BIOTECHNOLOGY - Facultatea de Biotehnologii

MINISTRY OF EDUCATION RESEARCH AND INNOVATIONUNIVERSITY OF AGRONOMICAL SCIENCES AND VETERINARYMEDICINE BUCHARESTSCIENTIFIC BULLETINSeries FXIII2008BIOTECHNOLOGYISSN 1224-7774B U C HAREST2008

Redactarea şi coordonarea buletinului de lucrări ştiinţifice:U.S.A.M.V.Bucureşti, Facultatea de BiotehnologiiB-dul Mărăşti nr.59, Bucureşti, sectorul 1,RomâniaTel: + 40 21 318 25 67Fax: + 40 21 318 28 88Web: http://www.biotehnologii.usamv.roPentru schimb de publicaţii va rugam sa contactati Prof Dr Petru Niculiţă, DecanulFacultăţii de BiotehnologiiTel: + 40 21 318 36 40; Mobil: + 40 740 11 44 01e-mail: petruniculita@agral.usamv.ro, petruniculita@yahoo.com∗Editorial board and coordination of the scientific bulletin:U.S.A.M.V.Bucuresti, Facultatea de BiotehnologiiB-dul Mărăşti nr.59, Bucureşti, sectorul 1,RomâniaTel: + 40 21 318 25 67Fax: + 40 21 318 28 88Web: http://www.biotehnologii.usamv.roFor the publications exchange please contact Prof Dr Petru Niculiţă, Dean of theFaculty of BiotechnologyTel: + 40 21 318 36 40; Mobile: + 40 740 11 44 01e-mail: petruniculita@agral.usamv.ro, petruniculita@yahoo.com∗La rédaction et la coordination de bulletin scientifique :U.S.A.M.V.Bucuresti, Facultatea de BiotehnologiiB-dul Mărăşti nr.59, Bucureşti, sectorul 1,RomâniaTel: + 40 21 318 25 67Fax: + 40 21 318 28 88Web: http://www.biotehnologii.usamv.roPour l’échange de publications prier de contacter Prof Dr Petru Niculiţă, Doyennede la Faculté de BiotechnologieTel: + 40 21 318 36 40; Portable: + 40 740 11 44 01e-mail: petruniculita@agral.usamv.ro, petruniculita@yahoo.com2

SCIENTIFIC COMMITTEEPetru NICULITA - Prof. univ. dr., Decanul Facultatii de Biotehnologii - USAMV Bucuresti, Membrutitular al Academiei de Stiinte Agricole si Silvice, Presedinte al sectiei deIndustrie Alimentara - ASAS- Prof. Dr., Dean of the Faculty of Biotechnologies - USAMV bucharest,Full-member of the Academy of Agricultural and Forestry Sciences,President of the Food Industry Department- ASASAcad. Cristian HERA - Prof. univ. dr., doctor Honoris Causa, Membru titular al Academiei Romane,Presedinte al Academiei de Stiinte Agricole si Silvice- Prof. Dr., Doctor Honoris Causa, Full member of the Romanian Academy,President of the Academy of Agricultural and Forestry Sciences,Acad. Ion Paun OTIMAN - Prof. univ. dr., doctor Honoris Causa, Membru titular al AcademieiRomane, Secretar General al Academiei Romane- Prof. Dr., Doctor Honoris Causa, Full member of the Romanian Academy,General Secretary of the Romanian AcademyConstantin BANU - Prof. univ. dr., Membru titular al Academiei de Stiinte Agricole si Silvice- Prof. Dr., Full member of the Academy of Agricultural and ForestrySciencesMona Elena POPA - Prof. univ. dr., Secretar Stiintific al Facultatii de Biotehnologii - USAMVBucuresti- Prof. Dr.,Scientific Secretary of the Faculty of Biotechnologies - USAMVBucharestCalina Petruta CORNEA - Prof. univ. dr., Facultatea de Biotehnologii - USAMV Bucuresti- Prof. Dr., Faculty of Biotechnologies, USAMV BucharestGheorghe CAMPEANU - Prof. univ. dr., Sef al Catedrei de Chimie, Facultatea de Biotehnologii -USAMV Bucuresti- Prof. Dr., Chef of the Chemistry department, Faculty of Biotechnologies -USAMV BucharestEDITORIAL BOARDFlorentina MATEI - Facultatea de Biotehnologii - USAMV Bucuresti; Faculty of Biotechnologies -USAMV BucharestAmalia MITELUT - Facultatea de Biotehnologii - USAMV Bucuresti; Faculty of Biotechnologies -USAMV BucharestMira TURTOI - Facultatea de Biotehnologii - USAMV Bucuresti; Faculty of Biotechnologies -USAMV BucharestMihaela GHIDURUS - Facultatea de Biotehnologii - USAMV Bucuresti; Faculty of Biotechnologies- USAMV Bucharest3

CONTENTMONICA ENACHE, NARCISA BĂBEANUMICROPROPAGATION OF GARDEN PLANTS (2): RARE PRIMULASADRIANA PETRUŞ – VANCEABIOCHEMICAL DETERMINATIONS MADE ON CYMBIDIUM HYBRIDUMEXVITROPLANTLETS, BEING ILLUMINATED DURING THEIRACCLIMATIZATION TO A SEPTIC MEDIUM, WITH DIFFERENT TYPES OFLIGHTM. POPA, P. NICULITA, A. MITELUT, M. TURTOI, M. GEICU, M.GHIDURUS, R.CRAMARIUC, A. KONTEK, A. TUDORACHE, E.BRINDUSE, A. KONTEKNON-THERMAL INNOVATIVE PASTEURIZATION TECHNOLOGY OF FOODUSING A COMBINATION BETWEEN PULSED ELECTRIC FIELD AND HIGHPRESSUREVAMANU EMANUEL, VAMANU ADRIAN, POPA OVIDIU, BĂBEANUNARCISAOBTAINING OF SYNBIOTIC PRODUCTS FROM APICULTURAL PRODUCTSAND PROBIOTIC BIOMASS OF BIFIDOBACTERIUM BIFIDUMFLORENTINA RADOI-MATEI, FLORENTINA ISRAEL-ROMING, ANCA RADU,CAMELIA DIGUTA, CRISTINA COCULESCU, R. DESPASTUDIES ON THE GROWTH AND MYCOTOXIN PRODUCTION OF MOULDSISOLATED FROM ROMANIAN FOOD PRODUCTSE. POPA, A. MUSCALU, A. DIHORU, V. HEBEANNUTRITIVE EFFECTS OF NON-CONVENTIONALLY PROCESSEDMEDICINAL PLANTS UPON MONOGASTRIC ANIMALS DEVELOPMENTCATALINA VOAIDES, GH. CAMPEANU, PETRUTA CORNEASTUDY REGARDING THE CONSUMER ACCEPTANCE OF GM FOODSLUMINIŢA VIŞAN, O. POPA, NICOLETA ARON, TH. SEROTANALYTICAL METHODS OF AROMA COMPOUNDS IN GRAPE JUICEOBTAINED FROM ROMANIAN WINE SORTS GALBENĂ DE ODOBEŞTI ANDŞARBĂIVAN DIMITROV, SVILEN RAYKOVPROBLEMS OF TECHNOLOGICAL AND CLIMATE NATURE ATRECULTIVATING DAMAGED AREAS OF THE OPEN COAL-MINING IN“MARITSA-IZTOK”ALINA ORTAN, MARIA LIDIA POPESCU, CRISTINA DINU-PIRVUANETHI AETHEROLEUM: CHEMICAL COMPOSITION AND BIOLOGICALEFFECTSALINA CULEŢU, ALINA CATRINEL ION, ION IONSHORT REVIEW ON APPLICATION OF ION-SELECTIVE SENSORS FORHEAVY METAL IONS IN FOOD AND ENVIRONMENTAL SAMPLESRĂZVAN PRISADA, CRISTINA DINU-PÂRVU, ALINA ORŢANCOMPLEX RESEARCHES REGARDING THE PREPARATION ANDCARACTHERIZATION OF SOME MICROCAPSULES OF ETHYLCELLULOSECULEA RODICA- ELENA , POPA NICOLAE-CIPRIAN, TAMBA-BEREHOIU RADIANACOMPARATIVE RESEARCH CONCERNING THE QUALITATIVECHARACTERISTICS OF THE SUPERIOR WINES DERIVED FROM WINE-GROWING CENTRE OSTROV51018243239505764697987974

Scientific Bulletin Biotechnology, U.Ş.A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p.5 - 9MICROPROPAGATION OF GARDEN PLANTS (2): RARE PRIMULASMICROPROPAGAREA PLANTELOR DE GRĂDINĂ (2): PRIMULERAREMONICA ENACHE, NARCISA BĂBEANUCuvinte cheie: in vitro, Primula aureata, micropropagareKey words: in vitro, Primula aureata, micropropagationABSTRACTMicropropagation has significant uses in vegetative propagation of horticultural importantspecies and cultivars. With a few exceptions most ornamentals can be micropropagated, and the needis for more and new basic research approaches. Although many of the techniques involved in theproduction and manipulation of plant in vitro cultures are in themselves relatively straightforward,there are problems in the view of the wide variation in behavior of different plant species in responseto chemical and physical culture parameters. In the present work, we extended our experience withmicropropagation of rare ornamentals (Enache, 2004) to the rare Asiatic petiolarid yellow species P.aureata, described by some primula growers as “the most desirable and beautiful of all primulas”(Richards, 2003).Micropropagarea este folosita cu succes in inmultirea vegetativa a speciilor si varietatilorcu importanta horticola. Cu cateva exceptii, majoritatea plantelor ornamentale pot fi micropropagatesi este nevoie de noi tehnici pentru diferite specii, intrucat exista o variatie mare in raspunsulacestora la conditiile de cultura in vitro. In lucrarea de fata, continuam prezentarea unor studiireferitoare la micropopagarea unor plante ornamentale rare (Enache, 2004), cu rezultatele obtinutein cazul unei primule asiatice rare cu flori galbene, Primula aureata, care este descrisa de uniicrescatori de primule ca fiind „cea mai dorita si frumoasa dintre toate primulele” (Richards, 2003).Primula is a genus of about 500 species in the family Primulaceae. Manyprimulas have beautiful flowers and are cultivated as garden plants and pot flowers.The first description of the genus was published by Carl Linné in 1735 who namedthem from the Latin prima (first) – a reflection of their early blooming in spring.Most Primula species are native from the Northern Hemisphere where theygrow in cool and wet areas, many of them are found in Himalayas and China.Although in natural settings some primulas can thrive in arid land or at highaltitude where they survive near 0°C temperatures during the night, the culture ofsome species could be quite challenging.5

The species native to Europe have been successfully used in horticulturefor hundreds of years and there are now numerous cultivars, in a huge range ofcolors. However, some of the most sought-after primulas are the petiolaridprimulas adapted to high alpine climates in Himalaya and China. These love acolder and more humid weather and many of them cannot be propagated by seedswhen cultured in Europe.Primula aureata (Figure 1) is a lovely evergreen perennial petiolaridprimula from the central Himalaya considered unique for its flower subsectioncolored yellow. Early in spring and in midseason it forms very short stalks thatbear umbels of 2 to 10 salverform primrose creamy white to yellow flowers withlarge, deeper yellow centers, which look fabulous against the foliage, lightly dustedwith farina.1. MATERIAL and METHODThe work started with one Primula aureata mother plant which wasprovided by a traveler who collected it from the wild, in his trip to Himalaya.Attracted by the beauty of this type of flower plant which he hadn’t seen before inhis native country (UK), he brought one living plant home, to try tomicropropagate.It is known that Himalaya is considered “a treasure house of plants that haslong held a magical attraction for botanists, plant hunters and garden lovers”(Jermyn, 2001). The impact of Primula aureata grown in its native habitat on theplant lovers is presented by Jermyn (2001) in his book “The Himalayan Garden.Growing Plants from the Roof of the World” as follows:“An ultimate reward for the plant-oriented trekkers to this well-known areain April and May, before the onslaught of monsoon, must be the sight of steep rockbluffs stained yellow with silvery, farinose rosettes crammed into the verticalcrevices and bursting into flower. The two forms seem to enjoy differing altitudinalrange and habitats. The type plant Primula aureata has been collected at altitudesbetween 3050-3750 m and always seeks shelter from the direct rain, whereas P.aureata subsp. fimbriata does not occur below 4180 m and is frequently found invery wet conditions, sometimes under waterfalls.”For multiplication, the Primula medium containing benzyl amino purine(BAP) and indole acetic acid (IAA) was used (Anderson, 1984) (Table 1). Themother plant was cleaned of soil debris by washing in tap water first. Then, outerleaves were cut away and discarded, while smaller pieces containing shoot budswere surface-sterilized as follows: an initial pre-sterilization in 70% alcohol wasfollowed by 1-2 % (available chlorine) sodium hypochlorite. An emulsifer (Tween-20) was added at the rate of 1 drop per 100 ml solution. The excised shoot budswere left into the bleach solution in small, screw-topped, sterile plastic tubs for a6

timed 15 minutes, with occasional inversion and gentle agitation to enhance theeffectiveness of the process. Then, with flame-sterilised forceps the pieces weretransferred briefly through 5 sterile distilled water washes, finally putting them in alidded Petri dish to await cutting. After removal from bleach solution all operationswere conducted in a laminar airflow cabinet.Fig. 1 - Primula aureataThe explants were cut and then placed into the surface of the agar-basedmedium in 30 ml universal tubs (one piece per container). The cultures were placedin incubators at 25ºC, about 3000 lx light level, on a 16h light-8h dark cycle.Transfer to fresh medium was repeated at irregular intervals for furthermultiplication. Shoot clusters were divided up into single and/or small groups ofshoots and transferred to fresh medium for further proliferation. After a fewpassages on the Primula medium, plantlets rooted and displayed normal, true-to-7

Aureata are perennial alpine plants, which will put up with the coldestweather conditions. Similarly to its relative P. auricula it may need some shelterfrom the heavy rain during the winter and spring, for example the use of a coldgreenhouse with sufficient ventilation or just a simple frame that could be left openon warm days. This would also help maintain the beauty of the leaves. They canstand outside in a cool, shady position only during the summer in temperateclimate. This approach could have helped the plants survive the humid weather ofwinter in Scotland and could be used in the future if the procedure will be repeated.3. CONCLUSIONSA simple micropropagation technique is presented for Primula aureata, arare garden plant, endemic to the central Himalaya. The method is based on theclonal proliferation of shoots on a tissue culture medium containing BAP and IAA.Multiplication was achieved through subdivision of shooting clumps and repeatedtransfer on this medium at irregular intervals. After rooting the plantlets weretransferred to soil directly into the greenhouse or after a period of time spent in soilin an incubator. Later they were moved and grown in a cold greenhouse thatprovided some ventilation and were commercialised over a period of one year.AcknowledgementsThe authors were involved with the latter part of the work, which wascarried out on a voluntary basis only during a placement at Liverpool JohnMoores University (UK) through a research grant SUCCESS/93-94.REFERENCES1. Anderson, W.C.: A revised tissue culture medium for shoot multiplication of Rhododendron.“Journal of the American Society for Horticultural Science” 109, 1984, 343-347.2. Enache, M.: Micropropagation of garden plants: Miscanthus sinensis “Yakushima”, Gentianatriflora. “Scientifical Papers USAMV Bucharest Ser. B Horticulture” (XLVII), 2004, 198-202.3. Jermyn, J.: “The Himalayan Garden. Growing Plants from the Roof of the World.” Portland,Oregon. Timber Press, 2001.4. RICHARDS, J.: “PRIMULA”, 2 ND ED. LONDON. B.T. BASTFORD LTD., 2003.9

Scientific Bulletin Biotechnology, U.Ş.A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 10 - 17BIOCHEMICAL DETERMINATIONS MADE ON CYMBIDIUMHYBRIDUM EXVITROPLANTLETS, BEING ILLUMINATED DURINGTHEIR ACCLIMATIZATION TO A SEPTIC MEDIUM, WITHDIFFERENT TYPES OF LIGHTADRIANA PETRUŞ – VANCEA 11 Depart. of Biology, Faculty of Science, University of Oradea, Romania, e – mail:adrianavan@yahoo.comKeywords: peroxidazic activity, assimilating pigments, color light, exvitroplantletsABSTRACTIn this experiments I studied the effect of lighting the Cymbidium hybridumexvitroplantlets with fluorescent light of different nature, namely: white (400 nm), red (660nm), yellow (580 nm), blue (430 nm) and green (544 nm), on the quantity of assimilatingpigments from the leaflets, as a meter of the photosynthesis process intensity, and also onthe peroxidazic activity at the rootlets level, as a marker of rhysogenesis process. After 30days from the acclimatization initiation of the Cymbidium exvitroplantlets, I shown thatgreen light illumination have determined the stimulation of creating assimilating pigments,especially of carotenoids (expressed in percent with 86% plusses) and peroxidazic activitywas stimulated with 10 – 15% by blue and green illumination.Adapting the vitroplantlets to the “ex vitro” life condition is a very delicateprocess, continuous, gradual and depends most of the performance through issucceed the release of photosynthesis (by unlock the chloroplast from the starchgranule accumulated in vitrocultures period and by the synthesis of assimilatingpigments) and establishing a hydric balances (by diminishing the excessiveperspiration and the functionality of the radicular system) (Petruş – Vancea, 2007).The a and b chlorophyll and carotenoids quantity gradually grows samewith the amplification of chloroplast structural complexity and is directlyproportional with the intensity of photosynthesis (Cachiţă & co., 2004). At Coleusvitrocultures, the stimulation of chlorophyll forming, was tried through maintainingthem under fluorescent tubes that give different types of light (Radoveţ – Salinschiand Cachiţă, 2004), noticing that the blue and green light, comparative to the redand orange one, had good effects in this case.10

During the exvitroplantlets acclimatization to septic medium, the lightorigin proved to be less important in the stemlets elongation phase, but the lightintensity (100 µmol m -2 s -1 ) conditioned their rooting process (Maene and Debergh,1985). The peroxidazic activity is a marker of rooting process (Moncousin andGaspar, 1983).As regarding the light effect on the rhysogenesis were made many studieson a lot of vegetable species (Druart & co., 1982; Lian & co., 2002). Tricoli andcollaborators (1985) said that the dark improve the percent of rooting and thenumber of root/shoot. Fuernkranz and collaborators (1990) had examined the effectof different light wave length and their conclusion was that yellow light (50µmol/m 2 /s, 475 - 750 nm with the peak at 575) determined the growth of root/shootnumber, on the other hand, the blue light (20 µmol/m 2 /s, 400 – 524 with the peak at436) completely stopped the rhysogenesis.The red light was used by Podwyszynska and Gabryszewska (2003) tostimulate the “ex vitro” rhysogenesis of the rose exvitroplantlets cv. “Sonia” andcv. “Sabrina” and African daisy cv. Rebecca. During the rhysogenesis process, thered light had a benefic effect in both rose species, in winter and summer period andto African daisy, this illumination increase the rooting with 86,1 – 98,6 %, from67,1% when was registered at control variant (exvitroplantlets illuminated withwhite light) in all month, with February exception.Studying the growth indexes and surviving same as the appearance ofleaflets epidermis during the acclimatization period, varying the light origin (white,red, yellow, blue or green) which illuminated the exvitrocultures, Petruş – Vanceaand Cachiţă (2005) observed that the Chrysanthemum exvitroplantlets survivedonly under white illumination (control) and at African violets the red lightdetermined decrease with 50% the survival percent and the growth indexesdiminished, comparatively with control. A decrease of growth rate was indentifiedat exvitroplantlets illuminated with yellow light. At Cymbidium hybridummaintaining the exvitro-plantlets under different type of light, on 30 days period,had stimulating effects, especially on caulogenesis process, and less on therhysogenesis and green light was the best having only beneficially consequences,namely: superior acclimatization surviving percent; increase data of growthparameter and less stomata density on inferior epidermis of leaflets (Petruş –Vancea and Cachiţă, 2005).In the case of African violets, Petruş and Cachiţă (2008) found that after30 days from the acclimatization of these exvitroplantlets, the biochemicaldeterminations have shown that, red light illumination have determined thestimulation of assimilating pigments accumulating, especially of chlorophyll a, atthe still living plantlets (half of the plantlets maintained under this type of lightfaded), on the other hand the peroxidazic activity was stimulated at all fourexperimental variants, reaching statistically significant values, expressed in percent11

with 20% plusses, in the case of the exvitroplantlets rootlets illuminated with bluelight.Starting from this studies, we proposed in this experiment to continue theresearches in this field by realizing some biochemical determination, namelyassimilating pigments from exvitroplantlets leaflets and peroxidazic activity fromrootlets of Cymbidium hybridum, at 30 days from their “ex vitro” transfer”,depending on origin of light with which was illuminated during the acclimatizationperiod.121. MATERIAL and METHODThe exvitroplantlets of orhideea Cymbidium was regenerated „in vitro”,during 365 days, on a basis culture medium (MB) Murashige – Skoog(MS)(1962), without growth regulators, having a number of 2 - 3 rootlets, with aheight of 2,5 cm, and approximately 5 leaflets – which we have planted,individually, into a mixture of “Top soil” soil and perlite, in a 3:1 report, preparedin incubators (Petruş - Vancea, 2007), substratum previously humidified with 300ml of tap water each time, brought at the laboratory’s temperature. The incubatorswere placed on shelves protected with aluminum foil, away from the incidence ofnatural light, the illumination of the cultures being made through fluorescent tubeswhich gave white (400 nm), red (660 nm), yellow (580 nm), blue (430 nm) andgreen light (544 nm) and the power from the neon was 13 V, the intensity was 103A and the light flow was 0.44 lm. The photoperiod was 16/24 h, and the substratumtemperature was 24 ο C ± 2 ο C, the one from the atmosphere being two degreeslower.At 30 days from the acclimatization, the post-acclimatization survivalpercent was calculated for the exvitroplantlets and it was determined the quantityof chlorophyll at the level of the foliar limb and the peroxidazic activity at theexvitroplantlets rootlets level.Establishing the assimilating pigments content form the leaflets,respectively the a, b chlorophylls and carotenoid pigments was made through theirextraction in N,N – dimethyl-formamide (DMF) 99.9% Merck, according to Moranand Porath method (1980).For the pigments extraction 0.5 g of foliar limb was submersed andmacerated in 2.5 ml N,N - dimethylformamide (DMF), for 72 hours, at 4 ºCtemperature and in the dark, for chlorophyll extraction, afterwards the supernatantbeing clarified and serving for the quantitative determination of the assimilatingpigments, through its photometration using a spectro-photometer Spekol 11 type,Carl Zeiss Jena, using filters with 480 nm bands (for the carotenoid pigments), with647 nm (for chlorophyll b) and with 664 nm (for chlorophyll a) (Wellburn, 1994).Adjusting the apparatus was made using in the control box a DMF solution, whichhelped the pointer to go to zero, after reading each test extinction.

