MINISTRY OF EDUCATION RESEARCH AND INNOVATION UNIVERSITY OF AGRONOMICAL SCIENCES AND VETERINARY MEDICINE BUCHAREST SCIENTIFIC BULLETIN Series F XIII 2008 BIOTECHNOLOGY ISSN 1224-7774 B U C HAREST 2008 Redactarea i coordonarea buletinului de lucrări tiinţifice: U.S.A.M.V.Bucure ti, Facultatea de Biotehnologii B-dul Mără ti nr.59, Bucure ti, sectorul 1,România Tel: + 40 21 318 25 67 Fax: + 40 21 318 28 88 Web: http://www.biotehnologii.usamv.ro Pentru schimb de publicaţii va rugam sa contactati Prof Dr Petru Niculiţă, Decanul Facultăţii de Biotehnologii Tel: + 40 21 318 36 40; Mobil: + 40 740 11 44 01 e-mail: [email protected], [email protected] ∗ Editorial board and coordination of the scientific bulletin: U.S.A.M.V.Bucuresti, Facultatea de Biotehnologii B-dul Mără ti nr.59, Bucure ti, sectorul 1,România Tel: + 40 21 318 25 67 Fax: + 40 21 318 28 88 Web: http://www.biotehnologii.usamv.ro For the publications exchange please contact Prof Dr Petru Niculiţă, Dean of the Faculty of Biotechnology Tel: + 40 21 318 36 40; Mobile: + 40 740 11 44 01 e-mail: [email protected], [email protected] ∗ La rédaction et la coordination de bulletin scientifique : U.S.A.M.V.Bucuresti, Facultatea de Biotehnologii B-dul Mără ti nr.59, Bucure ti, sectorul 1,România Tel: + 40 21 318 25 67 Fax: + 40 21 318 28 88 Web: http://www.biotehnologii.usamv.ro Pour l’échange de publications prier de contacter Prof Dr Petru Niculiţă, Doyenne de la Faculté de Biotechnologie Tel: + 40 21 318 36 40; Portable: + 40 740 11 44 01 e-mail: [email protected], [email protected] 2 SCIENTIFIC COMMITTEE Petru NICULITA - Prof. univ. dr., Decanul Facultatii de Biotehnologii - USAMV Bucuresti, Membru titular al Academiei de Stiinte Agricole si Silvice, Presedinte al sectiei de Industrie 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- ASAS Acad. 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 Academiei Romane, Secretar General al Academiei Romane - Prof. Dr., Doctor Honoris Causa, Full member of the Romanian Academy, General Secretary of the Romanian Academy Constantin BANU - Prof. univ. dr., Membru titular al Academiei de Stiinte Agricole si Silvice - Prof. Dr., Full member of the Academy of Agricultural and Forestry Sciences Mona Elena POPA - Prof. univ. dr., Secretar Stiintific al Facultatii de Biotehnologii - USAMV Bucuresti - Prof. Dr.,Scientific Secretary of the Faculty of Biotechnologies - USAMV Bucharest Calina Petruta CORNEA - Prof. univ. dr., Facultatea de Biotehnologii - USAMV Bucuresti - Prof. Dr., Faculty of Biotechnologies, USAMV Bucharest Gheorghe 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 Bucharest EDITORIAL BOARD Florentina MATEI - Facultatea de Biotehnologii - USAMV Bucuresti; Faculty of Biotechnologies USAMV Bucharest Amalia MITELUT - Facultatea de Biotehnologii - USAMV Bucuresti; Faculty of Biotechnologies USAMV Bucharest Mira TURTOI - Facultatea de Biotehnologii - USAMV Bucuresti; Faculty of Biotechnologies USAMV Bucharest Mihaela GHIDURUS - Facultatea de Biotehnologii - USAMV Bucuresti; Faculty of Biotechnologies - USAMV Bucharest 3 CONTENT MONICA ENACHE, NARCISA BĂBEANU MICROPROPAGATION OF GARDEN PLANTS (2): RARE PRIMULAS ADRIANA PETRU – VANCEA BIOCHEMICAL DETERMINATIONS MADE ON CYMBIDIUM HYBRIDUM EXVITROPLANTLETS, BEING ILLUMINATED DURING THEIR ACCLIMATIZATION TO A SEPTIC MEDIUM, WITH DIFFERENT TYPES OF LIGHT M. POPA, P. NICULITA, A. MITELUT, M. TURTOI, M. GEICU, M.GHIDURUS, R. CRAMARIUC, A. KONTEK, A. TUDORACHE, E.BRINDUSE, A. KONTEK NON-THERMAL INNOVATIVE PASTEURIZATION TECHNOLOGY OF FOOD USING A COMBINATION BETWEEN PULSED ELECTRIC FIELD AND HIGH PRESSURE VAMANU EMANUEL, VAMANU ADRIAN, POPA OVIDIU, BĂBEANU NARCISA OBTAINING OF SYNBIOTIC PRODUCTS FROM APICULTURAL PRODUCTS AND PROBIOTIC BIOMASS OF BIFIDOBACTERIUM BIFIDUM FLORENTINA RADOI-MATEI, FLORENTINA ISRAEL-ROMING, ANCA RADU, CAMELIA DIGUTA, CRISTINA COCULESCU, R. DESPA STUDIES ON THE GROWTH AND MYCOTOXIN PRODUCTION OF MOULDS ISOLATED FROM ROMANIAN FOOD PRODUCTS E. POPA, A. MUSCALU, A. DIHORU, V. HEBEAN NUTRITIVE EFFECTS OF NON-CONVENTIONALLY PROCESSED MEDICINAL PLANTS UPON MONOGASTRIC ANIMALS DEVELOPMENT CATALINA VOAIDES, GH. CAMPEANU, PETRUTA CORNEA STUDY REGARDING THE CONSUMER ACCEPTANCE OF GM FOODS LUMINIŢA VI AN, O. POPA, NICOLETA ARON, TH. SEROT ANALYTICAL METHODS OF AROMA COMPOUNDS IN GRAPE JUICE OBTAINED FROM ROMANIAN WINE SORTS GALBENĂ DE ODOBE TI AND ARBĂ IVAN DIMITROV, SVILEN RAYKOV PROBLEMS OF TECHNOLOGICAL AND CLIMATE NATURE AT RECULTIVATING DAMAGED AREAS OF THE OPEN COAL-MINING IN “MARITSA-IZTOK” ALINA ORTAN, MARIA LIDIA POPESCU, CRISTINA DINU-PIRVU ANETHI AETHEROLEUM: CHEMICAL COMPOSITION AND BIOLOGICAL EFFECTS ALINA CULEŢU, ALINA CATRINEL ION, ION ION SHORT REVIEW ON APPLICATION OF ION-SELECTIVE SENSORS FOR HEAVY METAL IONS IN FOOD AND ENVIRONMENTAL SAMPLES RĂZVAN PRISADA, CRISTINA DINU-PÂRVU, ALINA ORŢAN COMPLEX RESEARCHES REGARDING THE PREPARATION AND CARACTHERIZATION OF SOME MICROCAPSULES OF ETHYLCELLULOSE CULEA RODICA- ELENA , POPA NICOLAE-CIPRIAN, TAMBA-BEREHOIU RADIANA COMPARATIVE RESEARCH CONCERNING THE QUALITATIVE CHARACTERISTICS OF THE SUPERIOR WINES DERIVED FROM WINEGROWING CENTRE OSTROV 4 5 10 18 24 32 39 50 57 64 69 79 87 97 Scientific Bulletin Biotechnology, U. .A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p.5 - 9 MICROPROPAGATION OF GARDEN PLANTS (2): RARE PRIMULAS MICROPROPAGAREA PLANTELOR DE GRĂDINĂ (2): PRIMULE RARE MONICA ENACHE, NARCISA BĂBEANU Cuvinte cheie: in vitro, Primula aureata, micropropagare Key words: in vitro, Primula aureata, micropropagation ABSTRACT Micropropagation has significant uses in vegetative propagation of horticultural important species and cultivars. With a few exceptions most ornamentals can be micropropagated, and the need is for more and new basic research approaches. Although many of the techniques involved in the production 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 response to chemical and physical culture parameters. In the present work, we extended our experience with micropropagation 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 varietatilor cu importanta horticola. Cu cateva exceptii, majoritatea plantelor ornamentale pot fi micropropagate si este nevoie de noi tehnici pentru diferite specii, intrucat exista o variatie mare in raspunsul acestora la conditiile de cultura in vitro. In lucrarea de fata, continuam prezentarea unor studii referitoare la micropopagarea unor plante ornamentale rare (Enache, 2004), cu rezultatele obtinute in cazul unei primule asiatice rare cu flori galbene, Primula aureata, care este descrisa de unii crescatori 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. Many primulas 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 named them from the Latin prima (first) – a reflection of their early blooming in spring. Most Primula species are native from the Northern Hemisphere where they grow 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 high altitude where they survive near 0°C temperatures during the night, the culture of some species could be quite challenging. 5 The species native to Europe have been successfully used in horticulture for hundreds of years and there are now numerous cultivars, in a huge range of colors. However, some of the most sought-after primulas are the petiolarid primulas adapted to high alpine climates in Himalaya and China. These love a colder and more humid weather and many of them cannot be propagated by seeds when cultured in Europe. Primula aureata (Figure 1) is a lovely evergreen perennial petiolarid primula from the central Himalaya considered unique for its flower subsection colored yellow. Early in spring and in midseason it forms very short stalks that bear umbels of 2 to 10 salverform primrose creamy white to yellow flowers with large, deeper yellow centers, which look fabulous against the foliage, lightly dusted with farina. 1. MATERIAL and METHOD The work started with one Primula aureata mother plant which was provided 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 in his native country (UK), he brought one living plant home, to try to micropropagate. It is known that Himalaya is considered “a treasure house of plants that has long held a magical attraction for botanists, plant hunters and garden lovers” (Jermyn, 2001). The impact of Primula aureata grown in its native habitat on the plant 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 area in April and May, before the onslaught of monsoon, must be the sight of steep rock bluffs stained yellow with silvery, farinose rosettes crammed into the vertical crevices and bursting into flower. The two forms seem to enjoy differing altitudinal range and habitats. The type plant Primula aureata has been collected at altitudes between 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 in very 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). The mother plant was cleaned of soil debris by washing in tap water first. Then, outer leaves were cut away and discarded, while smaller pieces containing shoot buds were surface-sterilized as follows: an initial pre-sterilization in 70% alcohol was followed by 1-2 % (available chlorine) sodium hypochlorite. An emulsifer (Tween20) was added at the rate of 1 drop per 100 ml solution. The excised shoot buds were left into the bleach solution in small, screw-topped, sterile plastic tubs for a 6 timed 15 minutes, with occasional inversion and gentle agitation to enhance the effectiveness of the process. Then, with flame-sterilised forceps the pieces were transferred briefly through 5 sterile distilled water washes, finally putting them in a lidded Petri dish to await cutting. After removal from bleach solution all operations were conducted in a laminar airflow cabinet. Fig. 1 - Primula aureata The explants were cut and then placed into the surface of the agar-based medium in 30 ml universal tubs (one piece per container). The cultures were placed in 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 further multiplication. Shoot clusters were divided up into single and/or small groups of shoots and transferred to fresh medium for further proliferation. After a few passages on the Primula medium, plantlets rooted and displayed normal, true-to7 type rosette morphology. The cultures did not callus and the development proceeded via shoot and root production only. Once rooted, the plants grown in vitro were transferred to ex vitro conditions using soil and small pots. They were later moved into multi-purpose compost into 7 cm plastic pots. At the end of a 12month period, about 100 plants were obtained. The fully grown plants (about 10 cm height, 20 cm width) were sold to a small privately owned Nursery in SouthWest Scotland (UK). All plants were sold to the public as garden plants; however, later the nursery received complaints that the Himalayan primulas did not survive the first winter season in the UK. Table 1 Composition of the Anderson’s Rhododendron formula Compozitia mediului de cultura pentru Rhododendron a lui Anderson Ingredient NH4NO3 H3BO3 CaCl2.2H2O CoCl2.6H2O CuSO4.5H2O MgSO4.7H2O MnSO4.4H2O KI KNO3 Na2MoO4.2H2O Na3PO4 ZnSO4.7H2O FeSO4.7H2O Na2EDTA.2H2O Adenine sulfate Inositol Thiamine HCl IAA BAP Sucrose Agar pH 5.7 2. Concentration (mg l-1) 400 6.2 440 0.025 0.025 370 16.9 0.30 480 0.25 380 8.6 55.7 74.5 80 100 0.4 1.5 2.5 30,000 6,000 RESUSLTS and DISCUSSIONS Primroses are considered difficult to clean because the shoot tip area is in such close contact with the soil, yet one mother plant alone was used successfully for in vitro culture initiation here. 8 Aureata are perennial alpine plants, which will put up with the coldest weather conditions. Similarly to its relative P. auricula it may need some shelter from the heavy rain during the winter and spring, for example the use of a cold greenhouse with sufficient ventilation or just a simple frame that could be left open on warm days. This would also help maintain the beauty of the leaves. They can stand outside in a cool, shady position only during the summer in temperate climate. This approach could have helped the plants survive the humid weather of winter in Scotland and could be used in the future if the procedure will be repeated. 3. CONCLUSIONS A simple micropropagation technique is presented for Primula aureata, a rare garden plant, endemic to the central Himalaya. The method is based on the clonal proliferation of shoots on a tissue culture medium containing BAP and IAA. Multiplication was achieved through subdivision of shooting clumps and repeated transfer on this medium at irregular intervals. After rooting the plantlets were transferred to soil directly into the greenhouse or after a period of time spent in soil in an incubator. Later they were moved and grown in a cold greenhouse that provided some ventilation and were commercialised over a period of one year. Acknowledgements The authors were involved with the latter part of the work, which was carried out on a voluntary basis only during a placement at Liverpool John Moores University (UK) through a research grant SUCCESS/93-94. REFERENCES 1. 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”, Gentiana triflora. “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”, 2ND ED. LONDON. B.T. BASTFORD LTD., 2003. 9 Scientific Bulletin Biotechnology, U. .A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 10 - 17 BIOCHEMICAL DETERMINATIONS MADE ON CYMBIDIUM HYBRIDUM EXVITROPLANTLETS, BEING ILLUMINATED DURING THEIR ACCLIMATIZATION TO A SEPTIC MEDIUM, WITH DIFFERENT TYPES OF LIGHT ADRIANA PETRU – VANCEA1 1 Depart. of Biology, Faculty of Science, University of Oradea, Romania, e – mail: [email protected] Keywords: peroxidazic activity, assimilating pigments, color light, exvitroplantlets ABSTRACT In this experiments I studied the effect of lighting the Cymbidium hybridum exvitroplantlets with fluorescent light of different nature, namely: white (400 nm), red (660 nm), yellow (580 nm), blue (430 nm) and green (544 nm), on the quantity of assimilating pigments from the leaflets, as a meter of the photosynthesis process intensity, and also on the peroxidazic activity at the rootlets level, as a marker of rhysogenesis process. After 30 days from the acclimatization initiation of the Cymbidium exvitroplantlets, I shown that green light illumination have determined the stimulation of creating assimilating pigments, especially of carotenoids (expressed in percent with 86% plusses) and peroxidazic activity was stimulated with 10 – 15% by blue and green illumination. Adapting the vitroplantlets to the “ex vitro” life condition is a very delicate process, continuous, gradual and depends most of the performance through is succeed the release of photosynthesis (by unlock the chloroplast from the starch granule accumulated in vitrocultures period and by the synthesis of assimilating pigments) and establishing a hydric balances (by diminishing the excessive perspiration and the functionality of the radicular system) (Petru – Vancea, 2007). The a and b chlorophyll and carotenoids quantity gradually grows same with the amplification of chloroplast structural complexity and is directly proportional with the intensity of photosynthesis (Cachiţă & co., 2004). At Coleus vitrocultures, the stimulation of chlorophyll forming, was tried through maintaining them under fluorescent tubes that give different types of light (Radoveţ – Salinschi and Cachiţă, 2004), noticing that the blue and green light, comparative to the red and orange one, had good effects in this case. 10 During the exvitroplantlets acclimatization to septic medium, the light origin proved to be less important in the stemlets elongation phase, but the light intensity (100 µmol m-2 s-1) conditioned their rooting process (Maene and Debergh, 1985). The peroxidazic activity is a marker of rooting process (Moncousin and Gaspar, 1983). As regarding the light effect on the rhysogenesis were made many studies on a lot of vegetable species (Druart & co., 1982; Lian & co., 2002). Tricoli and collaborators (1985) said that the dark improve the percent of rooting and the number of root/shoot. Fuernkranz and collaborators (1990) had examined the effect of different light wave length and their conclusion was that yellow light (50 mol/m2/s, 475 - 750 nm with the peak at 575) determined the growth of root/shoot number, on the other hand, the blue light (20 mol/m2/s, 400 – 524 with the peak at 436) completely stopped the rhysogenesis. The red light was used by Podwyszynska and Gabryszewska (2003) to stimulate the “ex vitro” rhysogenesis of the rose exvitroplantlets cv. “Sonia” and cv. “Sabrina” and African daisy cv. Rebecca. During the rhysogenesis process, the red light had a benefic effect in both rose species, in winter and summer period and to African daisy, this illumination increase the rooting with 86,1 – 98,6 %, from 67,1% when was registered at control variant (exvitroplantlets illuminated with white light) in all month, with February exception. Studying the growth indexes and surviving same as the appearance of leaflets epidermis during the acclimatization period, varying the light origin (white, red, yellow, blue or green) which illuminated the exvitrocultures, Petru – Vancea and Cachiţă (2005) observed that the Chrysanthemum exvitroplantlets survived only under white illumination (control) and at African violets the red light determined decrease with 50% the survival percent and the growth indexes diminished, comparatively with control. A decrease of growth rate was indentified at exvitroplantlets illuminated with yellow light. At Cymbidium hybridum maintaining the exvitro-plantlets under different type of light, on 30 days period, had stimulating effects, especially on caulogenesis process, and less on the rhysogenesis and green light was the best having only beneficially consequences, namely: superior acclimatization surviving percent; increase data of growth parameter and less stomata density on inferior epidermis of leaflets (Petru – Vancea and Cachiţă, 2005). In the case of African violets, Petru and Cachiţă (2008) found that after 30 days from the acclimatization of these exvitroplantlets, the biochemical determinations have shown that, red light illumination have determined the stimulation of assimilating pigments accumulating, especially of chlorophyll a, at the still living plantlets (half of the plantlets maintained under this type of light faded), on the other hand the peroxidazic activity was stimulated at all four experimental variants, reaching statistically significant values, expressed in percent 11 with 20% plusses, in the case of the exvitroplantlets rootlets illuminated with blue light. Starting from this studies, we proposed in this experiment to continue the researches in this field by realizing some biochemical determination, namely assimilating pigments from exvitroplantlets leaflets and peroxidazic activity from rootlets of Cymbidium hybridum, at 30 days from their “ex vitro” transfer”, depending on origin of light with which was illuminated during the acclimatization period. 1. MATERIAL and METHOD The 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 a height 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, prepared in incubators (Petru - Vancea, 2007), substratum previously humidified with 300 ml of tap water each time, brought at the laboratory’s temperature. The incubators were placed on shelves protected with aluminum foil, away from the incidence of natural light, the illumination of the cultures being made through fluorescent tubes which gave white (400 nm), red (660 nm), yellow (580 nm), blue (430 nm) and green light (544 nm) and the power from the neon was 13 V, the intensity was 103 A and the light flow was 0.44 lm. The photoperiod was 16/24 h, and the substratum temperature was 24οC ± 2οC, the one from the atmosphere being two degrees lower. At 30 days from the acclimatization, the post-acclimatization survival percent was calculated for the exvitroplantlets and it was determined the quantity of chlorophyll at the level of the foliar limb and the peroxidazic activity at the exvitroplantlets rootlets level. Establishing the assimilating pigments content form the leaflets, respectively the a, b chlorophylls and carotenoid pigments was made through their extraction in N,N – dimethyl-formamide (DMF) 99.9% Merck, according to Moran and Porath method (1980). For the pigments extraction 0.5 g of foliar limb was submersed and macerated in 2.5 ml N,N - dimethylformamide (DMF), for 72 hours, at 4 ºC temperature and in the dark, for chlorophyll extraction, afterwards the supernatant being clarified and serving for the quantitative determination of the assimilating pigments, through its photometration using a spectro-photometer Spekol 11 type, Carl Zeiss Jena, using filters with 480 nm bands (for the carotenoid pigments), with 647 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, which helped the pointer to go to zero, after reading each test extinction. 12 The medium values of the assimilating pigments, determined in the extracts made from the exvitroplantlet leaflets kept under colored lights were reported 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 pphenylendiamine ( ipo & co., 2003 a and b, adapted after Lück method, 1974). The method principle consists in the fact that, in a vegetal extract that contains peroxidases, p-phenylendiamine is oxidized by them, phenomena that leads to changing the color of the obtained mixture into violet, and between the peroxidazic intensity (measured spectro-photometric, with a Spekol 11 Carl Zeiss Jena type spectrophotometer with a 483 nm filter) and activity, there is a proportionality direct report (Lück, 1974). Obtaining the peroxidazic extract was made trough triturating of the radicular tissues in a buffer solution made of phosphate 0.067 M, with pH 7. For this, 0.5 g rootlets (freshly ingathered), were crushed together with quartz sand in a grinding mortar (previously washed and sterilized through dry heat, in the stove at 120ºC), in the presence of 4 ml of ultra diluted phosphate buffer 1 : 9 (1 ml from the 0.067 M solution, with pH 7 plus 9 ml distillated water). The obtained extract was separated from the vegetal wastes through whizzing with a MSE centrifuge, at 6 000 rpm, for 25 minutes. Each specimen’s supernatant was collected and was kept in the refrigerator for 2 hours. The determination of the specimen’s extinction was made as a report with the distillated water, with a filter adjusted for a wave length of 483 nm. For reading the extinction in the box we introduced the mixture made of: 0.5 ml vegetal extract (supernatant), 0.05 ml oxygenated ultra diluted water, 1 ml phosphate buffer pH 7, 0.067 M, and after that it as added a 0.05 ml pphenylendiamine 1% solution. For each option (at 30 s, 60 s and 90 s from the obtaining of the final mixture) there were made three checking/readings for each. The oxygenated water used in our experiments was ultra diluted and was obtained from 33% hydrogen peroxide. In this way, for 100 ml distillated water there were added 0.3 ml of hydrogen peroxide; from this it was made a 1:9 distillated water dilution. Because the solutions are not stable in time, they are prepared only when the spectrophotometra-tion begins. The working temperature was 4 ºC. In order to compare the existing peroxidazic activity in the exvitroplantlet rootlets cultivated under different types of light, the average data of the extinctions read at 120 s, the moment when they got constant, came from the samples belonging to extracts that were obtained from the exvitroplantlet rootlets placed under different types of light, were reported to the similar photometric readings registered at the control lot (the exvitroplantlet rootlets set under white light), values considered as a reference, as being 100%. 