“Volatile organic compounds of plants and sheep milk at two mountainous areas of Greece:
a preliminary study”
Despina Bozoudi1, Zoi Parisi2, Salvatore Claps3, and Evanthia Litopoulou-Tzanetaki1
1
Laboratory of Food Microbiology and Hygiene, Faculty of Agriculture, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
2 Laboratory of Range Science, School of Forestry and Natural Environment, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
3 CRA-ZOE, Consilio per la Ricerca e Sperimentazione in Agricoltura, Unita di Ricerca per la Zootecnia Estensiva, Bella (PZ), Italy
Introduction. The sensory quality of cheeses is governed by a number of
factors linked to their production. Milk production factors is of particular
importance for cheeses under PDO status. The diet of grazing animals is very
different from season to season, according to the variability of pasture
botanical composition. This botanical diversification could be a source of
enrichment or impoverishment in secondary metabolites of milk and cheese.
The local forage-based diets are part of the basic link between dairy products
and their origin, “terroir”, a notion at the basis of the PDO labeling and image
of the product. Traditional Feta cheese is produced in small dairies or on
farms and the mountain farming system is based on local forage resources,
with a combination of preserved feeds and fresh pastures. The composition of
the cheese volatiles is dependent on the production altitudes. The flavor of
mountain cheeses is influenced by the presence of terpenoid compounds that
may be considered as biochemical markers, to characterize cheeses
originating from highlands. The present study, consists a part of a larger
project which was held in order to geographically discriminate PDO Feta
cheese produced in different areas of Greece. For this reason, reflects the first
attempt to study the particular plant species and establish the composition of
VOC contained in sheep pastures and the produced milk at two different areas
of mountainous Greece, which could influence Feta cheese. The analysis of
VOC and the detection of terpenoids in both feed and milk, would possibly
allow the discrimination of Feta cheese according to the production area.
Materials and Methods. The grazing study was conducted at northwest (NW)
~800m and southwest (SW) Greece, at 1300-1800m altitude. The plants were
collected at the flowering stage while the flock was grazing. Two bulk forage
samples from each area were gathered as representatives for what has been
grazed by sheep. Three subsamples, one each week, of milk deriving from 50
animals of each flock were used. The animals were of local herds and were
fed only by fresh pasture. VOC of plants, bulk and were studied using HS-GCMS technique.
Results and Discussion. The species composition of the grazing areas is
presented in Table 1. Forage from NW Greece indicated the predominance of
Graminaceae, Leguminosae and Compositae and the persistence of Trifolium
among Leguminosae. In SW area, Graminaceae, Caryophyllaceae, Rosaceae
and Convolvulaceae plant species were often found. Labiatae plants, often
present in the mountain flora, are rich in terpenes, which may be transferred
to milk (Bosset et al., 1994). Results of the GC-MS analysis imprinting the
profile of each plant species are shown in Figure 1.
Figure 1. VOC
profile of
different plants
from two
different
grazing areas of
Greece (see
Table 1 for
plant codes).
In general, comparison of the plant VOC composition revealed, that ~33.5
and 25% of the species collected in NW and SW area, respectively, were in
general richer in VOC, and contained aldehydes at amounts ≥10 A.U., and
ketones in 60% and 87.5% of the samples respectively. Esters and acids
were sporadically found. Hydrocarbons were found at levels 4.4-87.0AU
and 3.7-10.2, while terpenes detected at amounts 3.4-573.3 and 3.1-43.8,
for NW and SW area, respectively. In the majority of the plants collected
from NW area, terpenes predominated over the other VOC.
Figure 2. Score and loading plots of first two components after PCA based on the VOC
detected in the different plant species. A: different groups of compounds, B: Aldehydes, C:
Hydrocarbons, D: Alcohols.
Table 1. Plant species collected from two
mountainous Greek areas
All PCAs were formed in respect of grasses, legumes
and forbs. A PCA (Fig. 2A) was conducted on the
Code correlation matrix of aldehydes, ketones, esters,
Grasses
K1
hydrocarbons, alcohols and terpenes from the
K4
K6
different plant species. A 69.6% of total variance was
K9
explained and seemed that the two areas were
Legumes
K2
K5
clustered accordingly. In respect of the aldehydes (Fig.
K7
2B), hexanal, heptanal, nonanal, benzaldehyde and
K12
K14
decanal, differentiated the plant species from SW.
K15
Among aldehydes, hexanal and (E)-2-hexanal
Forbs
K3
predominated, and benzaldehyde, nonanal and
K13
decanal occurred frequently. The ketone 6-methyl-5K8
K10
hepten-2-one was the most commonly found among
K11
ketones. Toluene, p-xylene, nonane and benzene seem
Grasses
B3
to differentiate the plant species from SW area (Fig.
B5
2C). p-Xylene was detected in abundance among
B6
Forbs
B7
hydrocarbons from all plants of SW area. Heptane and
B8
ethylbenzene were often found in plants from both
B2
BB1
areas and benzene 1-methyl-4-(1-methylethyl)-, was
B4
exhibited by seven out of eight plants of SW area.
Hydrocarbons presence in milk and cheese is of plant derivation. The presence of 1-penten-3-ol
was frequent in the plant species of NW Greece (Fig. 2D). 1-Penten-3-ol, 2-penten-1-ol, 3-hexen-1ol, 1-octen-3-ol, 1-octanol also differentiated plants from SW area.
