50) and the content in total phenols and ascorbic acid in order to reveal the contribution of these biochemical compounds to the total antioxidant capacity of the berries fruits (figure 2).

Figure 1. Radical scavenging activity (%) of the tested berries fruits

Figure 2. Antioxidants content and radical scavenging activity in berries

The extract with the lowest scavenging capacity was strawberry, which required a higher concentration (727.63 ȝg/ml) to scavenge 50% of DPPH free radicals. However, this results are significant low compare to the standard (gluthatione) scavenging power (79.66 ȝg/ml), but we must

A strong linear correlation with Pearson’s correlation coefficient - 0.945 was obtained for total phenols content. For ascorbic acid the linear relationship had a lower value, respective - 0.8607. The obtained correlation coefficients were in accordance with those reported by Rufino M.S.

Franco D., Sineiro J., Rubilar M., Sanchez M., Jerez M., Pinelo M., Costoya M., Nunez M. J., 2008. EJEAFChe, 7(8), 3210-3216. Gutzeit D., Baleanu G., Winterhalter P., Jerz G., 2008. Vitamin C content in sea buckthorn berries (Hippophaë rhamnoides L. ssp. rhamnoides) and related products: a kinetic study on storage stability and the determination of processing effects. J Food Sci., 73(9). Kelly F.J., 1998. Use of antioxidants in the prevention and treatment of disease. J Int Fed Clin Chem 10 (1), 21– 3. Mayne S.T., 2003. Antioxidant nutrients and chronic disease: use of biomarkers of exposure and oxidative stress status in epidemiologic research. J. Nutr. 133 Suppl 3, 933S–940S. Nickavar B., Abolhasani F., 2009. Screening of antioxidant properties of seven Umbeliferae fruits from Iran. Pak. J. Pharm. Sci., vol 22, No 1., 30-35. Novruzov E., 2005. Biochemical Characteristics of Seabuckthorn (Hippophae rhamnoides L.) Growing in Azerbaijan. Seabuckthorn (Hippophae L.): A Multipurpose wonder Plant, Vol. II Singh V. (ed.) Daya Publishing House, New Delhi, India, 133–144. Padayatty S., Katz A., Wang Y., Eck P., Kwon O., Lee J. H., Chen S., Corpe C., Dutta A., Levine M., 2002. Vitamin C as an Antioxidant: Evaluation of Its Role in Disease Prevention, J. Am. Coll. Nutr. 22 (1), 18–35. Pellegrini N., Serafini M., Colombi B., del Rio D., Salvatore S., Bianchi M., Brighenti F., 2003. Total antioxidant capacity of plant foods, beverages and oils consumed in Italy assesed by three different in vitro assays. University of Parma, Italy. Pisoschi A.M., Negulescu G.P., 2011. Methods for Total Antioxidant Activity Determination: A Review. Biochem & Anal Biochem 1:106. Ribera A.E, Reyes-Díaz M., Alberdi M., Zuñiga G.E., Mora M.L., 2010. Antioxidant compounds in skin and pulp of fruits change among genotypes and maturity stages in highbush blueberry (Vaccinium corymbosum) grown in southern Chile. J. Soil. Sci. Plant Nutr. 10 (4), 509 - 536. Rufino M.S., Alves R.E., Brito E.S, Jiménez J.P., SauraCalixto F., Mancini-Filho J., 2010, Bioactive compounds and antioxidant capacities of 18 non-traditional tropical fruits from Brazil, Food Chemistry 121, 996–1002. Seglina D., Karklina D., Gailite I., Ruisa S., Krasnova I., Heidemane G., 2008. Changes of biochemical compounds in seabuckthorn marc during storage. Foodbalt, 109-113. Singleton V. L., Orthofer R., Lamuela-Raventos R. M., Lester P., 1999. Analysis of total phenols and other oxidation substrates and antioxidants by means of FolinCiocalteu reagent. Meth. Enzymol. 299, 152-178.

et al. (2010) and Arancibia-Avila P. et al. (2012) CONCLUSIONS Biochemical analysis performed indicated seabuckthorn fruits as the richest phenolics sources among the tested fruits (669.63 mg GAE/100g). Sea-buckthorn fruits reached also the highest amount of ascorbic acid content (1154.24 mg/100g). As expected, sea-buckthorn fruits manifested the highest radical scavenging activity expressed as EC50 value (512.76 ȝg/ml). The radical scavenging activity reached high values in the extracts rich in total phenols and ascorbic acid, so that a linear correlation was obtained for total phenols content (Pearson’s correlation coefficient -0.945) and for ascorbic acid (-0.8607). REFERENCES Arancibia-Avila P., Namiesnik J., Toledo F., Werner E., Martinez-Ayala A.L., Rocha-Guzmán N.E., GallegosInfante J.A., Gorinstein S., 2012. The influence of different time durations of thermal processing on berries quality, Food Control 26, 587-593. Battino M., Beekwilder J., Denoyes-Rothan B., Laimer M., McDougall G.M., Mezzetti B., 2009. Bioactive compounds in berries relevant to human health. Nutrition Reviews, Vol. 67(Suppl. 1), S145–S150. Blois M.S., 1958. Antioxidant determinations by the use of a stable free radical. Nature, 181, 1199-1200. Brand-Williams, W., Cuvelier M. E., Berset C., 1995. Use of free radical method to evaluate antioxidant activity. Lebensmittel-Wissenschaft und Technologie/Food Science and Technology, 28, 25-30. Christaki E., 2012. Hippophae Rhamnoides L. (Sea Buckthorn): a Potential Source of Nutraceuticals. Food and Public Health; 2(3), 69-72. Dvorakova M., Guido L.F., Dostalek P., Skulikova S., Moreira M., Barros A., 2008. Antioxidant properties of free, soluble ester and insoluble-bound phenolic compounds in different barley varieties and corresponding malts. J. Inst. Brew. 114(1), 27-33.

Scientific Bulletin. Series F. Biotechnologies, Vol. XVII, 2013 ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

THE INFLUENCE OF ENVIRONMENTAL CONDITIONS AND PLANTING DATE ON SUNFLOWER OIL CONTENT AND FATTY ACIDS COMPOSITION Mihaela POPA1, Narcisa BABEANU1, Georgeta DICU2, Andreea TEODORESCU2, Nicolae BOAGHE2 1

University of Agronomic Sciences and Veterinary Medicine of Bucharest, 59 Marasti Blvd, District 1, 011464, Bucharest, Romania 2 Procera Genetics SRL, 47 Muncii Street, Fundulea City, Calarasi, Romania Corresponding author email: [emailprotected] Abstract The sunflower oil is very important in human food because it contains a high percentage of unsaturated fatty acids. This oil ideally combines high nutritional value due to its content inlinoleic acid with the high stability resistance during cooking because of oleic acid. This paper aims to show the variation of the main fatty acids in sunflower oil depending on environmental conditions and sowing date. In the studied area, adjusting the planting date and considering climatic factors, we can obtain the desired fatty acid percentage in our inbreed lines, used further for obtaining valuable hybrids. The same set of sunflower inbred lines was planted in two stages: April 10 and the May 27. In this period the environmental factors varied from low temperatures and rainfalls up to very high temperatures and severe drought, year 2012 being marked by extreme phenomen. Keywords: fatty acids, linoleic acid, oleic acid, planting date, sunflower.

Burr and Burr's demonstrated in their experiences the importance of this specific group of polyunsaturated fatty acids called essential fatty acids, human and animal organisms being unable to synthesize them (Vles and Gottenboss, 1989). Unlike other vegetable oils, the sunflower oil has a great nutritional value due to its high linoleic acid content and it is very stable due to lack of linolenic acid, so it can be preserved for a long period of time. The linoleic/oleic ratio is not constant and it can be changed by many factors, the most important being temperature during oil accumulation and genotype. (Vrânceanu, 2000). This paper aims to show how the oil content and the main fatty acids varies depending on sowing date and climatic conditions of the year 2012 in Fundulea area.

INTRODUCTION Sunflower is a typical oleic plant due to its high content of oil from seeds, which often exceeds 50% of the dry matter. Whole seed oil content used to bedetermined as the relationship between the percentage of hulls and the percentage of kernel oil. Now, the oil content is expressed as percentage of whole seed weight. In cultivars with low percentage of hulls (2024%) the oil content exceeds 50% of dry matter. Sunflower oil is considered first-class edible oil due to its content of linoleic acid, followed by oleic acid, together representing approximately 90% of the fatty acids composition.The fats found in food represent a combination in different proportions of saturated, monounsaturated and polyunsaturated fatty acids. Sunflower oil contains saturated and monounsaturated fats but in much smaller amounts than polyunsaturated fats (Vrânceanu, 2000). The new mutant variety called oleic sunflower has a high content of monounsaturated fats, namely oleic acid and very low proportion of linoleic acid (Soldatov, 1976).

MATERIALS AND METHODS The biological material used for this experience consisted in a set of 5 sunflower lines which was sown on two different dates: on April 10 and May 27. The climatologically data were collected from a meteorological institute nearby

the studied area. The seeds were sown in 2 rows for each sunflower line. The distance between rows was 0.75 m and the length of a row was 4.8 m. Sunflower seeds were ground with laboratory mill Knifetech Foss, the oil content being determined with Foss 1241 equipment which uses NIR technology. For fatty acids the oil is extracted with diethyl-eter, which is an organic solvent, in Soxleth extractor. The obtained oil is subjected to a transesterification reaction and the fatty acid methyl esters will be determined by gas chromatography. The materials used for this methodare: nheptane, internal standard methyl heptadecanoat 10 mg/ml, gas chromatograph (Trace GC Ultra) with FID detector and split injector, DBWAX column (30 m-0.25 mm-0.25 μm), analytical balance, vials (with capacity of 10 ml), flask, pipette (5 ml). The chromatographic conditions are: oven temperature 210 ºC, injector temperature 250 ººC, gas pressure 80 kPa, gas flow 1-2 ml/min, injector flow 50 ml/min, analysis time 25 min. The work procedure consists in weighing of 250 mg of sample in a 10 ml vial then adding 5 ml of methyl heptadecanoate. Homogenized mixture is injected and the chromatogram is obtained. The peaks from C 14 to C 24:1 are integrated.

Figure 1. Evolution of temperatures and rainfalls during January-August 2012

The linoleic and palmitic acid contents increase, while the oleic acid content decreases in seed from the perimeter towards the centre of the head (Fick and Zimmerman, 1973; Zimmerman and Fick, 1973). Regarding the two main fatty acid of sunflower oil, it is well known that they closely depend on the environmental conditions. Temperature, especially day/night temperature differences are the most importantenvironmental factors driving seed oil percentage and oil chemical composition (Izquierdo et al., 2002; Rondanini et al., 2003, 2006; Qadir et al., 2006; Echarte et al., 2010). Data obtained after processing the samples are presented in table 1 and table 2.

RESULTS AND DISCUSSIONS Table 1. Oil and fatty acid composition for sunflower lines sown on April 10, 2012

The period Between January and August was marked by extreme phenomena from low to very high temperature and from heavy rain falls in May to severe draught and high temperatures in July. The ratio of the major fatty acids in sunflower oil is not constant, changing during the oil accumulation. Thus, the concentration of linoleic acid increases until the end of seed maturity, while oleic acid concentration decreases. There exists a negative correlation because oleic acid is a precursor of fatty acids with higher degree of unsaturation. This explains the variation of linoleic and oleic acid between the seeds of the same head (Canvin, 1965; Robertson et alii, 1978; Goyne et alii, 1979; Unger and Thompson, 1982; Downes and Tonnet, 1982; Simpson and alii. 1989; Connor and Sadras, 1992).

Line Oil Humidity V1 42.0 8.5 V2 46.8 7.9 V3 47.6 8.0 V4 48.0 8.4 V5 47.7 8.2

Palmitic 6.1 5.8 5.8 5.6 5.8

Stearic 2.5 4.4 2.6 3.6 4.1

Oleic Linoleic 36.4 53.1 33.0 55.3 28.7 61.7 32.0 57.4 30.5 58.0

Table 2. Oil and fatty acid composition for sunflower lines sown on May 27, 2012 Line V1 V2 V3 V4 V5

Oil 38.2 46.5 41.8 48.4 42.5

Humidity 7.9 8.5 8.6 7.5 8.6

Palmitic 6.1 5.9 5.4 6.3 6.1

Stearic 3.2 4.2 2.5 4.5 4.4

Oleic 31.7 33.7 33.6 29.1 28.7

Linoleic 57.1 54.4 57.2 57.6 58.1

Different planting dates and water regimes cause different environmental conditions during seed-filling and oil synthesis of

sunflower seed and therefore a possible alteration in oil content and fatty acid composition of the seed (Flagella et al., 2002). Thus, in our results we can observe that the concentration of oleic acid decreased for 4 of 5 lines until the end of seed maturity (figure 2), while for 4 of 5 lines the linoleic acid concentration increased for plants sown later (figure 3).

May 27 showed a significant decrease in oil content, even with 5% less than in the first set.

Figure 4. Oil content depending on sowing dat

CONCLUSIONS It was noted from results obtained for the five sunflower lines that the oil concentration varied up to 5% from one set to another, this trait being very strong affected by draught. Regarding fatty acids, there are also differences between the two set of sunflower lines; the linoleic acid increased in the second set sown later, while the oleic acid concentration was affected by late planting. In order to obtain optimal results, it is recommended to adjust sowing date according to the objectives pursued.

Figure 2. Variation of oleic acid depending on sowing date

REFERENCES Connor, D. J.,Sadras, V. O., 1992. Physiology of yield expression in sunflower. Field crop research, 30: 333389. Downes, R. W., Tonnet, M. L., 1982. Selection of sunflower plants containing high linoleic acid, and its agronomic significance. Proc. 10th Intern. Sunflower Conf., Surfers Paradise, Australia, pp 258-261. Fick, G.N. and Zimmerman, D.C. 1973. Variability in oil content among heads and seeds within heads of sunflowers (Helianthus annuus L). Journal of the American Oil Chemists’ Society 50: 529-531. Flagella, Z., Rotunno, T., Tarantino, E., Di Caterina, R. and De Caro, A. 2002. Changes in seed yield and oil fatty acid composition of high oleic sunflower (Helianthus annuus L.) hybrids in relation to the sowing date and the water regime. European Journal of Agronomy 17: 221-230. Izquierdo, N., Aguirrezábal, L., Andrade, F. and Pereyra, V. 2002. Night temperature affects fatty acid composition in sunflower oil depending on the hybrid and thephenological stage. Field Crops Research 77: 115-126. Qadir, G., Ahmad, S., Ul-Hassan, F. and Cheema, M.A. 2006. Oil and fatty acidaccumulation in sunflower as influenced by temperature variation. Pakistan Journal of Botany 38: 1137-1147 Rondanini, D., Savin, R. and Hall, A.J. 2003. Dynamics of fruit growth and oil quality of

Figure 3. Variation of linoleic acid depending on sowing date

It is known that the oil content varies very much, depending on genotype and environmental factors. The main factor in oil accumulation is water, thus in southern regions, that are more draughty, the oil content is lower than in humid regions. The rain fallen during seed formation have favorable effects on oil accumulation not only by improving the water supply but also because decrease temperature and increase atmospheric humidity. Even if it is a year with heavy rainfalls, but with high temperatures, the oil content decrease. In case of heavy rainfalls during the second half of summer they will lead to stimulate the secondary growth, which will also have negative effects on oil content (Vrânceanu et al., 1969). As can be seen in figure 2, the oil content varied very much, thus sunflower lines sown on

sunflower (Helianthus annuus L.) exposed to brief intervals of high temperatureduring grain filling. Field Crops Research 83: 79-90. Rondanini, D., Mantese, A., Savin, R. and Hall, A.J. 2006. Response of sunflower yield and grain quality to alternating day/night high temperature regimes during grain filling: Effects of timing, duration and intensity of exposure to stress. Field Crops Research 96: 48-62. Simpson, B. W., McLeod, C. m., George, D. L., 1989. Selection for high linoleic acid content in sunflower (Helianthus annuus L.) Aust. J. Exp. Agric., 29: 233-239. Soldatov, K. J., 1976.Chemical mutagenesis for sunflower breeding. Proc. 7th Intern. Sunflower Conf., Krasnodar, U.S.S.R., pp. 352-357.

Vles, R. O., Gottenboss, J. J., 1989. Nutritional characteristics and food used of vegetable oils. In: G. Robbelen, R. K. Downey, A. Ashri, Oil Crops of the world (Eds.), McGraw-Hill, U.S.A., pp. 63-86. Vrânceanu V., 2000. Hybrid sunflower. Ceres Publishing House, Bucharest. Vrânceanu, A. V., Istfan, D., Olteanu, F., 1969. Sunflower crop. Editura Agrosilvica Publishing House, Bucharest. Zimmerman, D.C. and Fick, G.N. 1973. Fatty acid composition of sunflower (Helianthusannuus L.) oil as influenced by seed position. Journal of the American Oil Chemists’Society 50: 273-275.

Scientific Bulletin. Series F. Biotechnologies, Vol. XVII, 2013 ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

EXTRACTION, PURIFICATION AND CHARACTERIZATION OF LECTIN FROM PHASEOLUS VULGARIS L. CV. WHITE SEEDS (WHITE KIDNEY BEAN) Basheer A. AL-ALWANI, Mohammed A. JEBOR, Yasser H. JALIL University of Babylon, College of Science, Department of Biology, Box.4, Hilla, Iraq Corresponding author email: [emailprotected] Abstract The purpose of the research was to study the purification and characterization of lectin from Phaseolus vulgaris L. cv.white seeds. The lectin was purified by sequence of steps , namly , first with ammonium sulfate precipitation followed by ion exchange (DEAE cellulose) and gel filtration (sephadex G-200) chromatographies , and finally by polyacrylamide electrophoresis (PAGE). Single band was observed in native PAGE . The lectin was shown to have molecular weight of 33 kDa in SDS PAGE and about 35 kDa in gel filtration and purified about 9.01 fold to final specific activity of 64 titer/mg of protein. The hemagglutination activity of the lectin was stable within the pH range from 4-11 and temperature range from 0°-50°C. Chemical modification results indicate that lysine and tryptophan were crucial for the hemagglutination activity of lectin. The results of carbohydrates specificity showed that the lectin was had complex sugar specifities, but not specific to xylose and mannose. Keywords: lactin, purification, characterization, Phaseolus vulgaris L.

maintaining the carbohydrate binding and hemagglutinating activities of lectins (Bao et al., 1996; Ba˘i˘miev et al., 2007). Identification of these amino acid residues is a prerequisite for investigating the structure-function relationships of lectins. Chemical modification with group-specific modifying agents provides a general approach for identification of the amino acid residues present in the functional or active site of proteins, including lectins (Bao et al., 1996; Nadimpalli, 1999). Hence, elucidation of biological activities of lectins and amino acid residues essential to these activities is a meaningful undertaking. Although lectins are found ubiquitously in plant species, they have variable structures and specific activities according to the plants they originate from cells (Sharon, 1993; Padma et al., 1998). Thus, purification and characterization of lectins from a variety of plant species interests researchers in the field of glycobiology. The more is known about the lectins, the wider the applications of this type of proteins that can be achieved. This study reports the purification and some properties of a new lectin isolated from seeds of the Phaseolus vulgaris L. cv.white cultivar (common name, white kidney bean). To date, the isolation of a lectin from the Phaseolus