13 2. RESULTS and DISCUSSIONS The survival percent of Cymbidium exvitroplantlets which was illuminated with 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 is difficult to acclimatization at septic medium conditions. A reason of this impediment was identified by Petru -Vancea and Cachiţă (2004), who observed that the Cymbidium hybridum vitroleaflets are amphystomatic and have the osteols permanently open, and to the natural medium plants are only hypostomatic. The Cymbidium exvitroplantlets placed under red light no surviving. The best surviving (50%) was presented the exvitroplantlets placed under green light. Survival percent % 100 day I 80 60 40 20 Green Blue Yellow Red White 0 Fig. 1 - The survival percent of the Cymbidium hybridum exvitroplantlets, at 30 days from their transfer into the septic medium, being illuminated with different types of fluorescent light: white (control), red, yellow, blue or green, reported to the situation from the moment of their passing „ex vitro”, reference values, considered 100%. As regarding assimilating pigments (Fig 2) was observed same differences to those leaflets which were maintained under different light colors comparative to those 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 those exvitroplantlets which were placed under yellow light and the pluses (+86%) in case of using the some filter to the samples obtained from exvitroplantlets acclimatized under green light. 14 chlorophyll „a” carotenoids %200 chlorophyll „b” 100 0 Yellow Blue G reen White light Fig. 2 - Expressing the chlorophyll pigments from the Cymbidium hybridum exvitroplantlet leaflets in percentage values, at 30 days after their transfer in a septic medium under different types of light: yellow, blue, green, as reference values there were taken homologous values registered at the exvitroplantlets kept under white light (control), these ones being considered 100%. % 140 120 White light 100 80 60 40 20 0 Yellow Blue Green Fig. 3 - Expressing the peroxidazic activity from the Cymbidium hybridum exvitroplantlet rootlets in percent values, at 30 days from their transfer into the septic medium under different types of light: yellow, blue, green, as reference values there were considered 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) presented rootlets obtained from exvitroplantlets placed “ex vitro” under blue and green light 15 on the same time, the exvitroplantlets rootlets maintained under yellow light were registered 18% lose in their peroxidazic activity, comparatively to the control. 3. CONCLUSIONS 1. Maintaining Cymbidium exvitroplantlets under different light types (yellow, blue or green) during the acclimatization period had various effects depending on their nature. The green light applied on Cymbidium exvitroplantlets had good consequences as: a higher percent of acclimatization surviving, higher values of assimilating pigments, especially of carotenoids but peroxidazic activity to. 2. The lowest results, biochemical point of view (of assimilating pigments accumulation and peroxidazic activity) were recorded to those Cymbidium exvitroplantlets which were placed during acclimatization period under yellow light, and under red light, the exvitroplantlets not survived. REFERENCES 1.Cachiţă, C.D., Deliu, C., Rakosy T.L., Ardelean, A.: Tratat de biotehnologie vegetală. Editura Dacia, Cluj – Napoca, 2004. 2.Druat, P., Kevers, C., Boxus, P., Gaspar, Th.: „In vitro” promotion of root formation by apple shoot through darkness effect on endogenous phenols and peroxidases. Zeitschr. Fűr Pflanzenphysiol., 108, 1982, p. 429–436. 3.Fuernkranz, H.A., Nowak, C.A., Maynard, C.A.: Light effects on „in vitro” adventitious root formation 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 CO2 & light improve acclimatization of in vitro propagated 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 elongation and 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 scolymus L. 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. Petru – Vancea, A.: Cercetări privind procesele morfofiziologice i biochimice care au loc în decursul aclimatizării plantulelor generate „in vitro”, la viaţa în mediul septic. Teză de doctorat, Universitatea din Oradea, 2007. 11. Petru – Vancea, A, Cachiţă, C.D., Studierea influenţei exercitate de natura luminii în aclimatizarea exvitroplantulelor. In: Lucrările celui de al XIII -lea Simpozion Naţional de Culturi de Ţesuturi i Celule Vegetale, „Vitroculturile la cormofite, modele experimentale în cercetările de biologie”. C.D. Cachiţă, A. Ardelean (vol. coords.), Satu – Mare, Editura Bion, 2005, p. 138–151. 12. Petru – Vancea, A., Cachiţă, C.D., Biochemical determinations made on African violets (Saintpaulia ionantha) exvitroplantlets, being illuminated during their acclimatization to a 16 13. 14. 15. 16. 17. 18. septic medium, with different types of light, Studia Univ. Vasile Goldi , t. Vieţii, Vol. 18, 2008 (in press). Podwyszyńska, M., Gabryszewska, A., Effect of red light on ex vitro rooting of rose and gerbera microcuting in rockwool. In: Proc. 1th Symp. on Accl.& Estab. Microprop. Plants. A.S. Economou, P.E. Read (vol. coords.). Acta Hort. 616, ISHS, 2003, p. 224– 237. Radoveţ-Salinschi, D., Cachiţă, C.D.: Conţinutul în pigmenţi asimilatori în frunzuliţele vitroplantulelor 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. ipo , M., Chiriiac, C., Flori , C.: Activitatea peroxidazică în embrionii i în plantulele de grâu (Triticum aestivum L. soiul Turda) rezultate prin germinaţia cariopselor submersate, în prealabil, în azot lichid (-196ºC). Analele Univ. Oradea, Fasc. Biologie, Tom X, 2003 a, p. 315-320. ipo , M., Chiriiac, C., Flori , C.: Activitatea peroxidazică în cariopsele de grâu (Triticum aestivum L. soiul Turda) după submersarea acestora în azot lichid (-196ºC). Analele Univ. Oradea, Fasc. Biologie, Tom X, 2003 b, p. 333-342. Tricoli, D.M., Maynard, C.A., Drew, A.P.: Tissue culture micropropagation of mature trees of Prunus serotonina Ehrh. I. Establishment, multiplication and rooting in vitro. Forest Sci, 31(1), 1985, p. 201–208. 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. .A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 18 - 23 NON-THERMAL INNOVATIVE PASTEURIZATION TECHNOLOGY OF FOOD USING A COMBINATION BETWEEN PULSED ELECTRIC FIELD AND HIGH PRESSURE M. 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: [email protected] ** Electrostatic and Electro-technologies Research Center – Bucharest, Romania *** Research Institute for Winery and Vinification – Valea Calugareasca, Romania Keywords: food pasteurization, pulsed electric field, high pressure ABSTRACT This paper is part of the national project results which has as main objective the elaboration of technologies and equipment for micro organisms’ inactivation by simultaneously applying a pulsed electric field and pressure to sterilize non-thermally food products (wine, fruit juices etc.). This technology will eliminate the deficiencies of the PEF technology applied in continuous flux. By simultaneously applying pressure treatment and PEF treatment, a PEF induced passive form of cellular plasmolysis will be obtained. The pressure will cause cell deterioration, will increase diffuse migration of humidity and will decrease cell recovery processes. The technology of simultaneously applying the electric field and pressure will lead to optimum results, will significantly increase the efficiency of wine and juice production and will increase their nutritional quality. This technology could be successfully applied to other food products too. Increased consumption demand for fresh products with minimum losses of nutrients stimulated research for new non-aggressive processing technologies. The development of alternatives for heat processing is also motivated by the real need for ecological technologies. Pulsed electric field (PEF), one of the nonthermal technology with minimum processing that for the specialized research represents an alternative process for liquid products preservation, fulfils these conditions. PEF processing is accomplished by introducing the food products in a chamber that contains two electrodes for the micro organisms’ inactivation and 18 for decreasing the enzymes activities as to increase shelf life of treated food products without causing any unwanted chemical and heat related effects. PEF applied to food products causes irreversible losses in the cell membrane functionality that leads to microbial cell inactivation. This process is known as electroporation and it effectively inactivates the micro organisms. PEF processing is a non-thermal pasteurization method of liquid food products. In the same time with the technological development of industrial applications, the system operation control becomes more and more important. In the recent years, many research groups demonstrated the inactivation possibility of some micro organisms also in buffer systems, in food products, using different systems, pressure and PEF. This paper is part of the national research project entitled “Multidisciplinary research for the development of an innovative non-thermal technology by combining 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 HPP and PEF treatment on micro organisms from young wines, respectively yeasts and lactic and acetic bacteria at the moment of wine raking from the solids. 1. MATERIAL and METHODS The raw material used in the experiment was a wine blending made from dry white wines obtained at Valea Calugareasca winery, after the wines were raked from the solids and sulphited. The purpose of wine blending use is to have a great micro organism’s diversity in the raw material subjected to treatment. The physico – chemical composition of wine blending used in the experiment was analyzed and the 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 SO2............................12,4 mg/l • Total SO2...........................44,6 mg/l • Sugar..................................1,47 • pH.......................................3,3 The wine blending was stored at 5oC for microbiological reasons (the inhibition of micro organism’s multiplication). The equipment used for HPP – PEF treatment was an experimental model designed in the second stage of the project by the Electrostatic and Electro technologies Research Center, as partner in the project. General scheme of the HPP 19 treatment equipment is presented in figure 1 and general scheme of PEF chamber is presented in figure 2. HPP equipment consists of: 1. CO2 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 of wine was introduced in the HPP experimental device at a hydrostatic pressure of 225 bars for 180 minutes. After the treatment, the wine was passed in aseptic conditions in thermo sterilized glass recipients and introduced immediately in the PEF chamber. PEF treatment was conducted at 30 kV/cm and 800 Hz for two periods 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 the microbiological point of view and the count of micro organisms remained active was assessed using the control method of active germs count. Cellulose acetate membranes with 0,45 pores diameter were used for microbiological filtration and the micro organisms were cultivated on specific media and incubated at 28oC. 2. RESULTS and DISCUSSIONS The inactivation effect of the two treatments applied subsequently on wine micro 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 4212 CFU/ml, in wine samples subjected to HPP treatment and then PEF treated for 1 minute, respectively 3 minutes. 20 Lactic bacteria were affected also by the combined HPP – PEF treatment, but in a gentler manner, their number decreasing from 2700 CFU/ml to 1830 CFU/ml for wine samples PEF treated for 1 minute and to 225 CFU/ml for wine samples PEF treated for 3 minutes. The most resistant wine micro organisms to the combined HPP – PEF treatment are acetic bacteria. Acetic bacteria load of wine samples decreased from 38 CFU/ml to 15 CFU/ml and to 8 CFU/ml, after 1 minute, respectively 3 minutes of PEF treatment. Considering the great diversity of wine micro flora, even if the grape juice is inoculated with a dominant yeast strain to be converted into wine, at the end the microbiological composition of wine consist of numerous species of yeasts, lactic bacteria 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 bar and the intensity of PEF above 30 kV/cm, the effect of HPP – PEF treatment will be an efficient sterilization of liquid products, such as fruit juices, grape juice and wines. 3. CONCLUSIONS The inhibitory effect of HPP – PEF treatment on wine natural micro flora was depending on the type of microorganism. Yeasts were the most sensitive to HPP – PEF treatment, the number of active yeasts in wine samples being reduced with more than 90% after 3 minutes of PEF treatment. Lactic bacteria were almost as sensitive as yeasts to HPP – PEF treatment, the number of active lactic bacteria in wine samples being reduced as well with more than 90% after 3 minutes of PEF treatment. The most resistant wine micro organisms to HPP – PEF treatment were acetic bacteria, the number of active acetic bacteria being reduced with less then 80% after 3 minutes of PEF treatment. Table 1 The effect of HPP – PEF treatment on wine micro organisms Experimental variant Treatment application period (min) Initial microbial load (CFU/ml) Microbial load after treatment (CFU/ml) Yeasts Lactic bacteria Acetic bacteria Yeasts Lactic bacteria Acetic bacteria HPP PEF 1 180 1 50400 2700 38 5208 1830 15 2 180 3 50400 2700 38 4212 225 8 21 Fig. 1 - General scheme of the equipment used for HPP treatment of wine for microbiological stabilization purposes: 1 – CO2 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 – glass cylinder; 3 – limiting Teflon fittings; 4 – stainless steel cylindrical electrode. 22 The e ffect of HPP-PEF treatme nt on lactic bacte ria 60000 3000 50000 2500 40000 Yeasts CFU/ml CFU/ml The effe ct of HPP-PEF tre atme nt on ye asts 30000 20000 10000 Lactic bacteria 2000 1500 1000 500 0 0 Initially 1 min 3 min Initially PEF treatment duration 1 min 3 min PEF treatme nt duration The effe ct of HPP-PEF tre atment on acetic bacte ria 40 Acetic bacteria CFU/ml 30 20 10 0 Initially 1 min 3 min PEF tre atment duration Fig. 3 - Effect of HPP – PEF treatment on wine micro organisms REFERENCES 1.Balestra P., Da Silva A A., Cug J.L., 1996 Inactivation of Escherichia coli by carbon dioxide under pressure., 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), Food Science 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 Conference on Electrostatics. 5.Daoudi L, Quevedo J.M, Trujillo A.J, Capdevila F.Bartra E, Minguez S, Guamis B, 2002 -Effects of high 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 Microbiological stabilization 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 Processing of Foods , ed. D.A. Ledward D.E. Johnston , R.G. Earnshaww A.P.M. Hastings , Nottingham University 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. .A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 24 - 31 OBTAINING OF SYNBIOTIC PRODUCTS FROM APICULTURAL PRODUCTS AND PROBIOTIC BIOMASS OF BIFIDOBACTERIUM BIFIDUM REALIZARE DE PRODUSE SINBIOTICE DIN PRODUSE APICOLE I BIOMASĂ PROBIOTICĂ DE BIFIDOBACTERIUM BIFIDUM VAMANU EMANUEL 1,2 , VAMANU ADRIAN 1,2 , POPA OVIDIU 1,2 , BĂBEANU NARCISA 1 1 – University of Agronomic Sciences and Veterinary Medicine, Faculty of Biotechnology, Bd. Mără ti no. 59, district 1, Bucharest, Romania, e-mail: [email protected] 2- Applied Biochemistry and Biotechnology Center – Biotehnol, Bd. Mără ti no. 59, district 1, Bucharest, Romania Key-words: Bifidobacterium, honey, pollen, CFU1, lactic acid Cuvinte cheie: Bifidobacterium, miere, pollen, UFC1, acid lactic ABSTRACT This study presents the multiplication of a Bifidobacterium bifidum b1 strain on media containing pollen and honey. The following parameters have been determined: the CFU 2 value, the production of lactic acid, the consumption of carbohydrates, and the variation of the kinetic parameters. The research works lasted 72 hours, with various rates of milled or non-milled pollen, in tightly closed recipients, at a temperature of 37 o C. The media were very well homogenized before inoculation. The inoculation was carried out only after the medium gained a homogenous consistency. The inoculum consists of Bifidobacterium bifidum b1 biomass. Acest studiu prezintă multiplicarea unei tulpini de Bifidobacterium bifidum b1 pe medii ce conţin polen i miere de albine. S-au determinat următorii parametrii : valoarea UFC, producţia de acid lactic, consumul glucidelor i variaţia parametrilor cinetici. Cercetările s-au desfă urat pe o perioadă de 72 de ore, cu procente diferite de polen măcinat sau nemăcinat, în recipiente închise etan , la o temperatură de 37 0 C. Mediile au fost omogenizate foarte bine înaintea inoculării. Inocularea s-a desfă urat numai după ce mediul a căpătat o consistenţă omogenă. Inoculul constă din biomasă de Bifidobacterium bifidum b1. The use both of probiotics and of prebiotics has much increased due to the high 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 protected] 24 Such a product is used for prophylaxis, not directly for its therapeutic effects, being considered a functional foodstuff. Bifidobacteria and the acidolactic bacteria are used as organisms with a probiotic effect in the fermented milk products and in the synbiotic 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 fibers ensures a normal functioning of the intestine and an improvement of the negative effects due to their absence in the ingested food (3, 4). The combination between prebiotic and probiotic has a direct effect on the increase of the number of anaerobic bacteria and on the decrease of the number of potentially pathogenic aerobic bacteria. These benefits are also due to the fact that the non-digestibility of the prebiotic component provides a plus of efficiency to the probiotic strains (5). The most frequent oligosaccharide encountered in the human diet is the insulin and, more recently, honey, which is very rich in oligosaccharides with a prebiotic 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, and particularly of the Bifidobacterium strains. This stimulation effect is caused by the composition of honey and by the used strain. Thus, the products that can be obtained are new products, which do not have a substitute on the market (8). Besides the broadening of the range of functional products that can be obtained, the effect on the bifidobacteria is highly improved, compared to other prebiotics. 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 kept under conditions not involving special requirements(9). The combination between honey and the probiotic biomass of bifidobacteria creates an optimal conservation medium without needing to add preservatives, which are extremely reprehended by all the norms and directives of the European Union in the field. 1. MATERIAL and METHOD Microorganism and culture media. In the practical experiments made for the purpose of this work, we used the Bifidobacterium bifidum b1 probiotic strain from the collection of the Faculty of Biotechnologies. It was conserved in the freezer, at -82oC, on a protective medium, enriched with 20% glycerol, and then revitalized 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, the culture medium is filtered in vacuum, then autoclaved at 115oC for 20 minutes, in an autoclave. The inoculation was made with 10% freshly reinvigorated culture. To obtain synbiotic products the following culture media are used: 20g non-milled pollen, 3g honey, 5ml distilled water; P2: 20g milled pollen, 3g honey, 5ml distilled water; P3: 20% non-milled pollen, 3% honey, 5ml 25 distilled water; P4: 20% milled pollen, 3% honey, 5ml distilled water. The inoculation was made with 10% Bifidobacterium bifidum b1 biomass. The milling of the pollen used in obtaining the P2 and P4 media was performed by using a mill, in series of 15 seconds, with a break of 10s; operation repeated 5 times. The P1 and P2 media are semi-solid media, obtained by mixing the components and adding a minimal quantity of water, just enough to break the pollen grains and obtain a relatively homogenous medium. The medium was distributed in tightly closed plastic recipients, and after inoculation they were subject to the temperature of 37 o C. The tubes were statically maintained (10). Determination of the quantity of carbohydrates by using the o-toluidine method. It was performed with the o-toluidine test, by the Chemical and Pharmaceutical Research and Development National Institute – ICCF of Bucharest. Determination of the quantity of lactic acid. The accumulation of lactic acid was determined by using an HPLC (11). Determination of the viability. To establish the number of colony forming units, the successive dilutions method was used, using the medium containing MRS+0.2% cysteine hydrochloride with various agar concentrations. 