“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.
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