The variability of fatty acids and terpenes in milk from dairy ewes depends on feeding management. In semi-extensive and extensive production systems, feeding management is dictated by seasonal availability of pastures. In Greece, as in many other Mediterranean countries, the majority of small ruminants are reared semi-extensively or extensively in mountainous or marginal regions of the country (Zervas, Reference Zervas1998). In such systems indoor housing takes place only during approximately three months in winter. At that time there is limited grazing and animals are offered supplementary feeding of Lucerne hay and concentrates. On the contrary, in spring and summer the animals are reared outdoors, thus allowing them to nourish exclusively from grazing pasture. Milk originating from pasture-grazed ewes has specific nutritional and organoleptic characteristics regarding its fatty acid and terpene composition (Chion et al. Reference Chion, Tabacco, Giaccone, Peiretti, Battelli and Borreani2010).
Although there have been several studies undertaken in Greece considering the fatty acid composition in sheep milk, not much work has been done concerning the terpene profile of sheep milk under real production conditions.
Therefore, the objective of this study was to investigate the effect of season on the profile of fatty acids (FA) and terpene composition in milk of ewe reared semi-extensively, under real production conditions, in the area of West Macedonia of Greece.
Materials and methods
The study was conducted from January to August, 2011. It took place in the area of West Macedonia of Greece and more specifically in the prefecture of Grevena. This region covers a total of 2291 Km2, the 85% of which is characterised as semi-mountainous and mountainous terrain. Mountains in the area (Vasilitsa, Pindos) reach altitudes up to 2250 m above sea level. These terrains are used by local sheep and goat breeders, for round-year grazing. The range of flock size was 150–1100 ewes. During winter months and until the end of March, animals were kept indoors and their diet was based mainly on concentrates with occasional grazing with different ratios each farmer, while from April onwards, grazing native pastures was the only nutritional source. The management and nutrition and also the grazing of each breeding remained the one each stockbreeder applied. Hence, we analysed the sheep's milk in the way it is traditionally produced.
Milk sampling
An effort was made to include farms located in the entire region of Grevena. Eventually, ninety commercial dairy sheep farms participated in the study. A total of 760 samples of bulk sheep milk were collected during winter (147 samples), spring (314 samples) and summer (299 samples) in 2011. The milk collection was made every 14 days in the morning, in approximately half to 1 h maximum after milking, from the bulk vat or from the small bulk tank. Two milkings (evening and morning) were done prior to bulk milk collection. The samples placed into 20 mL glass tubes that were filled almost to the top in order to prevent a large headspace hence reducing volatile losses (Blount et al. Reference Blount, Mc Elprang, Chambers, Waterhouse, Squibb and LaKind2010) due to equilibration with its environment. Immediately after the collection, all samples were stored at −20 °C for the fatty acid and terpene profile determination.
Fatty acid composition
Prior to analysis, the milk samples were allowed to thaw overnight at 4 °C. Milk lipids were extracted with a chloroform/methanol solution (1 : 2 v/v) according to Bligh & Dyer (Reference Bligh and Dyer1959) as described by Jensen & Nielsen (Reference Jensen and Nielsen1996). All used solvents contained 0·01% (wt/v) of t-butyl-hydroxytoluene (BHT) in order to avoid PUFA oxidation. The preparation of the fatty acid methyl esters was carried out using a base catalysed trans-esterification procedure according to International Organisation for Standardisation ISO (2002).