2. RESULTS and DISCUSSIONS Obtaining of the probiotic biomass of bifidobacteria To determine the effect of the corn extract on the biomass accumulation, the following fermentative parameters were determined first in parallel, at laboratory level: lactic acid, accumulated biomass, glucose consumption. The results 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 be ascribed to the supplementation of the medium with cysteine hydrochloride. The logarithmic increasing phase follows an almost constant layout, up to approximately 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. This also leads to a low productivity on this culture medium. When the microorganism is in the lag phase, there is no production of lactic acid. The first signs of existence of the lactic acid occur in the beginning of the logarithmic increasing phase. The production continues as long as the strain is in the logarithmic phase. The maximum level attained is of 0.5% lactic acid, every 36 hours of fermentation. Glucose gradually decreases during the entire period of 24 hours, the largest consumption being again during the logarithmic phase. Regardless of the quantity of glucose added to the medium, the production of lactic acid has lower values, because such strains do not produce significant quantities of this product. Anyway, even if the quantity of lactic acid were to increase, it should be neutralized because it decreases productivity. 26 2,5 D.O./O.D. (600 nm) Acid lactic/Lactic acid (%) Glucoză/Glucose (%) 2 1,5 1 0,5 Acid lactic/Lactic acid (%) D.O./O.D. (600 nm) 10 9 8 7 6 5 4 3 2 1 0 0 0 4 8 12 16 20 24 28 32 36 40 44 48 Timp/Time (ore/hours) Fig. 1 - Evolution of the fermentative parameters when cultivating the Bifidobacterium bifidum b1 strain on the medium MRS+0.2% cysteine hydrochloride at laboratory level If cultivation takes place under the conditions of a bioreactor (net volume of 2 liters), the results are shown in Figure 2. The accumulation of lactic acid increases linearly during the 48 hours of fermentation. Carbohydrates constantly decrease, more pronouncedly during the interval 4-24 hours. The lag phase is almost absent, while the exponential increase phase continues up to 32 hours of fermentation. Compared to the results obtained at laboratory level, we notice an increase of the logarithmic phase by approximately 8 hours. 30 6 25 5 20 4 15 3 10 2 5 1 0 0 0 4 Acid lactic/Lactic acid (%) D.O./O.D. (600 nm) D.O./O.D. (600 nm) Acid lactic/Lactic acid (%) Glucide/Glucides (%) 8 12 16 20 24 28 32 36 40 44 48 Timp/Time (ore/hours) Fig. 2 - Evolution of the fermentative parameters when cultivating the Bifidobacterium bifidum b1 strain at bioreactor level 27 Compared with the production of lactic acid obtained in the case of the inoculum, this medium determines the obtaining of significant quantities of lactic acid. The neutralization was made with Ca(OH)2 10%. It follows that the permanent maintaining of the pH value of the medium at 5-5.5 determines an important accumulation, but under the form of Ca lactate. It is worth mentioning that the accumulation of the biomass has values approximately 3 times larger, compared to the medium for the inoculum. Equally, the quantity of lactic acid also increases approximately 3 times, directly proportional to the quantity of accumulated biomass. Cultivation in media containing pollen and honey The multiplication and synthesis capacity of the lactic acid in the 4 formulas of medium containing pollen and honey was tested in tightly closed Falcon tubes and maintained at a constant temperature, up to 48 hours. 120 4 P1 3,5 P2 3 P3 2,5 P4 Glucide/Glucides (%) Acid lactic/Lactic acid (%) 4,5 2 1,5 1 0,5 P1 P2 P3 P4 100 80 60 40 20 0 0 0 8 16 24 32 40 48 Tim p/Tim e (ore/hours) Fig. 3 - Quantity of lactic acid synthesized when cultivating on media containing pollen and honey 0 8 16 24 32 40 48 Tim p/Tim e (ore/hours) Fig. 4 - Quantity of carbohydrates consumed when cultivating on media containing pollen and honey In Figure 3 you can notice that the media containing milled pollen determine a more powerful synthesis of the lactic acid, compared to the 2 media containing non-milled pollen. In case of medium P2, the quantity of lactic acid is approximately 43% larger, at the end of the 48 hours. For the medium P4, the quantity is approximately 27% larger than that obtained by using the P3 medium. Making the comparison between the 2 types of culture media used, the culture media 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 a consumption exceeding 30% for each type of culture medium. What needs to be mentioned 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. 70000000 60000000 Număr celule viabile/ Number of viable cells 50000000 40000000 P1 30000000 P2 20000000 P3 10000000 P4 Fig. 5 - Number of viable cells when cultivating on media containing pollen and honey 0 0 8 16 24 32 40 48 Timp/Time (ore/hours) The number of viable cells maintains the tendency noticed during the previous 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 factor in stimulating the multiplication of the microorganism, but also of the synthesis of the metabolic products. This finding is also noticed in the case of the second category of culture media, P3 and P4. For the medium P2, the number of cells that are formed is 3.5 times larger than of those that are formed by using medium P1. If we compared media P2 and P4, we would notice that, by using medium P2, 10 times more microorganisms would be obtained (Bifidobacterium bifidum) than by using medium P4. Table 1 Kinetic parameters for cultivating Bifidobacterium bifidum b1 on media P1, P2, P3 and P4 Mediu de cultură Culture medium P1 P2 P3 P4 Glucide iniţiale Original carbohydrates ( 103 107 51 56 Acid lactic Lactic acid g ) L ( 28.7 40 15.2 19 g ) L Viteză de cre tere maximă Maximum increase speed µmax (h-1) 0.09 0.11 0.02 0.03 Productivitate Productivity ( g ) L×h 1.08 1.32 0.24 0.36 29 In the fermentations that take place for obtaining the bifidobacteria probiotic biomass, it is noticed that using the corn extract determines a significant increase of the cellular productivity. This is also ascribed to using conditions similar to a classic bioreactor, but much easier to maneuver under laboratory conditions, for obtaining the biomass. The only parameter that can determine a decrease of the cellular concentration is the lactic acid formed, if it is not neutralized when its synthesis dramatically decreases the pH of the medium. This finding is particularly noticed when the biomass is obtained. The increased synthesis of the lactic acid, when cultivating on media containing pollen and honey, is not a very serious problem because the homogenization when using media P1 and P2 is more difficult because of the high consistency. Thus, due to this consistency of the culture medium, we believe that the diffusion of the lactic acid is more difficult and this way the formed cells are much less affected by the low pH. In exchange, for the media P3 and P4 the synthesis of the lactic acid is an important factor because they are culture media similar to the classic ones. This is also one of the reasons for which the two media are not so productive. When cultivating on media containing pollen and honey, only natural substances are used, without ingredients that could change the final composition of the product, and that is why, out of the 4 culture media, only medium P2 has the required features in the end. First of all, a viability of 107, which is over the minimal value of 106, which is characteristic to products with a probiotic effect. The acid synthesis and the high viability in the case of medium P2 can also be considered as an indicator of keeping the product sterile. This finding is extremely important due to the fact that no previously sterilized raw materials (pollen and honey) are used. Table 1 shows the kinetic parameters of cultivating the Bifidobacterium bifidum b1 strain on the 4 culture media containing pollen and honey. For each culture medium, the lactic acid formed is correlated to a significant consumption of the quantity of carbohydrates. The increase speed is significantly higher when using the medium P2. This can also be due to the increase in the concentration of oligosaccharides that are found in honey and that were completely homogenized with the milled pollen. Given the special cultivation conditions, productivity is the main indicator in this table. The increase of the water quantity does not lead to the increase of productivity, but to its dramatic decrease, which is also due to the dilution of the oligosaccharides contained by the medium, which are an important impulse for the increase of bifidobacteria. 3. CONCLUSIONS The cultivation of the Bifidobacterium bifidum b1 strain was performed with very good results under batch conditions in view of obtaining probiotic 30 biomass, which is used in obtaining a probiotic product based on honey and pollen. A large biomass quantity was obtained, according to the data provided by literature, by using the corn extract as an additional source of nitrogen and increase factors. By cultivating the Bifidobacterium bifidum b1 strain on media containing pollen and honey, we found that the medium P2 is optimal for obtaining such a probiotic product. Also, we found that the milling of the pollen, the low water quantity, 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. ACKNOWLEDGMENT The researches were financed through a project PNCDI II - PARTNERSHIPS IN PRIORITY S&T DOMAINS, Theme 61-047/2007. REFERENCES 1. Roy Sleator, Colin Hill (2007): Probiotics as therapeutics for the developing world. Journal of Infection in Developing Countries 1: 7-12. 2. Kingsley C. Anukam (2007): The potential role of probiotics in reducing poverty-associated infections in developing countries. Journal of Infection in Developing Countries 2: 81-83. 3. Almeghaiseeb Ebtissam (2007): Probiotics: An overview and their role in inflammatory bowel disease. 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. J Paediatr Child Health 5: 331-336. 6. Anukam K.C., Osazuwa E.O., Reid G. (2006): Knowledge of probiotics by Nigerian clinicians. Int J Probiotics Prebiotics 1: 57-62. 7. Pineiro M., Stanton C. (2007): Probiotic bacteria: legislative framework-- requirements to evidence basis. J. Nutr. 3: 850S-853S. 8. Paton A.W., Morona R., Paton J.C. (2006): Designer probiotics for prevention of enteric infections. Nat. Rev. Microbiol. 4: 193-200. 9. Schiffrin E.J., Donnet A., Blum S. (2005): How can we impact the immune system with pre- and probiotics? 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 Lactobacillus plantarum 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. .A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 32 - 38 STUDIES ON THE GROWTH AND MYCOTOXIN PRODUCTION OF MOULDS ISOLATED FROM ROMANIAN FOOD PRODUCTS STUDII ASUPRA CRESTERII SI PRODUCERII DE MICOTOXINE DE CATRE MUCEGAIURI IZOLATE DIN PRODUSE ALIMENTARE ROMANESTI FLORENTINA RADOI-MATEI1, FLORENTINA ISRAEL-ROMING1, ANCA RADU2, CAMELIA DIGUTA1, CRISTINA COCULESCU3, R. DESPA 3 1 - USAMV Bucharest, Faculty of Biotechnology, Romania - Institute of Food Bioresources, Bucharest, Romania 3 - Romanian-American University, Department of Mathematics, Bucharest 2 Key words: moulds, mycotoxin, predictive models Cuvinte cheie: mucegaiuri, micotoxine, modele previzionale ABSTRACT This research belongs to a national project regarding the prevention of the mycotoxin contamination in feed and food. At the very beginning of the project there have been isolated and identified 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 and deoxynivalenol production) by on-plate method and the toxins were quantified by Elisa Immunologic tests. 38 of those moulds showed toxinogenic activity under laboratory tests. From this toxinogenic collection, two strains (Fusarium graminearum MI 113 and Penicillium crysogenum MI 210) have been studied for their growth and mycotoxin (DON and OTA) production from a predictive point of view. Spores suspension of 103 - 104 was used as inoculum for natural (wheat) and synthetic (Czapek-Dox broth) media. The samples have been cultivated during 21 days under different temperatures conditions (4oC; 12oC; 16oC; 20oC; 23oC; 26oC; 30oC; 33oC; 36oC). Every 24 hours it has 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 (DON and 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 by Eviews program which has proved a linear evolution during the time in direct correlation with the temperature (similar to the Gompertz model). 32 By immunological test, the primary model of the DON production, showed a maximum production of mycotoxin at 26 oC; on this temperature level the maximum amount of DON was reached in the 17th 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 oC after 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 to guarantee the consumer health represent a very important objective in the agricultural and food fields. After a technological transformations succession, the final food product should present a minimal microbiological risk. In order to arrive to such of minimal microbiological risk they have been developed different control systems, as the HACCP system. An alternative method, complementary to the HACCP, it is the predictive microbiology which can evaluate de speed of the microbial development under different environmental conditions using mathematical models [1]. The biggest part of these moulds (Aspergillus, Penicillium, Fusarium, Claviceps and Alternaria) in the same time with the conidiation can produce toxic metabolites (aflatoxins, ochratoxines, DON, etc) having a higher termal stability comparing to their vegetative form, the mycelium. The prevention of the food products pollution with toxinogenic moulds depends mainly on the understanding of the fungal alteration phenomenon during the processing, conditioning and keeping [5, 10]. For that is necessary to have precise diagnostic methods in order to predict and describe in details the dynamic of the alteration and pollution with mycotoxins [8, 9]. The project proposes to elaborate a preventive diagnostic system of those pollutions, approaching the phenomenon by predictive microbiology and consist in a 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 the kinetic of the mycotoxins appearance starting from the development kinetics. 1. MATERIAL and METHOD Microorganisms From USAMVB – Biotechnology microorganism collection, two strains Fusarium graminearum MI 113 and Penicillium crysogenum MI 210 isolated from food products of intermediary humidity, have been taken into the study as proved mycotoxins producers (deoxynivalenol, respectively ochratoxin A). 33 Media and cultivation conditions Spores suspension of 103 - 104 obtained on Czapek – Dox broth was used as inoculum for natural (50 g of wheat + 10 ml DW) and synthetic (Czapek-Dox broth) media. The samples have been cultivated during 21 days under different temperatures conditions (4oC; 12oC; 16oC; 20oC; 23oC; 26oC; 30oC; 33oC; 36oC). Every 24 hours it has 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 (by extraction and centrifugation) for the mycotoxins (DON and OTA) detection. Myctoxins detection Two methods of DON and OTA detection have been used: Elisa type immuno-enzimatic tests and HPLC technique. The first one consisted in immunological Elisa kit Ridascreen – RBiopharm having a 18.5 ppb detection limit for DON, respectively Ridascreen OTA a 30/15 with a detection limit of 2,5 ppb. Fig. 1 - Sample preparation (filtration) for DON detection For HPLC detection (immunoafinity columns) it has been used a WatersAlliance HPLC system. For DON the detection was done at 218 nm, and for OTA the excitation was measured at 333 nm and the emission at 443 nm. For the separation it was employed a chromatographic columns made of octadecilsialns C18 – Spherisorb 4.6 x 150 mm, having 5 mm particles. The analyte separation was performed at 30oC, and the elution used the mixture of acetonytril + methanol + water (5:5:90). The data have been analyzed by a high performance program named EMPOWER. Mathematical tools Regarding their growth, the primary model of the mycelium development was analyzed by Eviews program. 34 2. RESULTS and DISCUSSIONS The growth evolution under different temperature conditions Former researchers showed that the growth and toxinogenesis doesn’t fit the same evolution curve under the same conditions. For example, for Aspergillus flavus, the optimum growth temperature was proved to be 30oC, while the highest aflatoxin production was measured at 33oC [3]. Starting from this results, the two strains were growth under different temperature conditions. Both strains, Penicillium MI 210 and Fusarium graminearum MI 113 didn’t show any growth at 4oC. As shown in figure 2, at 12oC, while the Penicillium strain had a linear growth, Fusarium showed an exponential growth. 8 3 2,5 6 2 4 1,5 1 2 0,5 13 11 9 7 5 3 1 13 11 9 7 3 5 0 1 0 Fig.2 – Growth evolution of P. crysogenum MI 210 (left) and F.graminearum MI 113 at 12oC (right) Same growth curves allure proved both strains at 16 and 20oC as at 12oC. Starting with 23oC and continuing with 26oC and 30oC, the Fusarium strain showd a linear growth curve, while Penicillium entered in an exponential evolution of the radial growth on plate (figure 3). 4 3 2,5 3 2 2 1,5 1 1 0,5 13 11 9 7 5 3 1 13 11 9 7 5 0 3 1 0 Fig.3 – Growth evolution of P. crysogenum MI 210 (left) and F.graminearum MI 113 at 12oC (right) at 33oC 35 For Fusarium gramineaurm MI113 the data growth has been analyzed from an econometrical point of view, using the Eviews program, a dedicated soft for the economical and statistical experimental data. An analysis of the temporal and a-temporal series have been done. An atypical model have been found for this strain at 33oC. From a statistical point of view, the Fusarium growth at different temperature involved a correlation coefficient of r = 0, 41575 , an experimental data of tstud = texp = 0, 3432 , while the table value is ttab = 2, 22814 . Comparing this values, it can be stated that there is a linear correlation between the development time and the mycelium growth, correlation proved also by other authors 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 tstud = texp = −0, 58186 , while the table value was ttab = 2, 22814 Mycotoxin evolution under different temperature conditions Fig.4 – DON evolution during the cultivation versus the temperature for Fusarium graminearum MI 113 36 Regarding the Elisa type tests, for all the inoculated wheat samples is has been measured the humidity. This one has varied between 28.89 and 48.39 %. For the final mycotoxin calculation, these values were taken into consideration. The DON formation on natural medium (wheat) have been measured every two days at a very significant quantity have been measured during all 17 days of cultivation at 26oC (starting from 89, 64 ppb to 741,05 ppb in the last cultivation day – Fig.4). As it was expected, this temperature is the optimum one for the DON formation as long as much the temperature grows from 26 to 36oC, less mycotoxin was detected. In the same conditions, on natural medium (wheat)The highest level of the OTA reached 24033.16 ppt at 36oC in the last day of cultivation (the 17th ). It was found interesting the fact that at lower temperature, as 30 or 33oC the OTA formation was maximum in the 5th day of cultivation, having values of about 1400 ppt. Another interesting result it was found by measuring the OTA formation in liquid synthetic medium. The data showed that the optimum temperature for OTA formation it was a lower temperature as on natural solid medium, respectively 23oC. Fig.5 – OTA evolution during the cultivation versus the temperature for Penicillium crysogenum MI 210 37 3. CONCLUSIONS Eviews program which has proved a linear evolution during the time in direct correlation with the temperature (similar to the Gompertz model). The primary model of the DON production, showed a maximum production of mycotoxin at 26 oC; on this temperature level the maximum amount of DON was reached in the 17th day of incubation (from 89ppb after 24 hours incubation to 741 ppb). Regarding the OTA production, the maximum amount of toxin was reached on 23 oC after 17 days of incubation. The maxium measured level was 24033 ppt. Further investigation for the OTA and DON gene expression will be performed by RT-RT-PCR, taking into consideration also the water activity influence. REFERENCES 1. 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 in food. Trends Food Sci. Technol. 8,353-358. 3. Judet D., Matei-Radoi F., Bensoussan M., Jurcoane S. (2006): Studies on Aspergillus flavus growth 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 cereals correlated 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 of Chemistry, Carleton University Otawa, Ontario - Mycotoxins and Food Safety, Kluwer Academic/Plenum Publishers, pag. 19 – 22. 6. Sautour M., Dantigny P., Divies C., Bensoussan M. (2000): A temperature - type model describing the relationship between fungal growth and water activity. International Journal of food Microbiology 67 63-69. 7. Sweeney M. J., Dobson D.W. Alan (1998): Mycotoxin production by Aspergillus, Fusarium and Penicillium 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 predictive mycology. 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 Applyed in Predictive Microbiology. Proceeding of the 31st ARA Congress, TRANSILVANIA University Brasov, Romania. 10. Weidenborner, M., 2001, Encyclopedia of Food Mycotoxins, Springer, Germania. 