INTRODUCTION Lectins are defined as proteins/glycoproteins possessing at least one non-catalytic domain which binds reversibly to a specific mono-or oligosaccharide (Van damme et al., 2003). Over the last few decades, lectins have become a topic of interest to a large number of researchers owing to their potentially exploitable biological properties including antitumor (Abdullaev et al., 1997; Ye et al., 2001), immunomodulatory and anti-insect (Rubinstein et al., 2004), antifungal (Barrientos et al., 2005), antibacterial (Pusztai et al., 1993), anti-HIV (Tsang et al., 2001; Barrientos et al., 2005; Pollicita et al., 2008), and mitogenic (Wimer, 1990) activities. Because of their sugar binding properties, lectins have been extensively studied and used as molecular tools for the study of carbohydrate architecture and dynamicson the cell surface, and have been exploited for such practical applications as distinguishing between normal and malignant cells (Sharon, 1993; Padma et al., 1998), purification of glycoconjugates (Yamamoto et al., 1984), and coating of drugs to enhance their gastrointestinal tract absorption (Naisbett & Woodley, 1990; Leher, 2000). Further, specific amino acid residues are essential for

saline (pH 7.2). Readings were recorded after about 30 minutes at room temperature, when the blank had fully sedimented. The hemagglutination titer, defined as the reciprocal of the highest dilution exhibiting hemagglutination, was treated as one hemagglutination unit. Specific activity was expressed as the number of hemagglutination units per mg protein (Yufang et al., 2010). 2.2.4. Purification of Phaseolus vulgaris L. cv.white lectin (White Kidney Bean). Purification of white kidney beans lectin firstly at precipitation by ammonium sulfate at 10%100% saturation rate to Crude extract from the soaking and then dialyzed against distilled water,then loaded on a DEAE cellulose column (2.6 cm × 60 cm) that had been equilibrated with the same buffer, and subjected to ion exchange chromatography. The column was washed initially with 0.1M NaCl in 0.02M Tris-HCl (pH 8.0) to remove proteins that had not specifically absorbed to the column, then washed with linear salt gradient elution. Fractions showing hemagglutinating activity were further purified by sieve chromatography on a Sephadex G-200 column in 0.15M NaCl in 0.02 M Tris-HCl (pH 8.0). 2.2.5. SDS-PAGE. SDS-PAGE. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) was performed in accordance with the method of Laemmli (Laemmli, et al., 1973) using a 15% separating and a 10% stacking gel. 2.2.6. Molecular mass determination. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in accordance with the procedure of Laemmli and Favre (Laemmli, et al., 1973). The molecular mass of the lectin was estimated from the standard curve plotting electrophoretic mobility against molecular mass. Gel filtration was carried out using a Sephadex G-200 column that had been calibrated with molecular-mass standards proteins (BSA, Alkaline phophatase, RNase and Trypsin) to estimate the molecular mass of the purified lectin. 2.2.7. Sugar specificity. The Sugar specificity investigate inhibition of lectin-induced hemagglutination by various carbohydrates were performed in a manner analogous to the hemagglutination test. Prepare 1-100 mM of each sugar samples was prepared

vulgaris L. bean and examining it for various potentially exploitable biological activities such as mitogenic, antitumor, immunomodulatory, and HIV-1 reverse transcriptase inhibitory activities have not been attempted. In this study, a lectin was isolated from Phaseolus vulgaris L. beans. It was assayed for the various aforementioned activities. In order to further characterize the lectin, a chemical modification study was undertaken to determine the involvement of different amino acid residues in its hemagglutinating activity. MATERIALS AND METHODS 2.1. Materials Phaseolus vulgaris L. cv.white (White kidney bean) from local source, Hilla, Babylon, Iraq, human red blood cells (healthy persons), ammonium sulfate, DEAE cellulose, sephadex G-200, PAGE, electrophoretic reagents were purchased from Sigma (USA), 5mMphenylmethylsulfonyl fluoride (PMSF), 5, 5-dithiobis- (2 nitrobenzoic acid (DTNB), NaBH4, Nbromosuccinimide (NBS) were obtained from Himedia (India), BSA, alkaline phophatase, RNase and Trypsin obtained from Biobasic . 2.2. Methods 2.2.1. Isolation of Phaseolus vulgaris lectin By using, the soaking Method white kidney beans were ground to a powder in filtered through 80-mesh grit. The powder (5 g) was mixed with 0.15M NaCl (1:8, w/v) for 48 h at 40C, and filtered through 80-mesh grid. Subsequently, the filtrate was centrifuged at 9168×g for 30 minutes, and the supernatant was fractionally precipitated with ammonium sulfate at 10%-100% saturation, respectively. The four pellets were combined, dissolved in a minimal volume of water, and dialyzed against distilled water at 4°C (Yufang et al., 2010). 2.2.2. Determination of protein concentration. Determination of Protein Concentration. Bradford’s method (Bradford, 1976) was used for protein quantification, using bovine serum albumin (BSA) as the standard. 2.2.3. Hemagglutinating activity assay. Serial two-fold dilutions of the lectin solution in microtiter v-plates (25 μL) was mixed with 25 μL 2% human red blood cell suspension in

in phosphate-buffered saline. All of the sugar samples were mixed with an equal volume (25 μL) of a solution of the lectin. The mixture was allowed to stand for 30 minutes at room temperature and then mixed with 50 μL of a 2% human erythrocyte suspension. The known sugar which gives agglutination with blood suspension and no precipitate of red blood cell that means the tested lectin specific to that sugar type (Wang et al., 2000). 2.2.8. Effect of temperature on purified lectininduced hemagglutination. The effect of temperature on hemagglutinating activity of the purified lectin was examined as previously described (Wang et al.., 2003). A solution of the lectin was incubated at various temperatures for 30 minutes: 0°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C and 70°C. The tubes were then put on ice, and assay of hemagglutinating activity was then carried out. 2.2.9. Effect of pH on purified lectin-induced hemagglutination. The pH stability of the lectin was determined by incubation of the lectin (1 mg/mL) in buffers of different pH values ranging from pH 4.0–12.0 for 60 minutes. The pH of the lectin solution was adjusted to 7.0 by the addition of 0.1N HCl or 0.1N NaOH before hemagglutination activity was determined (Yufang et al., 2010). 2.2.10. Effect of chemical modification of amino acid residues on hemagglutinating activity. For serine modification, the lectin (100 μg) in 0.1mL of 50mM Tris-HCl buffer (pH 7.4) was incubated with 5mMphenylmethylsulfonyl fluoride (PMSF) at 27°C for 1 hour (Habeeb, 1966). Aliquots were removed at 15 minutes intervals.followed by determination of residual hemagglutinating activity. Lectin incubated without PMSF served as a control. Reduction of the thiol groups of white kidney beanslectin was carried out by incubating the lectin (100 μg) in 0.1mL of 50mM phosphate buffer (pH 8.0) with 0.1mM 5, 5-dithiobis- (2nitrobenzoic acid (DTNB) at 27°C for 1 hour. Aliquots were removed at different time intervals, followed by determination of residual hemagglutinating activity. Lectin incubated in the absence of DTNB served as a control (Fraenkel, 1957). For lysine modification, 0.5 mg of NaBH4 was added to the lectin (5 mg) in 2mL of 0.2M

sodium borate buffer (pH 9.0) at 4°C, followed by six aliquots (5 μL each) of 3.5% formaldehyde at 10 minutes intervals. Excess reagent was removed by ultrafiltration. Lectin incubated in the absence of sodium borohydride (NaBH4) served as a control (Mean & Feeney, 1968). Modification of tryptophan residues was carried out according to the method of Spande and Witkop (Spande & Witkop, 2006). The lectin was dissolved in NaOAc buffer (0.1 M, pH 5.0) to 1mg/mL. The modification was carried out at 20°C. Nbromosuccinimide (NBS) (10 μL, 10mM) was added every 5 minutes and then assay of hemagglutinating activity was carried out. Lectin incubated in the absence of NBS served as a control. RESULTS AND DISCUSSIONS Soaking Method showed spicfic activity about 7.1 titer/mg of white kidney beans. The protein concentration was calculated based on the regression equation (Figure 1), and results showed protein concentration about 2.74 mg/ml by precipitation by ammonium sulfate at 70% saturation rate. Purification of white kidney beans lectin show two fractions were obtained from ion exchange chromatography with DEAE cellulose (Figure 2) just the first peak showed hemagglutination activity about 45.5 titer/mg,so just the first peak concentrated and loaded on sephadex G-200 to gel filtration step which show only one peak with hemagglutination activity about 57.07 titer/mg (Figure 3), the volume obtained from peak of sephadex G-200 was concentrated and reloaded on sephadex G-200 to improved the purification step of lectin and the result of second loading of white kidney beans lectin on sephadex G200 is also one peak show specific activity as 64 titer/mg (table 2). The peak of second loading of gel filtration was dialyzed and concentrated, then stored at-20°C. The purified lectin (fraction I in Figure 3) formed a single band with a molecular mass of about 33 kDa in SDS-PAGE electrophoresis and three bands was showed in results of ione exchange electrophoresis (Figure 4), which confirmed the effectiveness of the purification method used and about 35.5 kDa in gel filtration method to determination molecular mass.

Figure 1. Standard curve of bovine serum albumin

Figure 2. Fractionation of the crude extract of Phaseolus vulgaris L. cv.white bean lectin by DEAE cellulose ionexchange chromatography equilibrated with 0.1M NaCl in 0.02M Tris-HCl (pH 8.0). The column was washed initially with the same buffer to remove proteins that were not specifically absorbed to the column (data not shown on the figure) then washed with buffer in which the NaCl concentration increased linearly from 0.1M to 1.0M. Tow peaks were obtained, among which peak I exhibited hemagglutinating activity. Column: 2.5 cm × 50 cm; flow rate: 0.3 mL/min.

Figure 3. Fractionation of peak I on a Sephadex G-200 column with 0.15M NaCl in 0.02M Tris-HCl (pH 8.0). Column: 2 cm × 50 cm; flow rate: 0.5 mL/min. Only the major peakv exhibited hemagglutinating activity

The hemagglutinating activity of purified white kidney beans lectin could not be inhibited by many of the simple sugars tested at 1–100 mM of D (+) glucose, D (+) galactose, D (+) fructose, D (+) lactose, (+) ribose, D (+) arabinose, L (+) maltose, D (+) sucrose and N-acetylglucoseamine, but the hemagglutinating activity inhibited with D (+) xylose and D (+) mannose as show in figure (Figure 5). The hemagglutinating activity was completely stable between 0°C and 50°C. Considerable loss in activity occurred at 60°C. Some activity was discernible at 60°C (Figure 6a). The lectin exhibited remarkable stability over the range of pH 4–10 and loss the activity at pH 11 (Figure 6b). Residues, respectively, did not play any important role in its hemagglutinating activity. However, 85% loss of hemagglutinating activity after NBS treatment was noted, whereas no

change in the control was detected. These results strongly suggest a considerable involvement of tryptophan residues in hemagglutinating activity, and stability of the lectin. NaBH4 treatment resulted in also 86% loss in hemagglutination activity suggesting partial involvement of lysine in the lectin activity. A concomitant drop in lectin activity was clearly seen upon modification of tryptophan residues. The effects of various types of chemical modifications on hemagglutinating activity of the purified lectin are summarized in Table1, DTNB, reductive methylation and PMSF treatments did not produce any alterations in the hemagglutinating activity of white kidney beans lectin, suggesting that cysteine, cystine and serine

Figure 4. (a) Molecular weight determination of purified Phaseolus vulgaris L. cv.white bean lectin in SDS-PAGE electrophoresis. Line A: purified lectin. Line D: protein ladder line E: molecular weights (b) purification of lectin. Line C: crude lectin. Line B: purified lectine by gel filtration.

Figure 5. Sugar specificity of Phaseolus vulgaris L. cv.white bean lectin image showed the agglutination of red blood cell with (Glucose galactose lactose ribose arabinose xylose mannose fructose maltose sucrose Nacetylglucoseamine, respectively) xylose and mannose mean the tested lectine not specific for that simple sugar.

Figure 6. (a) Effect of temperature on hemagglutinating activity of Phaseolus vulgaris L. cv.white bean lectin. (b) Effect of pH on hemagglutinating activity of Phaseolus vulgaris L. cv.white bean lectin.

Figure 7. Effect of chemical modification on hemagglutinating activity of Phaseolus vulgaris L. cv.white beans lectin.

possess. There are five such isolectins: L4, L3E1, L2E2, L1E3, and E4 (Yufang et al., 2010). Although there is striking homology between white kidney beans lectin and other Phaseolus lectins in N-terminal sequence, white kidney beans lectin exhibit of some simple sugar specificity. Simple sugars as D (+) glucose, D (+) galactose, D (+) fructose, D (+) lactose, (+) ribose, D (+) arabinose, L (+) maltose, D (+) sucrose and N-acetylglucoseamine are not able to inhibit the hemagglutinating activity of white kidney beans. White kidney beans lectin is fairly thermostable because its hemagglutinating activity is stable at temperatures up to 40°C, and is reduced only at 50°C. Interestingly, some activity remained even after heating at 60°C for 30 minutes. However, have been shown to lose activity beyond 50°C in a temperature-dependent manner (Laemmli, et al., 1973; Wang et al., 2003). The lectin shows remarkable pH stability, its activity being unaffected throughout the entire range of pH from 4 to 10. This is in contrast to lectin from Parkia javanica beans which is stable in pH 7–10 (Utarabhand & Akkayanont, 1995). Chemical modification studies were carried out to investigate the role of specific amino acids in the hemagglutinating activity of white kidney beans lectin. The results disclose that tryptophan and lysine are important to the hemagglutinating activity, the contribution of tryptophan being more important. Previous studies have reported that lysine, tyrosine, and tryptophan (e.g., in Dolichos lab-lab bean) (Nadimpalli, 1999), or tryptophan alone are indispensable for the hemagglutinating activity of some legume lectins (Das, 1995). Specific amino acids may be involved in either direct interaction with the sugar or may have a role in maintaining conformation of the sugar binding pocket, and hence contribute to the hemagglutinating activity of lectins (Ba? i? imiev et al., 2007).

Figure 8. Purification table

Lectins possess many bioactivities that have important practical applications, but the high price resulting from the low extraction rate restricts practical application of lectins. In this study, a plant lectin has been purified by threestep chromatography from seeds of the white kidney beans. The homogeneity of the white kidney beans lectin preparation was evidenced by the presence of a single band in SDS-PAGE. The results of SDS-PAGE and gel filtration chromatography together revealed that the lectin exists as a monomer of one subunit. The molecularmass and monomeric nature of Phaseolus vulgaris L. cv.white lectin are similar to those of Anasazi bean lectin and most of the other Phaseolus lectins (Tsang et al., 2001; Reynoso et al., 2003). On the other hand, it differs from a tetrameric 115–120 kDa lectin from tepary bean (Phaseolus acutifolius) (Reynoso et al., 2003). Phaseolus acutifolius var. latifolius lectin from which consists of four subunits of 21 kDa molecular mass (Vargas et al., 1987), and a tetrameric 94 kDa immunosuppressive lectin isolated from seeds of Phaseolus vulgaris L. cv. Cacahuate. (Vargas et al., 1993). Lectins from some cultivars of Phaseolus vulgaris are oligomeric (Felsted et al., 1977), whereas Phaseolus vulgaris bean lectin is dimeric. Isolectins are absent in Phaseolus vulgaris L. cv.white beans but present in some cultivars of P. vulgaris such as red kidney bean (Felsted et al., 1977; Leavitt et al., 1977). The isolectins differ from one another by the number of erythrocytereactive (E) subunits and lymphotcyte-reactive (L) subunits that they

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Scientific Bulletin. Series F. Biotechnologies, Vol. XVII, 2013 ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

EXPERIMENTAL RESULTS ABOUT POTATO CALLUS INDUCTION Andreea NISTOR, Mihaela CIOLOCA, Nicoleta CHIRU, Monica POPA NIRDPSB BraƔov, Street Fundaturii no 2, Postal code: 500470, BraƔov, Romania Corresponding author email: [emailprotected] Abstract The callus is an unorganized mass of parenchymal proliferate cells that through cultivation, forming groups of meristematic cells, elements of leading system, pigmented cells, etc.. Using other explants than meristem, for regeneration neoplantlets require mandatory completion of a stage of callus culture. To obtain callus is need an agarose to support the cellular mass in growth. In 2012, at Brasov was fitted trifactorial experience, in which two clones of Christian variety were studied, 6 media for callus induction and 2 explants sources consisting of leaf disc and petiole segment. The following results were obtained: medium, explant source (foliar disc, petiole segment) and variety have different influences on callus proliferation. The callus explants responded better to the foliar disk (72.5%) than petiole segments (40%). Media containing 3 mg / l 2,4-D and 3 mg / l BAP x 3 mg / l 2,4-D favored callus induction rate of 90%. Differences obtained by using BAP citokinine are statistically assured, very significant, negative compared to 2,4-D auxine (2mg/l- concentration regarded as control), by -3.5 explants / that induced callus. The callus which was obtained will be used for plantlets regeneration and to identify any somaclonal variation. Keywords: leaf disc, petiole segment, callus.

Somaclonal variation, a common phenomenon in tissue culture includes all variations and derived from all tissue culture (Skirvin et al., 1993). Somaclonal variation is also called tissue variation or culture-induced (Kaeppler, et al., 2000). Somaclonal variation control represents a challenge (Amirato, 1991). Variations had as result: changes in structure and / or number of chromosomes, a gene changes as a result of structural change in chromosome (Rao et al., 1992; Kaeppler, et al., 2000). Evans & Sharp (1988) reported four critical variables for somaclonal variation: genotype, explant origin, period of cultivation and culture conditions. Plant genotype can have significant effects on somaclones regeneration. These effects are very obvious for potatoes: differences are observed in the number of plants regenerated from cultivars grown under identical conditions (Gunn & Shepard, 1981). Explant source is considered most essential variable in somaclonal variation. Because explants may present different stages of regeneration, selection procedures may differs between different types of explants (De Jong & Custers, 1986).

INTRODUCTION Callus is an unorganized mass by proliferate parenchymal cells that through cultivation, form centre of meristematic cells, elements of system leading, pigmented cells (Rodica Pop, 2008). Callus is a particular formation consisting by an undetermined mass of cell holding a uniform histological structure. Usually, when it is young, undifferentiated cells possess, actively dividing. Friable as possible callus induction its principal purpose when aiming is initiation of cell culture and from this a culture of protoplast. Callus growth is considered to be indefinite, because it can be multiplied and grown indefinitely when periodically is done subculturing, respectively fragmentation of it. Callus is often associated with somaclonal variability, variations derived from callus being called caliclones (Skirvin et al., 1976, quoted by Skirvin, 1994). At the beginning cultivars obtained „in vitro”, regardless of species, were called caliclones. Variation is associated, in caliclones case with direct regeneration from callus or cell suspension and not with micropropagation or meristem culture (Karp, 1989).

of 16 hours light and 8 hours dark at a temperature of 200C. Observations concerning callus growth were made weekly. Also, all sampling operations, inoculation subculturing were performed under sterile conditions in a laminar flow hood. The biological material used in the production experiences of clonal variability in laboratory conditions, consisted of two clones of the same variety (Christian) from NIRDPSB Brasov. All analyzes were performed in the laboratory plant tissue cultures from NIRDPSB Brasov. Other materials used in the experiments: -to initiate callus culture, the biological material used was represented by two types of explants (leaf discs of 1 cm2 and petiole segments of 1.5-2 cm). Explant source: When intended to make somaclonal variability for a new species or cultivar is better to use several types of explants and compare descendants from each. Not to all types of explants are supposed the same capacity for manifestation of variation. In general the variation is more difficult to observe in preformed shoots (derived from axillary buds, shoot tips and meristems), unless meristem explants were not preformed, such as leaves, roots or protoplasts (Rodica Pop, 2008). Regenerates obtained from organized explants with preformed meristems are genetically stable. For many plant species, cell differentiation is accompanied by qualitative and quantitative changes of genomic DNA. Therefore, when used as explants for callus obtained segments from leaf or root, cells begin to divide (Rodica Pop, 2008). The culture media used: To obtain callus is need by a medium with agarose to support cell mass in growth. Periodically, callus must be fragmented and subcultivated in order not enter into senescence. Callus formation can be induced and it proliferates, especially on culture media with 2,4-D. Also, other growth regulators: auxine or citokinine who are in high concentrations in the culture medium can generate callus. Vegetal explants can be kept alive by detaching their mother's body by growing and raising on aseptic media with a complex chemical composition. The success in vitro cultivation of

MATERIALS AND METHODS In laboratory Plant Tissue Cultures of NIRDPSB Brasov was assembled an experience that followed the influence of growth regulators and explants of Christian variety (two clones). The experience was by type 2x2x6, made by combining three experimental factors, placed by Latin rectangle method, the number of studied variants was 24, set in three repetitions, and on each experimental variant were inoculated 5 explants. - Experimental factor A-clone with two graduations: - a1- Christian Cl2 - a2- Christian CHR 01 ROU - Experimental factor B - explants, with two graduations: - b1- leaf discs; - b2- petiole segments. - Experimental factor C - nutrition environment, with six graduations: - c1 - MS medium and citokinine BAP (2 mg/l); - c2 - MS medium and citokinine BAP (3 mg/l); - c3 - MS medium and auxine 2,4-D (2 mg/l); - c4 - MS medium and auxine 2,4-D (3 mg/l); - c5 - MS medium and BAP x 2,4-D (2 mg/l x 2 mg/l); - c6 - MS medium and BAP x 2,4-D (3 mg/l x 3 mg/l). The proposed objective of this research is to determine the influence of hormonal composition of the culture medium on callus induction of different types of potato explants grown in vitro. Experience in which six medium were used to induce callus was organized in the laboratory on culture vessels, aiming on callus process from leaf discs and petiole segments. Experimental conditions: The experience was mounted in the laboratory using conditions required by „in vitro'' technology; experimental conditions were those specific to growth chamber of plantlets, provided in the working protocol, sterilization of culture vessels was performed in a drying chamber at 1800C and culture media was sterilized by autoclaving at 1210C for 20 minutes at pressure of 1.25 atmospheres. Cultures were transferred to growth chamber under conditions of darkness; after crossing this period light regime is 4000 lux, with a period

hiploclorit - 10 minutes, after which explants are rinsed with doubly distilled water; culture media preparing; sterilization of vessels and growing medium; preparing of plant material for explants sizing, inoculation, incubation either at dark or light, usually at 240C, regular observation and passing callus fragments (about 4-6 weeks) in fresh medium. Explants were represented by foliar disc (1 cm2) and petiole segments of 1.5 - 2 cm in length (Fig. 1), taken from 4 potato genotypes. After performing disinfection (Figure2), biological material was transferred to Erlenmeyer flask (Fig. 3) on a basic medium: MurashigeSkoog (1962) enriched with vitamins, 20 g / l sucrose, 9 g / l agar and different growth regulators, different concentrations.

vegetal explants depends very much on achievement of nutritional composition that best suits with vital requirements of cultured tissues. One of the most common culture media used Murashige-Skoog is (MS) (Rodica Pop, 2008). In our experience, basic medium used was Murashige-Skoog (1962) supplemented with different growth regulators. As a source of carbohydrates sucrose was used at a concentration of 2% and that phytoagar gelling agent was used in concentration of 0.9%. Growth regulators used: Growth regulators are organic compounds other than nutritive substances that in small amounts stimulate, inhibit or modify physiological processes in plants (Rodica Pop, 2008). Growth regulators, particularly 2,4-D (dichlorophenoxyacetic acid 2.4) and benzylaminopurine (BAP), are involved in inducing variability but their direct relation with this phenomenon is still discussed. Auxins Auxins are natural compounds that, in small doses, directly or indirectly, can stimulate the growth and development of plants, or forming of vegetative organs and regenerative. Between auxines, 2,4-dichlorophenoxyacetic acid (2,4-D), in concentrations ranging from 0.5 to 2.0 mg / l, has proven very efficient in callus induction and maintenance of various types of somatic tissues (Tang Ɣi Mullins, 1990; Castillo Ɣi colab., 1998, quotation by Rodica Pop, 2008). Citokinins Citochinins are substituted with a purine nucleus. They stimulate cell division and have important role in stimulating vegetal cells mature unmeristematic (Cachiԑĉ, 2000). Caulogenesis is stimulated by the presence in culture substrate of a particular ratio between auxine and citokinine. Specific technology applied in experiments From existing material from greenhouse during plant vegetation, aerial plant samples (leaves and stems) were harvested manually which constituted the biological material under experimentation. It was followed the influence of genotype on callus induction and influence of different medium variables used It was respected the following stages: vegetative material sterilization it requires a few minutes washing, sterilization with sodium

Figure 1. Fragment of leaf and petiole segment

Figure 2. Sterilization of explants

Figure 3. Inoculated explants for callus induction

After incubation of leaf and petiole segment in the dark for two weeks, were registered some features. These include:- Type of callus and callus color: After callus was initiated (Fig. 4), at about 4-6 weeks, from each recipient was fragmented into three segments and subcultivat on the same type of medium (Fig. 5). Callus was then exposed to UV radiation (Fig. 6), operation repeated three times at an interval of one week.