The fatty acid methyl ester analysis was performed on an Agilent Technologies 6890N GC equipped with a flame ionisation detector (FID) and a 60 m × 0·25 mm i.d., 0·25 µm film thickness DB-23 (50% Cyanopropyl 50% dimethyl polysiloxane) capillary column (Model Number: Agilent 122–2362). The injector temperature was set at 250 °C. The oven temperature was programmed from 110 °C (hold for 6 min), to 165 °C at 11 °C/min (hold for 13 min), to 195 °C at 15 °C/min (hold for 22 min) and to 230 °C at 7 °C/min (hold for 7 min). The carrier gas was helium at 0·7 ml/min and the injection volume was set at 3 µl and the split ratio was 1 : 50. The injection was performed using an Agilent 7683 Series auto-sampler. Individual FAMEs were identified by comparison with three commercial standard mixtures: (a) 37 component FAME mix (Supelco, 47885-U) (b) PUFA-2, Animal source (Supelco, 47015-U) and (c) a mixture of cis- and trans-9,11- and -10,12-octadecadienoic acid methyl esters (Sigma, O5632–250MG). Peak areas were determined with the GC Chemstation (Rev. B. 02·01-SR1 [260], Agilent technologies, 2001–2006). Each fatty acid methyl ester was expressed as a percentage of the total peak area of the chromatogram (without including the solvent peak). After analyses, the FAs were further grouped as saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), unsaturated fatty acids (UFA), n-3 PUFA, n-6 PUFA. Ratios between the different fractions, namely PUFA/SFA and n-6/n-3 were calculated. The atherogenicity (AI) and thrombogenecity (TI) indices were calculated as proposed by Ulbricht & Southgate (Reference Ulbricht and Southgate1991).
$$\eqalign{ ({\rm AI}) & = ({\rm C}_{12\;:\;0} + 4 \times {\rm C}_{14\;:\;0} + {\rm C}_{16\;:\;0} )/({\rm MUFA} + {\rm PUFA}) \cr ({\rm TI}) &= ({\rm C}_{14\;:\;0} + {\rm C}_{16\;:\;0} + {\rm C}_{18\;:\;0} )/(0,5 \times {\rm MUFA} + 0 \!\cdot\! 5 \cr & \quad \times n - 6{\rm PUFA} + 3 \times n - 3{\rm PUFA} \cr & \quad + n - 3{\rm PUFA}/n - 6{\rm PUFA})} $$
Terpenes extraction, separation and identification
Prior to SPME analysis, the milk samples were allowed to thaw overnight at 4 °C. The SPME procedure was performed using a manual SPME holder (Supelco) equipped with a divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS, 50/30 µm thickness) fibre coating (Supelco). Before the commencement of SPME analysis, each new fibre was preconditioned at 270 °C for 60 min in the GC injection port, in a helium flow, as suggested by the manufacturer. Five ml of milk sample were placed into a 15-mL vial. To ensure better volatile extraction, samples were heated for 60 min at 35 °C. After equilibration, the SPME fibre assembly, containing the DVB/CAR/PDMS fibre coating, was introduced, through the PTFE septum, and exposed to the headspace for 60 min to adsorb volatiles. After this period of time, the SPME fibre was retracted into the holder and the assembly was removed and then introduced in the GC injector port for thermal desorption. The GC/MS analyses were carried out using an Agilent 6890 GC coupled to an Agilent 5975 quartupole mass detector.
The chromatographic separation was achieved by a 60 m × 0·25 mm i.d. × 0·25 µm film thickness HP-5MS (5% Phenyl Methyl Siloxane) capillary column (Model Number: Agilent 19091S-433). The carrier gas was helium with a flow rate of 1 ml/min. The fibre was injected in splitless mode and left to be desorbed at 250 °C for 15 min in the injector, which was equipped with a 0·75 mm i.d. SPME injection sleeve (liner) (Supelco, Bellefonte, USA) to improve the GC resolution. The GC oven temperature was programmed to rise from 40 °C (held for 5 min) to 190 °C, at a rate of 4·5 °C/min and then rise to 230, at a rate of 15 °C/min and held there for 4 min. The overall temperature programme lasted for 45 min. The mass spectrometer detector was operated in the electron impact mode, in full scan mode from m/z 35 to 250, with a source temperature of 230 °C, quadrupole temperature of 150 °C, electron energy of 70 eV and a multiplier voltage of 1800 EMVolts. n-Alkanes (C9 to C24, Supelco) were analysed under the same conditions as the samples in order to calculate the LRI (linear retention indices) values of the compounds. Identification of terpenes was based on matching mass spectral data of volatiles with those of the NIST MS library (comparison quality >90%) obtained from the alkanes with those reported by Adams (Reference Adams2007). The peak area was obtained by integrating a specific ion for each of the identified terpene to avoid co elution problems (Sivadier et al. Reference Sivadier, Ratel, Bouvier and Engel2008). Therefore, ions 93 and 161 were selected for integrating monoterpenes and sesquiterpenes, respectively. The results were expressed as log10 of area arbitrary units (AAU) and the appearance of each terpene within each season, was presented as its corresponding relative frequency (percentage) of appearance (rFOA) in relation to the total number of samples collected in each season.