The medium values of the assimilating pigments, determined in theextracts made from the exvitroplantlet leaflets kept under colored lights werereported to the control ones (the exvitroplantlet leaflets kept under white light,considered of reference, 100%) and graphically represented.The determination of the peroxidazic activity was made with p-phenylendiamine (Şipoş & co., 2003 a and b, adapted after Lück method, 1974).The method principle consists in the fact that, in a vegetal extract that containsperoxidases, p-phenylendiamine is oxidized by them, phenomena that leads tochanging the color of the obtained mixture into violet, and between the peroxidazicintensity (measured spectro-photometric, with a Spekol 11 Carl Zeiss Jena typespectrophotometer with a 483 nm filter) and activity, there is a proportionalitydirect report (Lück, 1974).Obtaining the peroxidazic extract was made trough triturating of theradicular tissues in a buffer solution made of phosphate 0.067 M, with pH 7. Forthis, 0.5 g rootlets (freshly ingathered), were crushed together with quartz sand in agrinding mortar (previously washed and sterilized through dry heat, in the stove at120ºC), in the presence of 4 ml of ultra diluted phosphate buffer 1 : 9 (1 ml fromthe 0.067 M solution, with pH 7 plus 9 ml distillated water). The obtained extractwas separated from the vegetal wastes through whizzing with a MSE centrifuge, at6 000 rpm, for 25 minutes. Each specimen’s supernatant was collected and waskept in the refrigerator for 2 hours. The determination of the specimen’s extinctionwas made as a report with the distillated water, with a filter adjusted for a wavelength of 483 nm. For reading the extinction in the box we introduced the mixturemade of: 0.5 ml vegetal extract (supernatant), 0.05 ml oxygenated ultra dilutedwater, 1 ml phosphate buffer pH 7, 0.067 M, and after that it as added a 0.05 ml p-phenylendiamine 1% solution. For each option (at 30 s, 60 s and 90 s from theobtaining of the final mixture) there were made three checking/readings for each.The oxygenated water used in our experiments was ultra diluted and was obtainedfrom 33% hydrogen peroxide. In this way, for 100 ml distillated water there wereadded 0.3 ml of hydrogen peroxide; from this it was made a 1:9 distillated waterdilution. Because the solutions are not stable in time, they are prepared only whenthe spectrophotometra-tion begins. The working temperature was 4 ºC.In order to compare the existing peroxidazic activity in the exvitroplantletrootlets cultivated under different types of light, the average data of the extinctionsread at 120 s, the moment when they got constant, came from the samplesbelonging to extracts that were obtained from the exvitroplantlet rootlets placedunder different types of light, were reported to the similar photometric readingsregistered at the control lot (the exvitroplantlet rootlets set under white light),values considered as a reference, as being 100%.13

2. RESULTS and DISCUSSIONSThe survival percent of Cymbidium exvitroplantlets which was illuminatedwith different type of light during “ex vitro” acclimatization period was small,inclusive at control lot which registered 33,3% (Fig. 1). In generally, this specie isdifficult to acclimatization at septic medium conditions. A reason of thisimpediment was identified by Petruş-Vancea and Cachiţă (2004), who observedthat the Cymbidium hybridum vitroleaflets are amphystomatic and have the osteolspermanently open, and to the natural medium plants are only hypostomatic.The Cymbidium exvitroplantlets placed under red light no surviving. Thebest surviving (50%) was presented the exvitroplantlets placed under green light.Survival percent% 100dayI806040200WhiteRedYellowBlueGreenFig. 1 - The survival percent of the Cymbidium hybridum exvitroplantlets, at 30 daysfrom their transfer into the septic medium, being illuminated with different types offluorescent light: white (control), red, yellow, blue or green, reported to the situation fromthe moment of their passing „ex vitro”, reference values, considered 100%.As regarding assimilating pigments (Fig 2) was observed same differencesto those leaflets which were maintained under different light colors comparative tothose placed under white light (control lot).The only significant minuses (-17%) was registered at carotenoids level,recorded in case of reading to 480 nm filter to the samples obtained from thoseexvitroplantlets which were placed under yellow light and the pluses (+86%) incase of using the some filter to the samples obtained from exvitroplantletsacclimatized under green light.14

% 200100chlorophyll „a”carotenoidschlorophyll „b”0Yellow Blue G reenWhitelightFig. 2 - Expressing the chlorophyll pigments from the Cymbidium hybridumexvitroplantlet leaflets in percentage values, at 30 days after their transfer in a septicmedium under different types of light: yellow, blue, green, as reference values there weretaken homologous values registered at the exvitroplantlets kept under white light (control),these ones being considered 100%.%140120100806040200Yellow Blue GreenWhitelightFig. 3 - Expressing the peroxidazic activity from the Cymbidium hybridumexvitroplantlet rootlets in percent values, at 30 days from their transfer into the septicmedium under different types of light: yellow, blue, green, as reference values there wereconsidered the similar ones registered at the exvitroplantlets kept under white light(control), these ones being considered 100%.Peroxidazic activity (Fig. 3) superior with 10% and 15% to the control(rootlets provided from exvitroplantlets acclimatized under white light) presentedrootlets obtained from exvitroplantlets placed “ex vitro” under blue and green light15

on the same time, the exvitroplantlets rootlets maintained under yellow light wereregistered 18% lose in their peroxidazic activity, comparatively to the control.163. CONCLUSIONS1. Maintaining Cymbidium exvitroplantlets under different light types(yellow, blue or green) during the acclimatization period had various effectsdepending on their nature. The green light applied on Cymbidium exvitroplantletshad good consequences as: a higher percent of acclimatization surviving, highervalues of assimilating pigments, especially of carotenoids but peroxidazic activityto.2. The lowest results, biochemical point of view (of assimilating pigmentsaccumulation and peroxidazic activity) were recorded to those Cymbidiumexvitroplantlets which were placed during acclimatization period under yellowlight, and under red light, the exvitroplantlets not survived.REFERENCES1. Cachiţă, C.D., Deliu, C., Rakosy T.L., Ardelean, A.: Tratat de biotehnologie vegetală. EdituraDacia, Cluj – Napoca, 2004.2. Druat, P., Kevers, C., Boxus, P., Gaspar, Th.: „In vitro” promotion of root formation by apple shootthrough darkness effect on endogenous phenols and peroxidases. Zeitschr. FűrPflanzenphysiol., 108, 1982, p. 429–436.3. Fuernkranz, H.A., Nowak, C.A., Maynard, C.A.: Light effects on „in vitro” adventitious rootformation in axillary shoots of mature Prunus serotina. Physiol. Plant., 80, 1990, p. 337-341.4. Lian, M.-L., Murthy, H.N., Paek, K.-Y.: Hight CO 2 & light improve acclimatization of in vitropropagated plants. Scientia Hortic., 95, 2002, p. 239–249.5. Lück, H.: Peroxidaza. In: Methods of Enzymatic Analisys. H.U. Bergmeyer (vol. coord.). New –York, Academic Press, 1974, p. 895–897.6. Maene, L., Deberg, P.: Lichid medium addition to established tissue cultures to improve elongationand rooting. Plant Cell Tisssue Org. Cult. 5, 1985, p. 23–33.7. Moncousin, C., Gaspar, Th.: Peroxidase as a marker for rooting improvement of Cynara scolymusL. cultured „in vitro”. Bio. Physiol. Pflanzen, 178, 1983, p. 263–271.8. Moran, R., Porath, D.: Chlorophyll determination in intact tissue using N,N-dimetyl-formamide.Plant Physiol., 65, 1980, p. 487–479.9. Murashige, T., Skoog, F.: A revised medium for rapid growth bioassays with tobacco tissue cultures.Physiol. Plant., 15, 1962, p. 473-497.10. Petruş – Vancea, A.: Cercetări privind procesele morfofiziologice şi biochimice care au loc îndecursul aclimatizării plantulelor generate „in vitro”, la viaţa în mediul septic. Teză dedoctorat, Universitatea din Oradea, 2007.11. Petruş – Vancea, A, Cachiţă, C.D., Studierea influenţei exercitate de natura luminii înaclimatizarea exvitroplantulelor. In: Lucrările celui de al XIII -lea Simpozion Naţional deCulturi de Ţesuturi şi Celule Vegetale, „Vitroculturile la cormofite, modele experimentale încercetările de biologie”. C.D. Cachiţă, A. Ardelean (vol. coords.), Satu – Mare, Editura Bion,2005, p. 138–151.12. Petruş – Vancea, A., Cachiţă, C.D., Biochemical determinations made on African violets(Saintpaulia ionantha) exvitroplantlets, being illuminated during their acclimatization to a

septic medium, with different types of light, Studia Univ. Vasile Goldiş, Şt. Vieţii, Vol. 18,2008 (in press).13. Podwyszyńska, M., Gabryszewska, A., Effect of red light on ex vitro rooting of rose and gerberamicrocuting in rockwool. In: Proc. 1 th Symp. on Accl.& Estab. Microprop. Plants. A.S.Economou, P.E. Read (vol. coords.). Acta Hort. 616, ISHS, 2003, p. 224– 237.14. Radoveţ-Salinschi, D., Cachiţă, C.D.: Conţinutul în pigmenţi asimilatori în frunzuliţelevitroplantulelor de Coleus blumei Benth., culturi iluminate cu lumină de culori variate.Analale Societăţii Naţionale de Biologie Celulară. Vol. IX nr. 1. C. Crăciun, A. Ardelean (vol.coords.), Cluj – Napoca, Editura Risoprint, 2004, p. 387–391.15. Şipoş, M., Chiriiac, C., Floriş, C.: Activitatea peroxidazică în embrionii şi în plantulele de grâu(Triticum aestivum L. soiul Turda) rezultate prin germinaţia cariopselor submersate, înprealabil, în azot lichid (-196ºC). Analele Univ. Oradea, Fasc. Biologie, Tom X, 2003 a, p.315-320.16. Şipoş, M., Chiriiac, C., Floriş, C.: Activitatea peroxidazică în cariopsele de grâu (Triticumaestivum L. soiul Turda) după submersarea acestora în azot lichid (-196ºC). Analele Univ.Oradea, Fasc. Biologie, Tom X, 2003 b, p. 333-342.17. Tricoli, D.M., Maynard, C.A., Drew, A.P.: Tissue culture micropropagation of mature trees ofPrunus serotonina Ehrh. I. Establishment, multiplication and rooting in vitro. Forest Sci,31(1), 1985, p. 201–208.18. Wellburn, A.R.: The spectral determination of clorophylls a and b, as well as total carotenoides,using various solvents with spectrophotometers of different resolution. Plant Physiol., 144,1994, p. 307–313.17

Scientific Bulletin Biotechnology, U.Ş.A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 18 - 23NON-THERMAL INNOVATIVE PASTEURIZATION TECHNOLOGY OFFOOD USING A COMBINATION BETWEEN PULSED ELECTRIC FIELDAND HIGH PRESSUREM. POPA * , P. NICULITA * , A. MITELUT * , M. TURTOI * , M. GEICU * , M.GHIDURUS * , R. CRAMARIUC ** , A. KONTEK *** , A. TUDORACHE *** ,E. BRINDUSE *** , A. KONTEK **** Faculty of Biotechnology, Dept. of Industrial Biotechnologies, USAMV Bucharest, Romania, e-mail:monapopa@agral.usamv.ro** Electrostatic and Electro-technologies Research Center – Bucharest, Romania*** Research Institute for Winery and Vinification – Valea Calugareasca, RomaniaKeywords: food pasteurization, pulsed electric field, high pressureABSTRACTThis paper is part of the national project results which has as main objective theelaboration of technologies and equipment for micro organisms’ inactivation by simultaneouslyapplying a pulsed electric field and pressure to sterilize non-thermally food products (wine, fruitjuices etc.). This technology will eliminate the deficiencies of the PEF technology applied incontinuous flux. By simultaneously applying pressure treatment and PEF treatment, a PEF inducedpassive form of cellular plasmolysis will be obtained. The pressure will cause cell deterioration, willincrease diffuse migration of humidity and will decrease cell recovery processes.The technology of simultaneously applying the electric field and pressure will lead tooptimum results, will significantly increase the efficiency of wine and juice production and willincrease their nutritional quality. This technology could be successfully applied to other foodproducts too.Increased consumption demand for fresh products with minimum losses ofnutrients stimulated research for new non-aggressive processing technologies.The development of alternatives for heat processing is also motivated by the realneed for ecological technologies. Pulsed electric field (PEF), one of the nonthermaltechnology with minimum processing that for the specialized researchrepresents an alternative process for liquid products preservation, fulfils theseconditions. PEF processing is accomplished by introducing the food products in achamber that contains two electrodes for the micro organisms’ inactivation and18

for decreasing the enzymes activities as to increase shelf life of treated foodproducts without causing any unwanted chemical and heat related effects. PEFapplied to food products causes irreversible losses in the cell membranefunctionality that leads to microbial cell inactivation. This process is known aselectroporation and it effectively inactivates the micro organisms.PEF processing is a non-thermal pasteurization method of liquid foodproducts. In the same time with the technological development of industrialapplications, the system operation control becomes more and more important. Inthe recent years, many research groups demonstrated the inactivation possibility ofsome micro organisms also in buffer systems, in food products, using differentsystems, pressure and PEF.This paper is part of the national research project entitled “Multidisciplinaryresearch for the development of an innovative non-thermal technology bycombining pulsed electric field and pressure for food pasteurization” (contract no.61-004/2007).The aim of this study is to establish the effect of subsequently application of HPPand PEF treatment on micro organisms from young wines, respectively yeasts andlactic and acetic bacteria at the moment of wine raking from the solids.1. MATERIAL and METHODSThe raw material used in the experiment was a wine blending made fromdry white wines obtained at Valea Calugareasca winery, after the wines were rakedfrom the solids and sulphited. The purpose of wine blending use is to have a greatmicro organism’s diversity in the raw material subjected to treatment. The physico– chemical composition of wine blending used in the experiment was analyzed andthe results are presented as follow:• Alcohol....................12,7 vol.• Volatile acidity.........0.42 g/l acetic acid• Total acidity..............4,88g/l tartric acid• Free SO 2 ............................12,4 mg/l• Total SO 2 ...........................44,6 mg/l• Sugar..................................1,47• pH.......................................3,3The wine blending was stored at 5 o C for microbiological reasons (theinhibition of micro organism’s multiplication).The equipment used for HPP – PEF treatment was an experimental modeldesigned in the second stage of the project by the Electrostatic and Electrotechnologies Research Center, as partner in the project. General scheme of the HPP19

treatment equipment is presented in figure 1 and general scheme of PEF chamber ispresented in figure 2.HPP equipment consists of:1. CO 2 bottle;2. pressure reducer;3. piston screw;4. pressure cylinder;5. protection cylinders;6. source of low voltage continuous current;7. collecting recipient;8. high pressure manometer;9. manual and mechanical device for piston screw powering;10. three way cock.PEF chamber consist of:1. winding outside the cylinder (electrode);2. glass cylinder;3. limiting Teflon fittings;4. stainless steel cylindrical electrode.The work protocol used in the experiments was the following: 375 ml ofwine was introduced in the HPP experimental device at a hydrostatic pressure of225 bars for 180 minutes. After the treatment, the wine was passed in asepticconditions in thermo sterilized glass recipients and introduced immediately in thePEF chamber. PEF treatment was conducted at 30 kV/cm and 800 Hz for twoperiods of time, 1 minute and 3 minutes. Therefore, wine samples consisted of:- dry white wine blending HPP treated (225 bar, 180 min.) and then PEF treated(30 kV/cm, 800 Hz) for 1 minute;- dry white wine blending HPP treated (225 bar, 180 min.) and then PEF treated(30 kV/cm, 800 Hz) for 3 minutes.Immediately after treatment, wine samples were analyzed from themicrobiological point of view and the count of micro organisms remained activewas assessed using the control method of active germs count. Cellulose acetatemembranes with 0,45 µ pores diameter were used for microbiological filtration andthe micro organisms were cultivated on specific media and incubated at 28 o C.202. RESULTS and DISCUSSIONSThe inactivation effect of the two treatments applied subsequently on winemicro organisms is obvious, as is showed in table 1 and in graphs from figure 3.Yeast load was reduced from 50400 CFU/ml to 5208 CFU/ml and to 4212CFU/ml, in wine samples subjected to HPP treatment and then PEF treated for 1minute, respectively 3 minutes.

Lactic bacteria were affected also by the combined HPP – PEF treatment,but in a gentler manner, their number decreasing from 2700 CFU/ml to 1830CFU/ml for wine samples PEF treated for 1 minute and to 225 CFU/ml for winesamples PEF treated for 3 minutes.The most resistant wine micro organisms to the combined HPP – PEFtreatment are acetic bacteria. Acetic bacteria load of wine samples decreased from38 CFU/ml to 15 CFU/ml and to 8 CFU/ml, after 1 minute, respectively 3 minutesof PEF treatment.Considering the great diversity of wine micro flora, even if the grape juiceis inoculated with a dominant yeast strain to be converted into wine, at the end themicrobiological composition of wine consist of numerous species of yeasts, lacticbacteria and acetic bacteria, depending on genetic and enzymatic structure.Therefore, more studies are required.It can be assumed that increasing the pressure to approximately 2000 barand the intensity of PEF above 30 kV/cm, the effect of HPP – PEF treatment willbe an efficient sterilization of liquid products, such as fruit juices, grape juice andwines.3. CONCLUSIONSThe inhibitory effect of HPP – PEF treatment on wine natural micro florawas depending on the type of microorganism. Yeasts were the most sensitive toHPP – PEF treatment, the number of active yeasts in wine samples being reducedwith more than 90% after 3 minutes of PEF treatment. Lactic bacteria were almostas sensitive as yeasts to HPP – PEF treatment, the number of active lactic bacteriain wine samples being reduced as well with more than 90% after 3 minutes of PEFtreatment. The most resistant wine micro organisms to HPP – PEF treatment wereacetic bacteria, the number of active acetic bacteria being reduced with less then80% after 3 minutes of PEF treatment.The effect of HPP – PEF treatment on wine micro organismsTable 1ExperimentalvariantTreatmentapplicationperiod(min)HPPPEFYeastsInitial microbial load(CFU/ml)LacticbacteriaAceticbacteriaYeastsMicrobial load aftertreatment (CFU/ml)LacticbacteriaAceticbacteria1 180 1 50400 2700 38 5208 1830 152 180 3 50400 2700 38 4212 225 821

Fig. 1 - General scheme of the equipment used for HPP treatment of wine formicrobiological stabilization purposes:1 – CO 2 bottle; 2 – pressure reducer; 3 – piston screw; 4 – pressure cylinder; 5 – protection cylinders;6 – source of low voltage continuous current; 7 – collecting recipient; 8 – high pressure manometer;9 – manual and mechanical device for piston screw powering; 10 – three way cock.Fig. 2 - PEF treatment chamber:1 – winding outside the cylinder (electrode); 2 – glasscylinder; 3 – limiting Teflon fittings;4 – stainless steel cylindrical electrode.22

CFU/ml6000050000400003000020000The effect of HPP-PEF treatment onyeastsYeastsCFU/ml30002500200015001000The effect of HPP-PEF treatment onlactic bacteriaLactic bacteria100005000Initially 1 min 3 min0Initially 1 min 3 minPEF treatment durationPEF treatment durationCFU/ml403020100The effect of HPP-PEF treatment onacetic bacteriaInitially 1 min 3 minPEF treatment durationAcetic bacteriaFig. 3 - Effect of HPP – PEF treatment on wine micro organismsREFERENCES1. Balestra P., Da Silva A A., Cug J.L., 1996 Inactivation of Escherichia coli by carbon dioxide underpressure., J. Food Sci., 4/61, 829-836.2. Castro A. J, Barbosa – Canovas 1993. Microbial inactivation of foods by pulsed electric fields, J.Food Process Press 17:47-73.3. Cheftel J.C, 1995, High pressure, microbial inactivation and food preservation (review), FoodScience and Technology International, 1, 75-90.4. Cramariuc R., Tudorache A., Popa M., Branduse E., Nisiparu L., Mitelut A., Turtoi M., Fotescu L.,2007, Corona Discharge in Electroporation of Cell Membranes, 12th International Conferenceon Electrostatics.5. Daoudi L, Quevedo J.M, Trujillo A.J, Capdevila F.Bartra E, Minguez S, Guamis B, 2002 -Effects ofhigh pressure treatment on the sensory quality of white grape juice, High Pressure Research .6. Delfini C, Contreno L, Carpi G, Rovere P, Tabusso A, Cocitto C, Amati A 1995 Microbiologicalstabilization of grape musts and wines by high hydrostatic pressures . Journal 6, 2,143-151.7. Earnshaw R G 1995. High Pressure Microbial Inactivation Kinetics , in High Pressure Processingof Foods , ed. D.A. Ledward D.E. Johnston , R.G. Earnshaww A.P.M. Hastings , NottinghamUniversity Press, Nottinghasm , pp. 37-46.8. Haas G.J., Prescot, H E. 1989.- Inactivation of microorganisms by carbon dioxide under pressure,Journal of Food Safety, 9, 253, 265.23

Scientific Bulletin Biotechnology, U.Ş.A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 24 - 31OBTAINING OF SYNBIOTIC PRODUCTS FROM APICULTURALPRODUCTS AND PROBIOTIC BIOMASS OFBIFIDOBACTERIUM BIFIDUMREALIZARE DE PRODUSE SINBIOTICE DIN PRODUSE APICOLE ŞIBIOMASĂ PROBIOTICĂ DE BIFIDOBACTERIUM BIFIDUMVAMANU EMANUEL 1,2 , VAMANU ADRIAN 1,2 , POPA OVIDIU 1,2 ,BĂBEANU NARCISA 11 – University of Agronomic Sciences and Veterinary Medicine, Faculty of Biotechnology, Bd.Mărăşti no. 59, district 1, Bucharest, Romania, e-mail: emanuelvamanu@yahoo.com2- Applied Biochemistry and Biotechnology Center – Biotehnol, Bd. Mărăşti no. 59, district 1,Bucharest, RomaniaKey-words: Bifidobacterium, honey, pollen, CFU 1 , lactic acidCuvinte cheie: Bifidobacterium, miere, pollen, UFC 1 , acid lacticABSTRACTThis study presents the multiplication of a Bifidobacterium bifidum b1 strain onmedia containing pollen and honey. The following parameters have been determined: theCFU 2 value, the production of lactic acid, the consumption of carbohydrates, and thevariation of the kinetic parameters. The research works lasted 72 hours, with variousrates of milled or non-milled pollen, in tightly closed recipients, at a temperature of37 o C. The media were very well homogenized before inoculation. The inoculation wascarried out only after the medium gained a homogenous consistency. The inoculumconsists of Bifidobacterium bifidum b1 biomass.Acest studiu prezintă multiplicarea unei tulpini de Bifidobacterium bifidum b1 pemedii ce conţin polen şi miere de albine. S-au determinat următorii parametrii : valoareaUFC, producţia de acid lactic, consumul glucidelor şi variaţia parametrilor cinetici.Cercetările s-au desfăşurat pe o perioadă de 72 de ore, cu procente diferite de polenmăcinat sau nemăcinat, în recipiente închise etanş, la o temperatură de 37 0 C. Mediile aufost omogenizate foarte bine înaintea inoculării. Inocularea s-a desfăşurat numai după cemediul a căpătat o consistenţă omogenă. Inoculul constă din biomasă de Bifidobacteriumbifidum b1.The use both of probiotics and of prebiotics has much increased due to thehigh commercial interest regarding the supply of dietary supplements to people.2 Unităţi formatoare de colonii/Colony forming units* Corresponding author: Fax: +40215693492; web: www.emanuelvamanu.ro, e-mail: email@emanuelvamanu.ro24

Such a product is used for prophylaxis, not directly for its therapeutic effects, beingconsidered a functional foodstuff. Bifidobacteria and the acidolactic bacteria areused as organisms with a probiotic effect in the fermented milk products and in thesynbiotic products that are based on oligosaccharides obtained from honey (1, 2).The lack of fibers in the human diet is the reason of constipation, obesity,heart diseases, diabetes, colon cancer. Using foodstuffs containing such fibersensures a normal functioning of the intestine and an improvement of the negativeeffects due to their absence in the ingested food (3, 4). The combination betweenprebiotic and probiotic has a direct effect on the increase of the number ofanaerobic bacteria and on the decrease of the number of potentially pathogenicaerobic bacteria. These benefits are also due to the fact that the non-digestibility ofthe prebiotic component provides a plus of efficiency to the probiotic strains (5).The most frequent oligosaccharide encountered in the human diet is theinsulin and, more recently, honey, which is very rich in oligosaccharides with aprebiotic effect. Thus, these oligosaccharides are not present in a constant quantity,but the effect is identical, i.e. the selective stimulation of the anaerobic strains, andparticularly of the Bifidobacterium strains. This stimulation effect is caused by thecomposition of honey and by the used strain. Thus, the products that can beobtained are new products, which do not have a substitute on the market (8).Besides the broadening of the range of functional products that can beobtained, the effect on the bifidobacteria is highly improved, compared to otherprebiotics. The unique combination in honey may provide significant benefits,seeing that it is a natural product, with a long validity term and which can be keptunder conditions not involving special requirements(9). The combination betweenhoney and the probiotic biomass of bifidobacteria creates an optimal conservationmedium without needing to add preservatives, which are extremely reprehended byall the norms and directives of the European Union in the field.1. MATERIAL and METHODMicroorganism and culture media. In the practical experiments made forthe purpose of this work, we used the Bifidobacterium bifidum b1 probiotic strainfrom the collection of the Faculty of Biotechnologies. It was conserved in thefreezer, at -82 o C, on a protective medium, enriched with 20% glycerol, and thenrevitalized on a medium containing MRS+0.2% cysteine hydrochloride.To obtain the biomass, the following culture medium is used: glucose 5%,yeast extract 1%, corn extract 2.5% with 40% dry matter. After preparation, theculture medium is filtered in vacuum, then autoclaved at 115 o C for 20 minutes, inan autoclave. The inoculation was made with 10% freshly reinvigorated culture.To obtain synbiotic products the following culture media are used: 20gnon-milled pollen, 3g honey, 5ml distilled water; P2: 20g milled pollen, 3ghoney, 5ml distilled water; P3: 20% non-milled pollen, 3% honey, 5ml25

distilled water; P4: 20% milled pollen, 3% honey, 5ml distilled water. Theinoculation was made with 10% Bifidobacterium bifidum b1 biomass. Themilling of the pollen used in obtaining the P2 and P4 media was performedby using a mill, in series of 15 seconds, with a break of 10s; operationrepeated 5 times. The P1 and P2 media are semi-solid media, obtained bymixing the components and adding a minimal quantity of water, justenough to break the pollen grains and obtain a relatively homogenousmedium. The medium was distributed in tightly closed plastic recipients,and after inoculation they were subject to the temperature of 37 o C. Thetubes were statically maintained (10).Determination of the quantity of carbohydrates by using the o-toluidinemethod. It was performed with the o-toluidine test, by the Chemical andPharmaceutical Research and Development National Institute – ICCF of Bucharest.Determination of the quantity of lactic acid. The accumulation of lacticacid was determined by using an HPLC (11).Determination of the viability. To establish the number of colony formingunits, the successive dilutions method was used, using the medium containingMRS+0.2% cysteine hydrochloride with various agar concentrations.2. RESULTS and DISCUSSIONSObtaining of the probiotic biomass of bifidobacteriaTo determine the effect of the corn extract on the biomass accumulation,the following fermentative parameters were determined first in parallel, atlaboratory level: lactic acid, accumulated biomass, glucose consumption. Theresults obtained are shown in Figure 1. Following the layout of the climbing curve,a very short lag phase is noticed, which does not exceed 2 hours. This can beascribed to the supplementation of the medium with cysteine hydrochloride. Thelogarithmic increasing phase follows an almost constant layout, up toapproximately 20 hours, when it enters the stationary phase, which is very short.The half-life is quite long, much longer than that of the Lactobacillus strains. Thisalso leads to a low productivity on this culture medium. When the microorganismis in the lag phase, there is no production of lactic acid. The first signs of existenceof the lactic acid occur in the beginning of the logarithmic increasing phase. Theproduction continues as long as the strain is in the logarithmic phase. Themaximum level attained is of 0.5% lactic acid, every 36 hours of fermentation.Glucose gradually decreases during the entire period of 24 hours, thelargest consumption being again during the logarithmic phase. Regardless ofthe quantity of glucose added to the medium, the production of lactic acidhas lower values, because such strains do not produce significant quantitiesof this product. Anyway, even if the quantity of lactic acid were to increase,it should be neutralized because it decreases productivity.26