38 Scientific Bulletin Biotechnology, U. .A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 39 - 49 NUTRITIVE EFFECTS OF NON-CONVENTIONALLY PROCESSED MEDICINAL PLANTS UPON MONOGASTRIC ANIMALS DEVELOPMENT E. POPA*, A. MUSCALU*, A. DIHORU**, V. HEBEAN** * National Institute of Research - Development for Machines and Installations Designed to Agriculture and Food Industry ,INMA Bucharest, Romania, e-mail: [email protected] ** National Institute of Research - Development for Biology and Animal Feeding, IBNA Balote ti, Romania Keywords: non-ionized radiations, foddering additives, biomass. ABSTRACT The paper presents the processing of medicinal and aromatic plants by alternative method (with non-ionized radiations and continous flow) and the output of the utilization of resulted products as foddering additives for pigs food during the stress period after their nursing cessation. As a result of the necessity of shortening the processing time, in order to guarantee a high quality end product, as well as the achieving of enough biomass quantity for its capitalization for animal food, it is essential to obtain the forced drying of herb medicinal and aromatic plants, by state-of-the art efficient technologies. The medicinal plants herb drying represents a compulsory technological stage within the process of capitalization of this category of products. The forced desiccation of medicinal and aromatic plants herb is required as a result of necessity of shortening the process period and in order to obtain a quality end product. Therefore , the content of active substances of herbs must be preserved as well as possible. For vegetal products dessication alternative (non-conventional) methods have been developed, being designed at reducing the time and temperature effects, eliminating the chemical treatments and achieving an environmental-friendly processing. Among the non-conventional desiccation methods we have to notice:treatment with ionized electromagnetic radiations ( γ radiations, X radiations) and non-ionized (microwaves and radio waves, UV radiations), ultrasounds, processing at high pressure, oscillating magnetic field or these methodes combination with conventional desiccation methods.[1] 39 1. Introduction 1.1 Microwave heating process The most frequently used phenomenon of microwave processing is represented by dielectric materials heating, due to hysteresis of electric fields-variable in time, wich influences the conversion of electromagnetic energy in thermal energy. The source of energy 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 material depending on the air current speed. The energy concentration by little volumes of the material results in important temperature rises, wich can modify material properties. The dipolar dielectric materials are important for the microwave heating The phenomenon of polarization takes place inside a dielectric, situated within an alternative electric field The electric loads are oriented in terms of polarity of electric field, determining both a displacement of electrons around the nucleus (electronic polarization) and a displacement of atomic nucleus, due to uneven distribution of loads inside the molecule (atomic polarization). Fig.1 - Interface polarization (spatial load) (a) and reorientating polarization (b). It has been experimentally found out that the dielectric polarization depends 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 angle characterized by tg δ. The main factors wich determine the materials heating in the microwave field are: -ε´- dielectric permittivity, - value featuring the materials from their capacity’s point of view of becoming polarized; -ε´´ -loss factor including all the dissipation effects due to losses by Joule effect and 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 in which it is situated is given by the presence inside the medium (product) of electric dipols, tending to orient towards the electric field direction. The phenomenon is characterized by the dielectric constant ( dielectric permittivity) of material, definition starting from the vacuum permittivity: ε ' = ε 0ε r ' ( 1) where: ε′r = material relative permittivity. The product polarization phenomenon determines a displacement current as: JD = ∂D ∂t ( 2) where: D = vector of electric .induction. Having in view that poles’ rotation is performed by friction, the loads movement determines energy losses, in conclusion, material heating. In case of harmonic regime, ∂ = jω , the permittivity is a complex ∂t value. ε ' = ε '− j ε p ' ' ( 3) where: ε”p =represents the losses by dipolar relation. These losses join, in the particular case of humide bodies, the losses resulted from ionized conductance due to the presence of free loads which determine the loss factor: ε "= ε " p + σ ω ( 4) In this case the effective dielectric constant of a medium with losses, written as a complex value becomes: ε c∗ = ε '− jε " ( 5) from whitch it results the tangent of loss angle δ: ε' = tgδ ε" ( 6) The dielectric permittivity (ε´)as well as the loss factor (ε´´) defining the materials dielectric features in microwave field varies both in terms of temperature and humidity. For the dielectric free of losses tg δ is of10-4÷10-3 degree, and for wet earth of 10-2 ÷10-1 degree. 41 The main characteristic of dessications in high frequency field is the volumetric heating, the heat being generated in this particular case inside the material (product), in comparision with the conventional method, where the heat is supplied for the material outer side. The wave is attenuated during the medium crossing by energy losses. It results that the penetration depth Dp is the distance at wich the energy transported by wave is equivalent with e-1 out of the initial energy. Dp = 1 2α ( 7) where: α is the attenuation factor.for dielectrics with losses, this relation becomes:  ε'  D p = λ   2πε "  1/ 2 ( 8) Where : λ is the wavelength in free space. The penetration depth depends on the wavelength ( in inverse ratio to frequency). At frequencies used for microwave heating, the penetration depth is of degree of centimeteres and varies in accordance with temperature and material properties [2]. 1.2 Desiccation installation of medicinal and aromatic herbs by means of microwaves in continous flow The aim is to achieve the artificial (forced) desiccation of medicinal and aromatic plant herb – as chopped herb, in view of subsequent processing and capitalization of products at a highest quality. The scope comprises all species of medicinal and aromatic plants belonging to crops or spontaneous flora in Romania. 1.2.1. Installation general presentation The 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 transducer without contact, in infrared. B) Feeding/evacuation system of proccesed material - Transport system with conveying band; 42 - 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 0C; - 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: (3430x500 x522) mm; Personnel servicing:…………….max.3 people; Fig. 2 - Desiccation module and control assembly 1.2.2 Specific Working Regime The state parameters which define the dessication process are as follow: the temperature (°C); humidity (%); dessication speed (% humidity/min.). The development of the dessication process of the herb of medicinal and aromatic plants is done in three successive stages as follow: a) the preheating period during which the heat is consumed almost in totality for the heating of the material until the establishment of the regime temperature, at 43 which there is established an equilibrium between the transmitted heat quantity and that which is consumed for the water evaporation. b) the dessication period with constant speed which represents the dehydration periode for that matter; c) the dessication period with decreasing speed (the final period), in which the dessication speed is gradually reduced; The microwaves power regime can be continually adjusted starting from zero to the maximum power. In order not to be alterated and for conserving the active substances content and aspect, the medicinal and aromatic plant herb after desiccation has to contain, generally only 8...14% water. Taking into consideration the significant physical and chemical properties of the medicinal and aromatic plants species, we can notice two main groups: • a) plants containing volatile oils (culture thyme, hyssop, mint, etc.), which maximal dessication temperature must not be greater than 35 °C; • b) plants containing alkaloids and glycosids (artichoke, bay, etc.), which optimal dessication temperature is content between 50 and 65 °C. The using domain of the installation can be extended also to the forest fruits, which have a special situation. Like this, berriess (wild roses and hawthorn) have the optimum dessication temperature over 90 °C. Table 1 Dessication mode Natural drying Artificial (microwave) Dessication mode Natural drying Artificial ( microwave) 44 Active principles of volatile oils for following species (ml %) Salvia Mint Basil Thyme Hyssop officinalis 0.60 0.67 3.12 0.47 0.51 0.74 0.92 Polyphenols (%) 0.56 0.74 – 1.04 3.91 0.65 0.64 Table 2 Flavone (rutin) (%) 0.74 0.85 – 1.35 1.3 Utilization of medicinal plants in feeding of monogastric animals The intensive breeding of farm animals is based on using complex rations, respective concentrates and fodder additives to the monogastric animals. These fodder rations are potentiating the animals production, but can produce also unbalances on the environment by the ratio input/output.[3] The environment protection policy as well as the one of obtaining healthy animal products orientates the farm animals breeding activity towards applying alternative solutions to chemotherapy with pharmaceutical growing promoters or antibiotics. In this concept, the capitalizing of vegetal natural resources is an acceptable variant for the infusion of vitamins and minerals, for keeping the animals performances, for preserving their health and well being state, especially in their critical life periods, as it would be the ablactation.[4] At the same time it is raised the problem of assuring some corresponding quantities of quality vegetal resources, relationed with their harvesting at the optimal phenophases of plants development from the specific crops or from spontaneous flora, selecting and sampling the useful organs, preserving the vegetal material 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-test was realized on piglets in their after ablactating period, at Biobase INCDBNA – IBNA Balote ti, the animals allotments being lodged in growing facilities endowed with boxes with grates and permanent water alimentation. 2. MATERIAL and METHOD For being included within the combined fodder, the vegetal resources were conditioned via drying (with microwaves) and ground. Table 3 Specification Animals number Experiment duration (days) Angelica– Fruits (kg/100 kg CF) Oregano plant (kg/100 kg CF) Salvia officinalis. plant (kg/100 kg CF) Animals allotments E1 E2 M E3 8 18 - 8 18 3.0 8 18 3.0 - 8 18 0.5 - The aromatic and medicinal plants conditioning was effectuated differentially, in depending on the useful organs consistency and the water quantity they contain, applying to installation working regimes corresponding to the protection of the active principles. The biologic test was performed on a number of 32 piglets from the Great White breed, 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, 8 heads in each allotment and lodged in 4 heads boxes, according to the experimental scheme from table 3. Table 4 Ingredients (Kg) M Corn 46.15 Rice 15.00 Soy grit 16.00 Corn gluten 2.00 Powder milk 10.00 Oil 3.00 Fish flower 2.00 Monocalcium phosphate 2.00 Calcium carbonate 1.60 Salt 0.20 Methionine 0.25 Lysine 0.70 Sincaline 0,10 Premix vitamins-mineral P1+P2 1.00 Fruits ANGELICA Herba OREGANO Herba SALVIA OFF. Total (Kg) 100.00 Qualitative parameters Brute protein (%) 20.30 Metabolismic energy : - Kcal/kg NC 3400 - MJ/kg NC 14.23 Lysine (%) 1.61 Methionine + Cystine(%) 0.91 Calcium (%) 1.10 Phosphorus total (%) 0.90 46 Experimental allotments E1 E2 E3 43.15 15.00 16.00 2.00 10.00 3.00 2.00 2.00 1.60 0.20 0.25 0.70 0.10 1.00 3.00 100.00 45.65 15.00 16.00 2.00 10.00 3.00 2.00 2.00 1.60 0.20 0.25 0.70 0.10 1.00 0.50 100.00 43.15 15.00 16.00 2.00 10.00 3.00 2.00 2.00 1.60 0,20 0.25 0.70 0.10 1.00 3.00 100.00 21.41 3316 13.87 1.59 0.90 1.19 0.89 20.30 3300 13.81 1.58 0.90 1.19 0.89 21.10 3300 13.81 1.58 0.90 1.19 0.89 It was used an unique recipe of combined fodder for the 4 piglets allotments, reproduced in Table 4, whose structure has assured the quality indices for the nutritional requirements specific to the respective animal category. The differences between the experimental variants have consisted in the species and the useful part from the medicinal plant which was incorporated in the CF mixture, with the corresponding diminution of the corn 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 the distributed quantities and their consumption. The animals were weighted individually, at the beginning and at the end of the experimental period. Table 5 SPECIFICATION Initial weight ( kg.) Final weight ( kg.) * Daily average gain ( kg.) * Daily average consumption (kg.) Specific consumption ( kg./kg.gain ) M 12.00 20.81a 0.490 a 1.12 2.29 ALLOTMENT E1 E2 11.75 12.00 21.07 a 22.21 a a 0.504 0.552 a 1.23 1.35 2.44 2.45 E3 12.00 21.64 a 0.512 a 1.21 2.36 * the same small letter insignificant differences ( P > 0.05 ) During the experiment it was tracked the evolution of the following zootechnical parameters: -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 DISCUSSIONS The results obtained after the biological test are given in table 5. The initial average weight was established by individually weighting of the animals subjected to testing and estimated at 12.0 kg. 47 The final average weight, determined after 18 days from the experiment beginning, has marked out bigger values at the piglets from experimental allotments ( 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) between the experimental allotments for the final body weight, which indicates that the structure of the mark CF as well as of those supplemented with different phytoadditives did not influence this indicator. Regarding the daily average gain, is observed an increment of this parameter at the experimental allotments, the increment being bigger (12.65 %) at allotment E2, comparing with the mark allotment, but the difference isn't significant, the daily average gains being normal for the ablactating crisis. The daily average consumption of combined fodder is bigger in case of allotment E 2 namely of 1.35 kg,in comparison with the mark allotment M, of 1.12 kg. The daily average consumption of E 2, superior to the mark allotment, was reflected in the daily average gain too (0.552 kg) and the body weight (22.21 kg). The fodders specific consumption expressed by combined fodder kilogramme used for obtaining 1 kg meat gain, is correlated, on the one hand, with the daily average fodders consumption, and on the other with the daily average gain, this being very close for allotments M and E3 (2.29 respective 2.36 kg) and bigger 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) and implicitely a daily average gain with 12.65 % bigger than the mark allotment M. The lack of some significant differences between the experimental allotments and the mark allotment can be determined by the low participation quota of the vegetal phytoadditives, as well as to the complete structure of mark CF, with corn, powder milk, etc, which induce performances hard to beat by vegetal additivation. 4. CONCLUSIONS The utilization of the aromatic and medicinal plants drying installation with microwaves in continous flow presents the following advantages: - the heat generated by the microwaves field is manifested constantly in all the product'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 process efficiency and of the final product's quality; - the favourable impact on the environment, the technology being unpolluted unaffecting the ecological equilibrium and conserving the biodiversity of the environment factors. 48 The utilization as phytoadditives of some medicinal plants (Origanum, Angelica, Salvia) in the feeding process of piglets after ablactation, leads to the following conclusions: - the greater fodder daily consumtion, the final body weight and the daily average gain bigger at allotment E2 could indicate Oreganum as taste corrector and aromatizer; - the experimental results support the capitalizing of some vegetal species in piglets food in the ablactating stress period, as fodder additives, being a potential alternative for the modern nutritional management of monogastric animals, representing at the same time economical alternatives, in the exploitation of the local or regional resources. REFERENCES 1. Popa E.:New conservation techniques for vegetal agricultural products. Magazine INMATEH Nr 20, Bucharest march 2007, p. 103-104; 2. Voicu N. Sisteme cu microunde.Editura MATRIX ROM 2004. 3. Jarvis i Aarts: Nutrient management from a farming systems perspective in „Grassland Science in Europe” 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, Feed International, 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. .A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 50 - 56 STUDIES REGARDING THE CONSUMER ACCEPTANCE OF GM FOODS STUDII PRIVIND ACCEPTAREA ALIMENTELOR CU ORGANISME MODIFICATE GENETIC DE CATRE CONSUMATORI CATALINA VOAIDES1, GH. CAMPEANU1, PETRUTA CORNEA1 1 Faculty of Biotechnology, UASVM Bucharest, 59 Marasti Blvd., Bucharest, Romania Key woeds: GMO foods, soybean products, survey, special labeling Cuvinte cheie: alimente cu OMG, produse din soia, sondaj de opinie, etichetare speciala ABSTRACT Labeling of the genetically modified products has become a worldwide spread necessity associated with the decreased trust for the biotechnological products. The aim of this study was to reveal the necessity of a special labeling for products that contain or are made from GMO, so the consumers could choose what kind of products will consume. Also, the obtained experimental results emphasize 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 of 18 questions was used, each question having several possible answers. This study was 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 consumers regarding GM products/GM foods, the willingness to consume GM foods, the importance of the specific and explicit labeling as well as the importance of the price factor in the decision to buy or not to buy GM foods. The consumers’ awareness regarding GM foods reveals that 19% of the questioned group is uninformed, 75% are informed and in some degree informed and only 6% are very well informed (fig. 1). Taking into consideration the age distribution, it can be observed that into the group with age between 18 – 35 years old, only 18% have declared themselves as uninformed, meanwhile, into the group over 35 years old, 25% declared themselves uninformed about the subject. 50 1 . Ina inte de ace st chest iona r, c at de bine infor mat e rati in le ga tur a cu organisme le s au a lime ntle m odificate genetic? 1 9% Fig. 1 - Consumers’ information level regarding genetically modified foods 6% Boccaletti and Moro (2000) report in a study that 51% of the questioned group had heard about GM foods, and 39 % Blaine et al. (2002) reported in a review that among eight foa rte i nformat informat i ntrucatva ne informa t countries those with most knowledge of GM foods were Germany, UK and Japan with 95%, 94% and 89% of respondents having heard of them, respectively. Countries with least knowledge were Brazil, USA and Canada, with 39%, 66% and 78%, respectively. In our local survey regarding customers’ awareness about GMO, 64%, respectively 72% answered correctly at the following questions: “Genetically unmodified soybean does not contain genes, but genetically modified soybean does?”, respectively “By consuming genetically modified products, a person’s genes can be altered?” (fig. 2). 36 % Fig. 2 - Consumers’ knowledge regarding GMO 28 nu stiu 40 108 fals adevarat 97 14 13 Prin consumul alimentelor modificate genetic, genele unei persoane pot fi alterate. From the uninformed Soia nemodificata genetic nu contine gene, in timp ce soia modificata genetic contine. consumers’ category, 45% said that they would avoid consuming food products that contain genetically modified ingredients (fig. 3), and 87% of them consider the special labeling of GMO very important. Almost in the same proportion, the uninformed consumers would stand up for the mandatory labeling of all products, even if they contain or not GM ingredients. 51 14% 45% Fig. 3 - The willingness of uniformed consumers to consume GM foods 24% 17% dispus intrucatva nu foarte dispus din principiu, as evita produsele cu ingrediente modificate genetic In the same context, 98% of the Norwegian consumers and 87% of the US consumers demand the proper labeling of genetically modified foods (Chern and Rickertsen, 2002). These results are similar to the ones obtained in an European survey (2001), where 95% of the responders from 15 EU member countries, that participated to this study, wanted to have the right to choose between genetically modified and genetically unmodified foods. So, it can be observed that our study lines-up with the international needs, regarding the necessity of GMO foods labeling. Another aspect of our survey was to establish the consumers’ attitude regarding GM foods. In this context, 30% of the responders declared “I do not know” 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 alim entele m odificate genetic pentru sanatatea oam enilor? Fig. 4 - Consumers’ attitude regarding GM foods 7% 30% 24% 10% 1% 28% foarte riscante riscante nici una nici alta Also, 35% of the foarte sigure sigure nu stiu uninformed consumers emphasize a negative attitude to 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 this negative attitude (fig. 5). 52 12 nu stiu sigure 1 4 14 Fig. 5 - Consumers’ attitude regarding GM foods and their information level foarte sigure 1 1 nici una nici alta 5 riscante foarte riscante 25 6 4 consumatori neinformati 14 5 consumatori informati si foarte bine informati The responders willingness to consume foods that contain genetically modified ingredients showed that 37% are somewhat or are not very willing to consume them and 32% would avoid their consumption. Taking into consideration 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 willing to consume them. In the same time, 50% of the consumers over 35 years old would avoid to consume foods with GM ingredients (fig. 6). It can be noticed that young people, in generally, are more receptive to new, with good prior informing. 