All the above analysis concerning the determination of fatty acids and terpenes in ewe milk, were carried out at the Laboratory of Animal Husbandry of the Department of Agriculture (Aristotle University of Thessaloniki, Greece).
Statistical analysis
Fatty acids, totals, ratios, indices and terpenes were statistically studied by an analysis of variance using the one-way ANOVA procedure of SPSS ver.20. Significant differences among means were assessed using Tukey's test.
Results
Milk FA composition
Table 1 displays the FA composition of the ewe milk, as well as the comparison of each fatty acid, between the three studied seasons. Also Table 2 presents calculated ratios between groups and individual FA in the three studied periods. A quick glance at Tables 1 and 2 clearly reveals that the fatty acid profile of sheep milk significantly differs between the three examined seasons.
Table 1. Fatty acid composition† of ovine milk fat according to season, from farms in the area of Grevena
N.S., Non Significant.
**P < 0·01; ***P < 0·001.
† Fatty acid methyl ester expressed as a percentage of total peak area of the chromatogram, without including the solvent peak.
Table 2. Important ratios, indices and groups of FAs in ovine milk according to season, from farms in the area of Grevena
***P < 0·001.
† Saturated fatty acids = C4 : 0 + C6 : 0 + C8 : 0 + C10 : 0 + C11 : 0 + C12 : 0 + C13 : 0 + C14 : 0 + C15 : 0 + C16 : 0 + C17 : 0 + C18 : 0 + C20 : 0 + C22 : 0 + C24 : 0.
‡ Unsaturated fatty acids = C15 : 1 + C16 : 1 + C17 : 1 + C18 : 1n9t + C18 : 1n9 + C18 : 2n6 t-9, t-12 + C18 : 2n6, c-9, c-12 + C18 : 3n3 c-9, c-12, c-15 + C20 : 2 + C20 : 4n6 + C20 : 5n3 + C24 : 1 + C22 : 5n3 + C22 : 6n3 + CLA c-9, t-11.
§ Mono-unsaturated fatty acids = C15 : 1 + C16 : 1 + C17 : 1 + C18 : 1n9t + C18 : 1n9c + C24 : 1.
¶ Poly-unsaturated fatty acids = C18 : 2n6 t-9, t-12 + C18 : 2n6 c-9, c-12 + C18 : 3n3 c-9, c-12, c-15 + C20 : 2 + C20 : 4n6 + C20 : 5n3 + C22 : 5n3 + C22 : 6n3.
†† Atherogenic index.
‡‡ Thombogenic index.
The predominant FAs in ewe's milk were cis- oleic (cis- C18 : 1, OA) and palmitic (C16 : 0). Other abundant FAs were stearic (C18 : 0), myristic (C14 : 0), capric (C10 : 0) and lauric (C12 : 0). These prominent FAs accounted for approximately 73–76% of total FAs.
Saturated fatty acids (SFA)
Short and medium chain SFA
Regarding the short (C4) and medium (C6 : C12) chain saturated fatty acids, butyric acid (C4 : 0) caproic (C6 : 0), caprylic (C8 : 0), capric (C10 : 0) and lauric (C12 : 0) acids were found significantly lower in summer milk (P < 0·001). The higher concentrations were observed in winter milk.