D.O./O.D. (600 nm)109876543210D.O./O.D. (600 nm)Acid lactic/Lactic acid (%)Glucoză/Glucose (%)0 4 8 12 16 20 24 28 32 36 40 44 48Timp/Time (ore/hours)2,521,510,50Acid lactic/Lactic acid (%)Fig. 1 - Evolution of the fermentative parameters when cultivating the Bifidobacteriumbifidum b1 strain on the medium MRS+0.2% cysteine hydrochloride at laboratory levelIf cultivation takes place under the conditions of a bioreactor (net volumeof 2 liters), the results are shown in Figure 2. The accumulation of lactic acidincreases linearly during the 48 hours of fermentation. Carbohydrates constantlydecrease, more pronouncedly during the interval 4-24 hours. The lag phase isalmost absent, while the exponential increase phase continues up to 32 hours offermentation. Compared to the results obtained at laboratory level, we notice anincrease of the logarithmic phase by approximately 8 hours.D.O./O.D. (600 nm)302520151050D.O./O.D. (600 nm)Acid lactic/Lactic acid (%)Glucide/Glucides (%)0 4 8 12 16 20 24 28 32 36 40 44 48Timp/Time (ore/hours)Fig. 2 - Evolution of the fermentative parameters when cultivating the Bifidobacteriumbifidum b1 strain at bioreactor level6543210Acid lactic/Lactic acid (%)27

Compared with the production of lactic acid obtained in the case of theinoculum, this medium determines the obtaining of significant quantities of lacticacid. The neutralization was made with Ca(OH) 2 10%. It follows that thepermanent maintaining of the pH value of the medium at 5-5.5 determines animportant accumulation, but under the form of Ca lactate. It is worth mentioningthat the accumulation of the biomass has values approximately 3 times larger,compared to the medium for the inoculum. Equally, the quantity of lactic acid alsoincreases approximately 3 times, directly proportional to the quantity ofaccumulated biomass.Cultivation in media containing pollen and honeyThe multiplication and synthesis capacity of the lactic acid in the 4formulas of medium containing pollen and honey was tested in tightly closedFalcon tubes and maintained at a constant temperature, up to 48 hours.Acid lactic/Lactic acid (%)4,54 P13,5 P23 P32,5P421,510,500 8 16 24 32 40 48Timp/Time (ore/hours)Glucide/Glucides (%)1201008060402000 8 16 24 32 40 48Timp/Time (ore/hours)P1P2P3P4Fig. 3 - Quantity of lactic acid synthesizedwhen cultivating on media containing pollenand honeyFig. 4 - Quantity of carbohydrates consumed whencultivating on media containing pollen and honeyIn Figure 3 you can notice that the media containing milled pollendetermine a more powerful synthesis of the lactic acid, compared to the 2 mediacontaining non-milled pollen. In case of medium P2, the quantity of lactic acid isapproximately 43% larger, at the end of the 48 hours. For the medium P4, thequantity is approximately 27% larger than that obtained by using the P3 medium.Making the comparison between the 2 types of culture media used, the culturemedia P1 and P2 determine the synthesis of a much larger quantity of lactic acid.This finding is also noticed in the consumption of carbohydrates, which indicates aconsumption exceeding 30% for each type of culture medium. What needs to bementioned is that the carbohydrates usage rate is higher on media P3 and P4,28

compared to media P1 and P2 (Figure 4). The rate is 10% in favor of medium P4,compared to P2, and 12% in favor of medium P3, compared to medium P1.Numărceluleviabile/Number ofviable cells7000000060000000500000004000000030000000200000001000000000 8 16 24 32 40 48P1P2P3P4Fig. 5 - Number of viablecells when cultivating onmedia containing pollenand honeyTimp/Time (ore/hours)The number of viable cells maintains the tendency noticed during theprevious determinations, i.e. media P1 and P2 are those that cause the best results.The milling of the pollen in the case of medium P2 (Figure 5) is the essential factorin stimulating the multiplication of the microorganism, but also of the synthesis ofthe metabolic products. This finding is also noticed in the case of the secondcategory of culture media, P3 and P4. For the medium P2, the number of cells thatare formed is 3.5 times larger than of those that are formed by using medium P1. Ifwe compared media P2 and P4, we would notice that, by using medium P2, 10times more microorganisms would be obtained (Bifidobacterium bifidum) than byusing medium P4.Kinetic parameters for cultivating Bifidobacterium bifidum b1on media P1, P2, P3 and P4Table 1Mediu deculturăCulturemediumGlucide iniţialeViteză deAcid lacticOriginalcreştere maximăLactic acidcarbohydratesMaximumgg increase speed( )( ) L µ maxL (h -1 )P1 103 28.7 0.09 1.08P2 107 40 0.11 1.32P3 51 15.2 0.02 0.24P4 56 19 0.03 0.36ProductivitateProductivityg(L × h)29

In the fermentations that take place for obtaining the bifidobacteriaprobiotic biomass, it is noticed that using the corn extract determines a significantincrease of the cellular productivity. This is also ascribed to using conditionssimilar to a classic bioreactor, but much easier to maneuver under laboratoryconditions, for obtaining the biomass. The only parameter that can determine adecrease of the cellular concentration is the lactic acid formed, if it is notneutralized when its synthesis dramatically decreases the pH of the medium. Thisfinding is particularly noticed when the biomass is obtained. The increasedsynthesis of the lactic acid, when cultivating on media containing pollen andhoney, is not a very serious problem because the homogenization when usingmedia P1 and P2 is more difficult because of the high consistency. Thus, due tothis consistency of the culture medium, we believe that the diffusion of the lacticacid is more difficult and this way the formed cells are much less affected by thelow pH. In exchange, for the media P3 and P4 the synthesis of the lactic acid is animportant factor because they are culture media similar to the classic ones. This isalso one of the reasons for which the two media are not so productive. Whencultivating on media containing pollen and honey, only natural substances are used,without ingredients that could change the final composition of the product, and thatis why, out of the 4 culture media, only medium P2 has the required features in theend. First of all, a viability of 10 7 , which is over the minimal value of 10 6 , which ischaracteristic to products with a probiotic effect. The acid synthesis and the highviability in the case of medium P2 can also be considered as an indicator ofkeeping the product sterile. This finding is extremely important due to the fact thatno previously sterilized raw materials (pollen and honey) are used.Table 1 shows the kinetic parameters of cultivating the Bifidobacteriumbifidum b1 strain on the 4 culture media containing pollen and honey. For eachculture medium, the lactic acid formed is correlated to a significant consumption ofthe quantity of carbohydrates. The increase speed is significantly higher whenusing the medium P2. This can also be due to the increase in the concentration ofoligosaccharides that are found in honey and that were completely homogenizedwith the milled pollen. Given the special cultivation conditions, productivity is themain indicator in this table. The increase of the water quantity does not lead to theincrease of productivity, but to its dramatic decrease, which is also due to thedilution of the oligosaccharides contained by the medium, which are an importantimpulse for the increase of bifidobacteria.303. CONCLUSIONSThe cultivation of the Bifidobacterium bifidum b1 strain was performedwith very good results under batch conditions in view of obtaining probiotic

iomass, which is used in obtaining a probiotic product based on honey and pollen.A large biomass quantity was obtained, according to the data provided byliterature, by using the corn extract as an additional source of nitrogen and increasefactors. By cultivating the Bifidobacterium bifidum b1 strain on media containingpollen and honey, we found that the medium P2 is optimal for obtaining such aprobiotic product. Also, we found that the milling of the pollen, the low waterquantity, the high density of P2 – determine a significant increase of viability,comparable to the data provided by the literature regarding probiotic products,being compliant with such regulations.ACKNOWLEDGMENTThe researches were financed through a project PNCDI II - PARTNERSHIPS INPRIORITY S&T DOMAINS, Theme 61-047/2007.REFERENCES1. Roy Sleator, Colin Hill (2007): Probiotics as therapeutics for the developing world. Journal ofInfection in Developing Countries 1: 7-12.2. Kingsley C. Anukam (2007): The potential role of probiotics in reducing poverty-associatedinfections in developing countries. Journal of Infection in Developing Countries 2: 81-83.3. Almeghaiseeb Ebtissam (2007): Probiotics: An overview and their role in inflammatory boweldisease. Saudi Journal of Gastroenterology 3: 150-152.4. Zocco M.A., dal Verme L.Z., Cremonini F., Piscaglia A.C., Nista E.C., Candelli M. (2006):Efficacy of lactobacillus GG in maintaining remission of ulcerative colitis. Aliment.Pharmacol. Ther. 23: 1567-74.5. Lemberg D.A., Ooi C.Y., Day A.S. (2007): Probiotics in paediatric gastrointestinal diseases. JPaediatr Child Health 5: 331-336.6. Anukam K.C., Osazuwa E.O., Reid G. (2006): Knowledge of probiotics by Nigerian clinicians. Int JProbiotics Prebiotics 1: 57-62.7. Pineiro M., Stanton C. (2007): Probiotic bacteria: legislative framework-- requirements to evidencebasis. J. Nutr. 3: 850S-853S.8. Paton A.W., Morona R., Paton J.C. (2006): Designer probiotics for prevention of entericinfections. Nat. Rev. Microbiol. 4: 193-200.9. Schiffrin E.J., Donnet A., Blum S. (2005): How can we impact the immune system with pre- andprobiotics? J. Infect. Developing Countries 1: 7-12.10. Vamanu E., Vamanu A., Popa O., Câmpeanu Gheorghe, Albulescu Mihaela, Drugulescu Manuel(2006): Biotechnological researches concerning the multiplication of a Lactobacillusplantarum strain on media with pollen for the obtaining of a probiotic product. Roum.Biotechnol. Letters. 2: 2627 – 2635.11. Y.J. Yee et al. (2006): Lactic acid production with Lactobacillus sp. RKY2. Food Technol.Biotechnol. 2: 293 – 298.31

Scientific Bulletin Biotechnology, U.Ş.A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 32 - 38STUDIES ON THE GROWTH AND MYCOTOXIN PRODUCTION OFMOULDS ISOLATED FROM ROMANIAN FOOD PRODUCTSSTUDII ASUPRA CRESTERII SI PRODUCERII DE MICOTOXINE DECATRE MUCEGAIURI IZOLATE DIN PRODUSE ALIMENTAREROMANESTIFLORENTINA RADOI-MATEI 1 , FLORENTINA ISRAEL-ROMING 1 ,ANCA RADU 2 , CAMELIA DIGUTA 1 , CRISTINA COCULESCU 3 , R. DESPA 31 - USAMV Bucharest, Faculty of Biotechnology, Romania2 - Institute of Food Bioresources, Bucharest, Romania3 - Romanian-American University, Department of Mathematics, BucharestKey words: moulds, mycotoxin, predictive modelsCuvinte cheie: mucegaiuri, micotoxine, modele previzionaleABSTRACTThis research belongs to a national project regarding the prevention of the mycotoxincontamination in feed and food. At the very beginning of the project there have been isolated andidentified the main species involved in the alteration of food products of intermediary activity.Finally, 105 moulds strains have been insolated, belonging to the following genders:Aspergillus, Alternaria, Fusarium, Penicillium, Rhizopus, Cladosporium, Macrosporium.The strains have been screened for their toxinogenic activity (aflatoxins, ochratoxin anddeoxynivalenol production) by on-plate method and the toxins were quantified by Elisa Immunologictests. 38 of those moulds showed toxinogenic activity under laboratory tests.From this toxinogenic collection, two strains (Fusarium graminearum MI 113 andPenicillium crysogenum MI 210) have been studied for their growth and mycotoxin (DON and OTA)production from a predictive point of view.Spores suspension of 10 3 - 10 4 was used as inoculum for natural (wheat) and synthetic(Czapek-Dox broth) media. The samples have been cultivated during 21 days under differenttemperatures conditions (4 o C; 12 o C; 16 o C; 20 o C; 23 o C; 26 o C; 30 o C; 33 o C; 36 o C). Every 24 hours ithas been measured the colony diameter in order to obtain the primary model of the mycelia growth.On the same time interval the synthetic and the natural substrate were prepared for the toxin (DONand OTA) detection.Two methods of DON and OTA detection have been used: one by immunological Elisa test(Ridascreen – 18.5 ppb detection limit) and the other by HPLC detection (immunoafinity columns).Regarding their growth, the primary model of the mycelium development was analyzed byEviews program which has proved a linear evolution during the time in direct correlation with thetemperature (similar to the Gompertz model).32

By immunological test, the primary model of the DON production, showed a maximumproduction of mycotoxin at 26 o C; on this temperature level the maximum amount of DON wasreached in the 17 th day of incubation (from 89ppb after 24 hours incubation to 741 ppb).Instead, for the production of OTA the maximum amount of toxin was reached on 23 o Cafter 17 days of incubation. The maxium measured level was 24033 ppt.Data from the primary model have been confirmed by HPLC precision analysis.Maintaining the quality of the agricultural and food products in order toguarantee the consumer health represent a very important objective in theagricultural and food fields. After a technological transformations succession, thefinal food product should present a minimal microbiological risk. In order to arriveto such of minimal microbiological risk they have been developed different controlsystems, as the HACCP system. An alternative method, complementary to theHACCP, it is the predictive microbiology which can evaluate de speed of themicrobial development under different environmental conditions usingmathematical models [1].The biggest part of these moulds (Aspergillus, Penicillium, Fusarium,Claviceps and Alternaria) in the same time with the conidiation can produce toxicmetabolites (aflatoxins, ochratoxines, DON, etc) having a higher termal stabilitycomparing to their vegetative form, the mycelium.The prevention of the food products pollution with toxinogenic mouldsdepends mainly on the understanding of the fungal alteration phenomenon duringthe processing, conditioning and keeping [5, 10]. For that is necessary to haveprecise diagnostic methods in order to predict and describe in details the dynamic ofthe alteration and pollution with mycotoxins [8, 9].The project proposes to elaborate a preventive diagnostic system of thosepollutions, approaching the phenomenon by predictive microbiology and consist ina parallel modeling of the mycelian development (for the Aspergillus, Penicillium,Fusarium species) and of the toxinogenic secondary metabolite production(ochratoxina A, DON, aflatoxine).The obtained and validated model will allow, on an applied level, to predict thekinetic of the mycotoxins appearance starting from the development kinetics.1. MATERIAL and METHODMicroorganismsFrom USAMVB – Biotechnology microorganism collection, two strainsFusarium graminearum MI 113 and Penicillium crysogenum MI 210 isolated fromfood products of intermediary humidity, have been taken into the study as provedmycotoxins producers (deoxynivalenol, respectively ochratoxin A).33

Media and cultivation conditionsSpores suspension of 10 3 - 10 4 obtained on Czapek – Dox broth was usedas inoculum for natural (50 g of wheat + 10 ml DW) and synthetic (Czapek-Doxbroth) media.The samples have been cultivated during 21 days under differenttemperatures conditions (4 o C; 12 o C; 16 o C; 20 o C; 23 o C; 26 o C; 30 o C; 33 o C; 36 o C).Every 24 hours it has been measured the colony diameter in order to obtainthe primary model of the mycelia growth. On the same time interval the syntheticand the natural substrate were prepared (by extraction and centrifugation) for themycotoxins (DON and OTA) detection.Myctoxins detectionTwo methods of DON and OTA detection have been used: Elisa typeimmuno-enzimatic tests and HPLC technique.The first one consisted inimmunological Elisa kit Ridascreen – R-Biopharm having a 18.5 ppb detectionlimit for DON, respectively RidascreenOTA a 30/15 with a detection limit of2,5 ppb.Fig. 1 - Sample preparation (filtration) forDON detectionFor HPLC detection (immunoafinity columns) it has been used a Waters-Alliance HPLC system. For DON the detection was done at 218 nm, and for OTAthe excitation was measured at 333 nm and the emission at 443 nm.For the separation it was employed a chromatographic columns made ofoctadecilsialns C 18 – Spherisorb 4.6 x 150 mm, having 5 mm particles. The analyteseparation was performed at 30 o C, and the elution used the mixture of acetonytril +methanol + water (5:5:90).The data have been analyzed by a high performance program namedEMPOWER.Mathematical toolsRegarding their growth, the primary model of the mycelium developmentwas analyzed by Eviews program.34

11131111131311132. RESULTS and DISCUSSIONSThe growth evolution under different temperature conditionsFormer researchers showed that the growth and toxinogenesis doesn’t fitthe same evolution curve under the same conditions. For example, for Aspergillusflavus, the optimum growth temperature was proved to be 30 o C, while the highestaflatoxin production was measured at 33 o C [3]. Starting from this results, the twostrains were growth under different temperature conditions. Both strains,Penicillium MI 210 and Fusarium graminearum MI 113 didn’t show any growthat 4 o C.As shown in figure 2, at 12 o C, while the Penicillium strain had a lineargrowth, Fusarium showed an exponential growth.32,521,510,50864201357913579Fig.2 – Growth evolution of P. crysogenum MI 210 (left)and F.graminearum MI 113 at 12 o C (right)Same growth curves allure proved both strains at 16 and 20 o C as at 12 o C.Starting with 23 o C and continuing with 26 o C and 30 o C, the Fusarium strain showda linear growth curve, while Penicillium entered in an exponential evolution of theradial growth on plate (figure 3).4321032,521,510,501357913579Fig.3 – Growth evolution ofP. crysogenum MI 210 (left) and F.graminearum MI 113 at 12 o C (right) at 33 o C35

For Fusarium gramineaurm MI113 the data growth has been analyzedfrom an econometrical point of view, using the Eviews program, a dedicated softfor the economical and statistical experimental data. An analysis of the temporaland a-temporal series have been done. An atypical model have been found for thisstrain at 33 o C.From a statistical point of view, the Fusarium growth at differenttemperature involved a correlation coefficient of r = 0, 41575 , an experimentaldata of t = texp = 0,3432 , while the table value is t = 2,22814 . Comparingstudthis values, it can be stated that there is a linear correlation between thedevelopment time and the mycelium growth, correlation proved also by otherauthors using Gompertz model of the logistic equation [2, 6].In the case of Penicillium strain the correlation coefficient was r =0,18096, the experimental value of the test was t = texp = − 0,58186 , while thetable value was ttab= 2,22814 .Mycotoxin evolution under different temperature conditionsstudtabFig.4 – DON evolution during the cultivation versus the temperature for Fusariumgraminearum MI 11336

Regarding the Elisa type tests, for all the inoculated wheat samples is hasbeen measured the humidity. This one has varied between 28.89 and 48.39 %. Forthe final mycotoxin calculation, these values were taken into consideration.The DON formation on natural medium (wheat) have been measured everytwo days at a very significant quantity have been measured during all 17 days ofcultivation at 26 o C (starting from 89, 64 ppb to 741,05 ppb in the last cultivationday – Fig.4). As it was expected, this temperature is the optimum one for the DONformation as long as much the temperature grows from 26 to 36 o C, less mycotoxinwas detected.In the same conditions, on natural medium (wheat)The highest level of theOTA reached 24033.16 ppt at 36 o C in the last day of cultivation (the 17 th ). It wasfound interesting the fact that at lower temperature, as 30 or 33 o C the OTAformation was maximum in the 5 th day of cultivation, having values of about 1400ppt.Another interesting result it was found by measuring the OTA formation inliquid synthetic medium. The data showed that the optimum temperature for OTAformation it was a lower temperature as on natural solid medium, respectively23 o C.Fig.5 – OTA evolution during the cultivation versus the temperature forPenicillium crysogenum MI 21037

3. CONCLUSIONSEviews program which has proved a linear evolution during the time indirect correlation with the temperature (similar to the Gompertz model).The primary model of the DON production, showed a maximumproduction of mycotoxin at 26 o C; on this temperature level the maximum amountof DON was reached in the 17 th day of incubation (from 89ppb after 24 hoursincubation to 741 ppb).Regarding the OTA production, the maximum amount of toxin wasreached on 23 o C after 17 days of incubation. The maxium measured level was24033 ppt.Further investigation for the OTA and DON gene expression will beperformed by RT-RT-PCR, taking into consideration also the water activityinfluence.REFERENCES1. Allman E., Rhodes J. A. (2003): Mathematical Models in Biology, London.2. Gibson A .M., Hocking A.D. (1997) Advances in the predictive modelling of fungal growth infood. Trends Food Sci. Technol. 8,353-358.3. Judet D., Matei-Radoi F., Bensoussan M., Jurcoane S. (2006): Studies on Aspergillus flavusgrowth and toxicity. Roum Biotech. Lett. 11 (1), p. 2593-2597.4. Matei Radoi F., Avram M., Stanciu A, Correia D. (2007): Microbial charge of stocked cerealscorrelated to their aflatoxins content. Biotehcnology Sci. Bull.-serie F, vol. XII, p.41 – 46.5. Miller, J. David, (2002): Aspects of the ecology of Fusarium toxins in cereals. Department ofChemistry, Carleton University Otawa, Ontario - Mycotoxins and Food Safety, KluwerAcademic/Plenum Publishers, pag. 19 – 22.6. Sautour M., Dantigny P., Divies C., Bensoussan M. (2000): A temperature - type modeldescribing the relationship between fungal growth and water activity. International Journal offood Microbiology 67 63-69.7. Sweeney M. J., Dobson D.W. Alan (1998): Mycotoxin production by Aspergillus, Fusarium andPenicillium species . International Journal of Food Microbiology 43 141-158.8. Varga M., Matei F. (2008): An application of statistical modeling to a problem of the predictivemycology. Proceedings of the International Symposium “New Research in Biotechnology”,Bucharest, Nov 2008. p.165-172.9. Despa R., Folcut O., Radoi Matei F., Coculescu C. (2007) - Linear Regression Models Applyedin Predictive Microbiology. Proceeding of the 31st ARA Congress, TRANSILVANIAUniversity Brasov, Romania.10. Weidenborner, M., 2001, Encyclopedia of Food Mycotoxins, Springer, Germania.38

Scientific Bulletin Biotechnology, U.Ş.A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 39 - 49NUTRITIVE EFFECTS OF NON-CONVENTIONALLY PROCESSEDMEDICINAL PLANTS UPON MONOGASTRIC ANIMALSDEVELOPMENTE. POPA*, A. MUSCALU * , A. DIHORU ** , V. HEBEAN *** National Institute of Research - Development for Machines and Installations Designed to Agricultureand Food Industry ,INMA Bucharest, Romania, e-mail: amuscalis@yahoo.com** National Institute of Research - Development for Biology and Animal Feeding, IBNABaloteşti, RomaniaKeywords: non-ionized radiations, foddering additives, biomass.ABSTRACTThe paper presents the processing of medicinal and aromatic plants by alternative method (withnon-ionized radiations and continous flow) and the output of the utilization of resulted products asfoddering additives for pigs food during the stress period after their nursing cessation. As a result ofthe necessity of shortening the processing time, in order to guarantee a high quality end product, aswell as the achieving of enough biomass quantity for its capitalization for animal food, it is essentialto obtain the forced drying of herb medicinal and aromatic plants, by state-of-the art efficienttechnologies.The medicinal plants herb drying represents a compulsory technologicalstage within the process of capitalization of this category of products.The forced desiccation of medicinal and aromatic plants herb is required asa result of necessity of shortening the process period and in order to obtain aquality end product. Therefore , the content of active substances of herbs must bepreserved as well as possible.For vegetal products dessication alternative (non-conventional) methodshave been developed, being designed at reducing the time and temperature effects,eliminating the chemical treatments and achieving an environmental-friendlyprocessing. Among the non-conventional desiccation methods we have tonotice:treatment with ionized electromagnetic radiations ( γ radiations, Xradiations) and non-ionized (microwaves and radio waves, UV radiations),ultrasounds, processing at high pressure, oscillating magnetic field or thesemethodes combination with conventional desiccation methods.[1]39