Table 1. Effects of substances 2,4-D and BAP on callus induction Growth regulator (mg/l) 2,4-D BAP 0 2 0 3 2 0 3 0 2 2 3 3

Figure 4. Initiated callus

Callus texture Color callus friable friable strong strong

brownish brownish green green

Table 2. Influence of variety and medium in the callus culture Explant Callus induction/variety source, b Culture (clones) (%) medium, CHRISTIAN CHRISTIAN c Cl 2 CHR 01 ROU c3 100 40 80 100 Fragmen c4 t of leaf c5 60 20 c6 100 80 Average 85 60 Segment 40 40 of c4 petiole Note: c3= Medium MS – 2 mg/l 2,4-D c4= Medium MS – 3 mg/l 2,4-D c5= Medium MS - 2 mg/l BAPx2 mg/l 2,4-D c6= Medium MS - 3 mg/l BAPx3 mg/l 2,4-D

Figure 5. Recipents with callus fragmented

Figure 6. The exposure callus on UV radiation

RESULTS AND DISCUSSIONS Analyzing Table 1, we see that when using citokinine BAP, callus induction did not take place. In the case of auxine 2,4-D callus was brittle, with a brownish color, and if the used medium contained both growth regulators, callus was hard, its color being green. Clones explants of Christian variety were cultured on MS medium containing different concentrations of 2,4-D, BAP or in combination. Explants which produced callus were analyzed at 6 weeks, and the results show that there is a wide variation in the percentage of explants which initiated callus, callus texture, color callus, it depends on the culture. It was found a positive response of leaf fragments and auxine 2,4-D and its combination with citokinine BAP in callus proliferation. Reaction manifested callus after 8 weeks after inoculation. Medium did not contain 2,4-D (which contained the BAP 2mg, 3mg respectively media c1 and c2) did not produce callus induction (0%). Leaf segments and internode segments explants were cultured on MS medium containing different concentrations of 2,4-D, BAP and combinations thereof.

Average % variety Christian 70 90 40 90 72,5 40

From Table 2, it can be noted the superiority of the Christian variety clone 2 of the callus, in case of disc explant foliar (85%). About the influence of culture medium on callus, it appears that for clone 2, using media containing: 2.0 mg / l 2,4-D, and 3 mg / l BAP x 3 mg / l 2.4 -D is achieved highest percentage of callus (100%), followed by media containing 3 mg / l 2,4-D and 2 mg / l BAP x 2 mg / l 2,4D (80% and 60%). For CHR 01 ROU clone, of Christian variety, the best results were registered in callus induction on c4 medium using, leading to callus proliferation at 100%. On the variety, the best results were registered using c4 and c6 the media, achieving a rate of 90%. In case of using petiole segment, the callus induction had low intensity of only 40% (average of two clones), only at variants that experienced the quantity of 3 mg 2,4-D/l; the remaining samples were affected by infections. Statistical analysis of the influence of growth regulators on callus induction shows that all

data derived from experimentation showed statistically differences. Considering auxine 2,4-D (the quantity of 2mg / l) control, the results showed significant increases in the number of explants (1 explant) for citokinine BAP (in the case of both concentrations) decreases of explants numbers are significant of -3.5 explants; the combination of 2,4-D * BAP (2mg / l) are distinct differences significant negative (-1.5 explants) and for 2,4D * BAP (3 mg / l) increases of explants number are significantly positive (1explant), resulting that induction of callus was directly influenced by growth regulator and quantity of this (Table 3).

considered control), -3.5 explants / that induced callus. Medium that contained 3 mg / l 2,4-D * BAP determined obtaining an increase in the number of expants that induced callus (1 explant). REFERENCES AMMIRATO, P.V., 1991: Embriogênese somática e semente sintética. In: CROCOMO, O.J., SHARP, W.R., MELO, M. (Eds.) Biotecnologia para a produção vegetal. Piracicaba: CEBTEC/FEALQ, p.189-221. BADEA ELENA MARCELA, DANIELA SNDULESCU, 2001, Biotehnologii vegetale, Fundaԑia Biotech, BucureƔti. CACHIԐ– COSMA, DORINA, 1984: Culturi de celule Ɣi ԑesuturi vegetale,aplicaԑii în agriculturĉ, Editura Ceres, BucureƔti. CACHIԐ C. DORINA, 2000: Biotehnologie vegetalĉ. Vol. I Baze teoretice Ɣi practice, Editura Mira Design, Sibiu. DE JONG, J; CUSTERS, J.B.M, 1986: Induced changes and flowering of chrysanthemun after irradiation and in vitro culture of pedicels and petals epidermis. Euphytica, v.35, p.137-148. DE SILVA M., W. SENERATH, K. SUGATHADASA, 2006, In vitro callus production of Pterocarpus santalinus L. (Red Sandal) through nodal cuttings shoot tips and leaf discs, Forestry and Environment Symposium, Sri Lanka: In vitro callus production. RAO, I.M.; ROCA, W.M.; AYARZA, M.A.; TABARES, E.; GARCIA, R., 1992: Somaclonal variation in plant adaptation to acid soil in the tropical forage legume Stylosanthes guianensis. Plant and Soil, v.146, p.21-30. KARP, A., 1989. Can genetic instability be controlled in plant tissue culture? In: International Association for Plant Tissue Culture (IAPTC) Newsletter no. 58, pp. 211 KAEPPLER, S.M.; KAEPLLER, H.F.; RHEE, Y., 2000: Epigenetic aspects of somaclonal variation in plants. Plant Molecular Biology, v.43, p.179.188. GUNN, R.E.; SHEPARD, J.F., 1981: Regeneration of plants from mesophyll-derived protoplasts of British potato (Solanum tuberosum L.) cultivars. Plant Science Letters, v.22, p.97-101. POP RODICA, 2008: Studiul variabilitĉԑii somaclonale la viԑa de vie cu ajutorul markerilor moleculari, ISBN 978-973-88929-6-5, Bioflux, Cluj-Napoca. SKIRVIN, R.M.; NORTON, M.; MCPHEETERS, K.D., 1993: Somaclonal variation: has it proved useful for plant improvement? Acta Horticulturae, v.336, p.333340. SKIRVIN R.M., MC PHEETERS KD and NORTON M., 1994: Source and frequency of somaclonal variation. Hort Science 29: 1232-1237. XU X., J. LU, Z. REN, H. WANG, S. LEONG, 2005: Callus induction and somatic embryogenesis in muscadine and seedless bunch grapes (Vitis) from immature ovule culture, Proc. Fla. State Hort, Soc. 118, pag. 260-262.

Table 3. Influence of growth regulators used in the callus induction Number of Differences Growth Procent explants (explants Signification regulator (%) that induced number) used callus 2,4-D (2 mg) 3,5 100.00 (ct) 2,4-D 4,5 128.57 1 * (3 mg) BAP 0 0 -3.5 ooo (2 mg) BAP 0 0 -3.5 ooo (3 mg) 2,4D*BAP 2 57.14 -1.5 oo (2 mg) 2,4D*BAP 4,5 128.57 1 * (3 mg) DL 5% =0.80 (explants) DL 1% =1.13 (explants) DL 0,1% =1.64 (explants)

CONCLUSIONS Medium, explant surse (foliar disc, petiol segmente) and variety have different influences in callus proliferation. The callus induction responded better to explants from foliar disk (72.5%) than petiole segments (40%). C4 (3 mg / l 2,4-D) and c6 (3 mg / l BAP x 3 mg / l 2,4-D) media favored callus induction in proportion of 90 Differences obtained using BAP citokinine are statistically very significant negative towards auxine, 2,4-D (concentration of 2mg/l-

BIOTECHNOLOGY IN VETERINARY MEDICINE

Scientific Bulletin. Series F. Biotechnologies, Vol. XVII, 2013 ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

A STOCHASTIC EPIDEMIC MODELFOR DYNAMIC OF INFECTIOUS DISEASES Mioara VARGA Department of Mathematics, Physics and Terrestrial Measurements, University of Agronomic Sciences and Veterinary Medicine of Bucharest, 59 Mărăúti Blvd., 011464, Bucharest, Romania, Tel.: +40 723 256 154 Corresponding author: [emailprotected] Abstract After a short introduction of the deterministic SIS and SIR models, we present three types of stochastic epidemic models: discrete times Markov chain (DTMC) model, continuous times Markov chain (CTMC) model and stochastic differential equation (SDE) model. We discuss a stochastic epidemic model for dynamic of infectious diseases with variable population size, one which varies according to some population growth laws. Finally, we compare the stochastic differential equation of a SIS epidemic model having a constant population size with a stochastic differential equation having a variable population size. Keywords: model basic reproduction number, discrete times Markov chain (DTMC) model, continuous times Markov chain (CTMC) model and stochastic differential equation (SDE) model

a) S - susceptible: individuals who don’t have immunity to infectious agents and who have been exposed to the disease contact; b) I - infected: individuals who have been recently infected and who transmit the infection to susceptible individuals who are in contact with them; c) R- removed: individuals who are immune to the infection and who are not contaminated even if they come in contact with those categories a), b). An example of SIR Epidemic Model is represented by childhood diseases. If we denote S t , I t , R t , the number of

INTRODUCTION The beginnings of mathematical applications in epidemiology relate to the “smallpox” model, by Daniel Bernoulli (1760) and a primary theory was developed between 1900-1935. The research continued and recent progresses recorded in our days, a good example being "The SARS" (Severe Acute Respiratory Syndrome) epidemic (20022003). Establishing forecasts on the evolution of infectious disease and the comparison of different control methods maintain at a high level the interest of researchers in mathematical modeling of epidemiology.

individuals in each category in relation to time, the total population will be:

MATERIAL AND METHOD

N t S t I t R t (1)

1. The deterministic SIS and SIR Epidemic Models Most epidemic models are based on the dividing of target population into a small number of compartments, each of them containing members that are identical in terms of their relationship towards a certain disease. In a SIR model, the population is divided into three groups:

We observe that S t , I t , R t N and for a sufficiently large volume of the total population, they are continuous random variables, their variation is characterized by the following system of differential equations:

E dS ° dt N S I b I R ° ° dI E S I b J I (2) ® ° dt N dR ° J I bR ° dt ¯ where E ! 0 is the transmission rate, J ! 0 is the recover rate and b t 0 is the birth rate.

In a SIS Epidemic Model, a susceptible individual, after contact with an infected person in his turn becomes infected or infectious, but he doesn’t develop immunity, i.e., after recovery, he returns in the susceptible category, so in such a model, the population is divided only into two categories: Ssusceptible and I-infectious. An example of a SIS Epidemic Model is represented sexually transmitted diseases (STDs). With the same notations as in the SIR epidemic model and if we assume that all individuals are born susceptible and we don’t find the deaths caused by that epidemic, the dynamics of the SIS epidemic model is described by the following system of E dS °° dt N S I b J I differential equations: ® ° dI E S I b J I °¯ dt N (4) In this case, the population size is: N t S t I t (5)

The initial solution of the system (1) is:

S 0 ! 0, I 0 ! 0, R 0 t 0 , S 0 I 0 R 0 where N 0

(3)

N is the population volume at the

initial moment, when it downgraded the epidemic. Observation: If we note c , the death rate, we assume that in a SIR model, b c i.e. the birth rate is equal with the death rate, then the population volume is constant with respect to time, and

dN dt

Using

reproduction

the

E bJ

basic

Observation: If we assume as in the case of the SIR model b c , then in a SIS epidemic model the population size is

number,

, which represents the number of

constant with respect to time, i.e.

secondary infections caused by some infected individuals in the entire susceptible population (the

deaths and recovery rate ) the authorsJ. MenaLorca, H.W. Hethcote [6] characterize the system solution (2), in the following theorem:

Theorem 2 If S t , I t is the system solution (4), then: a) for R0 d 1,

Theorem 1 If S t , I t , R t is the system solution (2), then:

R0 d 1, lim I t 0

R0 ! 1, lim ( S t , I t , R t )

t of

1 · J N ¨¨ ¨1 ¸ , J R b R 0 ¹ bJ © © 0 ,

0 and R0

N , 0

(diseasefor

§N § 1 ·· ¨¨ R , N ¨ 1 R ¸ ¸¸ 0 ¹¹ © © 0

(endemic equilibrium) Observation: The interpretation of the first statements of the theorem is that if the number of secondary infections generated by the infected individuals is less than 1, then, I t o 0 si S t o N .

§ 1 ·· ¨1 ¸ ¸¸ R 0 ¹¹ ©

(endemic equilibrium) c) for b

t of

R0 ! 1, lim ( S t , I t )

equilibrium) b) for § N b N §

lim S t , I t

free equilibrium) b)

(disease-free

t of

0 . The

following theorem [6] characterizes the system dynamics model of differential equations (4) in relation to the variation of the basic reproduction number:

1 is the interval of infection relative to fraction bJ

a) for

dN dt

S 0 ! 1 , there is an N

initial increase of the number of infected cases I t and if R0

S 0 d 1 , then N

2. The Stochastic Epidemic Models In this section we present three types of stochastic modeling processes: (1) a discrete time Markov chain (DTMC) model, (2) a continuous time

I t monotone decreasing.

Markov chain (CTMC) model, and (3) a stochastic differential equation (SDE) model. The differences between these processes refer to time and to the set of states. In DTMC model, the time and the state are random discrete variables. In a CTMC model, time is continuous, but the state variable is discrete, finally, the SDE model is based on a diffusion process, where both the time and the state variables are continuous. One of the most important differences between the deterministic and stochastic epidemic models is their asymptotic dynamics. Eventually stochastic solutions (sample paths) converge to the disease-free state even though the corresponding deterministic solution converges to an endemic equilibrium.

Observation: If 't is a sufficiently small interval, the process I t can move from the state

i o i 1 , i o i 1 or i o i , i.e. the number of infected people can grow with one, or can be a birth, a death or a cure. In this case, the transition probabilities verify the relationships:

pij 't

The relations which are above can be interpreted as follows: x probability of the occurrence of another infected person in an interval 't ,

representing the number of individuals susceptible, infected, respectively immune to the time t, in relation to a specific infectious agent. In a DTMC epidemic model, t T ^0, 't , 2't ,...` and the discrete

( i o i 1 ) is

Further, we refer to a SIS Epidemic Model. the population size is We consider that N constant and I t is independent random variable.

N I t

process I t

and

the

has the probability function

So that the population size remains constant as with the deterministic case, b c meaning the birth rate must be equal to the death rate.

(6)

i 1

p t

p t , p t ,..., p t 0

Observation If a SIS epidemic model (DTMC) is seen as a process of birth and death, then the system (9) can be written more simple:

the

probability vector associated of stochastic process I t , then the process has the Markov

't ,

ª Ei N i º 1 « b J i » 't . N ¬ ¼

stochastic

¦ p t

x probability that not occur any change in state 't is ( i o i )in an interval

P I t i , i 0, N t T ,

pi t

Ei N i

x probability of the occurrence of death or recover ( i o i 1 ) in an interval 't is b J i 't ,

variables S t , I t , R t ^0,1,...N ` .

S t

(9)

2.1. Discrete Time Markov Chain Epidemic Models (DTMC) Let on consider S t , I t , R t random variables

Then

Ei N i 't , j i 1 ° N ° b J i 't , j i 1 °° ® °1 ª E i N i b J i º 't , j » ° «¬ N ¼ ° 0, j z i 1, i, i 1 ¯°

property:

P I t 't I t

P I t 't I 0 , I 't ,...I t

pij 't

(7)

p ji t 't , t

j is given by relationship:

P I t 't

j I t i

(10) with b(i ) 't it noted the probability of new infections and the probability of death or recover d (i) 't . If we apply the previous transition probabilities the Markov property, then pi t 't can be expressed in terms of

i.e. the process state (the number of infected individuals) at the moment t 't , depends only on the process state at the moment t. The probability of transition from the state I t i , to the state I t 't

b(i ) 't , j i 1 ° d (i ) 't , j i 1 ° ® °1 >b(i ) 't d (i ) 't @ , j °¯ 0, j z i 1, i, i 1

(8)

probabilities at the time t :

pi t 't

pi 1 t b i 1 't

pi 1 t d i 1 't pi t 1 >b(i ) 't d (i ) 't @ (11) where

Ei N i

i 1, N , b i

, d i

b J i .

(16) If we apply the Markov property to the previous transition probability and given that P I 0 i0 1 , then pi t 't can be

2.2 Continuous Time Markov Chain Epidemic Models (CTMC) In a CTMC process, time is a continuous random t > 0, f and variable,

S t , I t , R t ^0,1,..., N `

are

pi t 't

2.3. The Stochastic Differential Equations Epidemic Models (SDE) In a stochastic SDE epidemic model, time is a continuous random variable with T > 0, f and S t , I t , R t are also

p t , p t ,..., p t

p t

(12) with pi t

P I t i , i 0, N

continuous random variables, with the state-space interval > 0, N @ . Further we will stop to a SIS

The process has the Markov property:

P I tn 1 I t0 ,...I tn

stochastic epidemic model. The stochastic process I t t 0,f represents the number of individuals

P I tn 1 I tn ,

(13)

0 d t0 ... tn tn1

t of

³ p x, t dx . a

The process I t

t> 0, f

P I t d y I t n

n 1

(17) 0 d t0 ... tn1 tn T , and the transition

(14)

Ei N i ' t R 't , j i 1 ° N ° b J i 't R 't , j i 1 °° ® ª Ei N i b J i º 't R 't , j °1 « » ° ¬ N ¼ ° R 't , j z i 1, i, i 1 °¯

has the Markov property:

P I tn d y I t0 , I t1 ,...I tn 1

The infinitesimal transition probabilities are defined as:

p ji 't

with the property :

P a d I t d b

on the state of the process at the time tn . If in a DTMC, the transition probability refers to a short period of time 't , in the transition probabilities related CTMC process is included the term R 't ,

R 't

affected with respect to time. The random variable I t has the probability density p x, t and

The previous relationship indicates that the transition probability at the time tn 1 depends only

lim

pi1 t b i 1 't pi1 t d i 1 't

pi t 1 >b(i) 't d (i) 't @ R 't

discrete

expressed in terms of probabilities at the time t :

random variables. Further we characterize a CTMC SIS epidemic model. The vector of probability functions associated to stochastic process I t t 0,f is

notations as in the case of a DTMC process, the formulas (15) become: b(i ) 't R 't , j i 1 ° d (i ) 't R 't , j i 1 ° p ji 't ® 1 ( b i ) 't d (i ) 't @ R 't , j i > ° ° R 't , j z i 1, i, i 1 ¯

probability density is

p y, t 't; x, t I t

x , I t 't

(18) In the paper [1], it is shown that a construction of SDE SIS model epidemic, starting from the CTMC SIS epidemic model.