Medium and long chain SFA
Significant differences between seasons were detected concerning the long chain SFA and very long chain SFA in ewe milk fat. Stearic acid (C18 : 0) was detected in higher concentrations in summer milk while winter milk demonstrated the lowest concentration (P < 0·001). This was also the case for the rest of the fatty acids constituting the group of long and very long chain SFA, with the exception of C14 : 0 and C16 : 0. The latter were detected in lower concentrations in summer and higher in winter milk (P < 0·001), whilst C13 : 0 did not demonstrate any statistical differences between seasons (P > 0·05).
Unsaturated fatty acids (UFA)
Medium and long chain monounsaturated fatty acids
The cis monoenoic fatty acids with an 18-carbon chain were found in lower concentrations in winter rather than in spring and summer milk (P < 0·001). More specifically, Table 1 shows that trans-vaccenic acid (TVA) was significantly lower in winter in comparison with spring and summer, while OA was detected in higher concentrations in summer milk. The FAs C17 : 1 and C24 : 1 followed the same pattern as OA. On the other hand, the concentration of the monoenoic acids C10 : 1 and C16 : 1 where gradually reduced from winter to summer milk (P < 0·001).
Long chain poly-unsaturated fatty acids
Trans-9, trans-12 C18 : 2 n-6 and LA were found in higher concentrations during the spring months (P < 0·001) and lower in the winter while C20 : 4 n-6 exhibited the lowest value in winter. Τhe percentage of A-linolenic (ALA), C20 : 5, C22 : 5 and C22 : 6 was significantly higher in summer than in winter milk. Finally, the cis-9, trans-11 CLA (rumenic acid, RA) increased progressively, from winter to summer milk (P < 0·001).
Important ratios, indices and groups of FAs
The amount of total SFA detected in sheep milk was significantly reduced from winter to summer (P < 0·001) while the total UFA, PUFA and MUFA exhibited the adverse tendency (P < 0·001). As a consequence, the UFA/SFA and PUFA/SFA ratios in ewe milk were higher during summer and lower in winter (P < 0·001) (Table 2).
Due to the significantly higher n-3 FAs observed in summer milk, the n-6/n-3 ratio was lower in summer rather than in spring and especially in winter milk (P < 0·001). This was also the case for AI and TI, which were reduced significantly in summer milk (P < 0·001).
Milk terpenoid composition
Table 3, shows the terpene composition and profile of the ewe milk during the three seasons as well as the comparison between them. Furthermore, it presents the rFOA of each terpene, within each season. In total, thirty terpene compounds (12 monoterpenes and 18 sesquiterpenes) were identified, by the gas chromatograph-mass spectrometer.
Table 3. Composition (log10 of AAU) and rFOA (%) of terpenes in ovine milk according to season, from farms in the area of Grevena
AAU, Arbitrary Area Units.
N.S., Non Significant; N.D., Not Detected.
*P < 0·05; **P < 0·01; ***P < 0·001.
Monoterpenes
The chromatographic analysis revealed a wide range of monoterpenes occurring in all examined seasons. A-pinene, β-pinene and D-limonene were the most frequently detected terpenes in each season. From the aforementioned monoterpenes α-pinene and β-pinene were detected in a higher concentration in summer and lower in winter milk (P < 0·001). D-limonene was found in higher values during winter and lesser in spring milk (P < 0·05). This was also the case for tricyclene. On the other hand, the milks’ concentrations in camphene and 3-carene were found higher in summer and lower in spring (P < 0·01 and P < 0·01, respectively). As for their rFOA, it increased in summer in comparison to winter. Β-myrcene and γ–terpinolene were detected in higher concentrations in winter milk. Concerning the terpenes 3-thyjene, sabinene, 2-carene and terpinolene, these did not exhibit any statistical differences between seasons (P > 0·05) but noted a progressive rise in their rFOA, from winter to summer milk.