1. Introduction1.1 Microwave heating processThe most frequently used phenomenon of microwave processing is represented bydielectric materials heating, due to hysteresis of electric fields-variable in time, wichinfluences the conversion of electromagnetic energy in thermal energy. The source ofenergy is given by dielectric loss. The microwave heating is a volumetric heating,whose efficiency depends on the processed material’s characteristics.The thermal process rapidly evolves, the heating transfer inside the materialdepending on the air current speed. The energy concentration by little volumes of thematerial results in important temperature rises, wich can modify material properties.The dipolar dielectric materials are important for the microwave heating Thephenomenon of polarization takes place inside a dielectric, situated within analternative electric fieldThe electric loads are oriented in terms of polarity of electric field, determiningboth a displacement of electrons around the nucleus (electronic polarization) and adisplacement of atomic nucleus, due to uneven distribution of loads inside themolecule (atomic polarization).Fig.1 - Interface polarization(spatial load) (a) and reorientatingpolarization (b).It has been experimentally found out that the dielectric polarizationdepends on the following parameters:-the amplitude of electric field applied to product;-the field frequency;-the dielectric characteristics, such as ε the permittivity and the loss anglecharacterized by tg δ.The main factors wich determine the materials heating in the microwavefield are:-ε´- dielectric permittivity, - value featuring the materials from their capacity’spoint of view of becoming polarized;-ε´´ -loss factor including all the dissipation effects due to losses by Joule effectand the dielectric permittivity which have been produced inside the material;- microwave power dissipated in the material.40

The interaction which appears between the product and the electric field inwhich it is situated is given by the presence inside the medium (product) of electricdipols, tending to orient towards the electric field direction. The phenomenon ischaracterized by the dielectric constant ( dielectric permittivity) of material,definition starting from the vacuum permittivity:ε = ε ε '( 1)'0 rwhere: ε′ r = material relative permittivity.The product polarization phenomenon determines a displacement currentas:∂DJ D= ( 2)∂twhere: D = vector of electric .induction.Having in view that poles’ rotation is performed by friction, the loadsmovement determines energy losses, in conclusion, material heating.∂In case of harmonic regime, jω∂t = , the permittivity is a complexvalue.ε ' = ε'− jε ''( 3)pwhere: ε” p =represents the losses by dipolar relation.These losses join, in the particular case of humide bodies, the lossesresulted from ionized conductance due to the presence of free loads whichdetermine the loss factor:σε " = ε"p +( 4)ωIn this case the effective dielectric constant of a medium with losses,written as a complex value becomes:∗ε = ε' − jε"( 5)cfrom whitch it results the tangent of loss angle δ:ε'= tgδ( 6)ε"The dielectric permittivity (ε´)as well as the loss factor (ε´´) defining thematerials dielectric features in microwave field varies both in terms of temperatureand humidity. For the dielectric free of losses tg δ is of10 -4 ÷10 -3 degree, and forwet earth of 10 -2 ÷10 -1 degree.41

The main characteristic of dessications in high frequency field is thevolumetric heating, the heat being generated in this particular case inside thematerial (product), in comparision with the conventional method, where the heat issupplied for the material outer side.The wave is attenuated during the medium crossing by energy losses. Itresults that the penetration depth D p is the distance at wich the energy transportedby wave is equivalent with e -1 out of the initial energy.1Dp=( 7)2αwhere: α is the attenuation factor.for dielectrics with losses, this relation becomes:421/ 2⎛ ε'⎞Dp= λ⎜⎟( 8)⎝ 2πε" ⎠Where : λ is the wavelength in free space.The penetration depth depends on the wavelength ( in inverse ratio tofrequency). At frequencies used for microwave heating, the penetration depth is ofdegree of centimeteres and varies in accordance with temperature and materialproperties [2].1.2 Desiccation installation of medicinal and aromatic herbs by means ofmicrowaves in continous flowThe aim is to achieve the artificial (forced) desiccation of medicinal andaromatic plant herb – as chopped herb, in view of subsequent processing andcapitalization of products at a highest quality. The scope comprises all species ofmedicinal and aromatic plants belonging to crops or spontaneous flora in Romania.1.2.1. Installation general presentationThe installation (fig.2) comprises the follwing:A) Desiccation Module:- Desiccation precinct;- Supporting frame;- Microwave generators;- Wave guides;- Ventilation system of microwave generators;- Wet air evacuation system from the desiccation precinct;- control system of processed material temperature by temperature transducerwithout contact, in infrared.B) Feeding/evacuation system of proccesed material- Transport system with conveying band;

- Supporting frame of feeding/evacuation system;- Microwave recovering tunnel (at dehydrating module entrance);- Conveying band drum;- Stretching drum of conveying band;- Guiding elements of conveying band;- Microwave recovering tunnel (at dehydrating module exit);- Electric engine of transmission driving to conveying band.C) Main technical and functional characteristics of installation:- Type of desiccation installation:………..with continuos flow;-High frequency effective power…...10.2 KW;- Microwave source type:……..2M 107 A – 795 magnetrons (Toschiba);- Number of magnetrons/ installation 12 pieces;- Operating frequency:………………2450 MHz ;- Microwave power/ magnetron:……….850 W;-Supply voltage:…………………………380 V;- Temperature control range:………20…100 0 C;- Type of conveyer……with band PTFE with glass fiber insert;- Linear speed of conveying band........0.04...0.10 m/min.;Installation desiccating capacity...30...50 kg/h;Dimension of drying precinct: (3430x500x522) mm;Personnel servicing:…………….max.3 people;Fig. 2 - Desiccation module and control assembly1.2.2 Specific Working RegimeThe state parameters which define the dessication process are as follow:the temperature (°C); humidity (%); dessication speed (% humidity/min.). Thedevelopment of the dessication process of the herb of medicinal and aromaticplants is done in three successive stages as follow:a) the preheating period during which the heat is consumed almost in totality forthe heating of the material until the establishment of the regime temperature, at43

which there is established an equilibrium between the transmitted heat quantity andthat which is consumed for the water evaporation.b) the dessication period with constant speed which represents the dehydrationperiode for that matter;c) the dessication period with decreasing speed (the final period), in which thedessication speed is gradually reduced;The microwaves power regime can be continually adjusted starting fromzero to the maximum power. In order not to be alterated and for conserving theactive substances content and aspect, the medicinal and aromatic plant herb afterdesiccation has to contain, generally only8...14% water.Taking into consideration the significant physical and chemical properties ofthe medicinal and aromatic plants species, we can notice two main groups:• a) plants containing volatile oils (culture thyme, hyssop, mint, etc.), whichmaximal dessication temperature must not be greater than 35 °C;• b) plants containing alkaloids and glycosids (artichoke, bay, etc.), whichoptimal dessication temperature is content between 50 and 65 °C.The using domain of the installation can be extended also to the forestfruits, which have a special situation. Like this, berriess (wild roses and hawthorn)have the optimum dessication temperature over 90 °C.Table 1Active principles of volatile oils for following species (ml %)DessicationSalviamodeHyssopMint Basil ThymeofficinalisNatural drying 0.60 0.67 3.12 0.47 0.51Artificial(microwave)0.74 0.92 3.91 0.65 0.64Table 2Dessication mode Polyphenols (%) Flavone (rutin) (%)Natural drying 0.56 0.74Artificial ( microwave) 0.74 – 1.04 0.85 – 1.3544

1.3 Utilization of medicinal plants in feeding of monogastric animalsThe intensive breeding of farm animals is based on using complex rations,respective concentrates and fodder additives to the monogastric animals. Thesefodder rations are potentiating the animals production, but can produce alsounbalances on the environment by the ratio input/output.[3]The environment protection policy as well as the one of obtaining healthyanimal products orientates the farm animals breeding activity towards applyingalternative solutions to chemotherapy with pharmaceutical growing promoters orantibiotics. In this concept, the capitalizing of vegetal natural resources is anacceptable variant for the infusion of vitamins and minerals, for keeping theanimals performances, for preserving their health and well being state, especially intheir critical life periods, as it would be the ablactation.[4]At the same time it is raised the problem of assuring some correspondingquantities of quality vegetal resources, relationed with their harvesting at theoptimal phenophases of plants development from the specific crops or fromspontaneous flora, selecting and sampling the useful organs, preserving the vegetalmaterial for keeping the initial chemical composition as also the active principles.Within the experiment were used the following vegetal resources: angelica "fruits"flower, oregano flower, whole plant and salvia flower, whole plant[5] .The bio-testwas realized on piglets in their after ablactating period, at Biobase INCDBNA –IBNA Baloteşti, the animals allotments being lodged in growing facilities endowedwith boxes with grates and permanent water alimentation.2. MATERIAL and METHODFor being included within the combined fodder, the vegetal resources wereconditioned via drying (with microwaves) and ground.Table 3SpecificationAnimals allotmentsE 1 E 2 E 3MAnimals number 8 8 8 8Experiment duration (days) 18 18 18 18Angelica– Fruits (kg/100 kg CF) - 3.0 - -Oregano plant (kg/100 kg CF) - - 0.5 -Salvia officinalis. plant (kg/100 kg CF) - - - 3.0

The aromatic and medicinal plants conditioning was effectuateddifferentially, in depending on the useful organs consistency and the water quantitythey contain, applying to installation working regimes corresponding to theprotection of the active principles.The biologic test was performed on a number of 32 piglets from the Great Whitebreed, with two repetitions on allotment, in medium initial weight of 14 kg,repetition 1 and 10 kg repetition 2. the animals were distributed in 4 allotments, 8heads in each allotment and lodged in 4 heads boxes, according to the experimentalscheme from table 3.Ingredients (Kg)MTable 4Experimental allotmentsE 1 E 2 E 3Corn 46.15 43.15 45.65 43.15Rice 15.00 15.00 15.00 15.00Soy grit 16.00 16.00 16.00 16.00Corn gluten 2.00 2.00 2.00 2.00Powder milk 10.00 10.00 10.00 10.00Oil 3.00 3.00 3.00 3.00Fish flower 2.00 2.00 2.00 2.00Monocalcium phosphate 2.00 2.00 2.00 2.00Calcium carbonate 1.60 1.60 1.60 1.60Salt 0.20 0.20 0.20 0,20Methionine 0.25 0.25 0.25 0.25Lysine 0.70 0.70 0.70 0.70Sincaline 0,10 0.10 0.10 0.10Premix vitamins-mineral P1+P2 1.00 1.00 1.00 1.00Fruits ANGELICA - 3.00 - -Herba OREGANO - - 0.50 -Herba SALVIA OFF. - - - 3.00Total (Kg) 100.00 100.00 100.00 100.00Qualitative parametersBrute protein (%) 20.30 21.41 20.30 21.10Metabolismic energy : - Kcal/kg NC- MJ/kg NC340014.23331613.87330013.81330013.81Lysine (%) 1.61 1.59 1.58 1.58Methionine + Cystine(%) 0.91 0.90 0.90 0.90Calcium (%) 1.10 1.19 1.19 1.19Phosphorus total (%) 0.90 0.89 0.89 0.8946

It was used an unique recipe of combined fodder for the 4 pigletsallotments, reproduced in Table 4, whose structure has assured the qualityindices for the nutritional requirements specific to the respective animalcategory.The differences between the experimental variants have consisted inthe species and the useful part from the medicinal plant which wasincorporated in the CF mixture, with the corresponding diminution of thecorn quantity from the ration:-mark allotment (M) fed with CF without phytoaditives;-allotment E 1 fed with CF in which was included 3 % Angelica fruits;-allotment E 2 fed with CF in which was included 0.5 % Oregano herb;-allotment E 3 fed with CF in which was included 3 % Salvia herb.The fodders were fully administered in two rations, recording daily thedistributed quantities and their consumption. The animals were weightedindividually, at the beginning and at the end of the experimental period.Table 5SPECIFICATIONALLOTMENTM E1 E2 E3Initial weight ( kg.) 12.00 11.75 12.00 12.00Final weight ( kg.) * 20.81 a 21.07 a 22.21 a 21.64 aDaily average gain ( kg.) * 0.490 a 0.504 a 0.552 a 0.512 aDaily average consumption (kg.) 1.12 1.23 1.35 1.21Specific consumption ( kg./kg.gain ) 2.29 2.44 2.45 2.36* the same small letter insignificant differences ( P > 0.05 )During the experiment it was tracked the evolution of the following zootechnicalparameters:-body weight;-daily medium gain;-daily medium consume of combined fodder;-specific consume;The obtained results were processed statistically, applying the Student test.3. RESULTS and DISCUSSIONSThe results obtained after the biological test are given in table 5.The initial average weight was established by individually weighting of theanimals subjected to testing and estimated at 12.0 kg.47

The final average weight, determined after 18 days from the experimentbeginning, has marked out bigger values at the piglets from experimentalallotments ( 21.07 kg at allotment E1, 22.21 kg at E2 and 21.64 kg at E3).Statisticaly were not signaled significant differences (P > 0.05) betweenthe experimental allotments for the final body weight, which indicates that thestructure of the mark CF as well as of those supplemented with differentphytoadditives did not influence this indicator.Regarding the daily average gain, is observed an increment of thisparameter at the experimental allotments, the increment being bigger (12.65 %) atallotment E2, comparing with the mark allotment, but the difference isn'tsignificant, the daily average gains being normal for the ablactating crisis.The daily average consumption of combined fodder is bigger in case ofallotment E 2 namely of 1.35 kg,in comparison with the mark allotment M, of 1.12kg.The daily average consumption of E 2, superior to the mark allotment, wasreflected in the daily average gain too (0.552 kg) and the body weight (22.21 kg).The fodders specific consumption expressed by combined fodderkilogramme used for obtaining 1 kg meat gain, is correlated, on the one hand, withthe daily average fodders consumption, and on the other with the daily averagegain, this being very close for allotments M and E3 (2.29 respective 2.36 kg) andbigger for allotment E1 and E2 (2.44 and 2.45 kg).The fodders palatability with phytoadditives was good, especially at E2,which explains the fodders daily average consumption bigger (1.35 kg) andimplicitely a daily average gain with 12.65 % bigger than the mark allotment M.The lack of some significant differences between the experimental allotments andthe mark allotment can be determined by the low participation quota of the vegetalphytoadditives, as well as to the complete structure of mark CF, with corn, powdermilk, etc, which induce performances hard to beat by vegetal additivation.484. CONCLUSIONSThe utilization of the aromatic and medicinal plants drying installation withmicrowaves in continous flow presents the following advantages:- the heat generated by the microwaves field is manifested constantly in all theproduct's mass, during passing through the drying precinct;- the electro-thermal conversion of the electro-magnetic waves of high frequency,determines a rapid heating of the product in its entire volume;- the results are appropriate both from the point of view of drying processefficiency and of the final product's quality;- the favourable impact on the environment, the technology being unpollutedunaffecting the ecological equilibrium and conserving the biodiversity of theenvironment factors.

The utilization as phytoadditives of some medicinal plants (Origanum,Angelica, Salvia) in the feeding process of piglets after ablactation, leads to thefollowing conclusions:- the greater fodder daily consumtion, the final body weight and the daily averagegain bigger at allotment E2 could indicate Oreganum as taste corrector andaromatizer;- the experimental results support the capitalizing of some vegetal species in pigletsfood in the ablactating stress period, as fodder additives, being a potentialalternative for the modern nutritional management of monogastric animals,representing at the same time economical alternatives, in the exploitation of thelocal or regional resources.REFERENCES1. Popa E.:New conservation techniques for vegetal agricultural products. Magazine INMATEH Nr20, Bucharest march 2007, p. 103-104;2. Voicu N. Sisteme cu microunde.Editura MATRIX ROM 2004.3. Jarvis şi Aarts: Nutrient management from a farming systems perspective in „Grassland Science inEurope” in vol.5, Grassland farming - Balacing environmental and economic demands 2000, p.363 – 373.4. Clayton :More science behind < botanicals>: Herbs and plant extracts as growth enhancers, FeedInternational, 20 (4),1999, pp.20 – 23.5. Clayton G., Botanical feed additives. Mini directoy of suppliers, Feed International, 21 (4), 2000,pp.14 -16.49

Scientific Bulletin Biotechnology, U.Ş.A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 50 - 56STUDIES REGARDING THE CONSUMER ACCEPTANCE OF GMFOODSSTUDII PRIVIND ACCEPTAREA ALIMENTELOR CU ORGANISMEMODIFICATE GENETIC DE CATRE CONSUMATORICATALINA VOAIDES 1 , GH. CAMPEANU 1 , PETRUTA CORNEA 11 Faculty of Biotechnology, UASVM Bucharest, 59 Marasti Blvd., Bucharest, RomaniaKey woeds: GMO foods, soybean products, survey, special labelingCuvinte cheie: alimente cu OMG, produse din soia, sondaj de opinie, etichetare specialaABSTRACTLabeling of the genetically modified products has become a worldwide spread necessityassociated with the decreased trust for the biotechnological products. The aim of this study was toreveal the necessity of a special labeling for products that contain or are made from GMO, so theconsumers could choose what kind of products will consume. Also, the obtained experimental resultsemphasize the necessity of testing different products in order to certificate the date from the label.In order to determine the consumer’s perception regarding GMO, a set of18 questions was used, each question having several possible answers. This studywas made on a group of 150 persons, with age ranging from 18 to 65 years old.The aim of this study was to emphasize the information level of the consumersregarding GM products/GM foods, the willingness to consume GM foods, theimportance of the specific and explicit labeling as well as the importance of theprice factor in the decision to buy or not to buy GM foods.The consumers’ awareness regarding GM foods reveals that 19% of thequestioned group is uninformed, 75% are informed and in some degree informedand only 6% are very well informed (fig. 1).Taking into consideration the age distribution, it can be observed that intothe group with age between 18 – 35 years old, only 18% have declared themselvesas uninformed, meanwhile, into the group over 35 years old, 25% declaredthemselves uninformed about the subject.50

1. Inainte de acest chest ionar, cat de bine informaterati in legatura cu organismele sau alimentlemodificate genetic?Fig. 1 - Consumers’ information levelregarding genetically modified foods19%6%Boccaletti and Moro36% (2000) report in a study that 51%of the questioned group hadheard about GM foods, and39%Blaine et al. (2002) reported in areview that among eightfoarte informat informat intrucatva neinformat countries those with mostknowledge of GM foods wereGermany, UK and Japan with 95%, 94% and 89% of respondents having heardof them, respectively. Countries with least knowledge were Brazil, USA andCanada, with 39%, 66% and 78%, respectively.In our local survey regarding customers’ awareness about GMO, 64%,respectively 72% answered correctly at the following questions: “Geneticallyunmodified soybean does not contain genes, but genetically modified soybeandoes?”, respectively “By consuming genetically modified products, a person’sgenes can be altered?” (fig. 2).Fig. 2 - Consumers’ knowledgeregarding GMOnu stiufals284097108adevarat1413From the uninformedconsumers’ category, 45%said that they would avoidPrin consumul alimentelor modificate genetic, genele unei persoane pot fialterate.Soia nemodificata genetic nu contine gene, in timp ce soia modificata geneticcontine.consuming food products that contain genetically modified ingredients (fig. 3),and 87% of them consider the special labeling of GMO very important. Almostin the same proportion, the uninformed consumers would stand up for themandatory labeling of all products, even if they contain or not GM ingredients.51

14%45%17%24%Fig. 3 - The willingness ofuniformed consumers to consumeGM foodsdispusintrucatvanu foarte dispusdin principiu, as evita produsele cu ingrediente modificate geneticIn the same context, 98% of the Norwegian consumers and 87% of the USconsumers demand the proper labeling of genetically modified foods (Chernand Rickertsen, 2002). These results are similar to the ones obtained in anEuropean survey (2001), where 95% of the responders from 15 EU membercountries, that participated to this study, wanted to have the right to choosebetween genetically modified and genetically unmodified foods. So, it can beobserved that our study lines-up with the international needs, regarding thenecessity of GMO foods labeling.Another aspect of our survey was to establish the consumers’ attituderegarding GM foods. In this context, 30% of the responders declared “I do notknow” considering these products as being safe or risky, meanwhile 28%consider them as being neither safe nor risky (fig. 4).5. Cat de sigure sau riscante sunt alimentelemodificate genetic pentru sanatatea oamenilor?Fig. 4 - Consumers’ attituderegarding GM foods30%7%24%10%1%28%Also, 35% of thefoarte riscante riscante nici una nici altafoarte sigure sigure nu stiuuninformed consumersemphasize a negative attitudeto the GM foods, considering them as risky and very risky. In the same time,from the group of informed and very informed consumers, only 30% have thisnegative attitude (fig. 5).52

nu stiusigurefoarte sigurenici una nici altariscantefoarte riscante12 41 141 15 256 144 5Fig. 5 - Consumers’ attituderegarding GM foods and theirinformation levelconsumatori neinformaticonsumatori informati si foarte bine informatiThe responders willingness to consume foods that contain geneticallymodified ingredients showed that 37% are somewhat or are not very willing toconsume them and 32% would avoid their consumption. Taking intoconsideration the consumers’ age, 29% of the consumers less than 35 years old,would avoid the GM foods consumption and 16% of them are not very willingto consume them. In the same time, 50% of the consumers over 35 years oldwould avoid to consume foods with GM ingredients (fig. 6). It can be noticedthat young people, in generally, are more receptive to new, with good priorinforming.6. Cat sunteti de dispus sa consumati alimente cuingrediente modificate genetic?Fig. 6. - The willingness of theconsumers to consume foods withGM ingredients.din principiu, aş evita produsele cuingrediente modificate geneticnu foarte dispusintrucatva3514192835dispus436foarte dispus42peste 35 de anipana in 35 de aniIn the present study, among the participants that declared themselves asnot willing or prefer to avoid the consumption of the GM foods, 79% wouldpay a higher price for the products labeled as “genetically unmodified” (fig. 7).The tendency emphasized in this case could be explained as a sign of theincreased interest for healthier and safer nutrition but it does not reflect thelevel of informing regarding GMO.53

31%21%Fig. 7 - The price and thewillingness to consume GM foods48%From the possibility ofnureducing the quantities ofda, daca pretul este cu putin mai mareda, chiar daca pretul este mult mai marepesticides by using GMO pointof view, 57% of the respondersare somewhat willing to consume them (fig. 8). If genetically modified productswould be more valuable from nutritional point of view than similar unmodifiedproducts, 28% would still be unwilling to consume them and 13% are uninterestedof these aspects (fig. 9).7. Cat sunteti de dispus sa consumati alimentemodificate genetic, daca astfel sunt reduse cantitatilede pesticide aplicate culturilor?Fig. 8 - The willingness of theconsumers regarding GM foods13%17%13%34%23%foarte dispusdispusintrucatvanu foarte dispusdin principiu, as evita produsele cu ingrediente modificate genetic8. Cat sunteti de dispus sa consumati alimentemodificate genetic daca ele sunt mai valoroasenutritional decat cele similare nemodificate genetic?16%26%12%13%33%Fig. 9 - The willingness of theconsumers regarding GM foods54foarte dispusnici una nici altanu sunt deloc dispusintrucatva dispusoarecum nu sunt dispusIn a similar survey of theEuropean Commission, most of

the European citizens had a neutral attitude regarding GM foods, from theobtaining technology point of view. An interesting aspect is that the attitude ismore positive to the first GMO generation (for example herbicide resistant GMO)than the second one (for example increased protein content GMO, modified taste,increased nutritional values) (Frewer, 2004).Also, in another study (2002), Chern and Rickertsen showed that 65% ofthe Norwegian and 55% of the American respondents answered reduced use ofpesticides and below 10% answered reduced price, when they were asked which ofthese potential benefits was most important. More than half of Norwegians foundreduced price to be “extremely unimportant” for their decision to buy or not to buyGM foods.For most of the consumers (86%), labeling of the genetically modifiedfoods is very important and for 89% of them would stand up for a mandatorylabeling of all products, no matter if they contain or not genetically modifiedingredients.The European Commission stated in a previous study (2002) that most ofthe European consumers would prefer that the information regarding GMO to beavailable and an overwhelming majority would prefer to be able to make an“informed” choice, understanding by that the necessity of labeling GM foods(European Commission, 2002).The willingness to buy GM products, associated with the price factor, lead tothe conclusion that for 61% of the consumers the price factor is very important orimportant enough (fig. 10) and 68% of the consumers will pay a higher price forthe products labeled as “not genetically modified”.13. Cat este de important factorul pret cand vadecideti daca sa cumparati sau nu alimentemodificate genetic?6%21%19%Fig. 10 - The influence of theprice taking the decision to buyGM foods12%42%foarte important intrucatva important nici una nici altaintrucatva neimportantdeloc important55