(15) As 't is sufficiently small, there are three possibilities for mood swings: i o i 1 , i o i 1 or i o i . With the same

RESULTS AND DISCUSSIONS We assume that N - population size is not constant, but it varies in relation to the law of population growth. Formulation of an epidemic model requires

that the birth and death rates, which depend on population size. We suppose that the birth rate is:

O N b N

(19)

and the death rate:

N2 (20) k where k ! 0 is the carrying capacity. Then,the

P N b

number N checks differential equation:

dN dt

§ ©

O N P N bN ¨1

N· ¸ k ¹

(21)

According to [2] there are many forms of birth rates and death choice, depending on population dynamics that will be modeled. If we assume that the population size checks the differential equation (21), then a deterministic SIS epidemic model can be characterized by the system of differential equations:

dS °° dt ® ° °¯

S O N P N NE S I b J I N E dI I P N S I J I dt N N (22)

with S 0 ! 0 and I 0 ! 0 . The previous system solution depends on the basic reproduction number R0

E bJ

Next, we formulate a stochastic model of type EDS SIS solution and we compare it with the deterministic model [1], [2]. Let us consider S t , I t variables representing the number of

CONCLUSIONS In many cases these three stochastic formulations generate similar results, if the time step 't is small [2]. There are numerical advantages in applying the discrete time approximations (DTMC model) in that the discrete simulations generally have a shorter computational time than the CTMC model. Mode and Sleeman [7] discuss some computational methods in stochastic processes in epidemiology. The most important consideration in modeling, however, is to choose a model that best represents the demographics and epidemiology of the population being modeled. In the future we plan to continue studying to application of stochastic modeling in epidemiology to determine the final number of individuals of a population affected by an infectious agent but also for estimating the duration of an epidemic

individuals susceptible, infected respectively at and time t. Obviously S t I t N t

S t , I t > 0, f . If we apply the same method as for the model SDE SIS epidemic model, we obtain the system of differential equations In [4] Brauer, F., Driessche P present a graph of a SDE SIS epidemic model (a) with constant population size, N = 100 and (b) with variable population size, N(t). The parameter values are ȕ = 1, Ȗ = 0.25 = b, K = 100, and R0 2

Brauer, F., Driessche P.2008.Mathematical Epidemiology, Springer, 81-120. Isham, V., 1991.Assessing the variability of stochastic epidemics. Math. Biosci., 209–224. J. Mena-Lorca, H.W. Hethcote, 1992. Dynamic models of infectious diseases as regulators of population size. J. Math. Biol., 693–716 . Mode, C. J., Sleeman, C. K.2000: Stochastic Processes in Epidemiology. HIV/AIDS, Other Infectious Diseases and Computers. World Scientific, Singapore.

REFERENCES Allen, E. J. 1999. Stochastic differential equations and persistence time for two interacting populations.Dyn. Contin. Discrete Impulsive Syst., 271–281. Allen, L. J. S., Allen, E. J, 2003. A comparison of three different stochastic population models with regard to persistence time. Theor. Popul. Biol., 439–449. Allen, L. J. S., Burgin, A. M, 2000. Comparison of deterministic and stochastic SIS and SIR models in discrete time. Math. Biosci., 1–33.

Scientific Bulletin. Series F. Biotechnologies, Vol. XVII, 2013 ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

CHLAMYDIA PSITTACIIN THE PARROTS, PIGEONS AND CANARIES IN THE CITY OF TIRANA Gëzime SHEHU1, Kristaq BERXHOLI2, Zaçe MALAJ2, Luljeta QAFMOLLA3, Ymer ELEZI4 1

Department of Agriculture, district, Tirana, Albania University of Agriculture, district, Tirana, Albania 3 Institute of Food Safety and Veterinary, district, Tirana, Albania 4 Researcher of ornamental birds, district, Tiranë, Albania 2

Corresponding author email: [emailprotected] Abstract The growth of decorative birds in Albania in the recent years has brought an increased frequency of chlamydial infection not only in the flocks of birds but, also to humans. According to the data from the Public Health Institute (PHI) in Tirana, is it has been observed an increase in the vulnerability of people, mainly in to those who are in the young age who have been in contact with decorative birds. This study, the first of its kind conducted in Albania, was based on a serological control of 573 blood samples which were taken from pigeons, parrots and canaries in Tirana, Albania. Sampling was carried out in all four seasons of the year 2011 and their control was carried out by the Institute of Veterinary Food Safety (FSV) Tirana. In order to check them it was used an indirect immunofluorescence test (IFT), combined with chlamydia’s isolation in chicken egg embryo cells, as a comparative method, in which there were identified at least 51 cases with Ch. psittaci. Relative specificity of fluorescent antibody’s test in the serum was approximately 95.3% and the relative sensitivity was about 60.3%. Results of this study, which as mentioned above were conducted for the first time in Albania, showed that immunofluorescence tests performed using diagnostic kits of Medical servicce-2000, combined with primary isolation of chlamydia in embryo of chicken eggs,were very specific and very useful for the identification of useful option for veterinary service. Keywords: fluorescent antibody, immunodepression, immunofluorescence overhead, relative sensitivity seropositivity.

1993) and the presence of other infections in an interaction. Psittacosis’ soft “explosion’s“ can often go unnoticed because there are no clear symptoms. The most obvious are those of airways and diarrhea (Avian Disease Manual, 1983). In adult wild birds,C. psittaci is often seen without clinical signs and they can serve as asymptomatic carriers. The infection can develop in the acute form, sub-acute, or it can be chronic. In the acute form the disease can cause severe damages, which can be fatal, especially for some species, while at younger birds, it appears to be highly sensitive. The most typical sings are seen on young birds, which often appear weak, showing anorexia, they faint, lie in a special position, from their eyes and nose there is a purulent flow, they usually are contracted and stay with disheveled feathers (Gerlach 1986). Diagnostic methods used for detection of chlamydic infection in the birds are numerous. For specificity, sensitivity, speed and relativity

INTRODUCTION

Cage birds parrots, canaries, and pigeons showed different sensitivity to C.psittaci, causing their injuries, but also a risk for the spread of this disease with zoonotic nature. (Barnes, RC: 1989 and Pospisil, L., et al: 1996). Even in Albania from year to year, this category of birds has been increasing, serving as a potential risk in to the spread of the chlamydial infection not only in flocks of birds, but also in humans. (Pospisil, L., et al: 1996). Evidence shows that during the period 20012011 there have been reported about 635 cases of individuals infected with chlamydia (PHI: 2011) who mostly were pople in their young age or individuals involved decorative bird breeding. Expressed clinical signs were depending on chlamydia’s pathogenicity, type, race, and physiological condition, age of the bird, route and time frame of exposure, stressful factors, immunodepresive andsituation of the birds (Lublin, A., et al:

(Andersen, AA, 2008). The IFT control procedure was based on the principle of reaction of 573 blood samples, using IgG Antibody Kit, imported by Medical Servicce2000. The separation of serum was made using the usual metthod, by following rigorously all steps to preserve the kit, dilution, incubation, testing and control of the material prepared. At the end the reading of the small droplet shaped spots was made using a 400x magnification for each tile, and then they were compared with the visual intensity of basic troops, shown in the positive control well and negative.The tiles were stored in a dark room in a temp 2-8 degree celsius for a period of 24 hours. In the positive responses appeared a fluorescent glow, sharp, regular elementary troops and stained, which was rated at (1 +, 2 +). The isolation of chlamydia cells in chicken embryos was carried out according to standard procedures, injected into the viteline sac, up 0.5 ml.i emulsion prepared with positive material from positive birds and suspected, from the lungs, liver, spleen, trachea and air sacs injured birds and sacrificed, in chicken embryos aged 6-7 days. Then they were placed for incubation at 39 degree celsius. After that it was carefully observed the replication of chlamydia, and evaluation of results was done from the fetus state which, in case it results positive, usually dies within 5-12 days after inoculation. After the histopathological control of infected embryos with the typical chlamydie infection, the material was collected and homogenised with a 20% suspension sacus vitelinus. The Identification of the agent was carried out by preparation of an infected sakus vitelinus antigen.

and its simplicity, the identification of chlamydial antibodies, especially in subclinical cases, is made by using the indirect immunofluorescence test which is considered to be the most effective test, whereas the primary isolation of chlamydia of chicken embryos (Andersen, A.A.1991) can also be used as comperative method (Kennedy G, et al: 1985). In different species such as, pigeons, parrots and canaries, it has been seen that their immunological responses towards their organism can be different (Geens, T. et al. 2005). The study showed that organs taken from dead birds which were infected with C. psittaci were important in identifying the infection by using diagnostic kits. The aim of the study was to develop and evaluate a rapid antemortem diagnostic technique for detection of C. psittaci in the serum of birds. Also as a quick and accurate method it can allow a veterinary doctor to immediately begin treating the sickening birds thus, preventing the infection to spread from domestic birds to humans, Greub,G.2010)which is the basic objective of the veterinary medicine. MATERIALS AND METHODS

For conducting the study, parrots and canaries, were selected inclusive, and for the control of pigeons were included and those races where as indicated had cases of people affected by this infection.The perennial scope had as an objective the study of the dynamics of the disease, as well as climatic factors correlating with other environmental issues. The process of taking the blood samples from decorative birds was carried out in batches, at 0.5 ml / head, which were marked and have been monitored throughout the year, 573 samples which prevailed according to their species were: 118 heads of parrots, 348 pigeons and 107 canaries, of which 30% were selected from young birds, varying from 2-4 months old. As a laboratory method it was selected the indirect immunofluorescence test (IFT), which because of its high specificity helps in all sub-clinical cases, when a seropositive birds lack a complete clinical framework. It is fast and simple to implement in the field and laboratory procedures, and also combined with primary isolation method of chlamydia in the chicken embryo, it can obtain rapid and accurated data

RESULTS AND DISCUSSIONS

Study shows that in areas chlamydic infection monitoring circulating in the following levels: in parrots: 12.7%, pigeons: 8.4%, and canaries: 7.47%. Chlamydia focus in birds' bodies but, more sensitive and higher concentrations they are found in the lungs. IFT method, combined with the isolation of chlamydia’s in embryos of chicken eggs, as well as histopathological control of the heads of damage, proved to be effective for disease control, the practical implementation of field and laboratory procedures. Chlamydia chicken embryo grew

disease has been evident and obvious mainly in young birds, which make possible a veterinary service, whereas adults show a sub-clinical form, serving as an asymptomatic carrier, helping to permanent recycling of infection. The survey data show that chlamydia are ordinarily resident in the bodies of birds, which are activated when lowered their sustainability as a result of stress and other resources that accompany infection, findings that are compatible with those of many foreign authors. Recognition of favorable factors and the development cycle chlamydia’s can serve as options for preventive measures by the veterinary service to control the disease in poultry in general and particularly decorative, and as a zoonosis, prevention and protection of human health.

and multiplied in the first pass, where the largest concentration of them is in the core of an egg. Death of embryos, under the action of chlamydia is high in percentage; the most pathogenic were chlamydia which was separate from the parrots and less pigeons and canaries. Identification of C.psittaci is done by the clinically healthy birds, and those coupled other pathologies, where the intensity of touching the bodies, in the first case has been much lower than in cases with pathology combined with other pathology, aspergilosis, parasites etc. The morbidity rate of birds only by chlamydia are observed to be as 10, (7 parrots and 3 pigeons), but always accompanied with other causes, which have enabled C.psittaci where after histopathology 'Basic ' are found localized in the brains of damaged birds. The clinical

Table 1. Information about the positivity of chlamydiosis in parrots by the breeds (in%) Spring Race

Summer

Autumn

Winter

Annual

Birds Positive % Birds Positive % Birds Positive % Birds Positive % Birds Positive % Tested + infection Tested + infection Tested + infection Tested + infection Tested + infection

Total 28 Amazon 8 Ondule 8 Calopsitte 6 Cacatoe 6

4 2 1 1 0

14.2 25 12.5 16.6 0

26 7 7 6 6

6 3 2 1 0

23 42.8 28.5 16.6 0

34 10 10 7 7

3 1 1 1 0

8.8 10 10 14.3 0

30 10 10 5 5

2 1 1 0 0

6.6 10 10 0 0

118 35 35 24 24

15 7 5 3 0

12.7 20 14.3 12.5 0

Table 2. Information about the positivity of chlamidiosis in pigeons by breeds (in %) Spring Race

Summer

Autumn

Winter

Annual

% Birds Positive Birds Positive % Birds Positive % Birds Positive % Birds Positive % infecTested + Tested + infection Tested + infection Tested + infection Tested + infection tion

Total 84 Rancing 44 Other 40

8 5 3

8.5 11.3 7.5

89 44 45

11 7 4

12.3 15.9 8.8

95 41 54

6 3 3

6.3 7.3 5.5

80 40 40

3 2 1

3.3 5 2.5

348 169 179

28 17 11

8.04 10.05 6.14

Table 3. Information about the positivity of chlamydiosis in canaries by breeds (in%) Spring Race Birds Positive Tested

Total 29 Serina 19 Belge 10

3 2 1

Summer

Autumn

Winter

Annual

% Birds Positive % Birds Positive % Birds Positive % Birds Positive % infection Tested + infection Tested + infection Tested + infection Tested + infection

10.3 10.5 10

26 16 10

3 2 1

11.5 12.5 10

29 19 10

2 1 1

The presentation of data, in tables No. 1, 2, 3, in overall it has been identified a total of 51 heads of seropositive birds, of which 15 heads ofparrots, 28 heads of pigeons, 8 heads of canaries, and by using theprimary isolation method of chlamydia in chickens embryos, there were prepared 15 samples, of which 5 were parrots, 7 werepigeons and 3 canaries.

6.9 5.3 10

23 13 10

0 0 0

107 67 40

8 5 3

7.5 7.5 7.5

Selecting the method of indirect immunofluorescence in determining the positive samples resulted to be very useful especially in sub-clinical cases, when complete clinical signs were missing, mostly in adult birds, and combined with the method of isolation chlamydia in chicken embryos, because the death of embryos occurred when they were at the age of 12-18 days, making

races(Salinas, J., et al.1993), but also the use of uncontrolled food products with unsafe origin, low hygiene standards of breeding, stresses and strains circulation with high virulence, etc.. Should also be taken more into consideration (Geens, T., et al., 2005). During monitoring, it was noted that infection to young people is organized in acute form causing damage to former company because of their high sensitivity. Young birds have shown signs of weakness typical, anorexia, purulent leak from eyes and nose. Inactivity, stay in position to collect, disheveled feathers (Gerlach, 1986). In cases with mild to developments birds lacked clear symptoms, the most obvious would be those of the respiratory tract and diarrhea (Avian Disease Manual, 2007) making them serve as asymptomatic carriers, recycling permanently the infection. The isolation of chlamydia from chicken egg embryos aged 6-7 days, were made from 15 samples, of which 5 parrots, 7 pigeons and 3 canaries, which resulted seropositive IFT method. To avoid horizontal transmission of infection through eggs, before infecting, 6 embryos were checked aged 6-7 days, two for each species, with IFT method, which resulted in negative from chlamydia. Control embryos was carried out for 14-15 days in a row and the death rate of embryos infected with suspension by the parrots was 100% Infected embryos suspension pigeons was 4, or 57.7%, whereas embryos infected with suspension canary bodies 1, or 33%. Mortality dynamics was observed during the period 3-14 days after infection, and mortality to parrots, 70-80% of them occurred 3-8 days after infection, 10-12 pigeons after infection, while the canary in the day of 13 after infection. Histopathological control of deadembryos dominated by the presence of hemorrhage in the body, in the head region ofthe feet, thickening of the lining of an egg and the slowdown in their growth and development. Surviving embryos, especially those with suspension by pigeons and canaries, microscopic researches have been identified the 'Basic corpus'. By monitoring the people affected with C. psittaci, in the past three years, there have been a total of 132 cases, and after controlling almost 45 of them, it was found that the disease has been correlated directly from their contact with seropositive birds, parrots,

completely compatible with IFT score. (Andersen, A.A.2008). From the histologic control of typical chlamydia infection in chicken embryos, in general for all species it was observed vascular congestion of membranes sacus -vitelinus, (Andersen, AA 2008). An important element was observed and the dynamics of infection, which according to the species stands as follows : parrot’s, average annual level of infection has been 12.7%, in spring 14.2%, in summer 23%, in fall 8.8%,and in winter 6.6%. (Table.1). Pigeon’s, average annual level of infection was about 8.04%, 8.5% in spring, in summer 12.3%, in fall 6.3%, while in winter, 3.3%, where is almost is hidden completely. (Table.2) Canarie’s average annual level of infection was 7.47%, in spring 10.3%, in summer 11.5%, in autumn 6.89%, while in the winter it was 0, which means that is completely extinct (Table.3). Although in different species such as parrots, pigeons, canaries the frequency was different, it was strictly related to seasonal conditions of the weather. In spring the infection has shown a tendency to increase, in summer where the weather has been so hot it has shown it maximum value whereas in autumn, withthe weather cooling the value has tended to decrease, while in thewinter when the weather has been cold the infection is reduced, hidden or wiped out, facts which coincide with that of the foreign authors (Lublin, A., et al, 1995), etc. It is important to be considered as support for increasing the frequency of infection during the months with warm and hot weather there were also many other factors, individually or in the correlations between them have contributed to the situation. Those can be from the: increase of contact between birds with warm weather, activation of arthropods and hematophag insects which help spread the movement vectors of infection in decorative birds, which have also been observed on a case by case basis on the infected birds. While monitoring the incidence of pigeons (not decorative ones) and seropositive races, which make up about 30% of the samples, as a source of infection we should also evaluate the contact with water weeds, which have been polluted with eksements of porter birds. On the other hand unfamiliar areas affected by infection, and the transit during the

birds, and as a zoonosis, in prevention and protection of human health.

canaries and doves, and its frequency was variable, with age, the level of exposure and patogenicity were the determining factors for the occurrence and form infection clinic. (POSPISIL, L., et al., 1996)

REFERENCES Alexander D J., Bevan BJ., Lister SA., Bracewell CD., 1989. Chlamydia infections in racing pigeons in Great Britain: a retrospective serological survey. Vet. Rec. 125:239. Andersen A.A., 1991. Serotyping of Chlamidia psittaci isolates using serovar-specific monoclonal antibodies with the micro-immunofluorescence test Journal of clinical Microbiology 29, 707-711 Andersen AA., 1997. Two new serovar of Chlamidia psittaci from North Amerirican birds. Journal of Veterinary Diagnostic Investigation, 9, 159-164 Andersen AA., 2008. Avian chlamidiosis In OIE Manual of Diagnostic tests and Vaccines for terrestrial Animals .Sixth Edition OIE,Paris, France pp. 431-442 Instituti i Shëndetit Publik –ISHP, 2011. Tiranë, Batta MK., Dhingra PN., Mangat APS., 1993. Chlamydiosis in birds from Punjab: serological survey. Ind. J. Anim.Sci. 63:526-527. Bourke SJ., Carrington D., Frew CE., McSharry G., 1992. A comparison of the seroepidemiology ofchlamydial infection in pigeon fanciers and farmers in the U.K. J. Infect. 25 Suppl. 1:91-98. Barnes RC., 1989. Laboratory diagnosis of human Chlamydial infections.Clin.Microbiol.Rev.2:119-135. Bejleri J., Berxholi K., 1987. Klamidiet në shpendë Buletini nr. 2 i Shkencave Zooteknike dheVeterinare, Tiranë, faqe 63-70 El-Halawani ME., Waibel PE., Appel JR., Good AL., 1973. Effects of temperature stress oncatecholamines and corticosterone of male turkeys. Amer. J. Physiol. 224:384-388. Geens T., Dewitte A., Boon N., Vanrompay D., 2005. Development of a Chlamydophila psittaci speciesspecific and genotype specific real-time PCR Veterinary Research 36, 787-797 Grimes J., 1986.Chlamydia psittaci latex agglutination antigen for rapid detection of antibody activity inavian sera: comparison with direct complement fixation and isolation results. Avian Dis 30:60-66. Greub G., 2010a. Minutes of the Subcommittee on the Taxonomy of the Chlamidiae International Journal of Systamatic and Evolutionare Microbiology, 60, 26912693; 2694 Harrison GJ., 1989. A practitioner’s view of the problem of avian chlamidiosis J. Amer. Vet. Med. Assoc 195:1525-1528. Hobson D., Johnson F., Byng R., 1977. The growth of the ewe abortion Chlamydia1 agent in McCoy cellcultures. J Comp Pathol 87:155-l59. Kennedy G., Taylor F., Werdin R., et al., 1985. Laboratory diagnosis of chlamydia1 diseases. Proc Annu Meet Am Assoc Vet Lab Diagn 28:421-435. Lublin A., Mechani S., Malkinson M., Bendheim U. and Weisman Y., 1993. A 3-year survey indifferent avian species of frequency of detection Chlamydia psittaci antigens. Proceed. 1993 Europ. Conf.Avian Med. Surg. Utrecht. The Netherlands. pp. 478-492.