Sesquiterpenes
Concerning the sesquiterpenes, the results showed no concentration differences in milk between seasons for α-cubebene, copaene, β-caryophyllene, trans- bergamotene, α-caryophyllene and δ-cadinene (P < 0·05). Nevertheless, their rFOA noted a remarkable increase in summer milk compared to winter and spring milk. The analysis revealed the presence of a large number of sesquiterpenes in summer milk which were not detected in spring or winter samples. These terpenes were β-Cubebene, γ-Selinene, α-Muurolene, γ-Muurolene, D-Germacrene, a-Amorphene, α-Elemene, β-Panasinsene, Isodelene, β-Cadinene, γ-Cadinene and Valencene.
Discussion
FA composition of ewe milk
Feed management of ewes during each season has a large impact on the concentrations of the individual fatty acids. This was demonstrated thoroughly with cows and to a lesser extent with small ruminants (cows: Lock & Garnsworthy, Reference Lock and Garnsworthy2003; Elgersma et al. Reference Elgersma, Ellen, Horst, Boer, Dekker and Tamming2004; Talpur et al. Reference Talpur, Bhanger, Khooharo and Memon2008; Heck et al. Reference Heck, Valenberg, Dijkstra and Hooijdonk2009; Chion et al. Reference Chion, Tabacco, Giaccone, Peiretti, Battelli and Borreani2010; Kliem et al. Reference Kliem, Shingfield, Livingstone and Givens2013; goats: Renna et al. Reference Renna, Cornale, Lussiana, Malfatto, Mimosi and Battaglini2012; sheep: Nudda et al. Reference Nudda, Mcguire, Battacone and Pulina2005).
Saturated fatty acids
Short- and medium chain saturated fatty acids
The availability of long-chain unsaturated fatty acids with a high degree of unsaturation, lowers the concentration of short- and medium chain saturated fatty acids in milk fat (Chilliard et al. Reference Chilliard, Ferlay, Mansbridge and Doreau2000) due to the strong inhibitory effect on the de novo fatty acid synthesis (Couvrer et al. Reference Couvrer, Hurtaud, Lopez, Delaby and Peyraud2006). Long-chain unsaturated fatty acids exert direct and/or indirect effects on the lipogenic enzymes acetyl-CoA carboxilase (ACC) and fatty acid synthase (FAS) thus reducing acetate and 3-hydroxibutyrate bioavailability for mammary lipogenesis (Chilliard & Ferlay, Reference Chilliard and Ferlay2004). Pasture based diets offer large amounts of long-chain unsaturated fatty acids (ALA and LA). This could explain the decreased concentration of short- and medium chain fatty acid (C4 : C16) observed in this study for spring and summer milk in contrast with winter milk. Our observations are in accordance with other researchers who studied the seasonal changes or the changes in milk fatty acids during the transition from indoor to pasture diet (Tsiplakou et al. Reference Tsiplakou, Mountzouris and Zervas2006; Valvo et al. Reference Valvo, Bella, Scerra and Biondi2007; Biondi et al. Reference Biondi, Valvo, Di Gloria, Scinardo Tenghi, Galofaro and Priolo2008; De La Fuente et al. Reference De La Fuente, Barbosa, Carriedo, Gonzalo, Arenas, Fresno and San Primitivo2009). We noted significant decreases in C10 : 0 and C12 : 0 during spring and especially in summer milk, indicating less de novo synthesis of these FAs by the mammary glands. A similar tendency was also found by Kondyli et al. (Reference Kondyli, Svarnas, Samelis and Katsiari2012), while studying the Greek sheep breeds, Boutsiko and Karamaniko. They noted a decrease in most of the short- and medium chain fatty acids in ewe milk, from spring to summer.