REFERENCES1. Boccaletti S., Moro D., (2000), Consumer willingness-to-pay for GM food products in Italy,AgBioForum 3(4), p. 259-267.2. Blaine K., Kamaldeen S., Powell D., (2002), Public perception of biotechnology, Journal ofFood Science, 67(9), p. 3200-3208.3. Chern W.S., Rickertsen K., (2002), Consumer acceptance of GMO: Survey results from Japan,Norway, Taiwan and the United States, Agricultural, Environmental and DevelopmentEconomics.4. Frewer L., Lassen J., Kettlitz B., Scholderer J., Beekman V., Berdal K.G., (2004), Societalaspects of genetically modified foods, Food and chemical Toxicology 42, p. 1181-1193.5. European Commission, Eurobarometer 55.2, Europeans, Science andTechnology,http://europa.eu.int/comm/public_opinion/archives/eb/ebs_154_en,pdf, 2002.56

Scientific Bulletin Biotechnology, U.Ş.A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 57 – 63ANALYTICAL METHODS OF AROMA COMPOUNDS IN GRAPE JUICEOBTAINED FROM ROMANIAN WINE SORTSGALBENĂ DE ODOBEŞTI AND ŞARBĂIDENTIFICAREA AROMELOR PRIN METODE ANALITICE INMUSTURI OBŢINUTE DIN SOIURILE ROMÂNEŞTI DE VIŢĂ DE VIEGALBENĂ DE ODOBEŞTI ŞI ŞARBĂLUMINIŢA VIŞAN, O. POPA, NICOLETA ARON, TH. SEROTKey words: gas chromatography-olfactometry, Romanian sorts, odor analysisCuvinte cheie: gaz-cromatografie-olfactometrie, soiuri româneşti, analiza aromelorABSTRACTOlfactometric methods frequency of detection, time-intensity method and AEDA that allowto detected and evaluate the same odor-active compounds, were used to evaluate the main odorants oftwo musts obtained from Romanian wine sorts Galbenă de Odobeşti and Şarbă.A number of 15 compounds in grape musts from two analyzed varieties using this techniquewere identified. The volatile compounds identified into the analyzed musts came from differentorganic derivatives classes: alcohol, aldehydes, and ketones, acids and esters, aromatichidrocarbures and terpenoids which create the aroma profile of each variety.Among the other compounds, Ethyl-2-hydroxy propanoat, Ethyl-3-hydroxy butanoate, 3-methyl butan-1-ol, 2,6-nonadien-1-ol, Phénylacétaldéhyde and Linalol seemed to contribute activelyto the odor of Galbenă de Odobeşti must; 3-methyl-propanal, 1-α-terpineol and hexanal werecontributors to the Şarbă odor.Metodele olfactometrice FDT (frecvenţa detectării), TIM (metoda timp-intensitate) şi AEDA(analiza diluţiei extractului de aromă) au fost utilizate pentru detectarea şi evaluarea principalilorcompuşi de aromă din două musturi obţinute din soiuri româneşti de vinifera pentru vin, Galbenă deOdobeşti şi Şarbă.Utilizând metodele olfactometrice amintite au fost identificaţi 15 compuşi volatili de aromăcare aparţin diferitelor clase de compuşi organici: alcooli, aldehide şi cetone, acizi, esteri,hidrocarburi aromatice şi terpene, realizând profilul aromatic al fiecărui soi.Compuşii volatili 2-hidroxi-propanoatul de etil, 3-hidroxi-butanoatul de etil, 3-metilbutanolul, 2,6-nonadienolul, fenilacetaldehida şi linalolul contribuie activ la aroma mustului deGalbenă de Odobeşti iar 3-metil-propanalul, 1-α-terpineolul şi hexanalul contribuie la conturareaaromei mustului provenit din Şarbă.57

Romania is one of the famous viticultural country in production of thewines: Tămâioasă românească, Fetească albă, Şarbă, Grasă de Cotnari etc.Many studies have reported results on wine volatile compounds (1, 2).Many of these aromas are formed during grape processing (destalking, crushing,pressing) by chemical and enzymatic reactions.Gas chromatography/olfactometry (GC/O) methods have been extensivelyused in aroma research and allow the determination of odor-active compounds infood. Olfactometric methods used: time-intensity methods, detection frequencymethods and dilution methods.Dilution methods, Charm analysis and aroma extract dilution analysis(AEDA), are commonly applied and are suitable to screen the odorant compoundsin must or wine. The time-intensity method developed to characterize the Pinotnoir aroma. The frequency of detection has not been used in the determination ofactive-odorant compounds in grape wine or must.The most frequently used methods for isolation of flavor constituents frommust or wine involve extraction with solvent (3) or freon (4), dynamic headspaceanalysis or headspace solid phase microextraction (5).Two Romanian white grape were used for this study: Galbenă de Odobeşti(traditional varieties) and Şarbă (from the Riesling Italian sort).The aim of our study was to characterize the most odor-active compoundsin musts obtained from this varieties by using three GC/O analyses ofrepresentative extracts obtained by an appropriate liquid-liquid extraction method.581. MATERIALS and METHODSJuice preparationThe grapes from the two sorts were produced in the Valea Calugareascavitivinicole station. After harvesting (maturity was estimated by: refractometricdegree, titrable acidity and berry size), grapes were picked off the bunches, crushedwith a hand crusher then pressed at 40 kPa with a laboratory winepress. The mustswere homogenized and stored at –20°C prior to extraction of volatile compounds.Extraction of volatile compoundsSix liquid-liquid extraction methods were compared in order to optimizethe production of representative extracts.Method A: 200 ml of must, placed in a conical flask, were extracted with 5ml of freshly distilled dichloromethane (CH 2 CL 2 ) by stirring for 30 min at 0°C andthen centrifuged for 15 min at 10000 g. The organic extract was dried withanhydrous sodium sulfate and stored at –20°C until analysis.Method B: the same as method A, but NaCl 20% was added to the mustbefore extraction.

Method C: 200 ml of must, placed in a conical flask, were successivelyextracted (3x20 min) at 0°C with 3x25 ml of freshly distilled dichloromethane andthen centrifuged for 15 min at 10000 g. The three organic extracts were pooled,dried with anhydrous sodium sulfate and concentrated to 5 ml in a Danishconcentrator (45°C), then to 1 ml under a stream of nitrogen.Method D: the same as method C, but NaCl 20% was added to the mustbefore extraction.Methods E and F: The same as methods C and D respectively, butdichloromethane was replaced by 3 x 25 ml of dichloromethane:pentane (2:1).Sensory analysis/ representativeness of the extractsSample preparation and presentation. The must samples were thawedovernight at 4°C in a fridge. 5 ml were placed in 15 ml black coded flasks then thetemperature was raised to 25°C just before the musts were presented to the panel.An aliquot of each of the must extracts was adsorbed onto cardboard smelling stripand after 30s (time necessary for solvent evaporation) the ends of the strips werecut and placed in dark coded flasks. These flasks were hermetically closed andpresented to the panel after 30 min.Odor intensity evaluation. The odor intensities of the extracts wereevaluated by using an unstructured scale anchored with “no odor” on the left and“very strong odor” on the right. The positions of the extracts were read as distancein centimeters from the left anchor. Results were analyzed with an ANOVA andLSD test.Gas chromatography/mass spectrometry (GC/MS)The GC/MS system consisted of an HP5890 II gas chromatograph and anHP5971 Mass selective detector (Hewlett-Packard). Each extract was injected inthe splitless mode (250°C injector temperature, 30 sec valve delay) into a capillarycolumn (DBWax, 30 m length x 0.32 mm i.d. x 0.5 m film thickness). The flowrate of carrier gas (helium) was 1 ml/min. Oven temperature was programmed from50°C to 250°C at a rate of 5°C/min with an initial and final hold time of 5 and 10min respectively.Mass selective detector conditions were as follows: ion source temperature,180°C; ionization energy, 70 eV; mass range, 30-300 a.m.u., electron multipliervoltage, 2100 V.; and scan rate, 2 sec -1 .Volatile compound identification was based on comparison of GCretention indices (RI), mass spectra (comparison with MS spectra database, NBS75K and internal library of the laboratory) and odor properties.Gas chromatography-flame detection-olfactometry (GC-FID-O)The GC-FID-O system consisted of a 3400 Star GC (Varian), equippedwith an FID and a sniffing port supplied with humidified air at 40°C. GC effluentwas split 1/1 between the FID and the sniffing port. Each extract (2 µl) wasinjected in the splitless mode (250°C injector temperature) into a capillary column(DBWax, 30 m length x 0.32 mm i.d. x 0.5 m film thickness). The flow rate of59

carrier gas (helium) was 1 ml/min. Oven temperature was programmed from 50°Cto 250°C at a rate of 8°C/min.Frequency of detection (FDT)Sniffing of the chromatogram was performed for 23 min. The panelists (10judges) were asked to assign odor properties to each odorant area detected.Detection of an odor at the sniffing port by fewer than three of the ten assessorswas considered as noise. The ten individual aromagrams were summed, yieldingthe final aromagram (detection frequency versus RI).Time-intensity method (TIM)The time-intensity method was used to measure the perceived odorintensity of compounds in GC-O. The same panelists as before were trained toevaluate aroma intensity using a nine point intensity scale (1= very weak intensity,3= weak intensity, 5= moderate intensity, 7= strong intensity and 9= very strongintensity). Sniffing conditions were the same as for frequency of detection, exceptthat the panelists were also asked to assess intensity (according to a nine pointscale) for each odorous area. Times and intensities of areas which were detected byat least three judges were averaged and a consensus aromagram (averages versusRI) was created.Aroma Extract Dilution AnalysisThe method (AEDA) was used to assess the contribution of individualvolatile compounds to the aroma of grape juices. Serial dilutions (1:3 indichloromethane) of the extract were sniffed until no further odor-active area couldbe detected. AEDA was performed by two sensitive and repeatable panelistsselected during olfactometric global and time-intensity method analyses. The resultwas expressed as the flavor dilution factor. Data from AEDA were alsorepresented in graphs by plotting FD versus RI.602. RESULTS and DISCUSSIONSSensory analysisSimilarity test to measure the difference between the odors of the mustsand the corresponding extracts obtained the extraction methods (A, B, C, D, E andF) and the odor intensity of the extracts is to indicate in table 1.Table 2 shows that the attributes used by panelists to describe the mustsand associated extracts are very similar. The ten panelists described the two mustsand the three extracts principally by fruity notes such as grape, prune, red fruit, orwine-like odor.

Olfactometric analysisOdor active compounds detected in the two musts by three olfactometricmethods is to indicate in table 3. According to the three olfactometric methods, 15odorous areas were significantly perceived by the panelists, in at least one extract.Table 1Similarity test between the odors of the musts and the corresponding extractsTest de similaritate între aromele musturilor şi extractele corespondenteExtractsExtractSimilarity scale (cm)Scală de similaritate (cm)Intensity scale (cm)Scală de intensitate (cm)Galbenă de Odobeşti Şarbă Galbenă de Odobeşti ŞarbăA 5.2 a 4.9 a 4.9 a 5.6 aB 6.3 c 6.1 c 4.9 b 5.1 bC 6.7 c 6.2 c 5.1 c 5.7 cD 5.8 a 5.1 a 6.0 b 5.9 bE 4.8 b 4.9 b 5.0 a 4.9 aF 4.1 b 4.1 b 4.6 a 4.3 aAmong the odorants perceived, some alcohols and aldehydes wereidentified. These compounds are known to exhibit grass-like and green odors. Theyare mainly formed by enzymatic oxidation of polyunsaturated fatty acids. Theirconcentrations mainly depend on the grape cultivar although there were variationsdue to geographical origin or to the ripening stage..Hexanal was perceived with green and grass-like odor mainly in Şarbăextracts. 1-α-terpineol was perceived only in an extract from Şarbă; 2,6 nonadien-1-ol exhibited a melon, cucumber odor was perceived Galbenă de Odobeştiextracts; 1-octen-3-one, which exhibited a mushroom-like odor, was mainlyperceived in Galbenă de Odobeşti;Three compounds exhibited the sweaty aroma notes of phenylacetaldehyde, ethyl-3-hydroxybutanoate and an unidentified compound. Phenylacetaldehyde was perceived in an extract from Galbenă de Odobeşti. Phenylacetaldehyde was not detected in Şarbă. This compound, which exhibits a sweatyand honey-like odor, caramel and syrup odor has previously been identified inmuscadine juice. It may be formed by the decarboxylation of α-ceto-phenylacetate.61

Table 2Main odorant attributes to characterize the two musts and the corresponding volatileextracts obtained by method C.Atributele aromelor utilizate pentru caracterizarea celor două musturi şi extractelecorespondente obţinute prin metoda CGalbenă de OdobeştiŞarbăgrape juicemustExtractextractgrape juicemustExtractextractcaramel caramel wine pruneSweaty, honey honey fruity green, leafygrape grape prune red fruitswine prune apple applefruity grass, green grape grapegrass fruity grass, green woodyapple apple spicy spicy62Table 3Odor-active compounds detected in the musts of Galbenă de Odobeşti and ŞarbăCompuşii active de aromă detectaţi în musturile de Galbenă de Odobeşti şi ŞarbăPeakPicVolatile CompoundsCompuşi volatiliOdordescriptionDescriereaaromelorGalbenădeOdobeştiOlfactometric methodsMetode olfactometriceTIM FDT AEDAŞarbăGalbenădeOdobeştiŞarbăGalbenădeOdobeştiŞarbă1Ethyl-2-hydroxy Butterscotch,propanoatsweet9.5 1.1 8.5 0 540 02 Hexanal Grass, green 0 4.2 0 3.9 0 03 3-methyl butan-1-ol Buttery 5.9 0 8.3 1.1 820 1.24 1-Octen-3-onemushroom,woody3.9 0 4.5 1.1 380 05Ethyl-3-hydroxy hay-like,butanoatesweet8.2 2.2 7.5 1 221 0sweaty,6 Phénylacétaldéhyde caramel,syrup4.4 0 3.9 0 1.1 07 unknown fruity, sweet 1.8 4.2 0 3.2 0 08Ethyl-3-hydroxy hay-like,butanoatesweaty1.1 8.4 0 7.9 0 3249 2,3-butanedionecaramel,buttery0 7.9 0 6.5 0 7.2102-methyl-3-buten-2- buttery,onecannel1.2 1.9 1.1 3 0 011 (E)-3-hexen-1-ol Green, grass 2.1 4.9 1.8 3.5 1.1 32312 Linalol Floral, sweet 1.1 0 1.2 0 0 013 1-α-terpineol Floral 0 1.8 0 1.1 0 014 2,6-nonandien-1-ol15 1-Octen-3-oneMelon,cucumbermushroom,woody4.8 2.1 5.9 1.1 3.1 02.3 0 4.7 1.2 2.1 1.1

3. CONCLUSIONSAmong the odorants perceived, some alcohols and aldehydes wereidentified. These compounds are known to exhibit grass-like and green odors. Theyare mainly formed by enzymatic oxidation of polyunsaturated fatty acids, theconcentrations depend on the grape cultivar although there were variations due togeographical origin.Odor-active compounds detected in the musts of Galbenă de Odobeşti andŞarbă:- Hexanal was perceived with green and grass-like odor only in Şarbăextracts;- 2,3 butanedione, described by caramel, buttery odor, was perceived in anextract from Şarbă;- 2,6 nonadien-1-ol exhibited a melon, cucumber odor and ethyl 3-hydroxybutanoate was perceived with hay-like, sweaty odor, was perceivedGalbenă de Odobeşti extracts;- 1-octen-3-one, which exhibited a mushroom-like odor, was mainlyperceived in Galbenă de Odobeşti;- phenyl acetaldehyde which exhibits a sweaty and honey-like odor,caramel and syrup was perceived only in an extract from Galbenă de Odobeşti.REFERENCES1. Guth, H. (1997): Quantification and sensory studies of character impact odorants of different whitewine varieties. J. Agric. Food Chem. 45, 3027-3032.2. Ferreira,V., Lopez, R., Escudero,O., Cacho, J.F. (1998): The aroma of grenache red wine:hierarchy and nature of its main odorants. J. Sci. Food Agric., 77, 259-2673. Baek, H.H Cadwallader, E. Marroquin, E.and Silva,J.L. (1997): Identification of predominantaroma compounds in muscadine grape juice. J. Food Sci., 62, 249 - 252.4. Ferreira,V., Lopez, R., Cacho, J.F. (2000): Quantitative determination of the odorants of young redwines from different grape wine varieties. J. Sci. Food Agric., 80, 1659-1667.5. Mestres, M., Marti, M.P., Busto, O., Guasch, J. (1999): Simultaneous analysis of thiols, sulphidesand disulphides by headspace solid phase microextraction - gas chromatography. J. Chromtogr., 849, 293-297.63

Scientific Bulletin Biotechnology, U.Ş.A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 64 – 68PROBLEMS OF TECHNOLOGICAL AND CLIMATE NATURE AT RE-CULTIVATING DAMAGED AREAS OF THE OPEN COAL-MINING IN“MARITSA-IZTOK”e-mail: praykova@abv.bgIVAN DIMITROV, SSI; SVILEN RAYKOV, SHUKeywords: seed, coal mining area, climate,. Technology, damaging area, re-cultivationIn the region of Maritsa East Mines in open coal mining there are destroyedlarge areas of arable land. When the operation of the coal basin mop, dumps fromabove-coal revelation will take over 300,000 decares primary agricultural lands.Until now about half of that area is bombarded. To use these areas for agriculturalproduction or afforestation of forest types it is imposed their restoration through theapplication of technical and biological reclamation.Technical re-cultivation covers the arrangement of the materials on theireligibility, equalizing the ground and correcting areas. Biological re-cultivationincludes the selection of plant sorts, treatments, fertilization, introduction of regularsowing and other agricultural activities (Treykyashki, Hristov, 1982). In carryingout the re-cultivation ever more acute occurred at various problems. Thecompliance with them is mandatory and finding adequate solutions to overcomethem is related to the successful conduct of operations.In the splicing of clay materials and their leveling on according to nowaccepted practice often are used dumpers and bulldozers, which, because of thegreat weight and pressure on the soil surface substrate re-seal significantly the soilhorizons.A visual idea of the status of the land after technical re-cultivation given thedata from Figure 1 for the starting bulk density of the area planned for humus lessbiological re-cultivation.From the made experiments it was revealed that it is imperative to correct theestimates of technical re-cultivation. The alignment of the land instead of thebulldozer should be done with grader and the embanking of materials must beperformed only by the soil moisture content at which the additional sealing area isreduced to minimum size. This moisture content varies from 14% to 19% by soilweight depending on the texture of clay materials.64

Depth (cm)controlvariantvariantvariantBulk density (g/cm 3 )Fig. 1 - Bulk density in the betting experience on talus area MednikarovoAnother problem of technological nature is that there is no stability of themicro-relief of re-cultivated land. In order the effects of alignment on the groundwill not be short term there is necessary the bulk clay material to be with ahumidity below 60% of OPH and the splice of the layers to be accompanied bygradual rolling of fresh filled in area, not simultaneously. Less attention is paid tothe evaluation of granule-metric composition of materials from dumps andnamely it just lays down significantly the dynamics of many processes that givethe appearance of fertility.The major problem in the biological re-cultivation of land is a scarcity ofsoil humus material. Deposited quantities of soil humus horizons of arable landcovered by the coal industry can not cover about 40 percent of the estimates forcarrying out biological re-cultivation in humus method used so far. There arenecessary new technological solutions for making humus-less re-cultivation inwhich humus soil does not use or its participation in the soil substrate isnegligible. But particularly in the biological re-cultivation by humus-less methodthe problems of organizational issues, technology and agro-environmental65

easons increase.The final stage of technical re-cultivation (grounding level, introduction ofsubstrates and their mixture with clay) as the agro-technique during biological recultivationin a 10-20 year period thereafter, are crucial in humus-less methods torestore soils. According to some researchers (1,4,5,7), poor physical conditions areidentified as most limited factor for the successful cultivation of agricultural cropson mine dumps and their optimization problems can be resolved with providingadequate methods for their melioration at high values of their sealing or moving toother methods of technical re-cultivation.Compact humus-less re-cultivated soils have no continuous network ofmacro-pores, which facilitate the movement of water, aeration and growth of theroot system of crops. They are deprived of valuable agronomic structures of naturalsoils, which are created by decades. Until then it should be done through theprocessing of soil.Rainfall(mm/m 2 )Av. 10years200220012000199966MonthFig. 2 - Average rainfallDuring the past 10-15 years another problem attended the biologicalreclamation is relatively intensive aridization of the climate which leads to theinability of successfully growing of number of traditional for the area spring crops.There are presented on Fig. 2 the data of average rainfall statistics over time and

four years of the experiment with technology humus-less re-cultivation. With theexception of 2002 summer droughts are quite tangible, and in 2000 for July andAugust there is no receipt of rainfall water as the water stock in the 0-80 cm layeris about 1.5 times less than that in May 2002 (Table 1).Water stock (Lt) in layers up to 80 sm of the soil substratesTable 1Layer Soil substrates variants Soil substrates variantsdepth in 1 2 3 4 1 2 3 4cm2000 20020-10 15 13 12 10 27 23 23 1910 - 20 21 18 20 16 30 30 29 2320 - 30 23 17 19 20 32 32 33 2530 - 40 25 22 21 22 31 31 29 2540 - 60 54 52 52 50 62 63 60 4760 - 80 51 54 55 52 64 63 65 48Amount 189 176 179 171 245 242 239 186As a result of drought in one of the options with the soil substratecontaining 50 m3/da layer of ashes in supporting layer as a result of hydrophobiaproperties of ashes layer is formed of extensive density values within the range 1.9- 2.0 g/m3. In such values there obtains sealing, which prevents the penetration ofthe roots of cultivated plants in depth.Changed climate conditions require the development of a new structure ofcrops used for reclamation. Crops such as sunflower, corn and spring peas, widelyused in agricultural production in the region can not successfully develop theirreproductive capacity and obtained yields of grain and green mass are uneconomicfor manufacturers.Another problem arising from the soil water deficit is the quality of presowingpreparation areas for winter cereals. As a result of deteriorating physicalcondition of the soil it can not be reached the desired depth of processing andstructure of soil aggregates and the use of siderite crops in full ploughing plantresidues and creating conditions for their rapid decomposition.From the analysis of conditions for reclamation for agricultural purposes inthe region of "Maritsa East" and the prospects for its successful development byhumus-less means it was established that there are many problems, the mostsignificant of them is the importance of technological nature and climate .It is advisable to make adjustments to technological operations in technical andbiological re-cultivation. It is necessary to develop a specific system of farming forpurposes of biological reclamation as develop technologies for growing crops67

siderites, structure and sorts field crops grown subsequently tailored to the changedclimatic conditions and criteria to maintain the parameters of the soil substrates inthe optimal range according to the requirements of crops. Any agro-meliorationevent failed to conform to the specific conditions leads to the risk of failure of thewhole technology of re-cultivation of damaged areas of open coal mining area inthe Maritsa East.REFERENCES1. Banov, M., B. Hristov, S. Ruseva, 1992. General physical and hydro-physical properties of recultivatedlands without mold coverage in the region of "Maritsa East". XIV Colloquium"Physics in the protection of human rights and its surrounding environment, 77-90.2. Banov M., V. Marinkina, 2002. Conditions for biological re-cultivation of techno-gene soils builtwith humic material. Soil Science, Agrochemistry and Ecology, No 1-3208-210.3. E. Zheleva and colleagues. 1998. Comprehensive assessment of the results accomplished so far inthe biological reclamation Maritsa East Mines, fund Minproekt EAD / NIS - LTU, Sofia.4. Motorina V.L. 1973. К разработке теоретических основ рекультивации земель. ВСИМПОЗИУМ "Разработка способов рекулътивации ландшафта, нарушенногопромышленной деятельностью Development of theoretical bases of lands re-cultivation. InSYMPOSIUM " Development of re-cultivation methods of landscape broken by industrialactivity", Burgas, 80-87.5. Treykyashki P., Hristov B., 1982. Reclamation of disturbed lands for agriculture. Agriculture,No 1,53-56.6. Hristov B., M. Banov, 1996. Some changes in mineral mass of reclaimed land without moldcoverage in the region of "Maritsa East". Soil Science, Agrochemistry and Ecology, NQ 3,31-35.7. Darmer, G., 1991. Landscape and surface mining: Ecological guidelines for re-cultivation.Norman L. Ditrich. NY.68