ACKNOWLEDGEMENTS

The circulating levels of decorative birds with chlamydia infections are considered to be relatively low.C.psittaci are mainly focused on birds' bodies but, more sensitive and higher concentrations are to be found in the lungs. The IFT method, combined with the isolation of chlamydia in embryos of chicken eggs, as well as histopathological control of the damaged heads, proved to be effective for controlling the disease, the practical implementation of field and laboratory procedures. Identification of 'elementary bodies' can be done with materials which are taken from bodies stained with May-method Grynvald-Giemsa and by controlling them under a microscope, shows that they are located mainly within the cytoplasm of the cell in the form of meal pomegranate and being a little pink in color. It was observed that the death of the embryos was higher in chlamydia isolated from parrots and less from those in doves and canaries. Identification was done by the clinically healthy birds, where the intensity was much lower however, in cases with combined pathology with mycotic causes, aspergilosis, parasites etc intensity was higher. Morbidity cases of birds just by chlamydia alone are 10 in total, (7 parrots and pigeons 3) but, cases coupled with other causes have dominated. The clinical disease has been evident and obvious mainly in young birds which make possible orientation in veterinary service, whereas adults show a sub-clinical form, serving as asymptomatic carriers, helping to permanent recycling of infection. The study shows that chlamydia are ordinarily resident in the birds' bodies which, are activated when their sustainability is lowered as a result of stress and other resources that accompany infections. The findings are compatible with those of many foreign authors. Recognition of favorable factors and the development cycle in chlamydia can serve as options for preventive measures by the veterinary service to control the disease in poultry in general and particularly decorative

Lublin A., Mechani S., Malkinson M., Weisman Y. and Bendheim U., 1995. A 4-year survey of thedistribution of Chlamydia psittaci in 19 orders of birds in Israel with emphasis on seasonal variability.Proceed. 3rd Conf. Europ. Comm. Assoc. Avian Vet. Jerusalem. Israel. ECAMS. p. 1. Pospisil L., Veznik Z., Hirt M., Svecova D., Diblikova I. and Pejcoch M., 1996. Detection of Chlamydia in the

intestines and lungs in pigeons and humans. Epidemiol., Microbiol., Immunol. 45:123-126. Salinas J., Caro M.R. and Cuello F., 1993. Antibody prevalence and isolation of Chlamydia psittacifrom pigeons (Columba livia). Avian Dis. 37:523-527. Woods L., Woods D., 1986. Evaluation and development of a new antemortem diagnostic test of Chlamydia psittaci infection in psittacine birds. Proc Assoc Avian Vet, pp. 75-79.

FOOD BIOTECHNOLOGY

Scientific Bulletin. Series F. Biotechnologies, Vol. XVII, 2013 ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

IN VITRO EVALUATION OF ANTIOXIDATIVE PROPERTIES IN CAPSICUM ANNUUM, VACCINIUM VITIS-IDAEA AND MELISSA OFFICINALIS TINCTURES Emanuel VAMANU, Adrian VAMANU, Elena DOPCEA

Department of Industrial Biotechnology, Faculty of Biotechnologies, University of Agronomical Sciences and Veterinary Medicine Bucharest, 59 Marasti Blvd., 011464, Bucharest, Romania Corresponding author email: [emailprotected] Abstract Several commercial varieties of tinctures were analyzed for determining their antioxidant potential, since most knowledge relating to therapeutic properties of medicines are obtained from folk phytomedicine. Antioxidant potential was determined by the scavenging activity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3ethylbenzothiazoline-6-sulphonic acid) (ABTS) free radicals, reducing power, and chelating activity. The best results against scavenging of free radicals were obtained with tincture from Vaccinium vitis-idaea,followed by Capsicum annuum and Melissa officinalis. There were obvious significant differences between the free radicals’ scavenging activities. Results were correlated with values obtained for reducing power and chelating ability. This finding was also confirmed by the low values of the EC50. Also, the results were positively high when correlated with total phenolic and flavonoidic contents from the tinctures. The results given herein are the scientific proof that confirms the empirical medical knowledge on how to use the three tinctures in oxidative dysfunctions. Keywords: scavenging activity, phenolic content, tincture.

such circumstances, it is necessary to take antioxidative products which can restore the balance of a body’s antioxidant status. Polyphenols are the main secondary metabolites to be found in medicinal plants. Besides their antioxidant activity, such compounds are responsible for their antimicrobial actions (due to their content of flavonoids) and cytotoxic effects exerted against tumor cells Gharras, 2009). Their antimicrobial actions have been proven against potentially pathogenic strains, such as Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Candida strains, Listeria strains, or Bacillus cereus (Vamanu et al. 2011). On the other hand, their cytotoxic effects have also been associated with the content of alkaloids and triterpenes, which have been demonstrated in previous surveys as being directly related to the antioxidative response (Agarwal et al., 2012). This is the reason why the present research has undertaken to demonstrate the antioxidative properties of certain tinctures sold in Romania. Their properties have been linked to polyphenol and flavonoid contents. Highlighting the antioxidative effects has taken place by means

INTRODUCTION

In recent decades, interest in the use of medicinal plants has significantly increased due to the isolation of biologically active substances that have a wide range of pharmacological effects. The phytotherapeutical action of herbal products is generated by the rich content of such substances, including phenolic compounds, vitamins, alkaloids, saponins, essential oils, and various minerals (Ivan, 2007). Their main and most evident effect is antioxidative, that acts upon the characteristics of reactive oxygen and nitrogen species. The antioxidative molecules existing in herbal products can react with free radicals and they are able to neutralize the latter by donating their own electrons. This process helps prevent the damage of cell tissues. Each cell protects itself from the influence of free radicals by its own prevention mechanisms, but in the event of excessive formation, oxidative stress occurs (Sen et al., 2010). Oxidative stress has been defined as the imbalance between the production of various reactive species and the ability of a living body’s natural defense mechanisms (Oguntibeju et al., 2009). Under

of the DPPH and ABTS radicals’ scavenging activity, reducing power, and chelating properties.

±0.02. After adding 10 ʅL of the sample to 4mL of the diluted ABTS radical solution, the absorbance was measured at 30 min. The ABTS radical-scavenging activity of the samples was expressed as% = ( (Acontrol о Asample) /Acontrol) × 100, where Acontrol is the absorbance of the blank control (ABTS solution without test sample) and Asample is the absorbance of the test sample (Vamanu, 2012a). Standard antioxidant (TBHQ) was used for comparison as positive control. EC50 value (milligram extract/mL) is the effective concentration at which ABTS radicals were scavenged by 50% Reducing Power Each sample (2.5 mL) was mixed with 200 mM sodium phosphate buffer (2.5 mL, pH 6.6) and 1% potassium ferricyanide (2.5 mL), and the mixture was incubated at 50 ӑC for 20 min. Next, 10% trichloroacetic acid (2.5 mL) was added, and the mixture was centrifuged at 3,000 g for 10 min. The upper layer (2.5 mL) was mixed with distilled water (2.5 mL) and 0.1% ferric chloride (0.5 mL). Finally, the absorbance was measured at 700 nm and compared to a blank. The extract concentration providing 0.5 of absorbance (EC50) was calculated from the graph of absorbance at 700 nm plotted against the extract concentration. TBHQ was used as positive control (Vamanu, 2012b). Ferrous Ion Chelating Assay 1mL of the sample was mixed with 3.7mL of ultrapure water, following which the mixture was reacted with ferrous chloride (2 mmol/L, 0.1mL) and ferrozine (5 mmol/L, 0.2mL) for 20 min. The absorbance at 562nm was determined spectrophotometrically. TBHQ was used as a positive control. The chelating activity on the ferrous ion was calculated using the equation following: chelating activity (%) = [(Ab оAs) /Ab] ×100, where Ab is the absorbance of the blank without the extract or TBHQ and As is the absorbance in the presence of the extract or TBHQ (Vamanu and Nita, 2013). Determination of total phenolic and flavonoid content The total phenolic and flavonoid content of ethanolic extract, and several organic fractions, were determined using Folin-Ciocalteu reagent and aluminium chloride colorimetric method,

MATERIALS AND METHODS Materials The tinctures that have been used are: hot pepper tincture (Capsicum annuum), cranberry tincture (Vaccinium vitis-idaea), and lemon balm tincture (Melissa officinalis) manufactured by SC Dacia Plant SRL.

Figure 1. Marketing image of Capsicum annuum, Vaccinium vitis-idaea and Melissa officinalis tinctures

DPPH radical scavenging activity assay The sample (100 ʅl) was mixed with 3 ml of ethanol solution of 0.004% DPPH and the absorbance was read at 517 nm 30 min later. Standard antioxidant,tert-Butylhydroquinone (TBHQ), was used for comparison as positive control. EC50 value (milligram extract/mL) is the effective concentration at which DPPH radicals were scavenged by 50%. The DPPH radical-scavenging activity of the samples was expressed as% = ((Acontrol о Asample) /Acontrol) × 100, where Acontrol is the absorbance of the blank control (DPPH solution without test sample) and Asample is the absorbance of the test sample (Vamanu et al. 2011). ABTS Radical Scavenging Assay ABTS radical was obtained by adding 7mM of the ABTS stock solution to 2.45mM potassium persulfate. The mixture was left to stand in the dark, at room temperature, for 12–16 h before use. The ABTS radical solution was then diluted with 5mM phosphate-buffered saline (pH 7.4) to an absorbance at 730nm of 0.70

100

respectively (Vamanu, 2012c; Premanath et al., 2011). Statistical analysis All the essays were assessed in triplicate, and the results were expressed as mean ±SD values of three observations. The mean values and standard deviation were calculated with the EXCEL program from Microsoft Office 2010 package.

order of the scavenging activity against DPPH radicals has been: C. annuum >M. officinalis >V. vitis-idaea (Figure 2). Thus, in terms of the hot pepper tincture, the scavenging activity’s difference from the standard has been on average 10% higher. With respect to ABTS radicals, the descending order has been: V. vitis-idaea >M. officinalis >C. annuum (Figure 3). In this context, the set value of the TBHQ reaching 100 μg/mL has been on average 2% higher. The differences noticed in relation with the three tinctures for scavenging properties against the two free radicals have been generated by the existing molecules that possess antioxidative effects. The type and presence of certain molecules have caused specificity differences as compared with the DPPH or ABTS radicals which have been best monitored in the C. annuum tincture.

Figure 2. DPPH scavenging activity of Capsicum annuum, Vaccinium vitis-idaea and Melissa officinalis tinctures

RESULTS AND DISCUSSIONS

Evaluating antioxidative potential by the DPPH and ABTS determination of radicals’ scavenging activity requires a modern method to be applied due to the stability of these free radicals, and to the precise results that ensue thereof. The discoloration level of the reaction mixture (turning from mauve into beige for the DPPH scavenging assay; or turning from turquoise into colorless for the ABTS scavenging assay) is allocated in direct proportion to the ability of donating a hydrogen atom derived from the antioxidative compounds in the examined assay. The process leads to the reduction of existing free radicals, and also generating the cessation of the radical chain reaction which has determined the propagation of the oxidation mechanism (Kumaraswamy and Satish 2008). DPPH and ABTS free radicals accept hydrogen radical and become stable molecules (Nikhat et al., 2009). In this research, the standard (TBHQ) has proven a moderate scavenging activity against DPPH radicals, and a high scavenging activity against ABTS radicals. At a concentration of 100 μg/mL, the descending

Figure 3. ABTS scavenging activity of Capsicum annuum, Vaccinium vitis-idaea and Melissa officinalis tinctures

For reducing power assays, the antioxidative molecules to be found in the three tinctures will determin the reduction of Fe3+ into Fe2+ (Moein et al., 2008). The molecules’ reducing power in the three plant tinctures serves as a direct indicator of the extent of the antioxidative potential. According to the research in the field of natural supplements, reducing power is related to the existence of reductones (Li and Lin, 2010). Ascorbic acid is the best known reductone, and high reduction ability is generally associated with a significant amount of this compound which is frequently found in hydroalcoholic extracts. The absorbance increase has been set at 700 nm, as compared with the standard value (Figure 4). The value

101

set at the concentration of 100 μg/mL has ranged between 0.404 and 1.643. Compared with the standard, the reducing power value in the V. vitis-idaea tincture is 3.8 times higher which has indirectly confirmed the presence of molecules with obvious reduction properties. The same behavior has been noticed in the M. officinalis tincture as well. The difference between the two tinctures has been 28.4%, in favor of the cranberry tincture.

and 5.61% higher than the tincture obtained from the M. officinalis. The standard has shown a low ferrous chelating activity at a significantly lower value than the three assays, up to a maximum of 46.61% in the highest concentration of the assay.

Figure 5. Ferrous ion chelating activity of Capsicum annuum, Vaccinium vitis-idaea and Melissa officinalis tinctures

The results of the above tests have been relatively different based on each individual biochemical test performed. This finding proves that certain molecules can exert their antioxidative properties in their own individual ways. This confirmation has ensued from the inversely proportional values of the EC50 regarding its chelating capacity. From the perspective of this parameter, the maximum value has been reached for M. officinalis tincture (< 10 μg/mL). The EC50values of the other two tinctures have been у30 μg/mL in the C. annuum, and у65 μg/mL in V. vitis-idaea. The latter results confirm the high biological values of these tinctures, especially that of the cranberry one, because Ferrum immobilization within compounds stops its accumulation which has toxic effects at cellular level (GraceLynn et al., 2012). The same behavior has been assessed in the DPPH scavenging activity where the lemon balm tincture has shown its EC50value as being inferior to 90 μg/mL. On the contrary, the EC50values of the other two tinctures have been superior to the former, namely у95 μg/mL. The scavenging activity against free radicals is due to the amount of phenol compounds. Subsequent to all the measurements performed, the V. vitis-idaea tincture has contained an amount of 106 ± 8.15 mg gallic acid/g extract,

Figure 4. Reducing power of Capsicum annuum, Vaccinium vitis-idaea and Melissa officinalis tinctures

As far as the determination of ferrous chelating capacity is concerned, ferrozine forms Fe2+ compounds, whereas in the presence of chelating agents from the three tinctures, compound formation is disturbed. According to the sample concentration, the reaction mixture is colored in several shades of red and pink in an inverse proportion. The decrease in absorbance has been measured spectrophotometrically (Grace-Lynn et al., 2012). Ferrum is essential as it is very important for oxygen conduction and acts as an activating agent for various enzymes. The ferrous ion (or copper ion) intervenes in a range of mechanisms that may contribute in the emergence of oxidative processes. Its role is very well known in the lipid peroxidation of the cell membrane, and its action upon the protein content is also recognized (Karthika et al., 2012). In the present research, the capacity of the examined tinctures to link to Fe2+ in the presence of ferrozine as compared with the (TBHQ) standard has been shown in Figure 5. The maximum value of the ferrous ion chelating activity is 80.17%, at 100 μg/mL, with respect to C. annuum tincture. It has been 1.07% higher than the V. vitis-idaea tincture,

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M. officinalis tincture 96.4 ± 4.74 mg gallic acid/g extract, and 76.8 ± 3.98 mg gallic acid/g extract in the C. annuum tincture. The flavonoid content has proven a similar trend, with a maximum level of 4.43 ± 0.5 mg quercetin/g extract in the V. vitis-idaea tincture. This value has been 35.44% higher than the lemon balm tincture, and 64.78% higher than the hot pepper tincture. The calculated values of the main antioxidative compounds have corresponded with the increase in the EC50 index value in terms of the scavenging activity, reducing power and ferrous ion chelating activity (Zhu et al., 2006). Interpreting the results of the antioxidative properties’ analysis relating to the three tinctures has also taken place by calculating the index value of the correlation among various examination methods. In the hot pepper tincture, the R2 value has been 0.8811, while the same value among the remaining methods has ranged between 0.9711 and 0.9891 (p< 0.0005). The correlation coefficient has ranged from 0.828 to 0.9305, with a minimum value calculated for the ABTS ratio between radicals’ scavenging activity and ferrous ion chelating activity. The value ensuing thereof has confirmed the behavior of this tincture when expressing its antioxidative feedback.As to the lemon balm, there has been a low R2 value in the relationship between the ferrous ion chelating activity and the reducing power, a value of 0.6311. In the relation with the ABTS assay, a 0.7129 value of the correlation index has been measured, whereas, the correlation level has been high in the other outcomes, ranging between 0.8621 and 0.9446. Owing to the generally neutral pH contained in tinctures, the DPPH assay has been a less appropriate method where the sample’s correlation degree influences the final result. The differences when calculating the R2 value related to the ABTS assay more precisely indicate the distinctions among the three tinctures, as the method is valid irrespective of the pH value, and the results do not directly depend on the coloring level of the sample (Bhoyar et al., 2011; Zhu et al., 2006).

CONCLUSIONS

To conclude, the total phenolic content has been determined as significantly higher in the lemon balm tincture as compared with the other two. This ratio has been correlated in the case of flavonoid contents as well. According to the EC50 values, the V. vitis-idaea tincture has had a more obvious antioxidative activity in compliance with its correlation to the scavenging activity against free radicals and the chelating activity, than in the hot pepper (C. annuum) tincture. These measurements comply with similar research, thus confirming that a tincture’s expression of its antioxidative value is not automatically and directly connected with its phenolic content. The antioxidative feedback is very complex due also to the existence of additional compounds that exert specific effects. ACKNOWLEDGEMENTS

This work was partially supported by the project PNCDI II CNCSIS—Human Resources, Theme 9/2010 (http://www.emanuelvamanu.ro). REFERENCES Agarwal N., Majee C., Chakraborthy G.S., 2012. Natural Herbs as Anticancer Drugs. International Journal of PharmTech Research, Vol. 4 (3), p. 1142-1153. Bhoyar M.S., Mishra G.P., Naik P.K., Srivastava R.B., 2011. Estimation of antioxidant activity and total phenolics among natural populations of Caper (Capparis spinosa) leaves collected from cold arid desert of transHimalayas. Australian Journal of Crop Sciences, Vol. 5 (7), p. 912-919. Gharras H. E., 2009. Polyphenols: food sources, properties and applications – a review. International Journal of Food Science and Technology, Vol. 44 (12), p. 2512–2518. Grace-Lynn C., Darah I., Chen Y., Latha L.Y., Jothy S.L., Sasidharan S., 2012. In Vitro Antioxidant Activity Potential of Lantadene A, a Pentacyclic Triterpenoid of Lantana Plants. Molecules, Vol. 17 (9), p. 11185-11198. Ivan S., Medicina naturistĉ pentru toƜi, 2007. CNI Coresi Publishing House, p. 108. Karthika K., Paulsamy S., Jamuna S., 2012. Evaluation of in vitro antioxidant potential of methanolic leaf and stem extracts of Solena amplexicaulis (Lam.) Gandhi. Journal of Chemical and Pharmaceutical Research, Vol. 4 (6), p. 3254-3258. Kumaraswamy M.V., Satish S., 2008. Free radical scavenging activity and lipoxygenase inhibition of Woodfordia fructicosa Kurz and Betula utilis Wall.

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African Journal of Biotechnology, Vol. 7 (12), p. 20132016. Li C.C., Lin E.S., 2010. Antiradical capacity and reducing power of different extraction method of Areca catechu seed. African Journal of Biotechnology, Vol. 9 (46), p. 7831-7836. Moein M.R., Moein S., Ahmadizadeh S., 2008. Radical Scavenging and Reducing Power of Salvia mirzayanii Subfractions. Molecules Vol. 13 (11), p. 2804-2813. Nikhat F., Satynarayana D., Subhramanyam E.V.S., 2009. Isolation, Charectrisation and Screening of Antioxidant Activity of the Roots of Syzygiumcuminii (L) Skeel. Asian Journal of Research in Chemistry, Vol. 2 (2), p. 218-221. Oguntibeju O. O., Esterhuyse A.J., Truter E.J., 2009. Cardiovascular disease and the potential protective role of antioxidants. African Journal of Biotechnology, Vol. 8 (14), p. 3107-3117. Premanath R., Sudisha J., Devi N.L., AradhyaS.M., 2011. Antibacterial and anti-oxidant activities of fenugreek (Trigonella foenum graecum L.) leaves. Research Journal of Medicinal Plant, Vol. 5 (6), p. 695705. Vamanu E., 2012a. Biological Activities of the Polysaccharides Produced in Submerged Culture of Two

Edible Pleurotus ostreatus Mushrooms. Journal of Biomedicine and Biotechnology, Article ID 565974. Vamanu E., 2012b. In Vitro Antimicrobial and Antioxidant Activities of Ethanolic Extract of Lyophilized Mycelium of Pleurotus ostreatus PQMZ91109. Molecules, Vol. 17 (4), p. 3653-3671. Vamanu E., 2012c. Determination of antioxidant and antimicrobial properties of Agaricus bisporus from Romanian markets. Ovidius University Annals of Chemistry, Vol. 23 (1), p. 47-52. Vamanu E., Nita S., 2013. Antioxidant Capacity and the Correlation with Major Phenolic Compounds, Anthocyanin, and Tocopherol Content in Various Extracts from theWild Edible Boletus edulis Mushroom. Journal of Biomedicine and Biotechnology, Article ID 313905. Vamanu E., Vamanu A., Nita S., Colceriu S., 2011. Antioxidant and Antimicrobial Activities of Ethanol Extracts of Cynara Scolymus (Cynarae folium, Asteraceae Family). Tropical Journal of Pharmaceutical Research, Vol. 10 (6), p. 777-783. Zhu K., Zhou H., Qian H., 2006. Antioxidant and free radical-scavenging activities of wheat germ protein hydrolysates (wgph) prepared with alcalase. Process biochemistry, vol.41 (6),p.1296-1302.