6. Cat sunteti de dispus sa consumati alimente cu ingrediente modificate genetic? din principiu, a evita produsele cu ingrediente modificate genetic 4 2 peste 35 de ani 35 4 foarte dispus 36 5 dispus 28 intrucatva 19 3 nu foarte dispus 14 Fig. 6. - The willingness of the consumers to consume foods with GM ingredients. pana in 35 de ani In the present study, among the participants that declared themselves as not willing or prefer to avoid the consumption of the GM foods, 79% would pay 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 the increased interest for healthier and safer nutrition but it does not reflect the level of informing regarding GMO. 53 Fig. 7 - The price and the willingness to consume GM foods 21% 31% 48% From the possibility of reducing the quantities of da, daca pretul este cu putin mai mare da, chiar daca pretul este mult mai mare pesticides by using GMO point of view, 57% of the responders are somewhat willing to consume them (fig. 8). If genetically modified products would be more valuable from nutritional point of view than similar unmodified products, 28% would still be unwilling to consume them and 13% are uninterested of these aspects (fig. 9). nu 7. Cat sunteti de dispus sa consum ati alim ente m odificate genetic, daca astfel sunt reduse cantitatile de pesticide aplicate culturilor? 17% Fig. 8 - The willingness of the consumers regarding GM foods 13% 13% 34% 23% foarte dispus dispus intrucatva nu foarte dispus din principiu, as evita produsele cu ingrediente modificate genetic 8. Cat sunteti de dispus sa consum ati alim ente m odificate genetic daca ele sunt m ai valoroase nutritional decat cele sim ilare nem odificate genetic? 16% 26% 12% Fig. 9 - The willingness of the consumers regarding GM foods 13% 33% foarte dispus intrucatva dispus nici una nici alta oarecum nu sunt dispus nu sunt deloc dispus 54 In a similar survey of the European Commission, most of the European citizens had a neutral attitude regarding GM foods, from the obtaining technology point of view. An interesting aspect is that the attitude is more 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% of the Norwegian and 55% of the American respondents answered reduced use of pesticides and below 10% answered reduced price, when they were asked which of these potential benefits was most important. More than half of Norwegians found reduced price to be “extremely unimportant” for their decision to buy or not to buy GM foods. For most of the consumers (86%), labeling of the genetically modified foods is very important and for 89% of them would stand up for a mandatory labeling of all products, no matter if they contain or not genetically modified ingredients. The European Commission stated in a previous study (2002) that most of the European consumers would prefer that the information regarding GMO to be available 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 to the conclusion that for 61% of the consumers the price factor is very important or important enough (fig. 10) and 68% of the consumers will pay a higher price for the products labeled as “not genetically modified”. 13. Cat este de im portant factorul pret cand va decideti daca sa cum parati sau nu alim ente m odificate genetic? 19% 21% 6% Fig. 10 - The influence of the price taking the decision to buy GM foods 12% 42% foarte important intrucatva important intrucatva neimportant deloc important nici una nici alta 55 REFERENCES 1. 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 of Food 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 Development Economics. 4. Frewer L., Lassen J., Kettlitz B., Scholderer J., Beekman V., Berdal K.G., (2004), Societal aspects of genetically modified foods, Food and chemical Toxicology 42, p. 1181-1193. 5. European Commission, Eurobarometer 55.2, Europeans, Science and Technology,http://europa.eu.int/comm/public_opinion/archives/eb/ebs_154_en,pdf, 2002. 56 Scientific Bulletin Biotechnology, U. .A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 57 – 63 ANALYTICAL METHODS OF AROMA COMPOUNDS IN GRAPE JUICE OBTAINED FROM ROMANIAN WINE SORTS GALBENĂ DE ODOBE TI AND ARBĂ IDENTIFICAREA AROMELOR PRIN METODE ANALITICE IN MUSTURI OBŢINUTE DIN SOIURILE ROMÂNE TI DE VIŢĂ DE VIE GALBENĂ DE ODOBE TI I ARBĂ LUMINIŢA VI AN, O. POPA, NICOLETA ARON, TH. SEROT Key words: gas chromatography-olfactometry, Romanian sorts, odor analysis Cuvinte cheie: gaz-cromatografie-olfactometrie, soiuri române ti, analiza aromelor ABSTRACT Olfactometric methods frequency of detection, time-intensity method and AEDA that allow to detected and evaluate the same odor-active compounds, were used to evaluate the main odorants of two musts obtained from Romanian wine sorts Galbenă de Odobe ti and arbă. A number of 15 compounds in grape musts from two analyzed varieties using this technique were identified. The volatile compounds identified into the analyzed musts came from different organic derivatives classes: alcohol, aldehydes, and ketones, acids and esters, aromatic hidrocarbures and terpenoids which create the aroma profile of each variety. Among the other compounds, Ethyl-2-hydroxy propanoat, Ethyl-3-hydroxy butanoate, 3methyl butan-1-ol, 2,6-nonadien-1-ol, Phénylacétaldéhyde and Linalol seemed to contribute actively to the odor of Galbenă de Odobe ti must; 3-methyl-propanal, 1-α-terpineol and hexanal were contributors to the arbă odor. Metodele olfactometrice FDT (frecvenţa detectării), TIM (metoda timp-intensitate) i AEDA (analiza diluţiei extractului de aromă) au fost utilizate pentru detectarea i evaluarea principalilor compu i de aromă din două musturi obţinute din soiuri române ti de vinifera pentru vin, Galbenă de Odobe ti i arbă. Utilizând metodele olfactometrice amintite au fost identificaţi 15 compu i volatili de aromă care aparţin diferitelor clase de compu i organici: alcooli, aldehide i cetone, acizi, esteri, hidrocarburi aromatice i terpene, realizând profilul aromatic al fiecărui soi. Compu ii volatili 2-hidroxi-propanoatul de etil, 3-hidroxi-butanoatul de etil, 3-metil butanolul, 2,6-nonadienolul, fenilacetaldehida i linalolul contribuie activ la aroma mustului de Galbenă de Odobe ti iar 3-metil-propanalul, 1-α-terpineolul i hexanalul contribuie la conturarea aromei mustului provenit din arbă. 57 Romania is one of the famous viticultural country in production of the wines: Tămâioasă românească, Fetească albă, arbă, 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 extensively used in aroma research and allow the determination of odor-active compounds in food. Olfactometric methods used: time-intensity methods, detection frequency methods and dilution methods. Dilution methods, Charm analysis and aroma extract dilution analysis (AEDA), are commonly applied and are suitable to screen the odorant compounds in must or wine. The time-intensity method developed to characterize the Pinot noir aroma. The frequency of detection has not been used in the determination of active-odorant compounds in grape wine or must. The most frequently used methods for isolation of flavor constituents from must or wine involve extraction with solvent (3) or freon (4), dynamic headspace analysis or headspace solid phase microextraction (5). Two Romanian white grape were used for this study: Galbenă de Odobe ti (traditional varieties) and arbă (from the Riesling Italian sort). The aim of our study was to characterize the most odor-active compounds in musts obtained from this varieties by using three GC/O analyses of representative extracts obtained by an appropriate liquid-liquid extraction method. 1. MATERIALS and METHODS Juice preparation The grapes from the two sorts were produced in the Valea Calugareasca vitivinicole station. After harvesting (maturity was estimated by: refractometric degree, titrable acidity and berry size), grapes were picked off the bunches, crushed with a hand crusher then pressed at 40 kPa with a laboratory winepress. The musts were homogenized and stored at –20°C prior to extraction of volatile compounds. Extraction of volatile compounds Six liquid-liquid extraction methods were compared in order to optimize the production of representative extracts. Method A: 200 ml of must, placed in a conical flask, were extracted with 5 ml of freshly distilled dichloromethane (CH2CL2) by stirring for 30 min at 0°C and then centrifuged for 15 min at 10000 g. The organic extract was dried with anhydrous sodium sulfate and stored at –20°C until analysis. Method B: the same as method A, but NaCl 20% was added to the must before extraction. 58 Method C: 200 ml of must, placed in a conical flask, were successively extracted (3x20 min) at 0°C with 3x25 ml of freshly distilled dichloromethane and then 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 Danish concentrator (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 must before extraction. Methods E and F: The same as methods C and D respectively, but dichloromethane was replaced by 3 x 25 ml of dichloromethane:pentane (2:1). Sensory analysis/ representativeness of the extracts Sample preparation and presentation. The must samples were thawed overnight at 4°C in a fridge. 5 ml were placed in 15 ml black coded flasks then the temperature 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 strip and after 30s (time necessary for solvent evaporation) the ends of the strips were cut and placed in dark coded flasks. These flasks were hermetically closed and presented to the panel after 30 min. Odor intensity evaluation. The odor intensities of the extracts were evaluated 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 distance in centimeters from the left anchor. Results were analyzed with an ANOVA and LSD test. Gas chromatography/mass spectrometry (GC/MS) The GC/MS system consisted of an HP5890 II gas chromatograph and an HP5971 Mass selective detector (Hewlett-Packard). Each extract was injected in the splitless mode (250°C injector temperature, 30 sec valve delay) into a capillary column (DBWax, 30 m length x 0.32 mm i.d. x 0.5 m film thickness). The flow rate of carrier gas (helium) was 1 ml/min. Oven temperature was programmed from 50°C to 250°C at a rate of 5°C/min with an initial and final hold time of 5 and 10 min 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 multiplier voltage, 2100 V.; and scan rate, 2 sec-1. Volatile compound identification was based on comparison of GC retention indices (RI), mass spectra (comparison with MS spectra database, NBS 75K 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), equipped with an FID and a sniffing port supplied with humidified air at 40°C. GC effluent was split 1/1 between the FID and the sniffing port. Each extract (2 µl) was injected 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 of 59 carrier gas (helium) was 1 ml/min. Oven temperature was programmed from 50°C to 250°C at a rate of 8°C/min. Frequency of detection (FDT) Sniffing of the chromatogram was performed for 23 min. The panelists (10 judges) 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 assessors was considered as noise. The ten individual aromagrams were summed, yielding the final aromagram (detection frequency versus RI). Time-intensity method (TIM) The time-intensity method was used to measure the perceived odor intensity of compounds in GC-O. The same panelists as before were trained to evaluate aroma intensity using a nine point intensity scale (1= very weak intensity, 3= weak intensity, 5= moderate intensity, 7= strong intensity and 9= very strong intensity). Sniffing conditions were the same as for frequency of detection, except that the panelists were also asked to assess intensity (according to a nine point scale) for each odorous area. Times and intensities of areas which were detected by at least three judges were averaged and a consensus aromagram (averages versus RI) was created. Aroma Extract Dilution Analysis The method (AEDA) was used to assess the contribution of individual volatile compounds to the aroma of grape juices. Serial dilutions (1:3 in dichloromethane) of the extract were sniffed until no further odor-active area could be detected. AEDA was performed by two sensitive and repeatable panelists selected during olfactometric global and time-intensity method analyses. The result was expressed as the flavor dilution factor. Data from AEDA were also represented in graphs by plotting FD versus RI. 2. RESULTS and DISCUSSIONS Sensory analysis Similarity test to measure the difference between the odors of the musts and the corresponding extracts obtained the extraction methods (A, B, C, D, E and F) 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 musts and associated extracts are very similar. The ten panelists described the two musts and the three extracts principally by fruity notes such as grape, prune, red fruit, or wine-like odor. 60 Olfactometric analysis Odor active compounds detected in the two musts by three olfactometric methods is to indicate in table 3. According to the three olfactometric methods, 15 odorous areas were significantly perceived by the panelists, in at least one extract. Table 1 Similarity test between the odors of the musts and the corresponding extracts Test de similaritate între aromele musturilor i extractele corespondente Extracts Extract Similarity scale (cm) Scală de similaritate (cm) Galbenă de Odobe ti a Intensity scale (cm) Scală de intensitate (cm) arbă 4.9 a Galbenă de Odobe ti 4.9 arbă a 5.6 a A 5.2 B 6.3 c 6.1 c 4.9 b 5.1 b C 6.7 c 6.2 c 5.1 c 5.7 c D 5.8 a 5.1 a 6.0 b 5.9 b E 4.8 b 4.9 b 5.0 a 4.9 a F 4.1 b 4.1 b 4.6 a 4.3 a Among the odorants perceived, some alcohols and aldehydes were identified. These compounds are known to exhibit grass-like and green odors. They are mainly formed by enzymatic oxidation of polyunsaturated fatty acids. Their concentrations mainly depend on the grape cultivar although there were variations due to geographical origin or to the ripening stage.. Hexanal was perceived with green and grass-like odor mainly in arbă extracts. 1-α-terpineol was perceived only in an extract from arbă; 2,6 nonadien1-ol exhibited a melon, cucumber odor was perceived Galbenă de Odobe ti extracts; 1-octen-3-one, which exhibited a mushroom-like odor, was mainly perceived in Galbenă de Odobe ti; Three compounds exhibited the sweaty aroma notes of phenyl acetaldehyde, ethyl-3-hydroxybutanoate and an unidentified compound. Phenyl acetaldehyde was perceived in an extract from Galbenă de Odobe ti. Phenyl acetaldehyde was not detected in arbă. This compound, which exhibits a sweaty and honey-like odor, caramel and syrup odor has previously been identified in muscadine juice. It may be formed by the decarboxylation of α-ceto-phenylacetate. 61 Table 2 Main odorant attributes to characterize the two musts and the corresponding volatile extracts obtained by method C. Atributele aromelor utilizate pentru caracterizarea celor două musturi i extractele corespondente obţinute prin metoda C Galbenă de Odobe ti grape juice Extract must extract caramel caramel Sweaty, honey honey grape grape wine prune fruity grass, green grass fruity apple apple arbă grape juice must wine fruity prune apple grape grass, green spicy Extract extract prune green, leafy red fruits apple grape woody spicy Table 3 Odor-active compounds detected in the musts of Galbenă de Odobe ti and arbă Compu ii active de aromă detectaţi în musturile de Galbenă de Odobe ti i arbă Peak Pic Volatile Compounds Compu i volatili 2 3 Ethyl-2-hydroxy propanoat Hexanal 3-methyl butan-1-ol 4 1-Octen-3-one 5 Ethyl-3-hydroxy butanoate 6 Phénylacétaldéhyde 7 unknown Ethyl-3-hydroxy butanoate 1 8 9 2,3-butanedione 11 12 13 2-methyl-3-buten-2one (E)-3-hexen-1-ol Linalol 1-α-terpineol 14 2,6-nonandien-1-ol 15 1-Octen-3-one 10 62 Odor description Descrierea aromelor Butterscotch, sweet Grass, green Buttery mushroom, woody hay-like, sweet sweaty, caramel, syrup fruity, sweet hay-like, sweaty caramel, buttery buttery, cannel Green, grass Floral, sweet Floral Melon, cucumber mushroom, woody Olfactometric methods Metode olfactometrice TIM FDT AEDA Galbenă Galbenă Galbenă arbă arbă arbă de de de Odobe ti Odobe ti Odobe ti 9.5 1.1 8.5 0 540 0 0 5.9 4.2 0 0 8.3 3.9 1.1 0 820 0 1.2 3.9 0 4.5 1.1 380 0 8.2 2.2 7.5 1 221 0 4.4 0 3.9 0 1.1 0 1.8 4.2 0 3.2 0 0 1.1 8.4 0 7.9 0 324 0 7.9 0 6.5 0 7.2 1.2 1.9 1.1 3 0 0 2.1 1.1 0 4.9 0 1.8 1.8 1.2 0 3.5 0 1.1 1.1 0 0 323 0 0 4.8 2.1 5.9 1.1 3.1 0 2.3 0 4.7 1.2 2.1 1.1 3. CONCLUSIONS Among the odorants perceived, some alcohols and aldehydes were identified. These compounds are known to exhibit grass-like and green odors. They are mainly formed by enzymatic oxidation of polyunsaturated fatty acids, the concentrations depend on the grape cultivar although there were variations due to geographical origin. Odor-active compounds detected in the musts of Galbenă de Odobe ti and arbă: - Hexanal was perceived with green and grass-like odor only in arbă extracts; - 2,3 butanedione, described by caramel, buttery odor, was perceived in an extract from arbă; - 2,6 nonadien-1-ol exhibited a melon, cucumber odor and ethyl 3hydroxybutanoate was perceived with hay-like, sweaty odor, was perceived Galbenă de Odobe ti extracts; - 1-octen-3-one, which exhibited a mushroom-like odor, was mainly perceived in Galbenă de Odobe ti; - phenyl acetaldehyde which exhibits a sweaty and honey-like odor, caramel and syrup was perceived only in an extract from Galbenă de Odobe ti. REFERENCES 1. Guth, H. (1997): Quantification and sensory studies of character impact odorants of different white wine 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, 259-267 3. Baek, H.H Cadwallader, E. Marroquin, E.and Silva,J.L. (1997): Identification of predominant aroma 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 red wines 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, sulphides and disulphides by headspace solid phase microextraction - gas chromatography. J. Chromtogr. , 849, 293-297. 63 Scientific Bulletin Biotechnology, U. .A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 64 – 68 PROBLEMS OF TECHNOLOGICAL AND CLIMATE NATURE AT RECULTIVATING DAMAGED AREAS OF THE OPEN COAL-MINING IN “MARITSA-IZTOK” IVAN DIMITROV, SSI; SVILEN RAYKOV, SHU e-mail: [email protected] Keywords: seed, coal mining area, climate,. Technology, damaging area, re-cultivation In the region of Maritsa East Mines in open coal mining there are destroyed large areas of arable land. When the operation of the coal basin mop, dumps from above-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 agricultural production or afforestation of forest types it is imposed their restoration through the application of technical and biological reclamation. Technical re-cultivation covers the arrangement of the materials on their eligibility, equalizing the ground and correcting areas. Biological re-cultivation includes the selection of plant sorts, treatments, fertilization, introduction of regular sowing and other agricultural activities (Treykyashki, Hristov, 1982). In carrying out the re-cultivation ever more acute occurred at various problems. The compliance with them is mandatory and finding adequate solutions to overcome them is related to the successful conduct of operations. In the splicing of clay materials and their leveling on according to now accepted practice often are used dumpers and bulldozers, which, because of the great weight and pressure on the soil surface substrate re-seal significantly the soil horizons. A visual idea of the status of the land after technical re-cultivation given the data from Figure 1 for the starting bulk density of the area planned for humus less biological re-cultivation. From the made experiments it was revealed that it is imperative to correct the estimates of technical re-cultivation. The alignment of the land instead of the bulldozer should be done with grader and the embanking of materials must be performed only by the soil moisture content at which the additional sealing area is reduced to minimum size. This moisture content varies from 14% to 19% by soil weight depending on the texture of clay materials. 64 Depth (cm) control variant variant variant Bulk density (g/cm3) Fig. 1 - Bulk density in the betting experience on talus area Mednikarovo Another problem of technological nature is that there is no stability of the micro-relief of re-cultivated land. In order the effects of alignment on the ground will not be short term there is necessary the bulk clay material to be with a humidity below 60% of OPH and the splice of the layers to be accompanied by gradual rolling of fresh filled in area, not simultaneously. Less attention is paid to the evaluation of granule-metric composition of materials from dumps and namely it just lays down significantly the dynamics of many processes that give the appearance of fertility. The major problem in the biological re-cultivation of land is a scarcity of soil humus material. Deposited quantities of soil humus horizons of arable land covered by the coal industry can not cover about 40 percent of the estimates for carrying out biological re-cultivation in humus method used so far. There are necessary new technological solutions for making humus-less re-cultivation in which humus soil does not use or its participation in the soil substrate is negligible. But particularly in the biological re-cultivation by humus-less method the problems of organizational issues, technology and agro-environmental 65 reasons increase. The final stage of technical re-cultivation (grounding level, introduction of substrates and their mixture with clay) as the agro-technique during biological recultivation in a 10-20 year period thereafter, are crucial in humus-less methods to restore soils. According to some researchers (1,4,5,7), poor physical conditions are identified as most limited factor for the successful cultivation of agricultural crops on mine dumps and their optimization problems can be resolved with providing adequate methods for their melioration at high values of their sealing or moving to other methods of technical re-cultivation. Compact humus-less re-cultivated soils have no continuous network of macro-pores, which facilitate the movement of water, aeration and growth of the root system of crops. They are deprived of valuable agronomic structures of natural soils, which are created by decades. Until then it should be done through the processing of soil. Rainfall (mm/m2) Av. 10 years 2002 2001 2000 1999 Month Fig. 2 - Average rainfall During the past 10-15 years another problem attended the biological reclamation is relatively intensive aridization of the climate which leads to the inability 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 66 four years of the experiment with technology humus-less re-cultivation. With the exception of 2002 summer droughts are quite tangible, and in 2000 for July and August there is no receipt of rainfall water as the water stock in the 0-80 cm layer is about 1.5 times less than that in May 2002 (Table 1). Table 1 Water stock (Lt) in layers up to 80 sm of the soil substrates Layer depth in cm 0-10 10 - 20 20 - 30 30 - 40 40 - 60 60 - 80 Amount Soil substrates variants 2 3 4 2000 15 13 12 10 21 18 20 16 23 17 19 20 25 22 21 22 54 52 52 50 51 54 55 52 189 176 179 171 1 Soil substrates variants 2 3 2002 27 23 23 30 30 29 32 32 33 31 31 29 62 63 60 64 63 65 245 242 239 1 4 19 23 25 25 47 48 186 As a result of drought in one of the options with the soil substrate containing 50 m3/da layer of ashes in supporting layer as a result of hydrophobia properties 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 of the roots of cultivated plants in depth. Changed climate conditions require the development of a new structure of crops used for reclamation. Crops such as sunflower, corn and spring peas, widely used in agricultural production in the region can not successfully develop their reproductive capacity and obtained yields of grain and green mass are uneconomic for manufacturers. Another problem arising from the soil water deficit is the quality of presowing preparation areas for winter cereals. As a result of deteriorating physical condition of the soil it can not be reached the desired depth of processing and structure of soil aggregates and the use of siderite crops in full ploughing plant residues and creating conditions for their rapid decomposition. From the analysis of conditions for reclamation for agricultural purposes in the region of "Maritsa East" and the prospects for its successful development by humus-less means it was established that there are many problems, the most significant of them is the importance of technological nature and climate . It is advisable to make adjustments to technological operations in technical and biological re-cultivation. It is necessary to develop a specific system of farming for purposes of biological reclamation as develop technologies for growing crops 67 siderites, structure and sorts field crops grown subsequently tailored to the changed climatic conditions and criteria to maintain the parameters of the soil substrates in the optimal range according to the requirements of crops. Any agro-melioration event failed to conform to the specific conditions leads to the risk of failure of the whole technology of re-cultivation of damaged areas of open coal mining area in the Maritsa East. REFERENCES 1. Banov, M., B. Hristov, S. Ruseva, 1992. General physical and hydro-physical properties of recultivated lands 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 built with 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 in the biological reclamation Maritsa East Mines, fund Minproekt EAD / NIS - LTU, Sofia. 