Scientific Bulletin Biotechnology, U.Ş.A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 69 – 78ANETHI AETHEROLEUM: CHEMICAL COMPOSITION ANDBIOLOGICAL EFFECTS - reviewANETHI AETHEROLEOUM: COMPOZITIE CHIMICA SI EFECTEBIOLOGICE - reviewALINA ORTAN 1 , MARIA LIDIA POPESCU 2 , CRISTINA DINU-PIRVU 21 University of Agricultural Sciences and Veterinary Medicine, B-dul Marasti nr. 59 sector1,Bucharest, Romania. alina_ortan@hotmail.com2University of Medicine and Pharmacy, “Carol Davila”, Str. Traian Vuia nr 6, sect 2, Bucharest,Romania.INTRODUCTIONAnethum graveolens (dill, Apiaceae) is indigenous to the Mediterraneanregion, southern Russia, and cultivated throughout Europe, as well as North andSouth America. The main compounds are volatile oil, phthalides, coumarins,flavones and polyphenol acids. The fruit of the dill plant has an antispasmodiceffect on the gastrointestinal tract and bacteriosatict effect. Dill herb is used forprevention and treatment of diseases of the gastrointestinal tract, kidney andurinary tract, for sleep disorders and for spasm [1].Anethi aetheroleum is the volatile oil extracted by hidrodistilation or steamdistilation of leaves, fruits or flowers of Anethum graveolens, dill (Apiaceae).PHISICAL – CHEMICAL CHARACTERISTICSAnethi aetheroleum is of a pale yellow color, with an odour of the fruits anda hot, sweetish, acrid taste. By keeping, the color becomes darker and the tastepungent. Dill essential oils differ in odour and taste, depending on the part of theplant from which they were extracted. Dried leaves produce a volatile oil much lesspregnant in odour. The leaves yields about 0,4 - 0,8% oil, meanwhile in fruits theoil contents is significantly increased: 2,3 – 8 % [2]. In case of flowers, there are nodata presented in the consulted scientific literature.The specific gravity varies between 0,895 – 0,915, English distilled oilusually having the highest specific gravity, from 0.910 to 0.916. The oils from bothJapanese and Indian dill differ from European dill oil, in having a higher specificgravity (0,948 – 0,968), which is ascribed to the presence of dill apiol and incontaining much less carvone than the European oil.69

The refractive index of dill volatile oil is 1,483 – 1,488 at 25ºC, opticalrotation +70 - +80º, boiling point 155 – 260 ºC. It is soluble in 3 parts of alcohol(90%).EXTRACTION METHODSThe volatile oil of dill can be obtained by classical extraction methods(distillation, steam distillation, extraction with liquid carbon dioxide), from leaves,fruits and flowers. Presently, the most popular method for extraction is steamdistillation and the majority of essential oils used in aromatherapy are distilled inthis way.Steam distillation is a special type of distillation (a separation process) fortemperature sensitive materials like natural aromatic compounds. Many organiccompounds tend to decompose at high sustained temperatures. Separation bynormal distillation would then not be an option, so water or steam is introducedinto the distillation apparatus. By adding water or steam, the boiling points of thecompounds are depressed, allowing them to evaporate at lower temperatures,preferably below the temperatures at which the deterioration of the materialbecomes appreciable.The complete exhaustion of the product takes 2.5 - 4 hours in case of leaves,respectively 8 – 10 hours for fruits; fruits are first crushed into powder in order tofacilitate the extraction. During the first 2 hours, the distilled oil has a high contentof carvone, maximum ratio carvone: limonen being equal to 80(88) : 12(19);because carvone is more easy soluble in water and being higher boiling fraction, itis distilled more easily. This ratio is nevertheless decreasing in time.CHEMICAL COMPOSITIONSeveral studies presented scientific data concerning the composition of thevolatile oil of Anethum graveolens, extracted from Anethi fructus (fruits of dill) [3],or Anethi folium (dill leaves) [4], but there are no references about the chemicalcomposition of essential oil extracted from Anethi flores.Dill volatile oil from leaves contains: α-pinene (0.9%), β-pinene (0.1%),myrcene (0.4%), α-phellandrene (30.2%), limonene (22.5%), β-phellandrene(3.8%), p-cymene (1.0%), terpinolene (0.1%), α-p-dimenthylstyrene (0.1%), 3,9-epoxy-p-menthlene (5.6%), cis-p-mentha-2,8-dien-lol (0.1%), transdihydrocarvone(0.5%), cis-dihydrocarvone (1.2%), carvone (31.6%),dihydrocarveol (0.1%), cis-carvyl acetate (0.1%), trans-carveol (0.1%), cis-carvylacetate (0.1%), trans-carveol (0.1%), dihydrolimonene-10-ol (0.1%),dihydrolimenon e10-yhexanoate (0.1%), p-mentha-l, 3-dien-10-yl-hexanoate(0.1%) and p-mentha-1(7), 2-dien-10-yl butyrate (0.1%) besides a lot of othercompounds in traces.70

Dill volatile oil from fruits contains: limonene (44.0%), d-p, dimethylstyrene(0.2%), trans-hydrocarvone (0.4%), cis-dihydrocarvone (2.1%), neodihydrocarveol(0.2%), carvone (51.5%), dihydrocarveol (0.1%), isodihydrocarveol (0.4%), transanethole(0.1%), trans-carveol (0.1%) and cis-carveol (0.2%) besides many othercompounds in traces.In conclusion, Anethi aetheroleum obtained from fruits is characterized bythe presence of carvone (18 – 81%, depending on the soil, variety and extractionmethod), dihydrocarvone (0,1-62%), limonene (10 – 50%), besides other in traces,while in volatile oil extracted from leaves the compounds are: carvone (15 – 40%),alfa-phellandrene (25 – 65%), dillapiole (5-32%), anethofuran (dill ether), which ischaracteristic to leaves (2 – 10%). The chemical structures of the main componentsof volatile oil are presented in figures 1, 2, 3.OOS-carvonaR-carvonaFig. 1 - Chemical structures of the enantiomersof carvoneFig. 2 - Chemical structure oflimoneneOFig. 3 - Chemical structure of anethofuranFACTORS AFFECTING THE QUALITY OF THE ESSENTIAL OILVolatile oils existing on the market, for aromatherapy, cosmetic productsindustry, perfumery and alimentary industry have different qualities (different71

content in main compounds) depending on a lot of factors, such as geographicalarea, harvesting season, growing conditions and isolation procedure [5, 6, 7].Species. The importance of the accurate naming of the essential oils used inaromatherapy, by noting the precise species and variety prevents the possibleadverse effects or toxically phenomena, which can occur in case of someambiguities. For example, under name of Anethi aetheroleum we can find thevolatile oils from Anethum graveolens and Anethum sowa (Indian dill), whichpresent a significant different content in monotherpenic cetones, which have aneurotoxical and abortive activity.Vegetative stage. Pino et al [8] showed that there are differences in thecomposition of essential oils extracted from Anethum graveolens harvested indifferent stages of plant’s growth. It has been shown that withered, drid leaves havea lower content of carvone than the fresh ones; this is not happening to the oil ofAnethum fructus, in which the content of carvone is higher in dried, stored fruits. Inthis case, a part of terpines is lost and therefore the yield of carvone is higher.The content of carvone is increasing from 12% in vegetative stage, to 22%during maximum blooming and reaches 35% at the milky-wax stage.The fresh herb at vegetative stage contains 0.60% of oil, which progressivelyincreases with growth and is 0.78–0.99% at flowering, rises to 1.28–1.91% atmilky-wax seed ripening and 1.9–2.84% in the herb when the seed is nearingmaturity.Extrinsic factors (environmental and growing of aromatic plants) alsodetermine the quality of volatile oil. Temperature, relative humidity, sunny hours,air flows influence the quality of the essential oil.Geographical area. It has been observed that dill oil from Britain andSpain has a higher content of phelandrene than the one from Germany; the africandill has a higher yield of volatile oil than the oil from England. [9, 10, 11].An interesting feature of growing dill is that after successive generations, theEuropean dill develops higher oxygenated compounds in the oil, which includes asmall quantity of dillapiole. It was found to contain up to 3.0% of dillapiole [12]when grown under tropical climate. Gupta [13] explained this as being due to moresunlight hours combined with solar intensity in the tropics.COMPOUNDS INFLUENCING FLAVOURIn trade, the oils containing 20% or less carvone have been found to be offinest flavor [14]. The typical flavour of the oil is due to α-phellandrene (terpine)as the oil resembles the fresh herb in aroma. Haupalehti (1986) determined that α-phellandrene, limonene, myrsticin and dill furan were the most significantcontributors of dill herb aroma [15]. Later, Blank et al. (1991) determined that thearoma of dill herb was directly related to concentration of five components namelydill-furan, α-phellandrene, limonene, myrsticin and p-mentha dienbutyrate [16].72

The volatile dill oil extracted from fruits yields high quantities ofcarvone, which is the main constituent responsible for the aroma, but 4-vinyl-2-metoxiphenol (which confers the aroma of flavored meat), 4-hydroxi-3-methil-6lmethilethilciclohexanol (responsible of sweet note) and anethofuran stronglycontribute to the oil characteristic aroma.QUALITY INDICES AND STANDARDSThe presence of a minimum of 5.0% 3,9,epoxy-p-menthene in dill oil is agood indicator of its purity. Lawrence [17] has opined that examination of thepercentage ratio of, α-phellandrene to limonene to β-phellandrene is another testfor quality determination; the acceptable ratio was found to be 20:25:3 in dill herboil.Dill herb oil is easily differentiated from caraway oil as carvone content inthe latter ranges from 47.3% to 59.5%, while in dill oil it is 27.2 to 53.3%. Further,the α-pinene and α-phellandrene in the caraway oil is very meagre (traces to 0.1%)whereas these are between 0.1 to 0.2% and 1.0–2.3% respectively in dill herb oil.In contrast with Anethum graveolens volatile oil, the East Indian dill fruits(Sowa) oil contains large quantity of dillapiole (20%)The presence of high carvecrol is usually an indicator of an aged, partiallyoxydised, fruits oil.ACTIVITYSeveral studies have been performed in order to establish the antibacterial,antifungal, antiviral, anti-inflammatory, antioxidant properties of the essential oilsin general and of dill volatile oil in particular.• Antimicrobial activity (antibacterial and antifungal)The activity of Anethi aetheroleum have been demonstrated against a widevariety of microorganisms, including Gram-positive and Gram-negative bacteria.The antimicrobial activity has been attributed to a number of terpenoid andphenolic compounds [18, 19, 20, 21] and correlated with the proportions in whichthey are present and the interactions between them [22]. Additive, antagonistic, andsynergistic effects have also been observed between components of volatile oils[23]. Still, there are often important differences in the antimicrobial activity of thesame volatile oil. This variability can be atribute to the geografic areas, harvestingtime, genotype, climate, the part of the plant; all these factors can influencechemical composition and relative ratios of the components of the essential oil [24,25, 26, 27, 28, 29]. Some constituents of volatile oils showed a higherantimicrobial activity when tested alone [30, 31, 32]. Delaquis et al. [33] examined73

the antibacterial activity of crude oils and the distilled fractions of dill (Anethumgraveolens L.) against some common Gram-positive and Gram-negative foodspoilage bacteria: Salmonella typhimurium, Listeria monocytogenes,Staphylococcus aureus, Serratia grimesii, Enterobacter agglomerans, Yersiniaenterocolitica, Bacillus cereus). Results showed that crude dill volatile oil hadweak antimicrobial activity, while distilled fractions of dill essential oil containedhigher concentrations of the main chemical constituents, d-limonene and carvone,and exhibited higher antimicrobial activity.Sing et al [34] investigated the antifungal and antibacterial potential ofAnethum graveolens essential oil and they concluded that the volatile oilcompletely inhibited the growth of Fusarium graminearum at 6 µL dose.Moreover, using poison food technique, the essential oil was found to be highlyeffective for controlling the growth of Penicillium citrinum and Aspergillus niger.In antibacterial investigations, using agar well diffusion method, essential oil hasshown good activity for Pseudomonas aeruginosa.Essential oil from dill fruits from Bulgaria, stored for more than 35 years,showed high antimicrobial activity against the fungus Aspergillus niger and theyeasts Saccharomyces cerevisiae and Candida albicans [35].In case of the main component of Anethi aetheroleum, carvone, theantibacterial and antifungal potentials have been demonstrated (antifungal effect onFusarium sulphureum, Phoma exigua var. foveata and Helminthosporium solani[36, 37]. Moreover, some researchers consider that natural d-carvone has theadvantage of no toxicity on human, unlike the synthesis one [38].In a work carried out by Aggarwal et al. [39] it has been demonstrated thatboth optical isomers of carvone were effective against a wide spectrum of humanpathogenic fungi and bacteria; additionally, the antimicrobial properties of thesemonoterpenes were similar to those of the oil in which they were present. Friedmanet al [40] showed that d-carvone presented activity against Listeria monocytogenes.In a study of growth inhibition of E. coli O157:H7, Salmonella typhimurium andPhotobacterium leiognathi, d-carvone was less inhibitory than carvacrol, thymoland trans-cinnamaldehyde [41]. Carvacrol and thymol disintegrated the outermembrane which did not occur by action of carvone or trans-cinnamaldehyde.Pol and Smid [42] showed that a combination of compounds, such as nisin,carvone and lysozyme, is an effective method for decreasing the number ofcolonies of food-borne pathogens.In a study performed on Candida albicans, usind carvone as an inhibitoragent, it was demonstrated that carvone inhibits the transformation of C. albicansfrom a coccus to a filamentous form, which is associated with C. albicanspathogenicity, and thus carvone can be a potentially good therapeutic agent againstinfections caused by this fungus [43].74

• Antitumoral activityIn a study where a series of carvone-related compounds was assessed fortheir ability to induce increased activity of glutathione S-transferase in severaltissues of A/J mice [44], showed that d-carvone exhibited the highest activity as aninducer in all of the tested tissues. Carvone and its related compounds were provedto be a class of potential chemopreventive agents, since the anticarcinogenicactivity is correlated with the ability to induce increased activity of detoxifyingenzymes.D-limonene and d-carvone reduced, in female A/J mice, forestomach tumorformation by about 60% and pulmonary adenoma formation by nearly 35%, due totheir capacities to inhibit N-nitrosodiethylamine-induced carcinogenesis [45].• Other activities (on potatoe sprouting, insecticidal activity)Studies performed by Hartmans şi Diepenhorst [46] showed that, whencompared to the traditional chemical mixtures of isopropylphenylcarbamate andisopropyl-3- chlorophenylcarbamate, carvone was as good or even better duringlong-term storage,as a potato sprouting inhibitor. Carvone is alreadycommercialised in The Netherlands under the name ‘‘Talent’’ as an effectivesprout growth regulator [47].Carvone can be used as an insecticide against the fruit fly, Drosophilamelanogaster, although constituents of mint oil show higher insecticidal andgenotoxic activities [48].UTILIZATIONThe previously presented activities of Anethum graveolens volatile oilconduct to a series of utilization in several domains, such as aromatherapy,cosmetic and perfumery industry, alimentary industry, culinary art, etc.Aromatherapy uses Anethi aetheroleum in respiratory, cardio-vascular anddigestive disorders.In pharmacy, the dill oil can serve in obtaining hydrosols or aromatic water(Aqua Anethum), liposols, aerosols (fine sprayed in air and propulsive gas), indifferent pharmaceutical formulations, conferring to all, besides pleasant smell andaroma, its therapeutically properties.In cosmetic and perfumery industry, dill oil is used in preparation ofhygienically-sanitary products, creams, emulsions, lotions, gels, products withpreventive and curative role in dermal pharmacy.In alimentary industry and culinary art it is used as a spice.75

In other areas: because Anethi aetheroleum contain terpinene şi α–pinene, itcan be used in synthesis of terpin-hydrat, α-terpineol, linalol, borneol products.PRECAUTIONS AND ADVERSE REACTIONSPhotodermatosis appearance is possible after contact with the juice of thefreshly harvested plant; this could be explained by the presence of furanocoumarinsin the oil.Precautions: babies, children and pregnant women (neorotoxic and abortivebecause of the cetones content).REFERENCES1. XXX – PDR For Herbal Medicines, Third edition, ed Thomson, Montvale, 258 (2004).2. C.C.R. de Carvalho, M. M. R. da Fonseca – Carvone: why and how should one bother toproduce this terpene. J. of Food Chemistry, 95, 2006, p 413-4223. P.Schreier – Biogeneration of plant aromas. Developments in food flavours, Elsevier, New York,1986, 89-1064. B.M.Lawrence – A review of the world production of esssential oils. Perfum. Flavor., 10, 1985,1-16.5. H.Maarse – Volatile copounds in food and beverages, CRC Press, 1991, 463-4646. A Koedam, J. J. C. Scheffer and A. Baerheim Svendsen - Essential Oil Of Dill Herb. Chem.Mikrobiol. Technol. Lebensm.,. 6, 1 1979, 125.7. Huopalahti R., Linko R. R., 1983, Composition and content of aroma compounds in dill,Anethum graveolens L., at three different growth stages. J. Agric. Food Chem., 31, 331-333.8. J. Pino, A. Rosado, I. Goire, E. Roncal – Evaluation of flavour characteristics compounds in dillherb essential oil by sensory analysis and gas chromatography. J Agric. Food Chem, 1995, 43,1307 – 1309.9. V. Masotti, F. Juteau, J.M.Bessiere, J. Viano – Seasonal and phenological variations of theessential oil from the narrow endemic species Artemisia molinieri and its biological activities. J.Agric. Food Chem, 51, 2003, 7115-7121.10. A. Angioni, A.Barra, V. Coroneo, S.Dessi, P.Cabras – Chemical composition, seasonalvariability and antifungal activity of Lavandula stoechas L. Ssp. Stoechas essential oil fromstem/leaves and flowers. J. Agric. Food Chem, 54, 2006, 4364-4370.11. A. Koedam, N. Margaris, D. Vokou – Aromatic plants: basic and applied aspects. KluwerAcedemic publisher, 1995, 135.12. Baslas, R.K., Gupta, R. and Baslas, K.K. (1971) - Chemical examination of essential oil fromplants of genus Anethum (Umbelliferae): Oil of seed of Anethum graveolons. (Pt I) Flavour Ind.2(4), 241–5.13. Gupta, R. (1982) - Studies in cultivation and improvement of dill (Anethum graveolons) in India.In Cultivation and Utilization of Aromatic Plants, Eds. C.K. Atal and B.M. Kapur, RegionalResearch Laboratory, Jammu, pp. 545–8.14. E. Guenther - The Essential Oils. Van Nostrand Co, New York, 1950, pp. 619–34.15. R. Haupalehti - Gas chromatographic and sensory analysis in the evaluation of dill herb(Anethum graveolons L.) Lebensmitt. Wiss. Technol., 1986, 19, 27–30.16. I. Blank, W. Grosch - Evaluation of potent odorants in dill seed and dill herb Anethumgraveolens L.) by aroma extract dilution analysis. J. Food Sci. 1991, 56(1), 63–7.17. B.M.Lawrence – New trends in essential oils. Perfum. Flavor., 5, 1980, 6-16.76

18. Greathead, H., 2003. Plant and plant extract for improving animal productivity. Proc. Nutr. Soc.62, 279–290.19. Panizzi, L., Flamini, G., Cioni, P.L., Moreli, I., 1993. Composition and antimicrobial propertiesof essential oils of four Mediterranean Lamiaceae. J. Ethnopharmacol. 39, 167–170.20. Helander, I.M., Alakomi, H.-L., Latva-Kala, K., Mattila-Sandholm, T., Pol, L., Smid, E.J.,Gorris, L.G.M., von Wright, A., 1998. Characterization of the action of selected essential oilcomponents on Gram negative bacteria.. J. Agric. Food Chem. 46, 3590–3595.21. Chao, S.C., Young, D.G., Oberg, C.J., 2000. Screening for inhibitory activity of essential oils onselected bacteria, fungi and viruses. J. Essent. Oil Res. 12, 639–649.22. Dorman, H.J.D., Deans, S.G., 2000. Antimicrobial agents from plants: antibacterial activity ofplant volatile oils. J. Appl. Microbiol. 88, 308–316.23. Burt, S., 2004. Essential oils: Their antibacterial properties and potential applications in foods—a review. Int. J. Food Microb. 94, 223–253.24. Arrebola, M. L., Navarro, M. C., Jime´nez, J., & Oca´na, F. A. - Yield and composition of theessential oil of Thymus serpylloides subspserpylloides. Phytochemistry, 1994, 1, 67–72.25. Rhyu, H. Y. - Gas chromatographic characterization of sages of various geographic origins.Journal of Food Science, 1979, 44,758–762.26. Salgueiro, L. R., Vila, R., Tomi, F., Figueiredo, A. C., Barroso, J. G., Canigueral, S., et al. -Variability of essential oils of Thymus caespititius from Portugal. Phytochemistry, 1997, 45,307–311.27. Venskutonis, P. R. - Effect of drying on the volatile constituents of thyme (Thymus vulgaris L.)and sage (Salvia officinalis L.). Food Chemistry, 1996, 2, 219–227.28. Juliano, C., Mattana, A., & Usai, M. - Composition and in vitro antimicrobial activity of theessential oils of Thymus herba-barona Loisel growing wild in Sardinia. Journal of Essential OilResearch, 2000, 12, 516–522.29. McGimpsey, J. A., & Douglas, M. H. - Seasonal variation in essential oil yield and compositionfrom naturalized Thymus vulgaris L. in New Zealand. Flavour Flagrance Journal, 1994, 9, 347–352.30. Kim, J., Marshall, M. R., & Vei, C. - Antimicrobial activity of some essential oil componentsagainst five foodborne pathogens. Journal of Agricultural and Food Chemistry, 1995, 43, 2839–2845.31. Lambert, R. J. W., Skandamis, P. N., Coote, P., & Nychas, G.-J. E. - A study of the minimuminhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol.Journal of Applied Microbiology, 2001, 91, 453–462.32. Suresh, P., Ingle, V. K., & Vijayalakshmi, V. - Antibacterial activity of eugenol in comparisonwith other antibiotics. Journal of Food Science and Technology, 1992, 29, 254–256.33. Delaquis, R.J., Stanich, K., Girard, B., Massa, G. - Antimicrobial activity of individual andmixed fractions of dill, cilantro, coriander and eucalyptus essential oils. Int. J. Food Microbiol.2002, 74, 101–109.34. G. Singh , S. Maurya, M.P. de Lampasona, C. Catalan - Chemical Constituents, AntimicrobialInvestigations, and Antioxidative Potentials of Anethum graveolens L. Essential Oil and AcetoneExtract.35. Jirovetz, L., Buchbauer, G., Stoyanova, A. S., Georgiev, E. V., & Damianova, S. T. -Composition, quality control, and antimicrobial activity of the essential oil of long-time storeddill (Anethum graveolens L.) seeds from Bulgaria. Journal of Agricultural and Food Chemistry,2003, 51, 3854–3857.36. Hartmans, K. J., & Diepenhorst, P. - The use of carvone as a sprout inhibitor for potatoes. PotatoResearch, 1994, 37, 445–446.37. Hartmans, K. J., Diepenhorst, P., Bakker, W., & Gorris, L. G. M. - The use of carvone inagriculture – sprout suppression of potatoes and antifungal activity against potato-tuber and otherplant-diseases. Industrial Crops and Products, 1995, 4, 3–13.77

38. Kerstholt, R. P. V., Ree, C. M., & Moll, H. C. - Environmental life cycle analysis of potatosprout inhibitors. Industrial Crops and Products, 1997, 6, 187–194.39. Aggarwal, K. K., Khanuja, S. P. S., Ahmad, A., Kumar, T. R. S., Gupta, V. K., & Kumar, S. -Antimicrobial activity profiles of the two enantiomers of limonene and carvone isolated from theoils of Mentha spicata and Anethum sowa. Flavour and Fragrance Journal, 2002, 17, 59–63.40. Friedman, M., Henika, P. R., & Mandrell, R. E. - Bactericidal activities of plant essential oilsand some of their isolated constituents against Campylobacter jejuni, Escherichia coli, Listeriamonocytogenes, and Salmonella enterica. Journal of Food Protection, 2002, 65, 1545–1560.41. Helander, I. M., Alakomi, H. L., Latva-Kala, K., Mattila-Sandholm, T., Pol, I., Smid, E. J., et al.- Characterization of the action of selected essential oil components on gram-negative bacteria.Journal of Agricultural and Food Chemistry, 1998, 46, 3590–3595.42. Pol, I. E., & Smid, E. J. - Combined action of nisin and carvacrol on Bacillus cereus and Listeriamonocytogenes. Letters in Applied Microbiology, 1999, 29, 166–170.43. McGeady, P., Wansley, D. L., & Logan, D. A. Carvone and perillaldehyde interfere with theserum-induced formation of filamentous structures in Candida albicans at substantially lowerconcentrations than those causing significant inhibition of growth. Journal of Natural Products,2002, 65, 953–955.44. Zheng, G., Kenney, P. M., & Lam, L. K. T. - Effects of Carvone compounds on glutathione-Stransferaseactivity in A/J Mice. Journal of Agricultural and Food Chemistry, 1992a, 40, 751–755.45. Wattenberg, L. W., Sparnins, V. L., & Barany, G. - Inhibition of N-nitrosodiethylaminecarcinogenesis in mice by naturally occurring organosulfur compounds and monoterpenes.Cancer Research, 1989, 49, 2689–2694.46. Hartmans, K. J., & Diepenhorst, P. - The use of carvone as a sprout inhibitor for potatoes. PotatoResearch, 1994, 37, 445–446.47. Hartmans, K. J., Lenssen, J. M., & de Vries, R. G. Use of talent (carvone) as a sprout growthregulator of seed potatoes and the effect on stem and tuber number. Potato Research, 1998, 41,190–191.48. Franzios, G., Mirotsou, M., Hatziapostolou, E., Kral, J., Scouras, Z. G., & Mavragani-Tsipidou,P. - Insecticidal and genotoxic activities of mint essential oils. Journal of Agricultural and FoodChemistry, 1997, 45, 2690–2694.78