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Scientific Bulletin. Series F. Biotechnologies, Vol. XVII, 2013 ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

A COMPARATIVE STUDY ON MOUNTAIN AREA INFLUENCE OF MILK SAMPLES FROM COW AND SHEEP Elena MARCU1, Petru NICULITA1, Ramona IANCU2 1

Faculty of Biotechnologies, University of Agronomic Sciences and Veterinary Medicine of Bucharest, 59, Marasti Bvd., 011464, Bucharest, Romania 2 Faculty of Agricultural Sciences, Food Industry and Environmental Protection “Lucian Blaga” University, 5-7 Ion Ratiu Street, 550012, Sibiu, Romania Corresponding author email: [emailprotected]

Abstract With the current rapid growth of world population and prolongation of human life, the raise of the living level and the targeting of food to a growing extent of agro-food products, with high nutritional and biological value, the need for food, especially of animal origin have increased ever more. This research was carried out to investigate and compare the physicochemical and microbiological parameters of milk samples of two different species like cow and sheep, including: pH, fat, protein, total solids, density, and somatic cells. Results showed that maximum fat and protein content were observed at sample 3 and sample 4, indicate that in mountain area sheep’s are the most favorable animal breeding. The milk samples were collected nine month in the year 2011 from different farmers. The statistical analysis showed that the physicochemical and microbiological parameters of these milk samples were significantly different (p 1 indicate an antioxidant activity, the value of PF = 1 corresponds to no antioxidant activity and the values of PF < 1 mean prooxidative activity. Oils without any additives were analysed as blanks and the antioxidant efficiency was expressed as the protection factor: PF = IP0/IP, where: IP is the peroxide index of oil with addition of an antioxidant and, IP0 is the peroxide index of oil without the addition of the antioxidant. (Ixtaina et al., 2012). Analysing the results obtained it was observed that both extracts have antioxidant activity and increased oxidative stability of sunflower oil.

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extract in ethanol of Ginkgo Biloba the PF was 2,5. The results obtained can be summarized by starting that the added of 4 mg/mLof extract in ethanol of Ginkgo Biloba in sunflower oil has a better antioxidant activity. In Figure 3 and Figure 4 it can be seen that the greater stability of sunflower oil was obtained from Ginkgo Biloba extract in ethanol obtained in the laboratory at a concentration of 4 mg/mL in ethanol extract. At the addition of 40 mg of extract in oil it is noted that there has been a decrease in the value of the value of TBA in the oilwith addition of Ginkgo extract commercial with approximately 74% from the amount of TBA value of oil without added.

Figure 1. The values obtained from the analysis of peroxide value no oil addition and with the addition of a commercial extract of Ginkgo Biloba

Figure 2. The values obtained from the analysis of peroxide value no oil addition and with the addition of natural Ginkgo in ethanol. Figure 3. The values obtained as a result of determining absorbance of 2-tiobarbituric acid of the oil with no addition and with the addition of Ginkgo Biloba extract in ethanol.

In Figure 1 and Figure 2 it can be seen that the greater stability of sunflower oil was obtained from Ginkgo Biloba extract in ethanol obtained in the laboratory at a concentration of 4 mg/mL in ethanol extract. The addition of 40 mg of commercial extract in oil it is noted that there has been an increase in oxidative stability of oil with the addition of a commercial extract of Ginkgo with approximately 10% from the initial stability of oil without added. When was added a quantity of 2 mg/mL of extract of Ginkgo Biloba in ethanol, it is noted that the oxidative stability of oil has increased by about 25% relative to baseline stability of oil without added, and to the addition of 4mg/mL of extract of Ginkgo Biloba in ethanol, it was noted that the oxidative stability of oil has increased by approximately 60% relative to baseline of oil stability without added. As mentioned earlier, the protection factor (PF) for the commercial extract of Ginkgo Biloba in sunflower oil was 1.09, while for the added of 2mg/mLof extract in ethanol of Ginkgo Biloba the PF was 1,3 and for the added of 4mg/mLof

Figure 4. The values obtained as a result of determining absorbance of 2-tiobarbituric acid of the oil with no addition and with the addition of Ginkgo Biloba extract in ethanol in different concentration.

When was added a quantity of 2 mg/mL of extract of Ginkgo Biloba in ethanol, it is noted that there has been a decrease in the value of TBA by about 24% relative value of TBA of oil without added, and the addition of 4 mg/mL of extract of Ginkgo Biloba in ethanol, it is noted that there has been a decrease in the

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Papadopoulos V, Drieu K. 2003. Ginkgo biloba extracts and cancer: a research area in its infancy. Fundam Clin Pharmacol 17:405–17.3. Ellain Wojtaszek, M.Krucynski, Z.-Kasprzak, J.: Variations in free radical scavenging activity of Ginkgo biloba leaves in the period of complete development of green leaves to fall of yellow ones. Food Chemistry, 79, 2002, pp. 79-84.4. Farmacopeea Romana, 1993, Editura medicala, Bucuresti.5. Huisong Pharmaceuticals, (www.huisongpharm.com/manu.asp? id=82) 6. Ixtaina V.Y., Nolasco M.S., Tomas M.C., Oxiative Stability of Chia (Salvia hispanica L.) Seed Oil: Effect of antioxidants and storage Conditions, J.Am.Oil Chem. Soc. (2012) 89:1077-1090, DOI10.1007/s11746-0111990-x.5. Kobus J., Flaczyk E., Siger A., NogalaKalucka M., Korczak J., Pegg R.B., 2009. Phenolic compounds and antioxidant activity of extracts of Ginkgo leaves. Eur. J. Lipid Sci. Technol. 111.6. Kalisz O., Wolski T., Gerkowicz M., 2006. MiųorzČb japoŷski (Ginkgo biloba) i jego preparaty w terapii zaburzeŷ krČǏenia mózgowego i obwodowego [Ginkgo biloba (Ginkgo biloba) and its preparations in therapy of cerebral and peripheral circulation disorders]. Ann. Univ. Mariae Curie-Skųodowska 61, 2, 11-17.7. Mahady GB. 2002. Ginkgo biloba for the prevention and treatment of cardiovascular disease: a review of the literature. J Cardiovasc Nurs 16:21–32.8. Máriássyová, M.: Antioxidant activity of some herbal extracts in rapeseed and sunflower oils. Journal of Food and Nutrition Research, 45, 2006, 3, pp. 104-109.9. Rhee, D.-Myers, J.: Complementary and alternative medicine for glaucoma. Survey of Ophthalmology, 46, 2001, 1, pp. 43-55.10. Spence, K. E.-Jane, J.: Chemical and physical properties of ginkgo starch. Carbohydrate Polymers, 40, 1999, pp. 261-269.11. Trevisan Maurizio, Browne Richard, Ram Malathi, Muti Paola, Freudenheim Jo, Carosella Ann Marie, Armstrong Donald, Correlates of Markers of Oxidative Status in the General Population, Am. J. Epidemiol. 2000.

value of TBA by about 61% relative the initial stability of oil without added. As mentioned earlier, the protection factor (PF) for the value of TBA using the commercial extract of Ginkgo Biloba in sunflower oil was 3.7, while for the added of 2mg/mLof extract in ethanol of Ginkgo Biloba the PF was 0.7 and for the added of 4mg/mLof extract in ethanol of Ginkgo Biloba the PF was 1.4. The results obtained can be summarized by starting that the added of 4 mg/mLof extract in ethanol of Ginkgo Biloba in sunflower oil has a better antioxidant activity. CONCLUSIONS

The purpose of this work was to analyze the effects of the extracts of Ginkgo Biloba in terms of their active compounds over the sunflower oil, through two different methods: determination of hydrogen peroxide by titration and determination of TBA-standardization. The results obtained using our methods of analysis Ginkgo Biloba extract obtained by extraction with ethanol seems to be much better in the oxidative stability of sunflower oil. In terms of results, it was observed that both extracts have antioxidant action. REFERENCES 1. Byeoung-Soo, P.-Sung-Eun, L.: Antioxidative activity of Ginkgo biloba leaves – derived components on free radicals and active oxygen species. Food Science and Biotechnology, 9, 2000, pp. 317-321.2. DeFeudis FV,

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MISCELLANEOUS

Scientific Bulletin. Series F. Biotechnologies, Vol. XVII, 2013 ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

INTERACTION OF ASPERGILLUS NIGER HYPHAE AND SPORES WITHCOLLOIDAL SILVER NANOPARTICLES Ovidiu IORDACHE1, Calina Petruta CORNEA2 1

National Research and Development Institute for Textile and Leather, Lucretiu Patrascanu, No. 16, District 3, 030508, Bucharest, Romania 2 University of Agronomical Sciences and Veterinary Medicine, Faculty of Biotechnologies, 59 Marasti Blvd., District 1, 011464, Bucharest, Romania Corresponding author email: [emailprotected]

Abstract The experiments explored the interactions and antifungal properties of silver nanoparticles, against a model microorganism, Aspergillus niger. Scanning Electron Microscopy analysis were used for assessment of structural alterations done to fungal cells and hyphae, coupled with Energy Dispersive X-ray Spectroscopy (EDAX). The toxicity of the silver nanoparticles was tested using two methods: liquid exposure to the solution containing silver NPs, and spraying the NPs solution directly on the fungal culture. Analysis revealed significant cellular alteration due to the exposure to silver nanoparticles as well as effects on the growth of Aspergillus niger strain, in comparison to deionized water treatment, used at control sample. Microscopic SEM images revealed that silver nanoparticles treated hyphae were damaged on cell walls level, inducing plasmolysis, while EDAX analysis revealed strong silver depositions in the damaged areas of vegetative cells and spores walls, aspects that could be correlated with silver presence on the affected sites. Keywords: antimicrobial, silver, Aspergillus niger, SEM, EDAX.

extensively in many bactericidal applications (Singh et al., 2008). Generally, the antimicrobial mechanism of chemical agents depends on the specific binding with surface and metabolism of agents into the microorganism. As an inhibitory mechanism, it has been proposed, that the silver easily adders to the cell wall, which at the exterior has nucleophile centers and alters cell respiration process. Ionic silver strongly interacts with thiol groups of vital enzymes which lead to their inactivation (Matsumura et al., 2003; Gupta et al., 1998). Through the transmembranar transport of the silver nanoparticles inside the cell, the functions of proteins and DNA are altered, which leads to the microorganism not being able to reproduce himself anymore. Studies underlined structural changes in the cell membrane and formation of small electron-dense granules formed by silver and sulfur (Feng et al., 2000; Nover et al., 1983). The positive charge on the Ag ion is crucial for its antimicrobial activity through the electrostatic attraction between negative charged cell membrane of microorganism and positive charged nanoparticles.

INTRODUCTION

Nanotechnology is currently employed as a powerful tool in aiding biomedical applications regarding antimicrobial activity. As known, the smaller a particle is, the greater it is surface area to volume ratio and the higher its biological activity and chemical reactivity. Metal particles in the nanometer size range exhibit physical properties that are different from both the ion and the bulk material. This makes them exhibit remarkable properties such as increased catalytic activity due to morphologies with highly active facets (Singh et al., 2008). The use of metal nanoparticles represents a quick and straightforward way of fighting against different types of microorganisms, colloidal silver being an effective bacteria-fighting agent (Gibbs, 1999). The interaction between silver nanoparticles, and different microorganisms, is of outmost importance as a natural process that takes place in the nanometer scale region. Silver has long been known to exhibit a strong toxicity to a wide range of micro-organisms and for this reason silver-based compounds have been used

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MATERIALS AND METHODS

RESULTS AND DISCUSSIONS

Aspergillus niger (A. niger) was cultivated on a Czapek-Dox (2% w/v glucose, 2% w/v agar) media, at 29°C, for 14 days. The samples were kept on the agar media during the experimental procedure, in order to avoid mechanical damage that could influence the final results. After the culture was fully grown, the media containing the mature fungus was split in 2 equal halves, using a sterile spatula, one half for each method. The used colloid solution had a silver concentration of 1500 ppm, within a mix of styrenesulfonic acid and maleic anhydride. The silver nanoparticles were of colloidal shapes with an average size of 9 nm. For both methods, all samples were incubated 2 days at 29°C, with SEM and EDAX analysis being carried out after the incubation period. Before mounting, samples were washed carefully with deionized water. After stab mounting, the samples were allowed to dry in a desiccator at room temperature. For both methods, scanning electron microscopy (FEI Quanta 200) was used to observe changes in cell morphology after exposure to NPs. Also, EDAX (attached to a FEI Quanta 200 SEM) analysis was carried out to determine the deposition of silver nanoparticles on fungal cells. For liquid exposure method, the sample was fully sunken in the colloidal silver solution, thus being provided an equal coverage of the strain. For spraying method, the solution was sprayed over the media containing the mature fungus, using a pulveriser. In contrast to the previous method, this technique allows a much better O access. SEM images were taken after 2 days of exposure.

In this study the antifungal activity of colloidal silver against A. niger cells was evaluated. A. niger was chosen due to its ubiquitous character and aggressive growing properties. The SEM images collected demonstrated that the silver nanoparticles inhibit cell wall integrity, as silver nanoparticles present a highly reactive potential. A study carried out by Morones et al. (2005) stated that silver nanoparticles disrupt transport systems, including ion efflux. The dysfunction of ion efflux can cause rapid accumulation of silver ions, interrupting cellular processes at their lower concentrations such as metabolism and respiration by reacting with molecules (Seon Min, et al., 2009) Sulfur-containing proteins from the cell wall are likely to be preferential sites for silver nanoparticles binding. Also, following silver nanoparticles transmembranar passage process, involved in a possible DNA binding process, the nanoparticles may have a role in gene inhibition, which may result in the cell not being able to reproduce anymore. Reports on the mechanism of inhibitory action of silver ions on microorganisms show that upon Ag treatment, DNA loses its replication ability and expression of ribosomal subunit proteins as well as some other cellular proteins and enzymes essential to ATP production becomes inactivated (Pal et al., 2009).The two different methods presented slightly different action sites, as fungal hyphae were more affected by the liquid exposure method, Figure 1, while fungal spores responded more efficiently to the spraying method, Figure 2.

Figure 1. SEM images of sunken A. niger cells

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Figure 2. SEM images of sprayed A. niger cells

observed antibacterial property. For the witness samples treated with deionized water, no changes were noted varying the exposure period, Figure 3.

After silver treatment, microscopic images revealed that silver nanoparticles treated hyphae were damaged on hyphal walls, inducing hyphae plasmolysis, therefore cell lysis could be one of the reasons for the

Figure 3. SEM images of A. niger cells exposed to deionized water

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culture and silver NPs. The analysis allowed identification of silver depositions in the damaged areas of hyphal and spores walls, Figure 4.

The Energy Dispersive X-Ray spectroscopy (EDAX) was used as an analytical technique used for elemental analysis and chemical characterization of the interaction between the

Figure 4. SEM and EDAX analysis of damaged hyphae and spores

keV) and sulphur (S-2.307 keV) may be generated by proteins/enzymes present in the wall of the fungus, following cell lysis. Silver nanoparticles may have a crucial role in affecting the function of membrane-bound enzymes, those involved in the respiratory chain. This process can facilitate the generation of reactive oxygen species, which in the end can lead to cell death. The signal-peak found at 2.984 keV specific to silver (Ag) demonstrates the localization of silver in the damaged sites, thus strengthening the correlation between silver effect and hyphae and spores collapsing.

CONCLUSIONS

The slightly different effect of the two methods may be explained be explained by a possible formation of silver nanoparticles aggregates, which in the case of the liquid exposure method they gather at the bottom of the Petri dish where they have a better access at the inferior part of the fungus culture. On the other hand, for the spraying method, before spraying the culture, the solution was well stirred, to provide a homogeneous distribution of silver nanoparticles within the solution. Therefore, after spraying, the conidial heads were covered in a solution “capsule”, this way providing a better area of contact, in comparison with the fungus hyphae. The microscopic observations revealed that the silver nanoparticle solution clearly damaged fungal hyphae and spores, while the samples treated with deionized water appeared to remain intact. The EDAX data acquisition was made from the damage site of fungal hyphae. The area or intensity of a peak in the acquisition spectrum is proportional to the concentration of the corresponding element in the sample. The peaks specific to carbon (C – 0.277 keV) and oxygen (O – 0.523 keV) have the largest share due to the organic character of the sample. The signal-peaks characteristic to sodium (Na-1.040

REFERENCES Feng Q. L., Wu J., Chen G. Q., Cui F. Z., Kim T. N., Kim J. O., 2000, A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J Biomed Mater Res.; 52 (4) :662-8. Gupta A., Maynes M., Silver S., 1998, Effects of halides on plasmid-mediated silver resistance in Escherichia coli. Appl. Environ. Microbiol. 64:5042-5045. Gibbs Ronald J., 1999, Silver Colloids – Do they work? ISBN 0-9676992-0-7.Matsumura Y., Yoshikata K., Kunisaki S., Tsuchido T., 2003, Mode of bactericidal action of silver zeolite and its comparison with that of silver nitrate. Appl Environ Microbiol. 69 (7) :4278-81. Morones J.R., Elechiguerra J.L., Camacho A., Holt K., Kouri J.B., Ramirez J.T., Yacaman M.J., 2005, The nanoparticles. bactericidal effect of silver Nanobiotechnology.; 16:2346–2353.

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on Sclerotium-Forming Phytopathogenic Fungi. Plant Pathology Journal, Korean Society of Plant Pathology, Volume 25, No 4, pp. 376-380. Singh Mritunjai, Singh Shinjini, PrasaDa S., Gambhir I.S., 2008, Nanotechnology in Medicine and Antibacterial Effect of Silver Nanoparticles. Digest Journal of Nanomaterials and Biostructures, Vol. 3, No.3, p. 115 – 122.

Nover L., Scharf K. D., Neumann D., 1983, Formation of cytoplasmic heat shock granules in tomato cell cultures and leaves. Mol Cell Biol.; 3 (9) : 1648–1655. Pal Sukdeb, Tak Kyung Yu, Song Myong Joon, 2007, Does the Antibacterial Activity of Silver Nanoparticles Depend on the Shape of the Nanoparticle? A Study of the Gram-Negative Bacterium Escherichia coli. Appl Environ Microbiol.; 73 (6) : 1712–1720. Seon Min Ji, Kim Su Kyoung, Kim Woo Sang, Jung Hee Jin, Lamsal Kabir, Kim Bin Seung, Jung Mooyoung, Lee Su Youn, 2009, Effects of Colloidal Silver Nanoparticles

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Scientific Bulletin. Series F. Biotechnologies, Vol. XVII, 2013 ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

FUNGAL STRAINS ISOLATED FROM SEVERAL CASES OF HUMAN DERMATOPHYTOSES Mariana CALIN1, Iuliana RAUT1, Olguta DRACEA2, Luiza JECU1, Veronica LAZAR3 1

The National Institute for Research & Development in Chemistry and Petrochemistry- ICECHIM, 202 Independentei Spl., 060021, Bucharest, Romania 2 The National Institute of Research & Development for Microbiology and Immunology “Cantacuzino”, 103 Independentei Spl., 050096, Bucharest, Romania 3 University of Bucharest, Faculty of Biology, 91-95 Independentei Spl., Bucharest, Romania Corresponding author email: [emailprotected]

Abstract Dermatophytosis has become one of the most common human infectious diseases in the world so it is of interest to dedicate more studies to its etiological agent’s dermatophytes. These keratinophilic and keratinolytic filamentous fungi have the ability to invade and colonize keratinized layers of the skin and their appendages. Dermatophytes fungi group include three anamorphic genera namely Epidermophyton, Microsporum and Trichophyton. These three genera include geophilic, zoophilic and anthropophilic species. Usually these filamentous fungi are identified on the basis of conidia morphology and sometimes with specific physiological characters, such as the hair strand perforation and urea hydrolysis. The objective of present study was to isolate and to identify some filamentous dermatophytes fungi from human superficial mycoses. Isolated samples (scales, fragments of nails and subungual debris) were cultured on specific culture media during four months. After incubation time, morphological characters of cultured fungal were observed macroscopically and microscopically. The following cultural characteristics were analyzed: texture, surface and reverse color of the colony, the presence of pigmentation. Microscopic examination offered data on specific characters such as, the presence/absence of macroconidia and microconidia, theirs shape, their septa number, the presence of chlamydoconidia, spiral hyphae and nodular organs. The strains identification was completed with in vitro hair strand perforation and urea hydrolysis. The isolated microbial strains were identified as belonging to Trichophyton and Microsporum genera. Keywords: dermatophytosis, dermatophytes fungi, Microsporum, Trichophyton.

fungi namely Epidermophyton, Microsporum and Trichophyton (Weeks, 2003; Kayzer, 2005; http://www.doctorfungus.org/). They have the ability to invade and colonize keratinized layers (Sharma et al., 2011) and to produce enzymes (keratinases), endoproteases and exoproteases (Monod, 2008). Dermatophytes include geophilic, zoophilic and anthropofilic species, with a restricted or a worldwide geographical distribution (Achterman, 2012). Dermatophytosis can vary from acute to chronic forms, depending on many factors, including the host, species of fungus involved or lesions location on the body surface (Vermout et al., 2008, Bramono, 2012). Dermatophytosis can be transmitted directly by contact with infected person or indirectly through contact with infected products or objects (Gupta et al., 2003).