Hydrocarbons were the first most abundant group of Table 2. Terpenes detected in bulk forage and
VOC in milk from low altitude, followed by alcohols and milk from 2 mountainous Greek areas
aldehydes and their amounts differed significantly
NW Greece (~800m)
SW Greece (~1300-1800m)
(P<0.05) between the two areas. In high altitude milk, Terpenes
Bulk forage
Milk
Bulk forage
Milk
terpenes predominated (P<0.05), followed by
Tricyclene
*0.61a
0a
*0.38a
*0.57a
hydrocarbons and aldehydes (data not shown). The
α-Pinene
**0.90a ± 0.03 **0.86a ± 0.14 **1.25a ± 0.23 ***1.55a ± 0.52
aldehydes hexanal, benzaldehyde, nonanal and decanal
Camphene
**4.61a ± 3.78 **0.66a ± 0.20 **3.33a ± 0.59 ***2.68a ± 3.87
were found in bulk and milk of both areas, while 2**0.54a ± 0.01
*0.18b
hexanal, n-heptanal and octanal were significantly β-Pinene
**0.94a ± 1.06
**0.31a ± 0.08
higher (P<0.05) in bulk forage from high altitude. Six 2-β-Pinene
***1.07a ± 1.51
**0.24a ± 0.13
ketones were traced in bulk forage and milk samples 3-Carene
*11.34a
**5.21a ± 1.77
from both areas, with 1-octen-3-one being significantly Eucalyptol
*10.20a
***3.21a ± 3.01
0a
***9.18a ± 6.94
higher (P<0.05) in bulk forage from high altitude. In milk, dl-Limonene
0a
*0.16a
6-methyl-5-hepten-2-one was found in all samples γ-Terpinene
0a
**1.50a ± 0.70
tested and maybe considered as characteristic L-Fenchone
compound for milk produced in mountainous areas Camphor
**0.28a ± 0.16 **0.25a ± 0.13 **0.55a ± 0.31
*0.54a
since its presence was also confirmed in the different Menthone
0a
**0.60a ± 0.27
plant species. Alcohols in milk, constituted a significant Neo-iso-menthol
0a
**0.66a ± 0.28
part of the VOC representing ~17.3% and 12.9% for low α-Terpineol
*0.31a
0a
*0.26a
*0.41a
and high altitude area, respectively, and their amounts Nerylacetone **0.72a ± 0.16 ***0.87a ± 0.33 **0.44a ± 0.36 ***0.78a ± 0.13
differed significantly.
Hexanol 2-ethyl was higher (P<0.05) in NW and 1-octanol in SW Greece. 3-Hexen-1-ol was found in
higher (P<0.05) amounts at high altitude area. Toluene, ethyl-benzene and xylene were detected in
all samples and their amounts differed significantly between the two areas as well as hexane 3methyl and heptane. Hydrocarbon profile was very similar to that of the different plant species
analyzed. Those compounds are of plant origin, although some of them may result from the
oxidation of unsaturated fatty acids which were found in greater proportions in mountain milks.
Terpenes in milk produced at high altitude was significantly higher (Table 2) from that of the lower
altitude. In the milk samples examined, the terpenoid profile was similar to that of the plants, in
respect of the common and predominant terpenes (α-pinene, camphene, dl-limonene, camphor
and nerylacetone). However, 3-carene, which was not isolated from any plant and/or bulk and
nerylacetone, found in two plants only, were determined in all milk samples. There were also
terpenoid compounds exhibited by different plant species and bulk forage samples, which were not
detected in the milk produced by the grazing animals. Terpenes are of plant origin and they are
transferred to milk from the animals. Milk contained less terpenoids than the plants and/or bulk
forage. This could be due to the volatility of these compounds and/or to enzymatic reactions from
milk microflora. Terpenes detected only in milk but not in the bulk samples, may also be of plant
derivation. These compounds may derive from other plants of pasture consumed by the animals
and their presence in plants or bulk samples studied was possibly small or absent. A PCA was
conducted to support the findings on milk, as shown in Table 3. PC1 and PC2 explained 41 and 30%
of the variance, respectively.
~800m, North West Greece
Family
Species
Graminaceae
Aegilops sp.
Taehiatherum caput-medusae
Bromus sp. 1
Brachypodium sp.
Leguminosae
Trifolium campestre
Trifolium hirtum
Vicia sp.
Trifolium sp.1
Lathyrum sp.
Vicia villosa
Compositae
Anthemis cf tinctoria ssp.
parnassica
Crepis sp.
Scrophulariaceae Euphrasia sp.
Labiatae
Thymus sp.
Polygonaceae
Rumex acetosella
~1300-1800m, South West Greece
Graminaceae
Phleum sp.
Aegilops sp.
Taeniatherum caput-medusae
Caryophyllaceae Petrorhagia sp.1
Petrorhagia sp.2
Rosaceae
Ptentilla recta
Rosa sp.
Convolvulaceae
Convolvulus sp.1
Aldehydes
Ketones
Hydrocarbons
Terpenes
C1
-0.060
0.048
-0.871
0.927
C2
C3
-0.166 0.984
0.983 -0.177
-0.419 -0.255
-0.184 -0.326
Table 3. Correlations of different groups
of VOC tested in milk. Loadings >0.500
in absolute value, are boldfaced.
Conclusions: Differences in the plant composition of forages grazed by small ruminants at mountainous areas of Greece were shown. Each plant has its own unique profile in respect
of VOC. Common terpenoids in plants and milk, confirm that the type of diet is responsible for the presence of these marker compounds. The milk of the two areas is differentiated on
the basis of alcohols, hydrocarbons and some terpenes. Further studies are necessary, in order to detect the effect of grazing on the VOC profile of traditional Feta cheese produced at
various mountainous areas and its compositional characteristics.
Acknowledgement: This work is part of the " 08SMEs2009 project, funded by the GSRT, Ministry of Education, Greece.