4. Motorina V.L. 1973. К разработке теоретических основ рекультивации земель. В СИМПОЗИУМ "Разработка способов рекулътивации ландшафта, нарушенного промышленной деятельностью Development of theoretical bases of lands re-cultivation. In SYMPOSIUM " Development of re-cultivation methods of landscape broken by industrial activity", 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 mold coverage 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. .A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 69 – 78 ANETHI AETHEROLEUM: CHEMICAL COMPOSITION AND BIOLOGICAL EFFECTS - review ANETHI AETHEROLEOUM: COMPOZITIE CHIMICA SI EFECTE BIOLOGICE - review ALINA ORTAN1, MARIA LIDIA POPESCU2, CRISTINA DINU-PIRVU2 1 University of Agricultural Sciences and Veterinary Medicine, B-dul Marasti nr. 59 sector1, Bucharest, Romania. [email protected] 2 University of Medicine and Pharmacy, “Carol Davila”, Str. Traian Vuia nr 6, sect 2, Bucharest, Romania. INTRODUCTION Anethum graveolens (dill, Apiaceae) is indigenous to the Mediterranean region, southern Russia, and cultivated throughout Europe, as well as North and South America. The main compounds are volatile oil, phthalides, coumarins, flavones and polyphenol acids. The fruit of the dill plant has an antispasmodic effect on the gastrointestinal tract and bacteriosatict effect. Dill herb is used for prevention and treatment of diseases of the gastrointestinal tract, kidney and urinary tract, for sleep disorders and for spasm [1]. Anethi aetheroleum is the volatile oil extracted by hidrodistilation or steam distilation of leaves, fruits or flowers of Anethum graveolens, dill (Apiaceae). PHISICAL – CHEMICAL CHARACTERISTICS Anethi aetheroleum is of a pale yellow color, with an odour of the fruits and a hot, sweetish, acrid taste. By keeping, the color becomes darker and the taste pungent. Dill essential oils differ in odour and taste, depending on the part of the plant from which they were extracted. Dried leaves produce a volatile oil much less pregnant in odour. The leaves yields about 0,4 - 0,8% oil, meanwhile in fruits the oil contents is significantly increased: 2,3 – 8 % [2]. In case of flowers, there are no data presented in the consulted scientific literature. The specific gravity varies between 0,895 – 0,915, English distilled oil usually having the highest specific gravity, from 0.910 to 0.916. The oils from both Japanese and Indian dill differ from European dill oil, in having a higher specific gravity (0,948 – 0,968), which is ascribed to the presence of dill apiol and in containing much less carvone than the European oil. 69 The refractive index of dill volatile oil is 1,483 – 1,488 at 25ºC, optical rotation +70 - +80º, boiling point 155 – 260 ºC. It is soluble in 3 parts of alcohol (90%). EXTRACTION METHODS The 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 steam distillation and the majority of essential oils used in aromatherapy are distilled in this way. Steam distillation is a special type of distillation (a separation process) for temperature sensitive materials like natural aromatic compounds. Many organic compounds tend to decompose at high sustained temperatures. Separation by normal distillation would then not be an option, so water or steam is introduced into the distillation apparatus. By adding water or steam, the boiling points of the compounds are depressed, allowing them to evaporate at lower temperatures, preferably below the temperatures at which the deterioration of the material becomes 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 to facilitate the extraction. During the first 2 hours, the distilled oil has a high content of 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, it is distilled more easily. This ratio is nevertheless decreasing in time. CHEMICAL COMPOSITION Several studies presented scientific data concerning the composition of the volatile 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 chemical composition 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%), 3,9epoxy-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-carvyl acetate (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 other compounds 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 other compounds in traces. In conclusion, Anethi aetheroleum obtained from fruits is characterized by the presence of carvone (18 – 81%, depending on the soil, variety and extraction method), 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 is characteristic to leaves (2 – 10%). The chemical structures of the main components of volatile oil are presented in figures 1, 2, 3. O S-carvona O R-carvona Fig. 1 - Chemical structures of the enantiomers of carvone Fig. 2 - Chemical structure of limonene O Fig. 3 - Chemical structure of anethofuran FACTORS AFFECTING THE QUALITY OF THE ESSENTIAL OIL Volatile oils existing on the market, for aromatherapy, cosmetic products industry, perfumery and alimentary industry have different qualities (different 71 content in main compounds) depending on a lot of factors, such as geographical area, harvesting season, growing conditions and isolation procedure [5, 6, 7]. Species. The importance of the accurate naming of the essential oils used in aromatherapy, by noting the precise species and variety prevents the possible adverse effects or toxically phenomena, which can occur in case of some ambiguities. For example, under name of Anethi aetheroleum we can find the volatile oils from Anethum graveolens and Anethum sowa (Indian dill), which present a significant different content in monotherpenic cetones, which have a neurotoxical and abortive activity. Vegetative stage. Pino et al [8] showed that there are differences in the composition of essential oils extracted from Anethum graveolens harvested in different stages of plant’s growth. It has been shown that withered, drid leaves have a lower content of carvone than the fresh ones; this is not happening to the oil of Anethum fructus, in which the content of carvone is higher in dried, stored fruits. In this 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 progressively increases with growth and is 0.78–0.99% at flowering, rises to 1.28–1.91% at milky-wax seed ripening and 1.9–2.84% in the herb when the seed is nearing maturity. Extrinsic factors (environmental and growing of aromatic plants) also determine 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 and Spain has a higher content of phelandrene than the one from Germany; the african dill 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, the European dill develops higher oxygenated compounds in the oil, which includes a small 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 more sunlight hours combined with solar intensity in the tropics. COMPOUNDS INFLUENCING FLAVOUR In trade, the oils containing 20% or less carvone have been found to be of finest 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 αphellandrene, limonene, myrsticin and dill furan were the most significant contributors of dill herb aroma [15]. Later, Blank et al. (1991) determined that the aroma of dill herb was directly related to concentration of five components namely dill-furan, α-phellandrene, limonene, myrsticin and p-mentha dienbutyrate [16]. 72 The volatile dill oil extracted from fruits yields high quantities of carvone, which is the main constituent responsible for the aroma, but 4-vinyl-2metoxiphenol (which confers the aroma of flavored meat), 4-hydroxi-3-methil-6lmethilethil ciclohexanol (responsible of sweet note) and anethofuran strongly contribute to the oil characteristic aroma. QUALITY INDICES AND STANDARDS The presence of a minimum of 5.0% 3,9,epoxy-p-menthene in dill oil is a good indicator of its purity. Lawrence [17] has opined that examination of the percentage ratio of, α-phellandrene to limonene to β-phellandrene is another test for quality determination; the acceptable ratio was found to be 20:25:3 in dill herb oil. Dill herb oil is easily differentiated from caraway oil as carvone content in the 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, partially oxydised, fruits oil. ACTIVITY Several studies have been performed in order to establish the antibacterial, antifungal, antiviral, anti-inflammatory, antioxidant properties of the essential oils in general and of dill volatile oil in particular. • Antimicrobial activity (antibacterial and antifungal) The activity of Anethi aetheroleum have been demonstrated against a wide variety of microorganisms, including Gram-positive and Gram-negative bacteria. The antimicrobial activity has been attributed to a number of terpenoid and phenolic compounds [18, 19, 20, 21] and correlated with the proportions in which they are present and the interactions between them [22]. Additive, antagonistic, and synergistic effects have also been observed between components of volatile oils [23]. Still, there are often important differences in the antimicrobial activity of the same volatile oil. This variability can be atribute to the geografic areas, harvesting time, genotype, climate, the part of the plant; all these factors can influence chemical composition and relative ratios of the components of the essential oil [24, 25, 26, 27, 28, 29]. Some constituents of volatile oils showed a higher antimicrobial activity when tested alone [30, 31, 32]. Delaquis et al. [33] examined 73 the antibacterial activity of crude oils and the distilled fractions of dill (Anethum graveolens L.) against some common Gram-positive and Gram-negative food spoilage bacteria: Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Serratia grimesii, Enterobacter agglomerans, Yersinia enterocolitica, Bacillus cereus). Results showed that crude dill volatile oil had weak antimicrobial activity, while distilled fractions of dill essential oil contained higher 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 of Anethum graveolens essential oil and they concluded that the volatile oil completely inhibited the growth of Fusarium graminearum at 6 L dose. Moreover, using poison food technique, the essential oil was found to be highly effective for controlling the growth of Penicillium citrinum and Aspergillus niger. In antibacterial investigations, using agar well diffusion method, essential oil has shown 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 the yeasts Saccharomyces cerevisiae and Candida albicans [35]. In case of the main component of Anethi aetheroleum, carvone, the antibacterial and antifungal potentials have been demonstrated (antifungal effect on Fusarium sulphureum, Phoma exigua var. foveata and Helminthosporium solani [36, 37]. Moreover, some researchers consider that natural d-carvone has the advantage of no toxicity on human, unlike the synthesis one [38]. In a work carried out by Aggarwal et al. [39] it has been demonstrated that both optical isomers of carvone were effective against a wide spectrum of human pathogenic fungi and bacteria; additionally, the antimicrobial properties of these monoterpenes were similar to those of the oil in which they were present. Friedman et al [40] showed that d-carvone presented activity against Listeria monocytogenes. In a study of growth inhibition of E. coli O157:H7, Salmonella typhimurium and Photobacterium leiognathi, d-carvone was less inhibitory than carvacrol, thymol and trans-cinnamaldehyde [41]. Carvacrol and thymol disintegrated the outer membrane 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 of colonies of food-borne pathogens. In a study performed on Candida albicans, usind carvone as an inhibitor agent, it was demonstrated that carvone inhibits the transformation of C. albicans from a coccus to a filamentous form, which is associated with C. albicans pathogenicity, and thus carvone can be a potentially good therapeutic agent against infections caused by this fungus [43]. 74 • Antitumoral activity In a study where a series of carvone-related compounds was assessed for their ability to induce increased activity of glutathione S-transferase in several tissues of A/J mice [44], showed that d-carvone exhibited the highest activity as an inducer in all of the tested tissues. Carvone and its related compounds were proved to be a class of potential chemopreventive agents, since the anticarcinogenic activity is correlated with the ability to induce increased activity of detoxifying enzymes. D-limonene and d-carvone reduced, in female A/J mice, forestomach tumor formation by about 60% and pulmonary adenoma formation by nearly 35%, due to their capacities to inhibit N-nitrosodiethylamine-induced carcinogenesis [45]. • Other activities (on potatoe sprouting, insecticidal activity) Studies performed by Hartmans i Diepenhorst [46] showed that, when compared to the traditional chemical mixtures of isopropylphenylcarbamate and isopropyl-3- chlorophenylcarbamate, carvone was as good or even better during long-term storage,as a potato sprouting inhibitor. Carvone is already commercialised in The Netherlands under the name ‘‘Talent’’ as an effective sprout growth regulator [47]. Carvone can be used as an insecticide against the fruit fly, Drosophila melanogaster, although constituents of mint oil show higher insecticidal and genotoxic activities [48]. UTILIZATION The previously presented activities of Anethum graveolens volatile oil conduct 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 and digestive 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), in different pharmaceutical formulations, conferring to all, besides pleasant smell and aroma, its therapeutically properties. In cosmetic and perfumery industry, dill oil is used in preparation of hygienically-sanitary products, creams, emulsions, lotions, gels, products with preventive 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 i α–pinene, it can be used in synthesis of terpin-hydrat, α-terpineol, linalol, borneol products. PRECAUTIONS AND ADVERSE REACTIONS Photodermatosis appearance is possible after contact with the juice of the freshly harvested plant; this could be explained by the presence of furanocoumarins in the oil. Precautions: babies, children and pregnant women (neorotoxic and abortive because of the cetones content). REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 76 XXX – PDR For Herbal Medicines, Third edition, ed Thomson, Montvale, 258 (2004). C.C.R. de Carvalho, M. M. R. da Fonseca – Carvone: why and how should one bother to produce this terpene. J. of Food Chemistry, 95, 2006, p 413-422 P.Schreier – Biogeneration of plant aromas. Developments in food flavours, Elsevier, New York, 1986, 89-106 B.M.Lawrence – A review of the world production of esssential oils. Perfum. Flavor., 10, 1985, 1-16. H.Maarse – Volatile copounds in food and beverages, CRC Press, 1991, 463-464 A Koedam, J. J. C. Scheffer and A. Baerheim Svendsen - Essential Oil Of Dill Herb. Chem. Mikrobiol. Technol. Lebensm.,. 6, 1 1979, 125. 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. J. Pino, A. Rosado, I. Goire, E. Roncal – Evaluation of flavour characteristics compounds in dill herb essential oil by sensory analysis and gas chromatography. J Agric. Food Chem, 1995, 43, 1307 – 1309. V. Masotti, F. Juteau, J.M.Bessiere, J. Viano – Seasonal and phenological variations of the essential oil from the narrow endemic species Artemisia molinieri and its biological activities. J. Agric. Food Chem, 51, 2003, 7115-7121. A. Angioni, A.Barra, V. Coroneo, S.Dessi, P.Cabras – Chemical composition, seasonal variability and antifungal activity of Lavandula stoechas L. Ssp. Stoechas essential oil from stem/leaves and flowers. J. Agric. Food Chem, 54, 2006, 4364-4370. A. Koedam, N. Margaris, D. Vokou – Aromatic plants: basic and applied aspects. Kluwer Acedemic publisher, 1995, 135. Baslas, R.K., Gupta, R. and Baslas, K.K. (1971) - Chemical examination of essential oil from plants of genus Anethum (Umbelliferae): Oil of seed of Anethum graveolons. (Pt I) Flavour Ind. 2(4), 241–5. 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, Regional Research Laboratory, Jammu, pp. 545–8. E. Guenther - The Essential Oils. Van Nostrand Co, New York, 1950, pp. 619–34. R. Haupalehti - Gas chromatographic and sensory analysis in the evaluation of dill herb (Anethum graveolons L.) Lebensmitt. Wiss. Technol., 1986, 19, 27–30. I. Blank, W. Grosch - Evaluation of potent odorants in dill seed and dill herb Anethum graveolens L.) by aroma extract dilution analysis. J. Food Sci. 1991, 56(1), 63–7. B.M.Lawrence – New trends in essential oils. Perfum. Flavor., 5, 1980, 6-16. 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 properties of 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 oil components 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 on selected 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 of plant 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 the essential 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 the essential oils of Thymus herba-barona Loisel growing wild in Sardinia. Journal of Essential Oil Research, 2000, 12, 516–522. 29. McGimpsey, J. A., & Douglas, M. H. - Seasonal variation in essential oil yield and composition from 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 components against 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 minimum inhibitory 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 comparison with 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 and mixed 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, Antimicrobial Investigations, and Antioxidative Potentials of Anethum graveolens L. Essential Oil and Acetone Extract. 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 stored dill (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. Potato Research, 1994, 37, 445–446. 37. Hartmans, K. J., Diepenhorst, P., Bakker, W., & Gorris, L. G. M. - The use of carvone in agriculture – sprout suppression of potatoes and antifungal activity against potato-tuber and other plant-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 potato sprout 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 the oils 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 oils and some of their isolated constituents against Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, 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 Listeria monocytogenes. Letters in Applied Microbiology, 1999, 29, 166–170. 43. McGeady, P., Wansley, D. L., & Logan, D. A. Carvone and perillaldehyde interfere with the serum-induced formation of filamentous structures in Candida albicans at substantially lower concentrations 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-Stransferase activity 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-nitrosodiethylamine carcinogenesis 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. Potato Research, 1994, 37, 445–446. 47. Hartmans, K. J., Lenssen, J. M., & de Vries, R. G. Use of talent (carvone) as a sprout growth regulator 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 Food Chemistry, 1997, 45, 2690–2694. 