INTRODUCTION

Dermatophytosis are superficial skin infections confined to the stratum corneum, produced by filamentous fungi called dermatophytes. In a case of dermatophytosis the lesions are very characteristic and can affect different areas of the body surface. Depending on the area of the body affected by fungal infection, dermatophytosis (ringworm or tinea) are classified in several type, namely: tinea barbae (chin, mustache), tinea capitis (scalp, eyebrows, eyelashes), tinea corporis (glabrous skin), tinea cruris (groin), tinea faciei (on the face), tinea favosa (favus), tinea imbricata, tinea manuum (on hand), tinea unghium (on nails), tinea pedis (at legs) (Hainer, 2003; Vander Straten et al., 2003; Gupta et al., 2008). In the dermatophytes group are included three anamorphic genera of hyaline filamentous

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mounts mounted in a drop of lactophenol cotton blue and, respectively. Slide culture technique The technique was performed in a wet room made into a Petri plate by placing a fragment of sterile filter paper and moistening it with sterile distilled water. Over the filter paper a fragment of sterile glass in U-shape was placed and over which was placed a sterile microscope slide. A block of potato extract medium of about 1 cm2 was placed over the slide, in the center. The medium was inoculated with a mycelium from the fungal strain thereafter covered by a cover slip. The Petri plate was incubated at 30˚C until fungal growth was observed (Figure 1) Microscopic examination was made by wet mounts prepared in a drop of lactophenol cotton blue.

In recent years, the risk of fungal infections has been increasing drastically so it is of interest to solve these diseases. The objective of present study was to isolate and to identify some filamentous dermatophytes fungi from human superficial mycoses. Isolated samples (scales, fragments of nails and subungual debris) were cultured on specific culture media during four months. MATERIALS AND METHODS Samples A number of 34 samples were used in this study, represented by skin scales, hair strands, nails fragments and subungual debris. Samples used in study were collected in 2008-2009 in the Mycotic Infections laboratory of the National Institute of Research & Development for Microbiology and Immunology Cantacuzino from apparently healthy people. Sampling method A classic protocol was used for samples collection (Coman and Mares, 2000; Mares and Bazgan, 2008). The samples were collected after cleaning the affected area with 70% alcohol. From the skin surface, the samples were collected by scraping the lesion from the center to its edge using a sterile scalpel. The hair samples were plucked or shave (where the hair could not be plucked) using a sterile tuck. The nail fragments were collected using a sterile scissors, while the nail bed was scraped and the subungual debris have been collected. Culture method Collected samples were cultured on two solid media, potato extract medium and Sabouraud medium. After inoculation, the Petri plates were incubated at 25-30˚C, for 4 weeks. The microbial growth was monitored daily during the entire period. Identification methods Macroscopic observations Macroscopic observations of isolated strains in solid cultures were carried out weekly, noting the growth rate, colonies morphology and pigment formation in the culture medium. Microscopic observations The investigations were done using an Olympus BX51 microscope and consisted in direct microscopy in a drop of 10% potassium hydroxide (KOH) for skin scales examination and 20% for nails examination and in wet

Figure 1. Slide culture technique

Urea hydrolysis test The isolated fungi were incubated in urea liquid medium for 7 days at 25-30°C. The inoculated test tubes were examined daily to observe a possible color change of the culture medium in case of a positive test from straw to reddishpurple. Urea hydrolysis test was used to distinguish Trichoplyton mentagrophytes from Trichophyton rubrum. Trichophyton mentagrophytes is usually urease positive in 7 days and Trichophyton rubrum is usually urease negative In vitro hair strand perforation test The test was performed by placing a few hair strands fragments in a sterile Petri plate and adding 10 ml of sterile distilled water and 0.1 ml of yeast extract 10%. A piece of fungal mycelium was transferred in Petri plate and incubated at 25°C for 21 days. Periodically was done wet mounts, mounted in a drop of lactophenol cotton blue.

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RESULTS AND DISCUSSIONS

In the positive skin scraping samples, on direct microscopy (KOH), fungal elements as networks of branching fungal hyphae were observed (Figure 2). For 6 samples, the KOH test was negative and no fungal elements were relieved.

Figure 2. Skin scraping infected with septate, branching fungal hyphae

Collected samples were cultured on potato extract medium and Sabouraud medium in Petri plates. Of the 34 samples were obtained 28 fungal isolates and all were used for subsequent identification. The fungal colonies developed after 14 days of incubation were investigated through macroscopic and microscopic observations. Macroscopic observations of isolated strains offer information about growth rate, colonies morphology (color of colony obverse and reverse, shape of the edges, colony surface appearance, and texture) and pigment formation in the culture medium. Based on these informations, isolated fungi were identified as belonging to Trichophyton and Microsporum genera. Therefore, 22 strains belong to the genus Trichophyton, as Trichophytoninterdigitale, Trichophyton mentagrophytes, Trichophyton rubrum and Trichophyton sp. and 6 strains belong to the genus Microsporum, as Microsporum canis and Microsporum sp. (Figure 3). These fungal isolates are known to be involved in the etiology of human dermatophytosis (Jackson, 2006; Mahmoudabadi and Yaghoobi, 2008).

Figure 3. Macroscopic characteristics of isolated Trichophyton sp. and Microsporum sp. strains, on PDA medium at after 14 days of incubation

The preparations with lacthophenol cotton blue relieved some important characters for fungal strains identification, as shape and dimensions of macroconidia and microconidia, macroconidial septa number, presence of chlamydoconidia, spiral or raquet hyphae and nodular organs. The results of this investigation are presented in Figure 4. Characteristic of Trichophyton mentagrophytes culture are: rapid growth, granular surface, flat colonies, white to cream colour on obverse, irregular edge, brownish yellow to reddishbrown on reverse.

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clavate macroconidia and abundant pyriform microconidia (Figure 4c). Characteristic of Microsporum canis culture are: fast growth, flat colonies, white-yellowish surface; golden yellow colony reverse. Microscopic observation showed abundant long, rough, fusiform macroconidia and a few pyriform to clavate microconidia. (Figure 4d). For a more precise identification, more tests were carried out such as urea hydrolysis test and in vitro hair strand perforation test. Urea hydrolysis test was used to distinguish Trichoplyton mentagrophytes from Trichophyton rubrum. Trichophyton mentagrophytes is usually urease positive in 7 days and Trichophyton rubrum is usually urease negative. As it can be shown in Figure 5, the result for Trichophyton mentagrophytes is positive (left), the strain producing urease and breaking down urea. Meanwhile, Trichophyton rubrum has no hydrolytic activity against urea (right).

Figure 5. Urea hydrolysis test Figure 4. Microscopic characteristics of isolated Trichophyton sp. and Microsporum sp. strains in lacthophenol cotton blue-slide culture technique (400x)

The positive result at the in vitro hair strand perforation test for Microsporum canis is presented in Figure 6.

Microscopic observation showed smooth macroconidia (4-6 septa), numerous spherical or pyriform, microconidia, in clusters and spiral hyphae (Figure 4a). Characteristic of Trichophyton rubrum culture are: moderate growth, flat to slightly raised, white to cream colonies obverse, velvety with an wine red reverse. Microscopic observations showed pencil-shaped abundant macroconidia, often show an terminal appendages and pyriform microconidia (Figure 4b). Characteristic of Trichophyton interdigitale culture are: moderate growth, flat, white to cream colonies obverse, granular surface, with an yellow reverse. Microscopic observation showed few

Figure 6. The in vitro hair strand perforation test

Also, Trichophyton interdigitale, Trichophyton mentagrophytes are able to perforate the hair

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4. Gupta A.K., Chaudhry M., Elewski B., 2003. Tinea corporis, tinea cruris, tinea nigra, and piedra, Dermatologic Clinics, 21: 395–400. 5. Gupta A. K., Cooper E. A., 2008. Dermatophytosis (Tinea) and Other Superficial Fungal Infections. In: Infectious Disease Diagnosis and Treatment of Human Mycoses edited by D. R. Hospenthal, M. G. Rinaldi, Humana Press Inc.Totowa, New Jersey, 355-381. 6. Hainer B. L. 2003. Dermatophyte Infections, American Family Physician, 67 (1) :101-108. 7. Jackson C.J., Takashi Mochizuki T., Barton R.C., 2006. PCR fingerprinting of Trichophyton mentagrophytes var. interdigitale using polymorphic subrepeat loci in the rDNA nontranscribed spacer. Journal of Medical Microbiology, 55, 1349–1355. 8. Kayser F. H., Bienz K.A., Eckert J., Zinkernagel R.M. 2005. Fungi as humans pathogens, Coutaneous mycoses. In: Medical microbiology Thieme, cap. 6, pp. 372-376. 9. Mahmoudabadi A., Z., Yaghoobi R., 2008. Extensive tinea corporis due to Trichophyton rubrum on the trunk, Jundishapur Journal of Microbiology, 1 (1) : 35-37. 10. Mares M., Bazgan O., 2008. Diagnosticul de laborator al infectiilor produse de fungi. In: Tratat de Microbiologie Clinica D. Buiuc, M. Negut, Editia a II-a, Editura Medicala, Bucuresti, 953-1017. 11. Monod M., 2008. Secreted proteases from dermatophytes, Mycopathologia, 166:285-294 12. Seebacher C, Bouchara J-P., Mignon B., 2008. Updates on the epidemiology of dermatophyte infections, Mycopathologia,166:335–352. 13. Sharma M., Sharma M., Rao V.M., 2011. In vitro biodegradation of keratin by dermatophytes and some soil keratinophiles, African Journal of Biochemistry Research, 5 (1) :1-6. 14. Szepietowski J.C., Bielicka E., Maj J., 2002. Inflammatory tinea barbae due to Trichophyton rubrum infection – autoinoculation from fingernail onychomycosis?, Case Reports and Clinical Practice Review, 3 (4) : 254-256. 15. Venkatesan G., Ranjit Singh A.J.A., Murugesan A.G., C. Janaki C., Gokul Shankar S., 2007. Trichophyton rubrum – the predominant etiological agent in human dermatophytoses in Chennai, India. African Journal of Microbiology Research, 009-012. 16. Vander Straten M. R., Hossain M. A., Ghannoum M. A., 2003. Cutaneous infections, Dermatophytosis, onychomycosis and tinea versicolor, Infectious Disease Clinics of North America, 17: 87–112. 17. Vermout S., Tabart J., Baldo A., Mathy A., Losson B., Mignon B., 2008. Pathogenesis of dermatophytosis, Mycopathologia, 166: 267-275. 18. Weeks J., Moser S.A., Elewski B.E., 2003. Superficial cutaneous fungal infection. In: Clinical Mycology, edited by Dismukes W. E., Gappas P. G., Sobel J.D., Oxford University Press, 367-389. 19. Woodfolk J.A., 2005. Allergy and Dermatophytes, Clinical Microbiology Reviews. 18 (1) :30.*** http://www.doctorfungus.org/

strand. Trichophyton rubrum concerning the ability to perforate the hair strand in vitro and are negative (data not shown). The isolation and identification procedure finally provided two predominant fungal strains, namely Trichophyton rubrum and Trichophyton mentagrophytes.These results are similar to those reported by other authors (Szepietowski et al., 2002; Venkatesan, 2007; Seebacher et al., 2008; Woodfolk, 2012). CONCLUSIONS

In this study, several dermatophytes fungi were isolated from human infections. From the total number of samples, 82.35% were positive and the isolated strains belonged to dermatophytes fungi. The most frequently isolated strains belong to the Trichophyton genus, as Trichophyton rubrum and Trichophyton mentagrophytes; our results being in accordance with other similar studies. Of the total of 28 isolated strains Trichophyton rubrum reprezents 35,71%; Trichophyton mentagrophytes 21,43%; Trichophyton interdigitale 7,14%; Trichophyton sp. 14,29%; Microsporum canis 7,14% and Microsporum sp. 14,29%. The practical importance of the study is that the methods used to identify the fungal strains participate in improvement of dermatophytosis diagnostic algorithm. Also was performed the monitoring of incidence of etiologic agents involved in human dermatophytosis REFERENCES 1. Achterman R. R., White C. T., 2012. Dermatophyte virulence factors: Identifying and analyzing genes that may contribute to chronic or acute skin infections. International Journal of Microbiology, pp. 1-8. 2. Bramono K., 2012. Chronic Recurrent Dermatophytosis in the Tropics: Studies on Tinea Imbricata in Indonesia. Korean Journal of Medical Mycology,17 (1) :1-7. 3. Coman I., Mares M., 2000. Principalele genuri de micromiceti cu implicatii in patologie, Genul Microsporum, Genul Trichophyton; Diagnosticul bolilor produse de micromiceti. In: Micologie Medicala Aplicata; Editura Junimea, Iasi, 45-70; 154-165; 250260.

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Scientific Bulletin. Series F. Biotechnologies, Vol. XVII, 2013 ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

SCREEN-PRINTED CARBON ELECTRODES MODIFIED WITH PRUSSIAN BLUE AND A NON-CONDUCTING ELECTROPOLYMERIZED FILM FOR SELECTIVE DETERMINATION OF H2O2 IN BEVERAGES Florentina HUTANU1, 2, Emilia OCNARU2, Melania-Liliana ARSENE2, Mihaela BADEA-DONI2 1’

Stefan cel Mare’ University of Suceava - Universitatii 13, 720229, Suceava, Romania, e-mail: [emailprotected] 2 INCDCP- ICECHIM, 202 Splaiul Independentei, District 6, 06182, Bucharest, Romania Phone: +4021.315.32.99, Fax: +4021.312.34.93 Email: [emailprotected] Corresponding author email: [emailprotected] Abstract The development of a highly selective and sensitive sensor for H2O2 in beverages such as natural juices, is described in this work. The sensor is based on the deposition of Prussian Blue (PB) onto screen-printed carbon electrodes (SPCEs) followed by the electropolymerization of a non-conducting film. Several procedures for PB deposition on the SPE electrodes were tested: electrochemical deposition (potentiostatic, cyclic voltammetry) and chemical deposition. The electrochemical and analytical properties of the SPCE/PB sensors had been evaluated and the potentiostatic method for PB deposition was selected for the further development of the H2O2 sensor. In order to develop a robust sensor for H2O2 determination in samples with complex matrix, we covered the PB layer with an electropolymerized nonconducting film with a high permselectivity for H2O2. This film is a copolymer based on 2,6-DHN (2,6dihydroxynaphtalene) and APEA (2-(4-aminophenyl)-ethylamine). The SPCE/PB/copolymer sensor demonstrated improved stability in operational conditions and excellent interference rejection properties. This sensor may be successfully applied on-field, using a portable potentiostat-galvanostat and the chronoamperometry technique, as well as in a laboratory bench flow injection analysis system with amperometric detection. The developed sensor was able to measure H2O2 in the linear range 1 PM – 500 PM (R=0.9989), with a detection limit of 0.5 PM. The SPCE/PB/copolymer sensor maintained for a long period its response for H2O2 (94% response was retained after 60 days). Keywords:screen printed carbon electrode, Prussian Blue, nonconductive copolymer, H2O2, beverages

The first sensors for hydrogen peroxide based on PB modified glassy carbon electrode were reported by Karyakin et al [4]. The PB deposited on these sensors has an electrocatalytic effect on the reduction of H2O2, allowing its detection at potentials close to 0 V, thus making possible the coupling with oxidase enzymes while avoiding or reducing the electrochemical interference. Screen-printed electrodes are frequently used in analytical applications because of their unique properties such as small size, low detection limit, fast response, high reproducibility, etc. [5]. Screen-printed carbon electrodes (SPCEs) are devices that are produced by printing different inks on various types of plastic or ceramic

INTRODUCTION

The direct amperometric detection of hydrogen peroxide at conventional electrodes is possible only at 0.6 V vs. Ag/AgCl. At this potential, the presence of easily oxidizable compounds present in real samples (ascorbic acid, bilirubin, uric acid, etc.) can easily interfere in the measurement, being oxidized at the electrode together with hydrogen peroxide. For this reason the detection of H2O2 at potentials around 0 using electrodes modified with electrochemical modifiers, such as Prussian Blue (PB), has enormous advantages and applications in many fields [1-3].

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aminoethyl)aniline (APEA) were from Aldrich and respectively Fluka. Double-distilled water was used throughout. 2,6-DHN and APEA were dissolved in 0.1M phosphate buffer pH 7.4. Hydrogen peroxide (Fluka) was prepared daily in phosphate buffer, pH 6.5.

substrates. The composition of the inks used for printing on the electrodes determines the selectivity and sensitivity required for each sensor development. Screen-printed electrodes are inexpensive, simple to prepare, versatile and suitable for the mass-production of disposable electrodes [6]. The aim of this work was to develop a simple, robust and portable sensor based on PB and a non-conducting copolymer which are deposed on a SPCE for hydrogen peroxide determination in beverages (commercial juices).

Modification of SPCE with Prussian Blue

Three procedures were investigated for PB film formation on the working electrode of screen printed carbon electrodes in order to prepare sensitive and robust PB sensors for H2O2 determination. The tested procedure were based on: chemical, galvanostatic and cyclic voltammetry based deposition. Prior to Prussian Blue modification, the SPCEs were pretreated in the presence of 50 mM phosphate buffer in 0.1 M KCl, pH 7.4, by applying the potential of + 1.7 V versus Ag/AgCl for 3 minute. For the chemical deposition of PB films, two solutions were prepared. Solution 1: 100 mM K3[Fe(CN)6 in 10 mM HCl. Solution 2: 100 mM FeCl3 in 10 mM HCl. Prussian Blue modification of SPCE was then accomplished by placing 5 μl of precursor solution 1 and 5 μl of precursor solution 2 onto the working electrode area [7]. The solution was left onto the electrode for 10 min and then rinsed with a few millilitres of 10 mM HCl. The electrodes were then left 90 min in the oven at 100° C to obtain a more stable and active layer of PB. For electrochemical deposition of PB the galvanostatic and cyclic voltammetry techniques were tested. The galvanostatic deposition was made in a mixture (solution 3) of 2.5 mM K3[Fe(CN)6,], 2.5 mM FeCl3 and 1 mM AOT prepared in 100 mM KCl and 100 mM HCl solution by applying the potential of 0.4V for 40 sec [1]. After a gentle rinsing with water, the sensor was placed in a solution of 100 mM KCl in 100 mM HCl and a number of 20 cycles, between - 0.2 and 0.4 V, at a scan rate of 50 mV/s, was run. The cyclic voltammetrydeposition was carried out in the solution 3 (20 cycles, between - 0.2 and +0.4 V, at a scan rate of 50 mV/s) [14]. The deposition of the PB film was confirmed by performing cyclic voltammetry in 50 mM phosphate buffer, pH 7.4.

MATERIALS AND METHODS Apparatus

Electrochemical measurements were carried out using a μ Autolab type III potentiostat/galvanostat computer controlled by the GPES software, as well as a portable PalmSens potentiostat/ galvanostat controlled via the PalmSensPC software. The flow injection analysis system consisted from a four-channel Minipuls 3 Gilson peristaltic pump fitted with tygon tubing (1.52 mm id) used for the propulsion of fluids, an injection valve (Rheodyne, 7725i model) and a flow cell special for SPCE from Dropsens, Spain. The valve loop volume was 100μL. Fittings and connectors were used to connect the different components of the manifold. The optimum flow rate was 0.36 mL/minute. The detector was the same potentiostat/galvanostat used for voltammetric measurements. Electrodes Screen-printed carbon electrodes (SPCEs) model DRP-110 purchased from DropSens (Spain) were used for electrochemical measurements. In this case the electrochemical cell is composed by a graphite working electrode (d = 4mm), a graphite auxiliary electrode and a silver pseudoreference electrode, with silver electric contacts deposed on a ceramic substrate. Reagents All chemicals from commercial sources were of analytical grade. Iron chloride (FeCl3), potassium ferricyanide K3[Fe(CN)6],HCl 37%, sodium chloride, hydrogen peroxide (30%), were purchased from Sigma-Aldrich. AOT (Dioctyl sulfo-succinate sodium salt) was from Carlo Erba. The monomers 2,6dihydroxynapthalene (2,6-DHN) and 4-(2-

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The PB modified electrodes were stored dry at room temperature in the darkness. Electrodeposition of non-conducting films on SCPE/PB The SPCE/PB sensors were covered with a non-conducting copolymer electrodeposited using the cyclic voltammetry technique. The copolymer was synthesized from a solution of 0.9 mM 2,6-DHN and 10 mM APEA by cycling for 10 – 20 times the potential from + 0.2 V to +1.1 V with a scan rate of 5 – 10 mV/sec.

SPCE/PB - galvanostatic deposition of PB SPCE/PB - chemical deposition of PB SPCE/PB - cyclic voltammetry deposition of PB

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SPCE / PB sensors characterization After the preparation, the SPCEs modified with PB (SPCE / PB) where characterized by cyclic voltammetry, in 0.1 M KCl prepared 0.1M HCl, in order to prove the PB film formation.