Scientific Bulletin Biotechnology, U.Ş.A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 79 - 86SHORT REVIEW ON APPLICATION OF ION-SELECTIVE SENSORSFOR HEAVY METAL IONS IN FOOD AND ENVIRONMENTALSAMPLESO SCURTA PREZENTARE ASUPRA APLICARII SENZORILOR ION-SELECTIVI PENTRU DETERMINAREA IONILOR METALELORGRELE DIN ALIMENTE SI PROBE DE MEDIUALINA CULEŢU, ALINA CATRINEL ION, ION IONKey words: ion-selective electrodes, sensors, heavy metalsCuvinte cheie: electrozi ion-selectivi, senzori, metale greleABSTRACTChemical sensors are analytical instruments used for measurement of different ions invarious chemical, clinical or environmental samples. One of the most common classes of electrodes isthe one based on ionophores covering a broad area of organic or inorganic compounds that bind tocations and anions.Senzorii chimici sunt dispozitive analitice folosite pentru masurarea diferitilor ioni dindiverse probe chimice, clinice si de mediu. Una din cele mai cunoscute clase de electrozi este ceabazata pe ionofori ce acopera un domeniu larg de compusi organici si anorganici care leaga cationisi anioni.Ion-selective electrodes (ISEs) are part of a group of relatively simple andinexpensive analytical tools which are commonly referred to as sensors, the pHelectrode being one of the most well known and simplest member of this group.The good sensitivity, selectivity and speed of electrochemical sensors makethem applicable to the determination of food ingredients, as well as tracecompounds or contaminants in different foods and drinks.Heavy metals are very toxic environmental pollutants, thus the knowledgeof their real content in various matrices is very important. Such elements tend toconcentrate in all matrices in the environment involved in foods and food chainbecause of their irreversible toxic effects on man.The standard techniques for trace heavy-metal analysis include: AtomicAbsorption Spectrometry (AAS) and Inductively Coupled Plasma-MassSpectrometry (ICPMS). However, these methods require expensive equipment,79

which cannot be used in the field. Moreover, all of the methods involvecomplicated and time-consuming sample treatment and pre-concentration steps.Electrochemical methods are seen as complementary to the above mentionedtechniques, and are especially attractive because they allow the possibility ofcreating inexpensive and portable instrumentation.In recent years, the development of novel sensors for the detection ofheavy metals ions has been motivated by controlling the levels of environmentalpollutants in natural waterways and potable water.Potentiometric detectors based on ion-selective electrodes are suited, because theyoffer advantages such as high selectivity, sensitivity, good precision, simplicity,non destructive analysis, ability to monitor ion activity without extensivepreparation of sample and low cost.GENERAL PRESENTATION OF ISEsChemical sensors are miniaturized analytical devices, which can deliverreal-time and on-line information on the presence of specific compounds or ions incomplex samples. Usually the recognition process of the analytes takes placefollowed by the conversion of chemical information into an electrical or opticalsignal.External ReferenceElectrodeInternal ReferenceElectrodeION SELECTIVEELECTRODEInternalelectrolyteSample solutionMembraneFig.1 – Shematic diagram of an ion-selective potentiometric cell80

Among various classes of chemical sensors that one of ion-selectiveelectrodes is the most frequently used. Potentiometric sensors are used everywherefrom laboratory analysis till industry, process control, physiological measurements,and environmental monitoring.The ion-selective membrane is the key component of all potentiometric ionsensors. It establishes the preference with which the sensor responds to the analytein the presence of various interfering ions from the sample.In figure 1, a schematic diagram of an ion-selective potentiometric cellassembly is presented.Using a series of calibrating solutions, the response curve or calibrationcurve of an ion-selective electrode can be measured and plotted as the signal(electromotive force) versus the activity of the analyte.Typical calibration curve of a potentiometric sensor determined in this wayis shown in figure 2.Electromotive force, mVcationinterferenceanioninterferencelog aFig.2 – Typical calibration curve of ISEThe linear range of the calibration curve is usually applied to determine theactivity of the target ion in any unknown solution.ISE MEMBRANE COMPOUNDSThe polymeric membrane is used to separate the test solution from theinner compartment of the electrode, containing a solution of the target ion. Anypolymeric membrane ion selective sensor consists of some components. The natureand the amount of each component illustrate great effects on the nature and the81

characteristics of the sensor. These components are: the polymeric matrix, theionophore (membrane - active recognition), the membrane solvent (plasticizer) andthe ionic additives.The polymeric matrixThe application of polymers as homogeneous membrane matrices wasfirstly suggested for use with charged carriers in 1967 [1]. The first polymeric ISEmembranes, where the polymer was considered to provide the required physicalproperties, like elasticity and mechanical stability, were prepared in silicone rubberor polyvinyl chloride (PVC) [2].The ionophore (membrane-active recognition)The ionophore or the membrane-active recognition can be an ionexchanger or a neutral macrocyclic compound, having molecule-sized dimensionsand containing cavities or semi-cavities to surround the target ions. The ionophore,also named “ion carrier” is the most significant component of any polymericmembrane sensor with reference to the selectivity and sensitivity, since themolecular-level phenomenon reflected by the ISE is the binding between theionophore and the target ion.Many cyclic and linear macromolecules have been introduced asionophores in potentiometric membrane sensors for various cations and anions [3].Crown ethers, polyamines, calixarenes, metalloporphyrines are used as ionophoresin various sensors [3].The membrane solvent (plasticizer)Plasticizers are additives that increase the plasticity or fluidity of thematerial, to which they are added. Solvent polymeric membranes, used in ionsensors, are usually based on a matrix containing about 30 - 33% (w/w) PVC and60 - 66% from a membrane solvent [4]. Films with such a high plasticizer amountdemonstrate optimum physical properties and ensure relatively high mobilities oftheir constituents. In order to give a homogeneous organic phase, the membranesolvent must be physically compatible with the polymer in order to displayplasticizer properties. For various reasons, it also has an influence on the selectivitybehavior. Some of the common plasticizers are: benzyl acetate, bis(2- ethylhexyl)phthalate (dioctyl phthalate), bis-octyl sebacate, dibutyl phthalates, dibutylsebacate and 2 nitrophenyl phenyl ether [5].Ionic additivesIonic additives are ion exchangers, which themselves induce a selectiveresponse when no or only an insufficient ionophore amount is present. Therefore,their concentration must be adjusted carefully. Although the neutral-carrier-basedISE membranes may work properly, even when they contain only a very smallamount of ionic sites, the addition of a lipophilic ion salt is advisable and beneficialfor various other reasons, as well [6].82

THE PREPARATION OF MEMBRANE ISEA typical procedure to prepare the PVC membrane [4] is to mix thoroughly30 – 35 mg of the powdered PVC, 60 – 65 mg plasticizer, the suitable ionophoreamount (usually 1 – 10 mg) and 1 – 5 mg anionic additive in 5 mL tetrahydrofuran(THF).The resulting homogeneous mixture is poured into a glass ring with aninner diameter of 20 – 50 mm, resulting in a smooth glass plate. THF is evaporatedat room temperature. A transparent membrane is formed. A disc with a 10 mmdiameter is cut out from the PVC membrane and glued to the one end of the Pyrexglass tube. Afterwards, the tube is filled with the internal filling solution and isconditioned for 12 – 48 h by soaking in a solution which contains the analyte.PROPERTIES OF ISEsThe properties of an ISE [7] are characterized by parameters like:measuring range, detection limit, response time, selectivity, lifetime.Measuring rangeThe linear range of the electrode is defined as that part of the calibrationcurve through which a linear regression would demonstrate that the data points donot deviate from linearity by more than 2 mV.Detection limitThe detection limit of an ISE is calculated using the cross-section of thetwo extrapolated linear parts of the ion-selective calibration curve, according to theIUPAC recommendation [8].Response timeIt is defined as the time between the instant at which the ion-selectiveelectrode and a reference electrode are dipped in the sample solution and the firstinstant at which the potential of the cell becomes equal to its steady-state valuewithin 1 mV.SelectivitySelectivity is the most important characteristic of these devices. Itdescribes the ISE specificity toward the target ion in the presence of other ions,also called as “interfering ions”.LifetimeThe average lifetime for most of the reported ISEs is in the range of 4 - 10weeks. After this time, the detection limit of the sensor will decrease. It is acceptedthat the loss of plasticizer, carrier or ionic site from the polymeric film, as a resultof leaching into the sample, is the primary reason for the limited lifetime of thecarrier-based sensors.83

84APPLICATIONS OF ISEs FOR DETECTIONS OF SOME HEAVY METALSIonophore – incorporated PVC–membrane sensors are analytical tools usedfor the selective and direct measurement of a wide variety of different ions incomplex biological and environmental samples. The key ingredient of suchplasticized PVC-membranes is the involved ionophore, defining the selectivity ofthe electrodes complex formation with the cation of interest.Until now, a large number of ionophores with high selectivity for specificmetal ions have been developed for the use in potentiometric sensors selective forthe respective metal ions [5].Lead selective membrane sensorsBecause of the increased industrial use of lead and its serious hazardouseffect to human health, the preparation of the lead ion selective membraneelectrodes have been wide investigated.Examples of some lead ion selective sensors with biological orenvironmental applications are in: water samples [9], minerals [10], oil samples[11], etc.Copper selective membrane sensorsDue to the vital importance of copper in many biological systems and theurgent need for a copper-selective electrode for potentiometric monitoring of Cu 2+in environmental, medicinal and different industrial samples, there has beenincreasing interest in the development of novel membrane sensors for the detectionof copper ion, during the past. Some of the copper ion selective sensors withbiological or environmental applications are in milk powder samples [12], black teasamples and multivitamin capsules [13].Cadmium selective membrane sensorsCadmium is an extremely toxic metal commonly found in industrialworkplaces, particularly where ores are processed or smelted. The effect of itsacute poisoning is manifested in a variety of symptoms, including high bloodpressure, kidney damage, anemia, hypertension, bone marrow disorders, cancer andtoxicity to aquatic biota [14].Some of reported cadmium ion selective sensors with biological orenvironmental applications are in water samples [15], aqueous solutions [16], soils[17], etc.Chromium selective membrane sensorsChromium is known to be an essential element (Cr 3+ ) in the humannutrition and a toxic one in the hexavalent state, therefore its accumulation in thehuman body results in toxicity, chromium in hexavalent state being 100 – 1000times more toxic than the Cr 3+ .Some of the reported chromium ion selective sensors with biological orenvironmental applications are in: waste water and alloy samples [18], some foodmaterials and various types of plants [19].

Nickel selective membrane sensorsNickel is a silvery white metal that takes on a high polish. For manydecades, nickel was regarded as a potentially toxic element, since its concentrationin various foods was higher than that needed for living organisms. More recently[20], it is now considered a possible essential element for plants, althoughdeficiencies can occur under certain circumstances. However, nickel can be toxic athigh concentrations and can be a problem in some soils [21].Nickel ion selective sensors are used in biological and environmentalanalysis, for example in the determination of nickel content of chocolate [22], milkpowder samples [23], hydrogenated oils [24], industrial waters [25].CONCLUSIONSIon-selective electrodes represented in the past and are still developed assimple and efficient analytical tools for environmental and biological samples, dueto their advantages: rapidity and simplicity in operation and preparation, low cost,comparatively fast responses, very low detection limit, wide dynamic ranges andsatisfactory selectivity. It must be emphasized that their use is now increasing inspeciation studies in combination with AAS, because of the possibility ofdetermination of the concentration of the free metal ion (using ISEs) and the totalone (using AAS).This short review proposes a short approach of the characteristics andapplication of ISEs for heavy metal ions which are in progress due to the newionophores possible to be used in electrodic membranes.REFERENCES1. Shatkay, A. (1967): Ion specific membranes as electrodes in determination of activity of calcium.Anal. Chem. Vol. 39, 10: 1056 – 1065.2. Fiedler, U., Ruzicka, J. (1973): A Valinomycin-Based Potassium Inner Reference System. Anal.Chim. Acta. Vol. 67: 179 – 183.3. Ganjali, M. R., Norouzi, P., Rezapour, M., Faridbod, F., Pourjavid, M. R. (2006): SupramolecularBased Membrane Sensors. Sensors, Vol. 6: 1018 – 1086.4. Moody, G. J., Oke, R. B., Thomas, J. D. R. (1970): A calcium-sensitiveelectrode based on a liquidion exchanger in a poly(vinyl chloride) matrix. Analyst, Vol. 95: 910 – 918.5. Bakker, E., Buhlmann, P., Pretsch, E. (1997): Carrier-Based Ion-Selective Electrodes and BulkOptodes. 1. General Characteristics. Chem. Rev. Vol. 97, 8: 3083 – 3132.6. Bühlmann, P., Yajima, S., Tohda, K., Umezawa, K., Nishizawa, S., Umezawa, Y. (1995): Studies onthe phase boundaries and the significance of ionic sites of liquid membrane ion-selectiveelectrodes. Electroanalysis. Vol. 7: 811 – 816.85

7. Lindner, E., Umezawa, Y. (2008): Performance Evaluation Criteria for Preparation andMeasurement of Macro- and Microfabricated Ion-Selective Electrodes. Pure Appl. Chem., vol.80, 1: 85 – 104.8. Guilbault, G. G., Durst, R. A., Frant, M. S., Freiser, H., Hansen, E. H., Light, T. S., Pungor, E.,Rechnitz, G., Rice, N. M., Rohm, T. J., Simon, W., Thomas, J. D. R. (1976): Recommendationsfor nomenclature of ion-selective electrodes (IUPAC Recommendations). Pure Appl. Chem. Vol.48: 127-132.9. Rouhollahi, A., Ganjali, M. R., Shamsipur, M. (1998): Lead ion selective PVC membraneelectrode based on 5, 5 '-dithiobis-(2-nitrobenzoic acid). Talanta, vol. 46: 1341 – 1346.10. Vlasov, Y.G., Ermolenko, Y.E., Kolodnikov, V.V., Murzina, Y.G. (1999): Ion selectivepotentiometric determination of lead in minerals. J. Anal. Chem. Vol. 54: 1056-1062.11. Ganjali, M.R., Hosseini, M., Basiripour, F., Javanbakht, M., Hashemi, O.R., Rastegar, M.F.,Shamsipur, M., Buchanen, G.W. (2002): Novel coatedgraphite membrane sensor based on N,N'-dimethylcyanodiaza-18- crown-6 for the determination of ultra-trace amounts of lead. Anal.Chim. Acta. Vol. 464: 181-186.12. Firooz, A. R., Mazloum, M., Safari, J., Amini, M. K. (2002): Coated-wire copper(II)-selectiveelectrode based on phenylglyoxal based on phenylglyoxal-alpha-monoxime ionophore. Anal.Bioanal. Chem. vol. 372, 5-6: 718 – 722.13. Sadeghi, S., Vardini, M. T., Naeimi, H. (2006): Copper (II) ion selective liquid membraneelectrode based on new Schiff base carrier. Ann. Chim. vol. 96: 65 –74.14. Nordberg, G. F. (1984): Chelating agents and cadmium toxicity: problems and prospects. EnvironHealth Perspect. Vol. 54: 213–218.15. Singh, A. K., Mehtab, S., Singh, U. R., Aggarwal, V. (2007): Comparative studies of tridentatesulfur and nitrogen-containing ligands as Ionophores for construction of cadmium ion-selectivemembrane sensors. Electroanalysis, vol. 19: 1213 –1221.16. Mashhadizadeh, M.H., Sheikhshoaie, I., Saeid-Nia, S. (2005): Asymmetrical Schiff bases ascarriers in PVC membrane electrodes for cadmium (II) ions. Electroanalysis, Vol. 17: 648-654.17. Piñeros, M.A., Shaff J.E., Kochian L.V. (1998): Development, Characterization, and Applicationof a Cadmium-Selective Microelectrode for the Measurement of Cadmium Fluxes in Roots ofThlaspi Species and Wheat. Plant Physiol. Vol. 116, 4: 1393–1401.18. Gholivand, M.B., Raheedayat, F. (2004): Chromium (III) ion selective electrode based on oxalicacid bis (cyclohexylidene hydrazide). Electroanalysis. Vol. 16: 1330-1335.19. Khalil, S., Wassel, A. A., Belal, F. F. (2004): Coated graphite-epoxy ionselective electrode for thedetermination of chromium (III) in oxalic medium. Talanta, Vol. 63, 2: 303 –307.20. Sengar, R. S., Gupta, S., Gautam, M., Sharma, A., Sengar, K. (2008): Occurence, uptake,accumulation and physiological responses of nickel in plants and its effect on environment.Research J. Phytochem. Vol. 2, 2: 44 – 60.21. Gorbanov, S., Kostadinova, S., Gorbanova-Varatto, A. (2002): Manganase and nickel toxicity insome bulgarian soils and the effect of limiting on the soil properties. J. Environ. Prot. Ecology.Vol. 3, 1: 76 – 80.22. Gupta, V.K., Prasad, R., Kumar, P., Mangla, R. (2000): New nickel (II) selective potentiometricsensor based on 5, 7, 12, 14- tetramethyldibenzotetraazaannulene in a poly (vinyl chloride)matrix. Anal. Chim. Acta. Vol. 420, 1: 19-27.23. Mazloum, M., Salavati-Niasari, M., Amini, M.K. (2002): Pentacyclooctaaza as a neutral carrier incoated-wire ion-selective electrode for nickel (II). Sens. Actuators B-Chemical, Vol. 82: 259-264.24. Kumar, K.G., Poduva,l R., Augustine, P., John, S., Saraswathyamma, B. (2006): A PVCplasticized sensor for Ni (II) ion based on a simple ethylenediamine derivative. Anal. Sci. Vol.22: 1333-1337.25. Pleniceany, M., Isvoranu, M., Spinu, C. (2005): Liquid membrane ion selective electrodes forpotentiometric dosage of copper and nickel. J. Serbian Chem. Soc., Vol. 70: 269-276.86

Scientific Bulletin Biotechnology, U.Ş.A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 87 - 96COMPLEX RESEARCH REGARDING THE PREPARATION ANDCARACTHERIZATION OF SOME MICROCAPSULES OFETHYLCELLULOSECERCETĂRI COMPLEXE PRIVIND PREPARAREA ŞICARACTERIZAREA UNOR MICROCAPSULE DE ETILCELULOZĂRĂZVAN PRISADA 1 , CRISTINA DINU-PÂRVU 1 , ALINA ORŢAN 21.University of Medicine and Pharmacy ,,Carol Davila“,Faculty of Pharmacy, 6 Traian Vuia Street,Bucharest2 University of Agricultural Sciences and Veterinary Medicine, B-dul Marasti nr. 59 sector1,Bucharest, RomaniaKey words: microcapsules, ethylcellulose, xantinol nicotinateCuvinte cheie: microcapsule, etilceluloză, xantinol nicotinatABSTRACTUna dintre metodele de a realiza preparate cu eliberare modificată este microîncapsulareasubstanţelor medicamentoase.Acest procedeu poate fi utilizat si pentru modificarea aspectului unuiprodus, izolarea unor compuşi reactivi, mascarea mirosului sau gustului unor substanţe sau creştereastabilităţii unui produs. In acesta lucrare, prezentăm rezultatele unui studiu complex asupra unormicrocapsule de etilceluloză cu xantinol nicotinat.Propunem o modalitate de preparare şicaracterizăm fizico-chimic microcapsulele.One of the methods used for modifying the release of drugs is microencapsulation. Thismethod can be also used for modifying physical aspect of a product, isolation of reactive compounds,hiding bad smells or taste of certain substances or increasing their stability. In this paper, we arepresenting the results of a complex research regarding some ethylcellulose microcapsules withxantinol nicotinate. We are proposing a method of preparation and we are carrying out the complexcharacterization of the microencapsulation process.The microencapsulation processes can be performed by methods usingeither the evaporation of the solvent, or the emulsification in mineral oil. Therelease of the drug from microcapsules depends on the structure of their walls andthe polymeric material used for preparation. In the experimental study we assessedthat using the Jaselniack method (1), eventually in a modified way, as well as usingthe ethylcellulose as the polymeric material would correspond for the projection,87

preparation and further characterization of some tank microcapsules with xantinolnicotinate (2, 8).1. MATHERIAL and METHOD• Xantinol nicotinate (Merck);• Ethylcellulose(Fluka) containing 48 percent etoxy radicals, characterizedas a white powder, tasteless, odorless, melting point between 155-162Celsius, having a density of 1.17 g/cm 3 and a viscosity of a 5% solution intoluene/ethanol in a proportion of 40/60 is 5.4mPa.s(in standardconditions);• Cyclohexane (Merck).The microencapsulation process using the evaporation of the solventmethod results by emulsification in water of a polymeric solution in an appropriatesolvent or mixture of organic solvents (3, 4, 9). The polymeric solution contains thedrug in the form of microcrystals. The evaporation of the solvent is performedunder continuous stirring, either in an open jar at normal atmospherically pressure,or in a closed jar at lower pressure. After evacuating the solvent from the system,the microcapsules are separated by filtration, then washed and dried. Themodulation of the drug release from these microcapsules can be done by changingthe ratios of the drug and the polymer.The evaporation is influenced by some factors: the characteristics of thepolymer (such as molecular weight, structure), the type of the material to beencapsulated (shape of the crystals, solubility), the type of the used solvent, thetemperature and pressure during evaporation, the intensity of shaking, the nature ofthe washing solvent, the method used for drying (5,6,8).Our preliminary experiments of verifying some methods for obtainingsome microencapsulated, described in the literature, had shown that thereproducibility of the results (including structure, size distribution of microcapsulespopulation, yield) is rather limited (10).A general schedule of the process of preparation of tank microcapsules byevaporation of the solvent is presented in figure 1.The separation of the microcapsules, from different experimental lotsobtained in the same experimental conditions, in fraction of known dimensions isperformed by mechanical sieving, using a set of standard sieves with calibrateddimensions of the holes.The X-ray diffraction analysis is done using a diffractometer TUR M-61. Inadvance, the microcapsules are compressed in an aluminum support. Theexperimental conditions are the same ones used for the characterization of drugmicrocrystals (for the X-ray generator a voltage of 30KV, an electric intensity of20 mA).88

The determination of density for the microcapsules with xantinol nicotinateis made by a picnometric method, using as reference liquid cyclohexane (in whichthe microcapsules are not soluble)( b−a)ρρ= l. d − a − c − b( ) ( )where: ρ = density of the microcapsules; a = mass of the picnometer; b = mass ofthe picnometer with microcapsules; c = mass of the picnometer with microcapsulesand liquid; d = mass of the picnometer with liquid; ρ l = density of the liquid used020ciclohexan( ρ = 0,78g/cm 3 )(7).Fig.1 - Preparation of tank microcapsules by the evaporation of the solvent method89

In order to evaluate the total drug content of the microcapsules ofethylcellulose, a small quantity of 0.1g ethylcellulose powder was suspended in200ml of distilled water. The resulted suspension has been maintained at roomtemperature, occasionally shaken, for 72 hours. The saturated ethylcellulosesolution, resulted after filtering the suspension, has no absorbance in the wavelength domain in which the aqueous solution of xantinol nicotinate has a maximumabsorption (260-280 nm). The determinations were performed using aspectrophotometer UV-VIS Perkin Elmer Lambda2.Solutions of xantinol nicotinate of different concentrations (0- 0.01 g/100ml), prepared with a saturated solution of ethylcellulose, verify in this domainthe Bouguer-Beer-Lambert low. The obtained diagram can be used for computingthe apparent absorbance of xantinol nicotinate in saturated solutions ofethylcellulose A 1cm1%=276. This value is almost the same with the one of xantinolnicotinate in water.For detecting the content of the tank microcapsules with xantinolnicotinate, determined quantities of microcapsules of approx.0.10g havingdimensions in domains like 400-500 µm are grinded to pieces, the powder is thendissolved in 100ml water. The suspension is shaken until a constant absorbance isseen. The values computed can be used for a determination of the drug at 1g ofmicrocapsules. The values of the absorbance can be used to determine the quantityof xantinol nicotinate in 1g of microcapsules.2. RESULTS and DISCUSSIONSThe solvent evaporating method used to preparation of microcapsules withxantinol nicotinateAfter verifying the Jaselniack method for obtaining microencapsulated ofethylcellulose, we can assess that the eventual modification of that method couldassure a better reproducibility of the results (yield, structure, size distribution ofmicrocapsules population). In the same time, it is possible to modify the kinetic ofthe release of xantinol nicotinate from microcapsules of ethylcellulose by choosinga certain weight ratio of xantinol nicotinate and ethylcellulose, or by choosingsome technological parameters to assure the preparation of higher percentage frommicrocapsules of certain dimensions (for that it is necessary that the crystals ofdrug to have also certain well-known dimensions).The technological steps for preparation of such microcapsules are:- in a volume of 200ml cyclohexane warmed at 50 degrees Celsius, is brought adetermined quantity of ethylcellulose, under continuous stirring. For completing90

the dissolution, the solution is kept for 20 minutes at a higher temperature of 70degrees.- the drug, xantinol nicotinate, having a crystal dimension between 300-400µm, ofaccurate known weight, is brought under continuous stirring, and then the system iskept at 70 degrees Celsius for 20 minutes. Then the system is cooled to 20 degrees,and kept there for 10 minutes.- the resulted microcapsules are filtered, washed twice with cyclohexane at roomtemperature, and then at 30 degrees Celsius for 72 hours for losing all thecyclohexane.In order to obtain microcapsules with different thickness of the walls, thequantities of xantinol nicotinate and ethylcellulose can be modified (1:1),(4:1),(9:1).With a few exceptions, for depositing ethylcellulose on the microcrystals,the program of modifying the temperature was the one indicated in Jalseniackmethod. The intensity of stirring, depending on the geometry of the jar wasdetermined at 300 r.p.m., for resulting microcapsules with resembling dimensions,situated in the wanted domains of (400-500) µm and (500-630) µm .Characterization of ethylcellulose tank microcapsules with xantinolnicotinateRegardless of the weight ratio ethylcellulose/xantinol nicotinate, themicrocapsules populations obtained by the method we developed aremultidisperses. In previously described experimental conditions, it was found thatfor a stirring speed of 300 rpm, the majority of microcapsules populations havesizes in the domains (400-500) µm and (500-630) µm.30252015105D iame t rul o c hiurilo rs it e i d e re f uzD ia me t e r o f re je c t io nS1S2S30s e ries e rie sFig. 2 - Size distribution of microcapsules with xantinol nicotinate (average ofthree experiments in identical experimental conditions)91

