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