Figure 1 shows the specific voltammograms of the SPCE / PB sensors prepared according to the procedures described in ‘Materials and methods’ section. Two characteristic peak couples corresponding to conversion of high and low spin ions appear with formal potentials of 0.10 V and 0.80 V. The oxidation and reduction peaks centred at 0.10 V are much narrower and well shaped for the PB sensors obtained via electrochemical deposition compared with those obtained via chemical deposition. However, the SPCE / PB obtained by cyclic voltammetry showed the lowest oxidation and reduction peaks, and also some other experiments led to the conclusion that the PB film was too thin and fragile. For this reason, the most important further studies were done on the SPCE / PB sensors obtained by chemical and galvanostatic deposition. The influence of the thermal stabilization by keeping the electrodes at 100° for 90 min was also studied. No evident differences between the treated and nontreated PB electrodes were observed regarding the response of the electrodes in KCl, phosphate buffer or for H2O2, but the operational stability was greater improved for the electrodes stabilized via the thermal treatment.

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The effect of potential scan rate on the oxidation (Iox) and reduction peak (Ired) currents was studied for the redox couple present around 0.10 V. Plotting the Iox and Iredvs. square root of scan rate showed a linear relationship (figure 2), the result indicating a diffusion limited process. This behaviour was observed for all the three tested methods for PB deposition. SPCE / PB - galvanostatic deposition of PB Iox =-72.71 + 26.65 x ; R=0.9887 Ired = 27.67 - 25.55 x ; R=0.9974

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RESULTS AND DISCUSSIONS

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The pH value of the electrolyte solution is an important parameter for H2O2 determination using the PB modified SPCEs. The stability and sensitivity of the PB sensor may be affected by the hydroxide ions which can break the Fe-(CN)-Fe bonds, but also by the protons which may block the electrochemical reactivity of PB [8]. The pH influence on the electrochemical determination of 1 mM H2O2 using the SPCE / PB was studied at pH ranging from 6-7.4 (figure 3). For all the tested electrodes the highest reduction peaks were obtained for the

technique may be applied for on-field measurements using a portable detector. According to the measurement results, the linear range was from 1 PM to 100 PM H2O2 with the linear correlation of 0.9967 for the sensor obtained by chemical deposition of PB and, respectively, of 0.9985 for the sensor prepared by galvanostatic deposition. The sensitivity of the galvanostatic prepared sensors was with 50 % higher than obtained via the chemical deposition (figure 4). For both type of sensors the detection limit was 0.5 PM H2O2.

pH 6.5. For all the following studies, as optimum electrolyte solution, was used the 50 mM phosphate buffer, pH 6.5. The selection of the optimum working potential to be applied when measuring H2O2 was studied for the next electrochemical techniques: chronoamperometry, amperometry in stirred solution and amperometry in flow injection system (FIA). Potentials ranging from -100 mV to +200 mV were applied in presence of a selected concentration of hydrogen peroxide (100μM). The highest signal was obtained for the potentials of -50 mV when working in chronoamperometry and of -100 mV in amperometry (for both, stirred solutions and FIA). The lowest background noise and the highest signal for H2O2 measurement was recorded for the PB sensor prepared via the galvanostatic procedure. -5

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Characterization of SPCE / PB / copolymer sensors The PB exhibits excellent catalytic activity for the electroreduction of hydrogen peroxide, but the operational stability of PB is still a matter of concern in real samples with complex matrix, such as the food samples. In order to protect the PB layer, the coverage with nonconducting films as the poly(o-aminophenol) [1] or with ionomers as Nafion [9] was reported. In this work we report the use, for the first time, of the non-conducting copolymer electrosynthesized from a mixture of 2,6-DHN and APEA for protection of the PB layer. The copolymerization was performed via the cyclic voltammetry technique by cycling the potential from + 0.2 V to +1.1 V. In figure 5 one can observe that the irreversible oxidation peak at the +0.65 V present in the first three cycles disappears in the following cycles. The oxidation peak current decrease proves the

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Figure 3. Influence of electrolyte solution pH on the response of SPCE / PB to 1mM H2O2

Regarding the hydrogen peroxide determination, the amperometry under stirring technique gave the best results for the SPCE / PB sensor prepared via galvanostatic method in rapport with the background noise and with the response time. Even that the sensitivity recorded with sensor prepared by chemical deposition of PB is higher than that obtained for that obtained via electrochemical procedures, the linear range of concentration for PI shorter (data not shown). Cronoamperometry technique is very advantageous due to the fact that the screen printed electrodes allow working with a very low volume of sample (100 PL), the determination is fast and reproducible, and does not require a time for working electrode polarization. Also this electrochemical

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porous, which acts as a barrier especially for higher concentration of H2O2.

formation of a non-conducting film on the surface of the SPCE / PB sensor. -4

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The optimization of the copolymer formation related to the hydrogen peroxide determination was performed studying the influence of the scan rate and number of cycles. In figure 6, one can observe the influence of the number of cycles used for the copolymer electrodeposition on the calibration graphs obtained by chronoamperometry. A higher number of cycles led to a higher thickness of the copolymer layer and consequently to a more difficult diffusion of H2O2 across it.

By comparing figures 4 and 6, it can be concluded that an important feature achieved by the copolymer electrodeposition on the PB layer was the extending of the linear concentration range up to 500 PM in cronoamperometry. The optimum conditions for copolymer electrodeposition were: scan rate = 10 mV/s; number of cycles = 10; potential range = 0.2 V - 1.1 V. Another important characteristic of the SPCE / PB / copolymer sensors is represented by a higher operational stability even in flow injection analysis conditions. In figures 8 is presented the FIA amperogram recorded for the PB sensor covered with the poly(DHN – APEA) copolymer.

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Also, in FIA conditions the sensitivity for H2O2 detection was much higher comparing with those obtained in tcronoamperometric conditions. The possible interference of compounds present in beverages, such as ascorbic acid and glucose, was tested. Glucose showed no interference in H2O2 determination with SPCE/PB/copolymer sensor. Ascorbic acid may interfere, giving a false negative signal, only if the ascorbic acid concentration is much higher than of H2O2. The SPCE/PB/copolymer sensor maintained for a long period its response for H2O2 (94% response was retained after 60 days)

stability of the PB sensor. The SPCE/PB/ copolymer sensor has an excellent electrocatalytic activity for the reduction of H2O2, with a broad linear range from 1PM to 500 PM, and a detection limit of 0.5 PM. Furthermore, introduced into a FIA system, the sensor proved a great operational stability. The developed sensor was successfully applied to the determination of H2O2 in commercial juices. ACKNOWLEDGEMENTS

The authors acknowledge to the project POSDRU/CPP107/DMI1.5/S/78534 for financial support.

Real sample analysis Sometimes, for the aseptic packaging of natural fruit juices, hydrogen peroxide is used as a chemical agent for sterilization. However, the hydrogen peroxide residues in higher concentration are irritative for the skin and may affect the human health. The developed SPCE/PB/copolymer sensor was applied for the fast and simple determination of H2O2 in several commercial fruit juices. The sample treatment consisted only in dilution with 50 mM phospahate buffer, pH=6.5. In table 1 is presented the H2O2 concentration determined in the tested samples with the SPCE /PB/copolymer sensor by chronoamperometry.

REFERENCES 1. Ping, J., Wu, J., Fan, K., Ying, Y., 2011. An amperometric sensor based on Prussian blue and poly(ophenylenediamine) modified glassy carbon electrode for the determination of hydrogen peroxide in beverages. Food Chemistry 126, p. 2005–2009 2. de Mattos, I. L., da Cunha Areias, M. C., 2010. Automated determination of glucose in soluble coffee using Prussian blue–glucose oxidase–Nafion modified electrode. Talanta 66, p. 1281–1286. 3. Haghighi, B., Hamidi, H., Gorton, L., 2010. Electrochemical behavior and application of Prussian blue nanoparticle modified graphite electrode. Sensors and Actuators B 147, p. 270–276 4. Karyakin, A.A., Gitelmacher, O., Karyakina, E.E., 1994. A high-sensitive glucose amperometric biosensor based on Prussian Blue modified electrodes. Anal. Lett. 27 (15), p. 2861–2869. 5. Dominguez Renedo, O., Alonso-Lomillo, M.A., Arcos Martiınez, M.J., 2007. Recent developments in the field of screen-printed electrodes and their related applications. Talanta 73, p. 202–219 6. Pchelintsev, N.A., Millner, P.A., 2007. Development of surface activated screen-printed carbon transducers for biosensors application. Anal. Let. 40 (7), p. 1317 – 1332. 7. Ricci, F., Palleschi, G., 2007.Sensor and biosensor preparation, optimisation and applications of Prussian Blue modified electrodes. Biosens. Bioelectron. 21, p. 389–407. 8 de Mattos, I.L., Gorton, L., Ruzgas, T., Karyakin, u A.A., 2000. Sensor for hydrogen peroxide based on Prussian Blue modified electrode: improvement of the operational stability. Anal. Sci. 16, p. 795–798. 9. Ping, J., Mao, X., Fan, K., Li, D., Ru, S., Wu, J., Ying, Y., 2010. A Prussian blue-based amperometric sensor for the determination of hydrogen peroxide residues in milk. Ionics 16, p. 523-529.

Table 1. H2O2 concentration in several commercial fruit juices sample H2O2 (PM) Orange juice (Granini) 3.12 Peach juice (Prigat) n.d.* Apple Juice (Granini) n.d. Orange pulpy juice (Cappy) n.d. Orange juice (Fanta) 2.48 *n.d. = not detectable

The results demonstrated that the level of hydrogen peroxide concentration used in tested juices preservation is very low. CONCLUSIONS

In this work, it was developed a robust and cost-effective sensor based on SPCE modified with PB and an electropolymerized nonconducting film for H2O2 determination. The experimental results showed that the copolymer film remarkable improves the operational

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Scientific Bulletin. Series F. Biotechnologies, Vol. XVII, 2013 ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

SUPPORTING STUDENTS FOR A BIOTECH CAREER Florentina MATEI1, Mioara VARGA1, Silvana DANAILA-GUIDEA1, Mariana IORDACHE2, Stefana JURCOANE3, Camelia DIGUTA3 1

UASMV Bucharest, Faculty of Biotechnologies, 59 Marasti Blvd., Bucharest, Romania 2 Mari-Net 21 SRL, 21, Bucharest, Romania 3 CBM Biotehgen, 59 Marasti Blvd., Bucharest, Romania Corresponding author email: [emailprotected]

Abstract The biotech high-education in Romanian has started relative recently, the first accredited college being registered in 1996 in Bucharest, in the University of Agronomical Sciences and Veterinary Medicine. Since then, more than 1000 license students have graduated in different specializations as Agricultural Biotechnology, Industrial Biotechnology and Veterinary Biotechnology. Recently, during the implementation of a structural funds project for human resources (POSDRU/109/2.1/G/81570) has been conducted a survey on how the biotech graduates have been placed on the labour market. The statistics shows that less than 27% from the graduates have found jobs in biotech fields, such as scientific research and education, food and beverages production, environmental protection, including biofuel production, pharmaceutical products, instrumentation and suppliers. In this regard, educators, professionals and counsellors, on regional level, have started to take important measures to develop and implement activities to increase the absorption level of the biotech graduates. Aside the curricula improvement, an important step it has been the counselling of 100 students, between 20 and 30 years old about self-knowledge, neuro-linguistic programming, CV content, letter of intention writing, as well as about behaviour during an interview. Also, it has been underlined the importance of the continuous learning. More than 93% from the counselled students has found it very useful. Further, a network of 12 economical biotech operators have been developed and 38% of the counselled students have been involved in internships in fields as agriculture and food production, pharmaceutical industry, food safety and consumer protection, hygiene products design and biotech research. As preliminary result, all the graduated counselled students have decided to continue their studies in master courses, and 27% has already been employed in biotech field. The program will continue in the next years, with the aim to increase the employability for the biotech graduates. Keywords: biotech career, counselling, internship.

in the University of Agronomical Sciences and Veterinary Medicine having different specializations such as Agricultural Biotechnology, Industrial Biotechnology and Veterinary Biotechnology. In the mean time, the faculty leads to Master of Science degree in the fields of „Biotechnologies and Food Safety”, „Biotechnologies in Environment Protection”, as well as „Modern Applications of Biotechnology in Agriculture” (http://en.usamv.ro/faculty-ofbiotechnologies). Generally talking, Biotechnology is a growing industry and supposes to offer excellent opportunities, pay and benefits. These opportunities are available for people with a background in biological science with good laboratory and computer science skills (Frierman-Hunt G, 2008). In the past last years, the biotech graduates have faced difficult problems related to their

INTRODUCTION

By definition, biotechnology means the use of advances in life science to create products and services for our world (Frierman-Hunt G, 2008). Although biotechnology industries make many different products from vaccines to seeds to specialized equipment, many of the job duties and titles are similar across the industry. To understand the different job functions, the jobs can be grouped into five areas: research and development (which includes research and development, laboratory support and technician jobs); manufacturing and services; quality and regulatory affairs; sales and technical support; and administration and management. The biotech high-education in Romanian has started relative recently, the first accredited college being registered in 1996 in Bucharest,

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integration in the labour market in biotech fields (Matei, 2012). The causes are multiples and relate to biotech jobs market in Romania, student motivation, career counselling, don’t forgetting the last years of economical crisis. In 2011 our team have started to implement a structural funds project, financed by POSDRU (HRDSOP-Human Resources Development Sectorial and Operational Program) in the Bucharest-Ilfov region. The main objective of the project is to increase the biotech graduates employability developing counselling services and implementing good quality internships. The project financing lasts two years, but there are undertaken measures to sustain its activities after the end of EU funding. As mains activities, the project developed a survey on biotech graduated employability in the labour market, as well as organizing career counselling sessions for students and creating a partnership with economical agents for internships development.

2. Information, awareness and career counselling During March–May 2012 time frame and following the results’s survey the team has conducted a virulent campaign of biotech students information and awareness regarding the importance of their involvement in counselling sessions for their career development and in real and qualitative internships. The information and awareness tools were oral communication in front of the classes, distribution of flyers and brochures, electronic messages via e-mails and socialisation networks, as well as the use of the campus visibility spots. Between April 2012 and June 2012, 100 students (22 Master students and 78 licence students) have been involved in counselling sessions, where they have learned about selfknowledge, neuro-linguistic programming, CV content, letter of intention writing, as well as about behaviour during an interview. Also, it has been underlined the importance of the continuous learning. 3. Preparing and developing biotech internships Having in the team teachers from biotech high education and stakeholders from the field, it has been created a partners network with economic operators from the biotech fields. The economic operators by their legal representative or operational representatives, have been contacted via e-mail, phone or in person. Considering the sectors were the faculty members and former students have been employed, the dialogue have been conducted in the research field, food industry, pharmaceutical industry, food safety authorities and laboratories, environmental protection.

MATERIALS AND METHODS

The project is run by a multi-actor team, consisting in teachers from biotechnology high education field (UASMV Bucharest, Faculty of Biotechnologies), researchers from the same field (CBM Biotehgen), human resources and counselling professionals, former and actual students, stakeholders from biotech sector (research institutes, food production and food safety, pharmaceuticals, cosmetics, environmental protection). 1. Labour market insertion survey The target group for the survey consists of Biotechnology graduates between 2007 and 2011 in the region Bucharest-Ilfov. They questions addressed to their professional background and how they have passed the transition from school to active life on the market. The questionnaire consisted in eight questions batteries with answers on choice. The answers have been processed by tools of classical mathematical statistics. The questionnaire has been send to the graduates via e-mails existent in the Faculty of Biotechnologies database, as well as by socialisation website (Facebook) and groups created along the years by former and actual students from the biotechnology field.

RESULTS AND DISCUSSIONS 1. Labour market insertion survey We have received feedbacks from around 10% of the graduated students, from which 84% are living and working in Romania. The answers have come from licence graduates (54%) and from Master courses graduates (46%). The statistical results show the following facts: -67% from the licence students have follow further Master courses; -50% from the graduates have followed other types of professional training by their own

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yability, while only 10% consider useful doctoral and post-doctoral training; -38% from the graduates considered that the biotech job offer in Romanian labour market is very limited. 2. Information, awareness and career counselling During the conducted campaign regarding the importance of the biotech students’ involvement in counselling sessions and internships have been distribute around 350 flyers and 250 brochures. More than 200 students from licence and master courses have been informed and awarded about the opportunities given by the implementing project to get involved in counselling activities and to apply for an internship. As a result of the awareness campaign, 100 students, between 20 and 30 years old, got involved in counselling sessions with professional from human resources (Mari Net 21 SRL). They have received a brochure regarding their possible professional biotech pathway and a mini-guide for the young employees. They have learned about selfknowledge, neuro-linguistic programming, CV content, letter of intention writing, as well as about behaviour during an interview. Regarding the satisfaction grade, more than 93% from the counselled students considered that the sessions were very interesting and useful, as well as the received materials (table 1).

funds, the main reason being the career advancement; - 96% consider that they have a high level knowledge in the biotech field, while only 19% stated relevant knowledge in complementary fields; - from the graduates sample, 40% have been already employed in the labour market, 29% were following other type or studies, mainly Master courses and 31% were in job hunting; -regarding the way of finding a job, 14% graduate consider that the university network is the most important; - very interesting fact, only 50% from them have been interested to find a job during the study, while the other half started the job hunting only after the graduation; -from the employed graduates, 93% have a single job, while 7% have parallel jobs; - 58% from the employed graduates have contracts on indeterminate time, while the rest have contract on determinate time; - only for 35% of graduates the stated that the employers takes into account the licence or dissertation paper field and the marks obtained during the school; - for the employer final decision, 37% considered that the university reputation has been important, while 44% consider that their personality was the most important; -33% from the graduates consider that the Master courses are relevant for their employ-

Table 1. Statistic on the students’ satisfaction grade after the counselling sessions Quality indicator* The training content Lecturer effort for the material accessibility Lecturer efficiency in teaching Lecturer expression clarity Lecturer ability for alternative explanation, when applied Used examples and illustration Students’ encouragement to express themselves Overall assessment results are given in %

Very week 0 0 0 0 0 0 0 0

Weak 0 0 0 0 0 0 0 0

Good 7.5 1.1 3.2 3.2 3.2 15.1 4.3 8.6

Very good 41.9 21.5 29.0 21.5 34.4 38.7 33.3 38.7

Excellent 50.6 77.4 67.8 75.3 62.4 46.2 62.4 52.7

has been created a partners network with 12 economic operators from the biotech fields, respectively agriculture and food and beverages production (Angst Buftea, VelPitar Bucharest, United Romanian Breweries BereprodTuborg), pharmaceutical industry (Medica Group, Slavia Pharma, LabormedPharma), food

As a special remark, all the counselled students from the licence level have decided to continue their further studies in Master courses. 3. Preparing and developing biotech internships Having in the team teachers from biotech high education and stakeholders from the field, it

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as the received materials. All the counselled students from the licence level have decided to continue their further studies in Master courses. From the 38 students involved in internships, in less than one year 27% have been already employed in the existent network or in very similar companies related to the existent network. The others are continuing their studies. It has been demonstrated that the internships firstly, give experience to the student. Secondly, the company gets to “look over” a prospective employee. Employers prefer to hire people they know over strangers. Thirdly, internships count as job experience. Listing an internship and the skills used in it on a resume will help get a job. After meetings involving the project team, students and former students, as well as stakeholders, it has been concluded that the practical competences acquired during an internship have a huge impact on finding an adequate job in the biotech field. The project activities, counselling and internships, will last one more year with EU funding. The team has already foreseen some measures to assure the activities continuation, by enlarging the partnership with economical operators and by proposing on the university level the establishment of a counselling bureau for the students, as well as for their parents.

safety and consumer protection (DSVSA Bucharest, S.C. ICA Research & Development S.R.L.), hygiene products design (Evic Romania – Bio High Tech SRL) and biotech research (NARDI Fundulea, NICPRI Bucharest, Institut of Food Bioresources Bucharest). Finally, 38% from the counselled students have been involved in 3 weeks of internships implemented with the help of the 12 economic operators, totalising 3420 internship hours. When choosing the internship sector, the first choice of the students it was the pharmaceutical industry, being motivated by the higher jobs opportunities, as well as the higher salaries in the field. The second choice was the food industry, which in Romania is well developed and still offers job positions of high interest. In a foreseeable manner, the last students’ options were towards the national research institutes, because of the actual low financing opportunities. After the internship a strong relation have been developed between the students and the hosts, and by March 2013, 27% of them have been employed in the existent network or in very similar companies related to the existent network. CONCLUSIONS

The statistical survey shows that about 40% from the 2006-2011 biotech graduates have found a job, while 29% were following other type or studies; less than 25% from the graduates have found jobs in biotech fields, such as scientific research and education, food and beverages production, environmental protecttion, including biofuel production, pharmaceutical products, instrumentation and suppliers. In this regard, more effort should be done to create the partnerships between university, research institutes and economic operators leading to the increase of the absorption rate of biotech graduates into the labour market and to counsel the students from their early study years in finding a job. Regarding the counselling sessions, more than 93% from the counselled students considered the training very interesting and useful, as well

ACKNOWLEDGEMENTS

The study has been financed by structural funds project POSDRU/109/2.1./G/81570– BIOPRACT. REFERENCES Frierman-Hunt G., Solberg J., 2008, Careers in Biotechnology-A Counselor’s Guide to the Best Jobs in the United States, 3rd Edition. Matei F., 2012, Studentii si consilierea în cariera, http://www.earticlestore.com/articles/ Matei B, Matei F., 2012, Stagii practice în domeniul biotehnologiilor prin POSDRU. http://publicaarticol.com/educatie

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