78 Scientific Bulletin Biotechnology, U. .A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 79 - 86 SHORT REVIEW ON APPLICATION OF ION-SELECTIVE SENSORS FOR HEAVY METAL IONS IN FOOD AND ENVIRONMENTAL SAMPLES O SCURTA PREZENTARE ASUPRA APLICARII SENZORILOR IONSELECTIVI PENTRU DETERMINAREA IONILOR METALELOR GRELE DIN ALIMENTE SI PROBE DE MEDIU ALINA CULEŢU, ALINA CATRINEL ION, ION ION Key words: ion-selective electrodes, sensors, heavy metals Cuvinte cheie: electrozi ion-selectivi, senzori, metale grele ABSTRACT Chemical sensors are analytical instruments used for measurement of different ions in various chemical, clinical or environmental samples. One of the most common classes of electrodes is the one based on ionophores covering a broad area of organic or inorganic compounds that bind to cations and anions. Senzorii chimici sunt dispozitive analitice folosite pentru masurarea diferitilor ioni din diverse probe chimice, clinice si de mediu. Una din cele mai cunoscute clase de electrozi este cea bazata pe ionofori ce acopera un domeniu larg de compusi organici si anorganici care leaga cationi si anioni. Ion-selective electrodes (ISEs) are part of a group of relatively simple and inexpensive analytical tools which are commonly referred to as sensors, the pH electrode being one of the most well known and simplest member of this group. The good sensitivity, selectivity and speed of electrochemical sensors make them applicable to the determination of food ingredients, as well as trace compounds or contaminants in different foods and drinks. Heavy metals are very toxic environmental pollutants, thus the knowledge of their real content in various matrices is very important. Such elements tend to concentrate in all matrices in the environment involved in foods and food chain because of their irreversible toxic effects on man. The standard techniques for trace heavy-metal analysis include: Atomic Absorption Spectrometry (AAS) and Inductively Coupled Plasma-Mass Spectrometry (ICPMS). However, these methods require expensive equipment, 79 which cannot be used in the field. Moreover, all of the methods involve complicated and time-consuming sample treatment and pre-concentration steps. Electrochemical methods are seen as complementary to the above mentioned techniques, and are especially attractive because they allow the possibility of creating inexpensive and portable instrumentation. In recent years, the development of novel sensors for the detection of heavy metals ions has been motivated by controlling the levels of environmental pollutants in natural waterways and potable water. Potentiometric detectors based on ion-selective electrodes are suited, because they offer advantages such as high selectivity, sensitivity, good precision, simplicity, non destructive analysis, ability to monitor ion activity without extensive preparation of sample and low cost. GENERAL PRESENTATION OF ISEs Chemical sensors are miniaturized analytical devices, which can deliver real-time and on-line information on the presence of specific compounds or ions in complex samples. Usually the recognition process of the analytes takes place followed by the conversion of chemical information into an electrical or optical signal. External Reference Electrode Internal Reference Electrode ION SELECTIVE ELECTRODE Internal electrolyte Membrane Sample solution Fig.1 – Shematic diagram of an ion-selective potentiometric cell 80 Electromotive force, mV Among various classes of chemical sensors that one of ion-selective electrodes is the most frequently used. Potentiometric sensors are used everywhere from laboratory analysis till industry, process control, physiological measurements, and environmental monitoring. The ion-selective membrane is the key component of all potentiometric ion sensors. It establishes the preference with which the sensor responds to the analyte in the presence of various interfering ions from the sample. In figure 1, a schematic diagram of an ion-selective potentiometric cell assembly is presented. Using a series of calibrating solutions, the response curve or calibration curve 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 way is shown in figure 2. anion interference cation interference log a Fig.2 – Typical calibration curve of ISE The linear range of the calibration curve is usually applied to determine the activity of the target ion in any unknown solution. ISE MEMBRANE COMPOUNDS The polymeric membrane is used to separate the test solution from the inner compartment of the electrode, containing a solution of the target ion. Any polymeric membrane ion selective sensor consists of some components. The nature and the amount of each component illustrate great effects on the nature and the 81 characteristics of the sensor. These components are: the polymeric matrix, the ionophore (membrane - active recognition), the membrane solvent (plasticizer) and the ionic additives. The polymeric matrix The application of polymers as homogeneous membrane matrices was firstly suggested for use with charged carriers in 1967 [1]. The first polymeric ISE membranes, where the polymer was considered to provide the required physical properties, like elasticity and mechanical stability, were prepared in silicone rubber or polyvinyl chloride (PVC) [2]. The ionophore (membrane-active recognition) The ionophore or the membrane-active recognition can be an ion exchanger or a neutral macrocyclic compound, having molecule-sized dimensions and containing cavities or semi-cavities to surround the target ions. The ionophore, also named “ion carrier” is the most significant component of any polymeric membrane sensor with reference to the selectivity and sensitivity, since the molecular-level phenomenon reflected by the ISE is the binding between the ionophore and the target ion. Many cyclic and linear macromolecules have been introduced as ionophores in potentiometric membrane sensors for various cations and anions [3]. Crown ethers, polyamines, calixarenes, metalloporphyrines are used as ionophores in various sensors [3]. The membrane solvent (plasticizer) Plasticizers are additives that increase the plasticity or fluidity of the material, to which they are added. Solvent polymeric membranes, used in ion sensors, are usually based on a matrix containing about 30 - 33% (w/w) PVC and 60 - 66% from a membrane solvent [4]. Films with such a high plasticizer amount demonstrate optimum physical properties and ensure relatively high mobilities of their constituents. In order to give a homogeneous organic phase, the membrane solvent must be physically compatible with the polymer in order to display plasticizer properties. For various reasons, it also has an influence on the selectivity behavior. Some of the common plasticizers are: benzyl acetate, bis(2- ethylhexyl) phthalate (dioctyl phthalate), bis-octyl sebacate, dibutyl phthalates, dibutyl sebacate and 2 nitrophenyl phenyl ether [5]. Ionic additives Ionic additives are ion exchangers, which themselves induce a selective response when no or only an insufficient ionophore amount is present. Therefore, their concentration must be adjusted carefully. Although the neutral-carrier-based ISE membranes may work properly, even when they contain only a very small amount of ionic sites, the addition of a lipophilic ion salt is advisable and beneficial for various other reasons, as well [6]. 82 THE PREPARATION OF MEMBRANE ISE A typical procedure to prepare the PVC membrane [4] is to mix thoroughly 30 – 35 mg of the powdered PVC, 60 – 65 mg plasticizer, the suitable ionophore amount (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 an inner diameter of 20 – 50 mm, resulting in a smooth glass plate. THF is evaporated at room temperature. A transparent membrane is formed. A disc with a 10 mm diameter is cut out from the PVC membrane and glued to the one end of the Pyrex glass tube. Afterwards, the tube is filled with the internal filling solution and is conditioned for 12 – 48 h by soaking in a solution which contains the analyte. PROPERTIES OF ISEs The properties of an ISE [7] are characterized by parameters like: measuring range, detection limit, response time, selectivity, lifetime. Measuring range The linear range of the electrode is defined as that part of the calibration curve through which a linear regression would demonstrate that the data points do not deviate from linearity by more than 2 mV. Detection limit The detection limit of an ISE is calculated using the cross-section of the two extrapolated linear parts of the ion-selective calibration curve, according to the IUPAC recommendation [8]. Response time It is defined as the time between the instant at which the ion-selective electrode and a reference electrode are dipped in the sample solution and the first instant at which the potential of the cell becomes equal to its steady-state value within 1 mV. Selectivity Selectivity is the most important characteristic of these devices. It describes the ISE specificity toward the target ion in the presence of other ions, also called as “interfering ions”. Lifetime The average lifetime for most of the reported ISEs is in the range of 4 - 10 weeks. After this time, the detection limit of the sensor will decrease. It is accepted that the loss of plasticizer, carrier or ionic site from the polymeric film, as a result of leaching into the sample, is the primary reason for the limited lifetime of the carrier-based sensors. 83 APPLICATIONS OF ISEs FOR DETECTIONS OF SOME HEAVY METALS Ionophore – incorporated PVC–membrane sensors are analytical tools used for the selective and direct measurement of a wide variety of different ions in complex biological and environmental samples. The key ingredient of such plasticized PVC-membranes is the involved ionophore, defining the selectivity of the electrodes complex formation with the cation of interest. Until now, a large number of ionophores with high selectivity for specific metal ions have been developed for the use in potentiometric sensors selective for the respective metal ions [5]. Lead selective membrane sensors Because of the increased industrial use of lead and its serious hazardous effect to human health, the preparation of the lead ion selective membrane electrodes have been wide investigated. Examples of some lead ion selective sensors with biological or environmental applications are in: water samples [9], minerals [10], oil samples [11], etc. Copper selective membrane sensors Due to the vital importance of copper in many biological systems and the urgent need for a copper-selective electrode for potentiometric monitoring of Cu2+ in environmental, medicinal and different industrial samples, there has been increasing interest in the development of novel membrane sensors for the detection of copper ion, during the past. Some of the copper ion selective sensors with biological or environmental applications are in milk powder samples [12], black tea samples and multivitamin capsules [13]. Cadmium selective membrane sensors Cadmium is an extremely toxic metal commonly found in industrial workplaces, particularly where ores are processed or smelted. The effect of its acute poisoning is manifested in a variety of symptoms, including high blood pressure, kidney damage, anemia, hypertension, bone marrow disorders, cancer and toxicity to aquatic biota [14]. Some of reported cadmium ion selective sensors with biological or environmental applications are in water samples [15], aqueous solutions [16], soils [17], etc. Chromium selective membrane sensors Chromium is known to be an essential element (Cr3+) in the human nutrition and a toxic one in the hexavalent state, therefore its accumulation in the human body results in toxicity, chromium in hexavalent state being 100 – 1000 times more toxic than the Cr3+. Some of the reported chromium ion selective sensors with biological or environmental applications are in: waste water and alloy samples [18], some food materials and various types of plants [19]. 84 Nickel selective membrane sensors Nickel is a silvery white metal that takes on a high polish. For many decades, nickel was regarded as a potentially toxic element, since its concentration in various foods was higher than that needed for living organisms. More recently [20], it is now considered a possible essential element for plants, although deficiencies can occur under certain circumstances. However, nickel can be toxic at high concentrations and can be a problem in some soils [21]. Nickel ion selective sensors are used in biological and environmental analysis, for example in the determination of nickel content of chocolate [22], milk powder samples [23], hydrogenated oils [24], industrial waters [25]. CONCLUSIONS Ion-selective electrodes represented in the past and are still developed as simple and efficient analytical tools for environmental and biological samples, due to their advantages: rapidity and simplicity in operation and preparation, low cost, comparatively fast responses, very low detection limit, wide dynamic ranges and satisfactory selectivity. It must be emphasized that their use is now increasing in speciation studies in combination with AAS, because of the possibility of determination of the concentration of the free metal ion (using ISEs) and the total one (using AAS). This short review proposes a short approach of the characteristics and application of ISEs for heavy metal ions which are in progress due to the new ionophores possible to be used in electrodic membranes. REFERENCES 1. 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): Supramolecular Based Membrane Sensors. Sensors, Vol. 6: 1018 – 1086. 4. Moody, G. J., Oke, R. B., Thomas, J. D. R. (1970): A calcium-sensitive electrode based on a liquid ion 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 Bulk Optodes. 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 on the phase boundaries and the significance of ionic sites of liquid membrane ion-selective electrodes. Electroanalysis. Vol. 7: 811 – 816. 85 7. Lindner, E., Umezawa, Y. (2008): Performance Evaluation Criteria for Preparation and Measurement 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): Recommendations for 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 membrane electrode 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 selective potentiometric 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)-selective electrode 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 membrane electrode 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. Environ Health Perspect. Vol. 54: 213–218. 15. Singh, A. K., Mehtab, S., Singh, U. R., Aggarwal, V. (2007): Comparative studies of tridentate sulfur and nitrogen-containing ligands as Ionophores for construction of cadmium ion-selective membrane sensors. Electroanalysis, vol. 19: 1213 –1221. 16. Mashhadizadeh, M.H., Sheikhshoaie, I., Saeid-Nia, S. (2005): Asymmetrical Schiff bases as carriers 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 Application of a Cadmium-Selective Microelectrode for the Measurement of Cadmium Fluxes in Roots of Thlaspi Species and Wheat. Plant Physiol. Vol. 116, 4: 1393–1401. 18. Gholivand, M.B., Raheedayat, F. (2004): Chromium (III) ion selective electrode based on oxalic acid bis (cyclohexylidene hydrazide). Electroanalysis. Vol. 16: 1330-1335. 19. Khalil, S., Wassel, A. A., Belal, F. F. (2004): Coated graphite-epoxy ionselective electrode for the determination 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 in some 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 potentiometric sensor 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 in coated-wire ion-selective electrode for nickel (II). Sens. Actuators B-Chemical, Vol. 82: 259264. 24. Kumar, K.G., Poduva,l R., Augustine, P., John, S., Saraswathyamma, B. (2006): A PVC plasticized 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 for potentiometric dosage of copper and nickel. J. Serbian Chem. Soc., Vol. 70: 269-276. 86 Scientific Bulletin Biotechnology, U. .A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 87 - 96 COMPLEX RESEARCH REGARDING THE PREPARATION AND CARACTHERIZATION OF SOME MICROCAPSULES OF ETHYLCELLULOSE CERCETĂRI COMPLEXE PRIVIND PREPARAREA I CARACTERIZAREA UNOR MICROCAPSULE DE ETILCELULOZĂ RĂZVAN PRISADA1, CRISTINA DINU-PÂRVU1, ALINA ORŢAN2 1.University of Medicine and Pharmacy ,,Carol Davila“,Faculty of Pharmacy, 6 Traian Vuia Street, Bucharest 2 University of Agricultural Sciences and Veterinary Medicine, B-dul Marasti nr. 59 sector1, Bucharest, Romania Key words: microcapsules, ethylcellulose, xantinol nicotinate Cuvinte cheie: microcapsule, etilceluloză, xantinol nicotinat ABSTRACT Una dintre metodele de a realiza preparate cu eliberare modificată este microîncapsularea substanţelor medicamentoase.Acest procedeu poate fi utilizat si pentru modificarea aspectului unui produs, izolarea unor compu i reactivi, mascarea mirosului sau gustului unor substanţe sau cre terea stabilităţii unui produs. In acesta lucrare, prezentăm rezultatele unui studiu complex asupra unor microcapsule de etilceluloză cu xantinol nicotinat.Propunem o modalitate de preparare i caracterizăm fizico-chimic microcapsulele. One of the methods used for modifying the release of drugs is microencapsulation. This method 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 are presenting the results of a complex research regarding some ethylcellulose microcapsules with xantinol nicotinate. We are proposing a method of preparation and we are carrying out the complex characterization of the microencapsulation process. The microencapsulation processes can be performed by methods using either the evaporation of the solvent, or the emulsification in mineral oil. The release of the drug from microcapsules depends on the structure of their walls and the polymeric material used for preparation. In the experimental study we assessed that using the Jaselniack method (1), eventually in a modified way, as well as using the ethylcellulose as the polymeric material would correspond for the projection, 87 preparation and further characterization of some tank microcapsules with xantinol nicotinate (2, 8). 1. MATHERIAL and METHOD • • • Xantinol nicotinate (Merck); Ethylcellulose(Fluka) containing 48 percent etoxy radicals, characterized as a white powder, tasteless, odorless, melting point between 155-162 Celsius, having a density of 1.17 g/cm3 and a viscosity of a 5% solution in toluene/ethanol in a proportion of 40/60 is 5.4mPa.s(in standard conditions); Cyclohexane (Merck). The microencapsulation process using the evaporation of the solvent method results by emulsification in water of a polymeric solution in an appropriate solvent or mixture of organic solvents (3, 4, 9). The polymeric solution contains the drug in the form of microcrystals. The evaporation of the solvent is performed under 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. The modulation of the drug release from these microcapsules can be done by changing the ratios of the drug and the polymer. The evaporation is influenced by some factors: the characteristics of the polymer (such as molecular weight, structure), the type of the material to be encapsulated (shape of the crystals, solubility), the type of the used solvent, the temperature and pressure during evaporation, the intensity of shaking, the nature of the washing solvent, the method used for drying (5,6,8). Our preliminary experiments of verifying some methods for obtaining some microencapsulated, described in the literature, had shown that the reproducibility of the results (including structure, size distribution of microcapsules population, yield) is rather limited (10). A general schedule of the process of preparation of tank microcapsules by evaporation of the solvent is presented in figure 1. The separation of the microcapsules, from different experimental lots obtained in the same experimental conditions, in fraction of known dimensions is performed by mechanical sieving, using a set of standard sieves with calibrated dimensions of the holes. The X-ray diffraction analysis is done using a diffractometer TUR M-61. In advance, the microcapsules are compressed in an aluminum support. The experimental conditions are the same ones used for the characterization of drug microcrystals (for the X-ray generator a voltage of 30KV, an electric intensity of 20 mA). 88 The determination of density for the microcapsules with xantinol nicotinate is made by a picnometric method, using as reference liquid cyclohexane (in which the microcapsules are not soluble) . ρ= ( b−a)ρl ( d−a) −( c−b) where: ρ = density of the microcapsules; a = mass of the picnometer; b = mass of the picnometer with microcapsules; c = mass of the picnometer with microcapsules and liquid; d = mass of the picnometer with liquid; ρl = density of the liquid used 0 20 ( ρ ciclohexan = 0,78g/cm3)(7). Fig.1 - Preparation of tank microcapsules by the evaporation of the solvent method 89 In order to evaluate the total drug content of the microcapsules of ethylcellulose, a small quantity of 0.1g ethylcellulose powder was suspended in 200ml of distilled water. The resulted suspension has been maintained at room temperature, occasionally shaken, for 72 hours. The saturated ethylcellulose solution, resulted after filtering the suspension, has no absorbance in the wave length domain in which the aqueous solution of xantinol nicotinate has a maximum absorption (260-280 nm). The determinations were performed using a spectrophotometer 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 domain the Bouguer-Beer-Lambert low. The obtained diagram can be used for computing the apparent absorbance of xantinol nicotinate in saturated solutions of ethylcellulose A 1cm1% =276. This value is almost the same with the one of xantinol nicotinate in water. For detecting the content of the tank microcapsules with xantinol nicotinate, determined quantities of microcapsules of approx.0.10g having dimensions in domains like 400-500 µm are grinded to pieces, the powder is then dissolved in 100ml water. The suspension is shaken until a constant absorbance is seen. The values computed can be used for a determination of the drug at 1g of microcapsules. The values of the absorbance can be used to determine the quantity of xantinol nicotinate in 1g of microcapsules. 