Microcapsules morphologyThe surface of the microcapsules can be studied by electronic microscopyor by X rays diffraction. The morphological analysis using electronic microscopyof the ethylcellulose microcapsules with xantinol nicotinate obtained by thedescribed method, shows they have a spherical or almost spherical form, and thatthere are no nicotinic xantinol crystals present on their surface.The X ray analysis shows that ethylcellulose microcapsules with xantinolnicotinate have a diffraction spectrum identical with the ones of pure xantinolnicotinate.Fig. 3 - X-ray diffraction spectrumof pure xantinol nicotinate (CuKα1radiation, Ni filter)Density of tank ethylcellulose microcapsules with xantinol nicotinateThe densities for the tank ethylcellulose microcapsules with xantinolnicotinate with described before sizes, resulting from different ratios ofethylcellulose/xantinol nicotinate were determined by picnometric method, and aredescribed in table 1.As a reference liquid was used the cyclohexane at 20 degrees Celsius, inwhich ethylcellulose is soluble only at higher temperatures.The table shows that the density of tank microcapsules with xantinolnicotinate increases at higher values for the ethylcellulose/xantinol nicotinate ratio;their density decreases with the increase of their sizes.Drug content of the tank ethylcellulose microcapsules with xantinol nicotinateThe total drug content of microcapsules was determined after eachdissolution test. The microcapsules samples were stirred with ultrasounds, in orderto obtain complete breaking of the microcapsule walls and to release the drugcontent. The samples were spectrophotometrically analyzed. We used the fact thatethylcellulose has no relevant absorption at characteristic wave lengths for our drug( λ XN =270.4 nm, λ XN =205.6 nm).92

Table 1Densities of different sizes ethylcellulose microcapsules with xantinol nicotinateTipes of microcapsuleswith different proportions polymer/drugD ∈ (400-500)µmρ(g/cm 3 )D ∈ (500-630)µmρ(g/cm 3 )Tank microcapsulesethylcellulose/xantinol nicotinate 1 : 1 1,036 1,023Tank microcapsulesethylcellulose/xantinol nicotinate 4 : 1 1,124 1,096Tank microcapsulesethylcellulose/xantinol nicotinate 9 : 1 1,195 1,181Liquide medium : cyclohexane ; t = 20 0 CFig. 4 - Absorption spectrum UV of xantinol nicotinate in aqueous solution ofethylcellulose (C=0,005g/100ml, h=1cm)93

Screening of the Lambert-Beer law for concentrations domain (0-0.005)g/100ml for xantinol nicotinate permitted the evaluation of percentageabsorbance:Abs. =276.24C + 0.0012 (r 2 =0.999)Fig. 5 - Screening of the Bouguer-Lambert-Beer law for xantinol nicotinate(C=0-0.005g/100ml)The value of percentage absorbance for xantinol nicotinate A 1% 1cm=276.24(λ XN =270.4 nm) is similar to the one presented in the literature: A 1% 1cm=270(λ XN =273 nm).For the tank ethylcellulose microcapsules with xantinol nicotinate, theaverage drug content resulted from three independent determinations is presentedin table 2.3. CONCLUSIONSThe tank ethylcellulose microcapsules obtained by the solvent evaporationmethod are represented by a multidispersional population, but evaluating theinfluence of some factors (such as type of drug, the nature of polymeric material,polymer / drug substances ratios, sizes of drug crystals on the surface, nature andintensity of stirring, the modification of the temperature) we succeeded in reducingthis inconvenient.94

The shape of ethylcellulose microcapsules obtained by the solventevaporation method is spherical or almost spherical (due to the different shapes ofdrug microcrystals).The drug substance from tank ethylcellulose microcapsules has a similarX-ray diffraction spectrum with the one of pure microcrystalline xantinolnicotinate.Density of tank ethylcellulose microcapsules has over unitary values. Thevalues are influenced by the proportion xantinol nicotinate/ethylcellulose.For tank ethylcellulose microcapsules obtained by evaporation of thesolvent method the average drug content computed from three differentexperiments was approx. 0.5g XN /g microcaps. ( xantinol nicotinate/ethylcellulose=1:1) ;0.2g XN /g microcaps. ( xantinol nicotinate/ethylcellulose=1:4); 0.1g XN /g microcaps. ( xantinolnicotinate/ethylcellulose=1:9).In conclusion, these results regarding the preparation and thecharacterization of some tank ethylcellulose microcapsules could be developed insome sustained release dosage forms.Average drug content of ethylcellulose microcapsules with xantinol nicotinateTable 2Covering proportion Average dimension ofxantinol nicotinate : the microcapsules (µm)ethylcellulose1 : 1 400 – 500500 – 6301 : 4 400 – 500500 – 6301 : 9 400 – 500500 - 630Average drug content in xantinol nicotinate(g)/g microcapsules( three determinations)experimentaltheoretical0,4840,5000,4920,5000,1910,2000,1880,2000,0970,1000,0960,100REFERENCES1. Senjkovič R., Jalšenjak I., Apparent diffusion coefficient of sodium phenobarbitone inethylcellulose microcapsules: effects of capsule size, J.Pharm.Pharmacol., 33, 279-282, 1981.2. Kristmundsdóttir T., Ingvarsdóttir K., Ibuprofen microcapsules: the effect of production variableson microcapsule properties, Drug Dev. Ind. Pharm., 20(5), 769-778, 1994.95

3.Luzzi A.L., Encapsulation technique for pharmaceuticals: Consideration for the microencapsulationof drugs, Microencapsulation, Marcel Dekker Inc., New York, 193 – 207, 1976.4. Zinutti C., Kedzierewicz F., Hoffman M., Maincent P., Preparation and characterization of ethylcellulose microspheres containing 5- fluorouracil, J. Microencapsulation, 11, 555 – 563, 19945. Yamakawa Y., Tsushima Y., Machida R., Watanabe S., Preparation of neurotensin analoguecontainingpoly(dl-lactic acid) microspheres formed by oil-in-water solvent evaporation,J.Pharm.Sci., 81, 899 – 903, 1992.6. Yamakawa Y., Tsushima Y., Machida R., Watanabe S., In vitro and in vivo release of poly(dllacticacid) microspheres containing neurotensin analogue prepared by novel oil-in-water solventevaporation method, J.Pharm.Sci., 81, 808 – 811, 1992.7. Pîrvu C., Moisescu Şt., Formularea unor medicamente cu acţiune vasodilatatoare cerebrală şiperiferică, Comunicare prezentată în cadrul celui de-al XI-lea Congres Naţional de Farmacie, 8 –10 octombrie Iaşi, 1998.8. Pîrvu C, Chemical caracterization and applications of microcapsules, Farmacia nr6,61-67,2005.9.Pîrvu C., Microencapsules in pharmacy and medicine, Farmacia nr4, 54-59,2005.10. Sood A., Panchagnula R., Role of dissolution studies in controlled release drug relivery system,STP Pharma, 9, 157 – 168, 1999.96

Scientific Bulletin Biotechnology, U.Ş.A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 97 - 104COMPARATIVE RESEARCH CONCERNING THE QUALITATIVECHARACTERISTICS OF THE SUPERIOR WINES DERIVED FROMWINE-GROWING CENTRE OSTROVCULEA RODICA- ELENA *, POPA NICOLAE-CIPRIAN**,TAMBA-BEREHOIU RADIANA** University of Agronomic Sciences and Veterinary Medicine of Bucharest(rodica.culea@gmail.com)**S.C. FARINSAN S.A. GiurgiuKey words: White superior wines, physical and chemical characteristics, variability estimates,statistic differencesABSTRACTSo as to realize the whole image of the quality evolution of the superior, dry, white wines,derived from wine-growing centre Ostrov, we established the variability estimates of the physical andchemical parameters concerning White Fetească, Italian Riesling and Sauvignon grapes variety ofwine, along three succesive crops: 2004, 2005 and 2006. The most increased variability wasregistered by the Free Sugar parameter (between 34 and 81%), no matter which grapes variety ofwine was analysed. The most stable parameters of the three grapes variety of wines, were Density(with a medium variation coefficient, smaller than 1%) and Alcohol content (with a medium variationcoefficient, smaller than 4%), parameters which can be considered typical for this category of whitewines. The sort of wine which presented the most increased homogenity of the physical and chemicalparameters, along the three years investigated, was the Italian Riesling sort. The most heterogeneousvalues were registered by the Sauvignon sort of wine.The wines produced to SC Ostrovit SA carry the specific nature of thisplace, with calcareous soil and droughty climate, with humid atmosphere, due tothe Danube influence. In the wine-growing centre Ostrov, the grapes varieties forwine (especially White Feteasca, Italian Riesling, Royal Feteasca) are cultivated ona surface counting 245 ha and the grapes varieties for consumption (Italia, AfuzAli, Muscat, Hamburg, Sultanina, Moldova) are cultivated on a surface of 936 ha.The wines for current consumption have an Alcohol content of 10-10,4 vol. % andthe superior quality wines, obtained of White Feteasca, Italian Riesling, Merlot,Cabernet, Sauvignon sorts, have an Alcohol content between 10,5 -10,4 vol. % [1,5].The climatic peculiarities, as well as some anthropic elements, whichmodulate the action of the environmental agents (applied phytotechny, thecropping moment, the vinification technology applied), determine the annualvariability of the wines’ physical and chemical parameters [3, 4, 6].97

This study recommends a comparative qualitative characterization of thedry wines, obtained of the White Feteasca, Italian Riesling and Sauvignon grapesvarieties, made by S.C. OSTROVIT S.A., in 2004, 2005 and 2006 productionyears.981. MATERIAL and METHODThe determinations were made in preceding stage of the wines bottling andcommercialization. There were comparatively anlysed the physical and chemicalparameters of White Feteasca (n=33), Italian Riesling (n = 30) and Sauvignon (n =31), ingathered from 2004, 2005 and 2006 crops. The following quality parametersof the wine have been analysed: d 20 20 (picnometric method STAS 6182/8-71),Alcohol % vol. (picnometric method STAS 6182/6-70), total dry extract mg/l(densimetric method STAS 6182/9-80), Free Sugar g/l (iodometric method STAS6182/18-81), Unreducing Extract g/l, Total Acidity g/l C 4 H 6 O 6 (titrimetric methodSTAS 6182/1-79), Free SO 2 mg/l (iodometric method STAS 6182/13-72) and TotalSO 2 mg/l (iodometric method STAS 6182/13-72) [2,3,4,7]. The obtained resultswere statistical processed using the professional program COHORT [2, 7, 8].2. RESULTS and DISCUSSIONSThe first table shows the variability estimates of physical and chemicalparameters, belonging to White Feteasca sort of wine, concerning the wholeinvestigated period (2004 -2006).Table 1The variability estimates of White Feteasca sort of wine samplescorresponding to 2004, 2005 and 2006 crops cumulatively (n = 33)Parameter X±s s CV %D 20 20 0,9937 ± 0,001 0,000001 0,110Alcohol (vol %) 11,363 ± 0,489 0,239 4,304Total dry extract (g/l) 23,012 ± 2,150 4,625 9,346Free sugar (g/l) 2,184 ± 0,749 0,561 34,305Unreducing extract (g/l) 20,600 ± 2,388 5,703 11,592Total acidity (g/l C 4 H 6 O 6 ) 5,491 ± 0,386 0,149 7,038Free SO 2 (mg/l) 38,696 ± 4,149 17,217 10,722Total SO 2 (mg/l) 129,279 ± 17,731 314,392 13,716Generally, it is noticed that the physical and chemical parameters of WhiteFeteasca grapes variety of wine, are not characterized by excesiv values of thevariation coefficient, with one exception, namely the Free sugar parameter (34,305%).

The second table shows the obtained results in the case of Italian Rieslinggrapes variety of wine, concerning the whole investigated period (2004 -2006).Table 2The variability estimates of Italian Riesling sort of wine samplescorresponding to 2004, 2005 and 2006 crops cumulatively (n = 30)Parameter X±s s CV %D 20 20 0,9914 ± 0,011 0,000121 1,113Alcohol (vol %) 11,206 ± 0,297 0,088 2,650Total dry extract (g/l) 21,493 ± 1,077 1,160 5,012Free sugar (g/l) 1,268 ± 0,448 0,201 35,358Unreducing extract (g/l) 20,243 ± 0,948 0,899 4,684Total acidity (g/l C 4 H 6 O 6 ) 5,895 ± 0,274 0,075 4,664Free SO 2 (mg/l) 39,000 ± 5,717 32,689 14,660Total SO 2 (mg/l) 128,733 ± 11,057 122,271 8,589In this case too, the main parameter which was affected of excesiv variationis also the Free sugar (35,358 %). In the case of Italian Riesling grapes variety ofwine, distinguishes a bit increased variation coefficient, accordingly to the FreeSO 2 parameter (14,66 %).The results, concerning the variability estimates of the analyzed physicaland chemical parameters, in the case of the wine obtained from the Sauvignongrapes variety of 2004-2006 crops, are presented in the table 3.Table 3The variability estimates of Sauvignon sort of wine samplescorresponding to 2004, 2005 and 2006 crops cumulatively (n = 31)Parameter X±s s CV %D 20 20 0,9937 ± 0,001 0,000001 0,170Alcohol (vol %) 12,251 ± 0,559 0,313 4,568Total dry extract (g/l) 25,580 ± 3,135 9,830 12,157Free sugar (g/l) 3,389 ± 2,770 7,768 81,763Unreducing extract (g/l) 22,097 ± 0,927 0,859 4,195Total acidity (g/l C 4 H 6 O 6 ) 5,280 ± 0,307 0,094 5,830Free SO 2 (mg/l) 39,966 ± 6,365 40,516 15,926Total SO 2 (mg/l) 145,700 ± 26,241 688,631 18,010The Free sugar parameter was characterized by a variation coefficient value,higher than 81 % and the parameters connected to the wine sulphitation operations,had variation coefficients moreover than 15 %.99

Observing the first figure, we noticed that the most stable parameters of thethree wines, were Density (with a medium variation coefficient smaller than 1 %)and the Alcohol content (with a medium variation coefficient smaller than 4 %).The Free sugar parameter was the most exposed to variations, no matter the grapesvariety, having a medium variation over 50 %.Averag e o f variatio n coefficients (% )60.0050.0040.0030.0020.0010.000.000.46D ens ity(D 2020)3.84Alc ohol(v ol % )8.84T otal dryex trac t (g/l)50.48F ree s ugar(g/l)6.82U nreduc ingex trac t (g/l)5.84T otal ac idity(g/l C 4H 6O 6)13.77F ree SO 2(m g/l)13.44T otal SO 2(m g/l)Fig .1 - The variation coefficients average of the physical andchemical parameters, which characterized the analysed winesThe grapes variety of wine, which presented the most higher homogenity forphysical and chemical parameters, along the three years investigated, was ItalianRiesling (the lowest medium variation coefficient = 9,59 %). The most increasedvalues were registered in the case of Sauvignon wine, for which it was establisheda medium variation coefficient of 17,83 % (figure 2). We can say that the ItalianRiesling grapes variety was the best adapted to climatic conditions, which variedalong the analysed period. This sort of wine pointed out the most homogeneouscharacteristics, while the Sauvignon wine proved to be the most exposed andmisfit.100

Average of variation coefficients (%)2018161412108642017.8311.399.59White Feteasca Italian Riesling SauvignonFig. 2 - The variation coefficients average of all the parameters, which characterized everysort of wine (White Feteasca, Italian Riesling and Sauvignon)The table 4 presents the meaning of averages differences, following theapplication of the t test (Student), for all the physical and chemical parameters, inthe case of the three studied wines.As noticeable from the table 4, the Density parameter did not differsignificantly between neither one of the analysed wine pairs. This fact shows thatthe parameter Density is rather typical for the wines category (white, dry wines),than for the grapes variety of wines, respective White Feteasca, Italian Riesling orSauvignon.One can also see, that the Alcoholic Content % did not differ significantbetween the White Feteasca and Italian Riesling grapes varieties of wines.However, the White Feteasca and Italian Riesling grapes varieties of wines, had avery significant low alcoholic concentration, comparative to the Sauvignon grapesvariety of wine.The parameter Total dry extract was very significant different between thethree grapes varieties of wine. The highest value was registered to Sauvignongrapes variety (25,58 %), followed by White Feteasca grapes variety (2,184 g/l)and Italian Riesling grapes variety (21,493 %).The parameter Free sugar presented a grapes variety specificity, havingsignificant and very significant different values between the wines. The highestvalue one can see in the case of Sauvignon grapes variety (3,389 %), followed by101

White Feteasca grapes variety (2,184 g/l) and Italian Riesling grapes variety (1,268g/l).The meaning of averages differences (t test) of the physical andchemical parameters concerning the three superior winesTable 4Pairs D 20 20 Average (a) Average (b) tWhite Feteasca (a) – Italian Riesling (b) 0,9937 0,9914 1,144White Feteasca (a) – Sauvignon (b) 0,9937 0,9937 0Italian Riesling (a) – Sauvignon (b) 0,9914 0,9937 1,163Pairs Alcohol (%) Average (a) Average (b) tWhite Feteasca (a) – Italian Riesling (b) 11,363 11,206 1,563White Feteasca (a) – Sauvignon (b) 11,363 12,251 6,791***Italian Riesling (a) – Sauvignon (b) 11,206 12,251 9,154***Pairs Total dry extract (g/l) Average (a) Average (b) tWhite Feteasca (a) – Italian Riesling (b) 23,012 21,493 3,592**White Feteasca (a) – Sauvignon (b) 23,012 25,580 3.797***Italian Riesling (a) – Sauvignon (b) 21,493 25,580 6,852***Pairs Free Sugar (g/l) Average (a) Average (b) tWhite Feteasca (a) – Italian Riesling (b) 2,184 1,268 5,950***White Feteasca (a) – Sauvignon (b) 2,184 3,389 2,394*Italian Riesling (a) – Sauvignon (b) 1,268 3,389 4,181***Pairs Unreducing extract (g/l) Average (a) Average (b) tWhite Feteasca (a) – Italian Riesling (b) 20,600 20,243 0.792White Feteasca (a) – Sauvignon (b) 20,600 22,097 3.342*Italian Riesling (a) – Sauvignon (b) 20,243 22,097 7,722***Pairs Total acidity (g/l C 4 H 6 O 6 ) Average (a) Average (b) tWhite Feteasca (a) – Italian Riesling (b) 5,491 5,895 4,744***White Feteasca (a) – Sauvignon (b) 5,491 5,280 2,411*Italian Riesling (a) – Sauvignon (b) 5,895 5,280 8,252***Pairs Free SO 2 Average (a) Average (b) tWhite Feteasca (a) – Italian Riesling (b) 38,696 39,000 0,243White Feteasca (a) – Sauvignon (b) 38,696 39,966 0,939Italian Riesling (a) – Sauvignon (b) 39,000 39,966 0,692Pairs Total SO 2 Average (a) Average (b) tWhite Feteasca (a) – Italian Riesling (b) 129,279 128,733 0,148White Feteasca (a) – Sauvignon (b) 129,279 145,700 2,914**Italian Riesling (a) – Sauvignon (b) 128,733 145,700 3,309**Regarding the Unreducing extract parameter, it was noticed that registeredvalues, in the case of White Feteasca and Italian Riesling grapes varieties of wines,did not differ significant. Instead, the Sauvignon and White Feteasca, respectiveItalian Riesling grapes varieties of wines, differed significant and very significant.102

So, the highest value of the Unreducing extract parameter was specific toSauvignon grapes variety of wine, followed by White Feteasca and Italian Rieslinggrapes varieties of wine.The Total Acidity parameter could also be considered as being peculiar toevery grapes varieties of wines. Matter of fact, all grapes varieties of winespresented at least significant differences between the values of this parameter.White Feteasca grapes variety of wine presented a very significant decreased valueof Total Acidity, comparative to Riesling Italian grapes variety of wine and asignificant grown value, comparative to Sauvignon grapes variety of wine. TheItalian Riesling grapes variety of wine presented a very significant grown value ofthe Total Acidity, comparative to Sauvignon grapes variety of wine. The highestvalues of this parameter were peculiar to the Italian Riesling grapes variety ofwine, followed in order by the White Feteasca and Sauvignon grapes varieties ofwines.The analysed grapes varieties of wine did not differ concerning the FreeSO 2 quantities, though appeared some distinctly significant differences betweenSauvignon and White Feteasca or Italian Riesling grapes varieties of wines. Thesedifferences due to the higher quantity of Total SO 2 present in the Sauvignon grapesvariety of wine, comparative with other two sorts of wines.Our analysis showed that the least peculiar parameters of the grapes varietyof wine were the Density and the Free SO 2 content. In practice, the values of theseparameters seem rather characteristics of the wine category, respective superior,white, dry wines, then grapes variety characteristics. Instead, prameters as: TotalDry Extract, Free Sugar or Total Acidity presented significant differences betweeneach pair of analysed grapes varieties. The fact is showing that the value of theseparameters could be considered typical for every sort of wine.3. CONCLUSIONS♦ The wine obtained of White Feteasca grapes variety had the qualityparameters values specific to superior, white and dry wines. The physical andchemical parameters did not present excessive values of the variation coefficients,single exception being the Free Sugar parameter (CV=34,305 %).♦ Concerning the Italian Riesling grapes variety, the values of the qualityparameters were typical for superior, white and dry wines. The main parameterwhich was affected by excessive variations was also, the Free sugar (CV=35,358%). Concomitantly, we can observe the existence of an easily growning variation,in the case of Free SO 2 parameter (8,589 %).♦ The Sauvignon grapes variety of wine, pointed out quality parameterssuitable to superior, white and dry categories of wines, but there were affected by103

increased variations. So, the Free Sugar parameter showed a variation coefficientvalue higher than 81 %. A rather increased variation coefficient was observed atthe parameters connected to the wine sulphitation, moreover than 15 %.♦ The Italian Riesling grapes variety was the best adapted to climaticconditions, peculiar to 2004-2006 period. So, the wine produced of Italian Rieslinggrapes variety, revealed homogenous characteristics.♦ The Sauvignon grapes variety of wine proved to be the most exposedand misfit.♦ We may assert that the Density, Alcoholic content and Free SO 2parameters are peculiar to superior, white and dry wines, while the Total DryExtract, Free Sugar and Total Acidity parameters could be considered typical forevery sort of wine.REFERENCES1. COTEA, V.D., GRIGORESCU, C., BARBU, N., COTEA, V.V., 2000, Podgoriile si vinurileRomâniei, Editura Academiei Române2. COTOFREI, S. C., 2004, Cercetari privind optimizarea metodologiei de calcul matematic inoenologie, Teză de doctorat, Iaşi3. IONESCU, I.A., 1968, Factorii ce determină calitatea vinurilor, Revista Horticultura şi Viticulturanr. 2, Bucureşti4. KONTEK, A., KONTEK, Ad., 1999, Măsuri tehnologice care contribuie la îmbunătăţirea calităţiivinurilor albe seci, Concepţii moderne în cercetarea horticolă Românească, Editura Medro.5. POMOHACI, N.,NĂMOLOŞANU, I., NĂMOLOŞANU, A., 2000, Producerea şi îngrijireavinurilor, Editura Ceres, Bucureşti, pg. 27-31.6. POMOHACI, N., SÎRGHI,C.., STOIAN, V., COTEA, V.,NĂMOLOŞANU, I., 2000, Oenologie,Editura Ceres, Bucureşti, pg 132.7. SNEDECOR, G.W., COCHRAN, W.G., 1989. Statistical Methods. Iowa State University Press,Ames, IA, USA.8. **** Culegere de Standarde Române comentate / Metode de analiză, I.R.S. Institutul Român destandardizare, Bucureşti, 1997.104