2. RESULTS and DISCUSSIONS The solvent evaporating method used to preparation of microcapsules with xantinol nicotinate After verifying the Jaselniack method for obtaining microencapsulated of ethylcellulose, we can assess that the eventual modification of that method could assure a better reproducibility of the results (yield, structure, size distribution of microcapsules population). In the same time, it is possible to modify the kinetic of the release of xantinol nicotinate from microcapsules of ethylcellulose by choosing a certain weight ratio of xantinol nicotinate and ethylcellulose, or by choosing some technological parameters to assure the preparation of higher percentage from microcapsules of certain dimensions (for that it is necessary that the crystals of drug 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 a determined quantity of ethylcellulose, under continuous stirring. For completing 90 the dissolution, the solution is kept for 20 minutes at a higher temperature of 70 degrees. - the drug, xantinol nicotinate, having a crystal dimension between 300-400µm, of accurate known weight, is brought under continuous stirring, and then the system is kept 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 room temperature, and then at 30 degrees Celsius for 72 hours for losing all the cyclohexane. In order to obtain microcapsules with different thickness of the walls, the quantities 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 Jalseniack method. The intensity of stirring, depending on the geometry of the jar was determined 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 xantinol nicotinate Regardless of the weight ratio ethylcellulose/xantinol nicotinate, the microcapsules populations obtained by the method we developed are multidisperses. In previously described experimental conditions, it was found that for a stirring speed of 300 rpm, the majority of microcapsules populations have sizes in the domains (400-500) µm and (500-630) µm. 30 25 20 15 10 5 D i a me t rul o c hiuri l o r s i t e i d e re f uz D ia me t e r o f re je c t i o n 0 S1 S2 S3 s e ri e s e ri e s Fig. 2 - Size distribution of microcapsules with xantinol nicotinate (average of three experiments in identical experimental conditions) 91 Microcapsules morphology The surface of the microcapsules can be studied by electronic microscopy or by X rays diffraction. The morphological analysis using electronic microscopy of the ethylcellulose microcapsules with xantinol nicotinate obtained by the described method, shows they have a spherical or almost spherical form, and that there are no nicotinic xantinol crystals present on their surface. The X ray analysis shows that ethylcellulose microcapsules with xantinol nicotinate have a diffraction spectrum identical with the ones of pure xantinol nicotinate. Fig. 3 - X-ray diffraction spectrum of pure xantinol nicotinate (CuKα1 radiation, Ni filter) Density of tank ethylcellulose microcapsules with xantinol nicotinate The densities for the tank ethylcellulose microcapsules with xantinol nicotinate with described before sizes, resulting from different ratios of ethylcellulose/xantinol nicotinate were determined by picnometric method, and are described in table 1. As a reference liquid was used the cyclohexane at 20 degrees Celsius, in which ethylcellulose is soluble only at higher temperatures. The table shows that the density of tank microcapsules with xantinol nicotinate 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 nicotinate The total drug content of microcapsules was determined after each dissolution test. The microcapsules samples were stirred with ultrasounds, in order to obtain complete breaking of the microcapsule walls and to release the drug content. The samples were spectrophotometrically analyzed. We used the fact that ethylcellulose has no relevant absorption at characteristic wave lengths for our drug ( λXN=270.4 nm, λXN=205.6 nm). 92 Table 1 Densities of different sizes ethylcellulose microcapsules with xantinol nicotinate Tipes of microcapsules with different proportions polymer/drug Tank microcapsules ethylcellulose/xantinol nicotinate 1 : 1 D ∈ (400-500)µm ρ(g/cm3) D ∈ (500-630)µm ρ(g/cm3) 1,036 1,023 Tank microcapsules ethylcellulose/xantinol nicotinate 4 : 1 1,124 1,096 Tank microcapsules ethylcellulose/xantinol nicotinate 9 : 1 1,195 1,181 Liquide medium : cyclohexane ; t = 200C Fig. 4 - Absorption spectrum UV of xantinol nicotinate in aqueous solution of ethylcellulose (C=0,005g/100ml, h=1cm) 93 Screening of the Lambert-Beer law for concentrations domain (00.005)g/100ml for xantinol nicotinate permitted the evaluation of percentage absorbance: Abs. =276.24C + 0.0012 (r2=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 A1%1cm=276.24 (λXN=270.4 nm) is similar to the one presented in the literature: A1%1cm=270 (λXN=273 nm). For the tank ethylcellulose microcapsules with xantinol nicotinate, the average drug content resulted from three independent determinations is presented in table 2. 3. CONCLUSIONS The tank ethylcellulose microcapsules obtained by the solvent evaporation method are represented by a multidispersional population, but evaluating the influence 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 and intensity of stirring, the modification of the temperature) we succeeded in reducing this inconvenient. 94 The shape of ethylcellulose microcapsules obtained by the solvent evaporation method is spherical or almost spherical (due to the different shapes of drug microcrystals). The drug substance from tank ethylcellulose microcapsules has a similar X-ray diffraction spectrum with the one of pure microcrystalline xantinol nicotinate. Density of tank ethylcellulose microcapsules has over unitary values. The values are influenced by the proportion xantinol nicotinate/ethylcellulose. For tank ethylcellulose microcapsules obtained by evaporation of the solvent method the average drug content computed from three different experiments was approx. 0.5gXN/gmicrocaps.( xantinol nicotinate/ethylcellulose=1:1) ; 0.2gXN/gmicrocaps.( xantinol nicotinate/ethylcellulose=1:4); 0.1gXN/gmicrocaps.( xantinol nicotinate/ethylcellulose=1:9). In conclusion, these results regarding the preparation and the characterization of some tank ethylcellulose microcapsules could be developed in some sustained release dosage forms. Table 2 Average drug content of ethylcellulose microcapsules with xantinol nicotinate Covering proportion Average dimension of Average drug content in xantinol nicotinate xantinol nicotinate : the microcapsules (µm) (g)/g microcapsules ethylcellulose 1:1 1:4 1:9 ( three determinations) experimental theoretical 400 – 500 0,484 0,500 500 – 630 0,492 0,500 400 – 500 0,191 0,200 500 – 630 0,188 0,200 400 – 500 0,097 0,100 500 - 630 0,096 0,100 REFERENCES 1. Senjkovič R., Jalšenjak I., Apparent diffusion coefficient of sodium phenobarbitone in ethylcellulose microcapsules: effects of capsule size, J.Pharm.Pharmacol., 33, 279-282, 1981. 2. Kristmundsdóttir T., Ingvarsdóttir K., Ibuprofen microcapsules: the effect of production variables on microcapsule properties, Drug Dev. Ind. Pharm., 20(5), 769-778, 1994. 95 3.Luzzi A.L., Encapsulation technique for pharmaceuticals: Consideration for the microencapsulation of drugs, Microencapsulation, Marcel Dekker Inc., New York, 193 – 207, 1976. 4. Zinutti C., Kedzierewicz F., Hoffman M., Maincent P., Preparation and characterization of ethyl cellulose microspheres containing 5- fluorouracil, J. Microencapsulation, 11, 555 – 563, 1994 5. Yamakawa Y., Tsushima Y., Machida R., Watanabe S., Preparation of neurotensin analoguecontaining poly(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(dllactic acid) microspheres containing neurotensin analogue prepared by novel oil-in-water solvent evaporation method, J.Pharm.Sci., 81, 808 – 811, 1992. 7. Pîrvu C., Moisescu t., Formularea unor medicamente cu acţiune vasodilatatoare cerebrală i periferică, Comunicare prezentată în cadrul celui de-al XI-lea Congres Naţional de Farmacie, 8 – 10 octombrie Ia i, 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. .A.M.V.Bucharest, Serie F, Vol. XIII, 2008, p. 97 - 104 COMPARATIVE RESEARCH CONCERNING THE QUALITATIVE CHARACTERISTICS OF THE SUPERIOR WINES DERIVED FROM WINE-GROWING CENTRE OSTROV CULEA RODICA- ELENA *, POPA NICOLAE-CIPRIAN**, TAMBA-BEREHOIU RADIANA* * University of Agronomic Sciences and Veterinary Medicine of Bucharest ([email protected]) **S.C. FARINSAN S.A. Giurgiu Key words: White superior wines, physical and chemical characteristics, variability estimates, statistic differences ABSTRACT So 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 and chemical parameters concerning White Fetească, Italian Riesling and Sauvignon grapes variety of wine, along three succesive crops: 2004, 2005 and 2006. The most increased variability was registered by the Free Sugar parameter (between 34 and 81%), no matter which grapes variety of wine 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 variation coefficient, smaller than 4%), parameters which can be considered typical for this category of white wines. The sort of wine which presented the most increased homogenity of the physical and chemical parameters, along the three years investigated, was the Italian Riesling sort. The most heterogeneous values were registered by the Sauvignon sort of wine. The wines produced to SC Ostrovit SA carry the specific nature of this place, with calcareous soil and droughty climate, with humid atmosphere, due to the Danube influence. In the wine-growing centre Ostrov, the grapes varieties for wine (especially White Feteasca, Italian Riesling, Royal Feteasca) are cultivated on a surface counting 245 ha and the grapes varieties for consumption (Italia, Afuz Ali, 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. % and the 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, which modulate the action of the environmental agents (applied phytotechny, the cropping moment, the vinification technology applied), determine the annual variability of the wines’ physical and chemical parameters [3, 4, 6]. 97 This study recommends a comparative qualitative characterization of the dry wines, obtained of the White Feteasca, Italian Riesling and Sauvignon grapes varieties, made by S.C. OSTROVIT S.A., in 2004, 2005 and 2006 production years. 1. MATERIAL and METHOD The determinations were made in preceding stage of the wines bottling and commercialization. There were comparatively anlysed the physical and chemical parameters of White Feteasca (n=33), Italian Riesling (n = 30) and Sauvignon (n = 31), ingathered from 2004, 2005 and 2006 crops. The following quality parameters of the wine have been analysed: d2020 (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 STAS 6182/18-81), Unreducing Extract g/l, Total Acidity g/l C4H6O6 (titrimetric method STAS 6182/1-79), Free SO2 mg/l (iodometric method STAS 6182/13-72) and Total SO2 mg/l (iodometric method STAS 6182/13-72) [2,3,4,7]. The obtained results were statistical processed using the professional program COHORT [2, 7, 8]. 2. RESULTS and DISCUSSIONS The first table shows the variability estimates of physical and chemical parameters, belonging to White Feteasca sort of wine, concerning the whole investigated period (2004 -2006). Table 1 The variability estimates of White Feteasca sort of wine samples corresponding to 2004, 2005 and 2006 crops cumulatively (n = 33) Parameter D2020 Alcohol (vol %) Total dry extract (g/l) Free sugar (g/l) Unreducing extract (g/l) Total acidity (g/l C4H6O6) Free SO2 (mg/l) Total SO2 (mg/l) X±s 0,9937 ± 0,001 11,363 ± 0,489 23,012 ± 2,150 2,184 ± 0,749 20,600 ± 2,388 5,491 ± 0,386 38,696 ± 4,149 129,279 ± 17,731 s 0,000001 0,239 4,625 0,561 5,703 0,149 17,217 314,392 CV % 0,110 4,304 9,346 34,305 11,592 7,038 10,722 13,716 Generally, it is noticed that the physical and chemical parameters of White Feteasca grapes variety of wine, are not characterized by excesiv values of the variation coefficient, with one exception, namely the Free sugar parameter (34,305 %). 98 The second table shows the obtained results in the case of Italian Riesling grapes variety of wine, concerning the whole investigated period (2004 -2006). Table 2 The variability estimates of Italian Riesling sort of wine samples corresponding to 2004, 2005 and 2006 crops cumulatively (n = 30) Parameter X±s s CV % D2020 Alcohol (vol %) Total dry extract (g/l) Free sugar (g/l) Unreducing extract (g/l) Total acidity (g/l C4H6O6) Free SO2 (mg/l) Total SO2 (mg/l) 0,9914 ± 0,011 11,206 ± 0,297 21,493 ± 1,077 1,268 ± 0,448 20,243 ± 0,948 5,895 ± 0,274 39,000 ± 5,717 128,733 ± 11,057 0,000121 0,088 1,160 0,201 0,899 0,075 32,689 122,271 1,113 2,650 5,012 35,358 4,684 4,664 14,660 8,589 In this case too, the main parameter which was affected of excesiv variation is also the Free sugar (35,358 %). In the case of Italian Riesling grapes variety of wine, distinguishes a bit increased variation coefficient, accordingly to the Free SO2 parameter (14,66 %). The results, concerning the variability estimates of the analyzed physical and chemical parameters, in the case of the wine obtained from the Sauvignon grapes variety of 2004-2006 crops, are presented in the table 3. Table 3 The variability estimates of Sauvignon sort of wine samples corresponding to 2004, 2005 and 2006 crops cumulatively (n = 31) Parameter X±s s CV % D2020 Alcohol (vol %) Total dry extract (g/l) Free sugar (g/l) Unreducing extract (g/l) Total acidity (g/l C4H6O6) Free SO2 (mg/l) Total SO2 (mg/l) 0,9937 ± 0,001 12,251 ± 0,559 25,580 ± 3,135 3,389 ± 2,770 22,097 ± 0,927 5,280 ± 0,307 39,966 ± 6,365 145,700 ± 26,241 0,000001 0,313 9,830 7,768 0,859 0,094 40,516 688,631 0,170 4,568 12,157 81,763 4,195 5,830 15,926 18,010 The 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 60.00 50.48 50.00 40.00 30.00 13.77 13.44 3.84 F ree s ugar (g/l) T otal dr y ex trac t (g/l) Alc ohol (v ol % ) 0.00 T otal SO 2 (m g/l) 0.46 5.84 F ree SO 2 (m g/l) 10.00 6.82 T otal ac idity (g/l C 4H 6O 6) 8.84 U nreduc ing ex trac t (g/l) 20.00 D ens ity (D 2020) A verag e o f variatio n co efficien ts (% ) Observing the first figure, we noticed that the most stable parameters of the three 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 grapes variety, having a medium variation over 50 %. Fig .1 - The variation coefficients average of the physical and chemical parameters, which characterized the analysed wines The grapes variety of wine, which presented the most higher homogenity for physical and chemical parameters, along the three years investigated, was Italian Riesling (the lowest medium variation coefficient = 9,59 %). The most increased values were registered in the case of Sauvignon wine, for which it was established a medium variation coefficient of 17,83 % (figure 2). We can say that the Italian Riesling grapes variety was the best adapted to climatic conditions, which varied along the analysed period. This sort of wine pointed out the most homogeneous characteristics, while the Sauvignon wine proved to be the most exposed and misfit. 100 Average of variation coefficients (%) 20 17.83 18 16 14 12 11.39 9.59 10 8 6 4 2 0 White Feteasca Italian Riesling Sauvignon Fig. 2 - The variation coefficients average of all the parameters, which characterized every sort of wine (White Feteasca, Italian Riesling and Sauvignon) The table 4 presents the meaning of averages differences, following the application of the t test (Student), for all the physical and chemical parameters, in the case of the three studied wines. As noticeable from the table 4, the Density parameter did not differ significantly between neither one of the analysed wine pairs. This fact shows that the parameter Density is rather typical for the wines category (white, dry wines), than for the grapes variety of wines, respective White Feteasca, Italian Riesling or Sauvignon. One can also see, that the Alcoholic Content % did not differ significant between the White Feteasca and Italian Riesling grapes varieties of wines. However, the White Feteasca and Italian Riesling grapes varieties of wines, had a very significant low alcoholic concentration, comparative to the Sauvignon grapes variety of wine. The parameter Total dry extract was very significant different between the three grapes varieties of wine. The highest value was registered to Sauvignon grapes 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, having significant and very significant different values between the wines. The highest value one can see in the case of Sauvignon grapes variety (3,389 %), followed by 101 White Feteasca grapes variety (2,184 g/l) and Italian Riesling grapes variety (1,268 g/l). Table 4 The meaning of averages differences (t test) of the physical and chemical parameters concerning the three superior wines Pairs D2020 White Feteasca (a) – Italian Riesling (b) White Feteasca (a) – Sauvignon (b) Italian Riesling (a) – Sauvignon (b) Pairs Alcohol (%) White Feteasca (a) – Italian Riesling (b) White Feteasca (a) – Sauvignon (b) Italian Riesling (a) – Sauvignon (b) Pairs Total dry extract (g/l) White Feteasca (a) – Italian Riesling (b) White Feteasca (a) – Sauvignon (b) Italian Riesling (a) – Sauvignon (b) Pairs Free Sugar (g/l) White Feteasca (a) – Italian Riesling (b) White Feteasca (a) – Sauvignon (b) Italian Riesling (a) – Sauvignon (b) Pairs Unreducing extract (g/l) White Feteasca (a) – Italian Riesling (b) White Feteasca (a) – Sauvignon (b) Italian Riesling (a) – Sauvignon (b) Pairs Total acidity (g/l C4H6O6) White Feteasca (a) – Italian Riesling (b) White Feteasca (a) – Sauvignon (b) Italian Riesling (a) – Sauvignon (b) Pairs Free SO2 White Feteasca (a) – Italian Riesling (b) White Feteasca (a) – Sauvignon (b) Italian Riesling (a) – Sauvignon (b) Pairs Total SO2 White Feteasca (a) – Italian Riesling (b) White Feteasca (a) – Sauvignon (b) Italian Riesling (a) – Sauvignon (b) Average (a) 0,9937 0,9937 0,9914 Average (a) 11,363 11,363 11,206 Average (a) 23,012 23,012 21,493 Average (a) 2,184 2,184 1,268 Average (a) 20,600 20,600 20,243 Average (a) 5,491 5,491 5,895 Average (a) 38,696 38,696 39,000 Average (a) 129,279 129,279 128,733 Average (b) 0,9914 0,9937 0,9937 Average (b) 11,206 12,251 12,251 Average (b) 21,493 25,580 25,580 Average (b) 1,268 3,389 3,389 Average (b) 20,243 22,097 22,097 Average (b) 5,895 5,280 5,280 Average (b) 39,000 39,966 39,966 Average (b) 128,733 145,700 145,700 t 1,144 0 1,163 t 1,563 6,791*** 9,154*** t 3,592** 3.797*** 6,852*** t 5,950*** 2,394* 4,181*** t 0.792 3.342* 7,722*** t 4,744*** 2,411* 8,252*** t 0,243 0,939 0,692 t 0,148 2,914** 3,309** Regarding the Unreducing extract parameter, it was noticed that registered values, in the case of White Feteasca and Italian Riesling grapes varieties of wines, did not differ significant. Instead, the Sauvignon and White Feteasca, respective Italian Riesling grapes varieties of wines, differed significant and very significant. 102 So, the highest value of the Unreducing extract parameter was specific to Sauvignon grapes variety of wine, followed by White Feteasca and Italian Riesling grapes varieties of wine. The Total Acidity parameter could also be considered as being peculiar to every grapes varieties of wines. Matter of fact, all grapes varieties of wines presented at least significant differences between the values of this parameter. White Feteasca grapes variety of wine presented a very significant decreased value of Total Acidity, comparative to Riesling Italian grapes variety of wine and a significant grown value, comparative to Sauvignon grapes variety of wine. The Italian Riesling grapes variety of wine presented a very significant grown value of the Total Acidity, comparative to Sauvignon grapes variety of wine. The highest values of this parameter were peculiar to the Italian Riesling grapes variety of wine, followed in order by the White Feteasca and Sauvignon grapes varieties of wines. The analysed grapes varieties of wine did not differ concerning the Free SO2 quantities, though appeared some distinctly significant differences between Sauvignon and White Feteasca or Italian Riesling grapes varieties of wines. These differences due to the higher quantity of Total SO2 present in the Sauvignon grapes variety of wine, comparative with other two sorts of wines. Our analysis showed that the least peculiar parameters of the grapes variety of wine were the Density and the Free SO2 content. In practice, the values of these parameters seem rather characteristics of the wine category, respective superior, white, dry wines, then grapes variety characteristics. Instead, prameters as: Total Dry Extract, Free Sugar or Total Acidity presented significant differences between each pair of analysed grapes varieties. The fact is showing that the value of these parameters could be considered typical for every sort of wine. 3. CONCLUSIONS ♦ The wine obtained of White Feteasca grapes variety had the quality parameters values specific to superior, white and dry wines. The physical and chemical 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 quality parameters were typical for superior, white and dry wines. The main parameter which 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 SO2 parameter (8,589 %). ♦ The Sauvignon grapes variety of wine, pointed out quality parameters suitable to superior, white and dry categories of wines, but there were affected by 103 increased variations. So, the Free Sugar parameter showed a variation coefficient value higher than 81 %. A rather increased variation coefficient was observed at the parameters connected to the wine sulphitation, moreover than 15 %. ♦ The Italian Riesling grapes variety was the best adapted to climatic conditions, peculiar to 2004-2006 period. So, the wine produced of Italian Riesling grapes variety, revealed homogenous characteristics. ♦ The Sauvignon grapes variety of wine proved to be the most exposed and misfit. ♦ We may assert that the Density, Alcoholic content and Free SO2 parameters are peculiar to superior, white and dry wines, while the Total Dry Extract, Free Sugar and Total Acidity parameters could be considered typical for every sort of wine. REFERENCES 1. COTEA, V.D., GRIGORESCU, C., BARBU, N., COTEA, V.V., 2000, Podgoriile si vinurile României, Editura Academiei Române 2. COTOFREI, S. C., 2004, Cercetari privind optimizarea metodologiei de calcul matematic in oenologie, Teză de doctorat, Ia i 3. IONESCU, I.A., 1968, Factorii ce determină calitatea vinurilor, Revista Horticultura i Viticultura nr. 2, Bucure ti 4. KONTEK, A., KONTEK, Ad., 1999, Măsuri tehnologice care contribuie la îmbunătăţirea calităţii vinurilor albe seci, Concepţii moderne în cercetarea horticolă Românească, Editura Medro. 5. POMOHACI, N.,NĂMOLO ANU, I., NĂMOLO ANU, A., 2000, Producerea i îngrijirea vinurilor, Editura Ceres, Bucure ti, pg. 27-31. 6. POMOHACI, N., SÎRGHI,C.., STOIAN, V., COTEA, V.,NĂMOLO ANU, I., 2000, Oenologie, Editura Ceres, Bucure ti, 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 de standardizare, Bucure ti, 1997. 104