Unsaturated fatty acids
The positive effect of fresh herbage in ruminant diet, on the increase of proportion of UFAs of milk fat has been well documented (Dewhurst et al. Reference Dewhurst, Shingfield, Lee and Scollan2006; Elgersma et al. Reference Elgersma, Wever and Nalecz-Tarwacka2006; Kalac & Samkova, Reference Kalac and Samkova2010). Plants have the unique ability of synthesising de novo ALA, which is the building block of the n-3 series of essential fatty acids. Even though this polyunsaturated fatty acid is usually present in a percentage smaller than 0·05% of forage's total fatty acids, its presence in grasses can substantially affect the fatty acid composition of dairy products (Dewhurst et al. Reference Dewhurst, Fisher, Tweed and Wilkins2003). The concentration of ALA found in ewe milk in this study, raised from winter to summer. It is notable that the concentration of ALA increased twofold in summer in relation to winter milk. This is attributed mostly to the pasture-based feeding system during spring and summer. As far as LA is concerned – the second most abundant fatty acid found in grasses – it was found slightly reduced in winter milk, just as in other studies (De La Fuente et al. Reference De La Fuente, Barbosa, Carriedo, Gonzalo, Arenas, Fresno and San Primitivo2009; Chion et al. Reference Chion, Tabacco, Giaccone, Peiretti, Battelli and Borreani2010).
The proportion of TVA is highly correlated with RA concentration in ruminant milk (Sanz Sampelayo et al. Reference Sanz Sampelayo, Chilliard, Schmidely and Boza2007; Carta et al. Reference Carta, Casu, Usai, Addis, Fiori, Fraghí, Miari, Mura, Piredda, Schibler, Sechi, Elsen and Barillet2008; Mele et al. Reference Mele, Conte, Serra, Pollicardo, Buccioni and Secchiari2008; Nudda et al. Reference Nudda, Palmquist, Battacone, Fancellu, Rassu and Pulina2008; Larsen et al. Reference Larsen, Nielsen, Butler, Leifert, Slots, Kristiansen and Gustafsson2010; Toral et al. Reference Toral, Frutos, Hervás, Juárez and Fuente2010; Moioli et al. Reference Moioli, Contarini, Pariset, Marchitelli, Crisà, Catillo and Napolitano2012). TVA serves as a precursor in the ruminant mammary gland for the Δ9-desaturase to act upon, thus producing RA (Bauman et al. Reference Bauman, Mather, Wall and Lock2006). In comparison to winter milk, increased concentrations of TVA were found in spring and summer milk. These findings are in accordance with a number of other studies (Valvo et al. Reference Valvo, Bella, Scerra and Biondi2007; Biondi et al. Reference Biondi, Valvo, Di Gloria, Scinardo Tenghi, Galofaro and Priolo2008; Soják et al. Reference Soják, Blasko, Kubinec, Górová, Addová, Ostrovsky and Margetín2013).
RA is by far the most abundant conjugated linoleic isomer, usually representing 75 to 90% of total CLA in milk fat (Lock & Bauman, Reference Lock and Bauman2004). More than 70% of the RA of ruminants’ milk is produced in the mammary tissue by the activity of Stearoyl-CoA (SCD) (Bauman et al. Reference Bauman, Mather, Wall and Lock2006). This fatty acid has been shown to possess health-enhancing properties such as anticarcinogenic and antidiabetogenic activities. So, efforts have been made to increase milk CLA content (Bauman et al. Reference Bauman, Mather, Wall and Lock2006). Various authors (Tsiplakou et al. Reference Tsiplakou, Mountzouris and Zervas2006; De La Fuente et al. Reference De La Fuente, Barbosa, Carriedo, Gonzalo, Arenas, Fresno and San Primitivo2009; Ostrovský et al. Reference Ostrovský, Pavlíková, Blaško, Górová, Kubinec, Margetín and Soják2009) found substantial seasonal differences in RA in milk fat, being consistently higher in the spring-summer period, which coincides with the grazing season. Similar observations were made when ewes were turned out to pasture, in relation to indoors feeding stradegies (Valvo et al. Reference Valvo, Bella, Scerra and Biondi2007; Biondi et al. Reference Biondi, Valvo, Di Gloria, Scinardo Tenghi, Galofaro and Priolo2008; Soják et al. Reference Soják, Blasko, Kubinec, Górová, Addová, Ostrovsky and Margetín2013). In general, RA concentration is higher in milk from animals fed pasture than those fed indoors. Our findings concur with the latter statement. The RA presented a twofold increase from winter to summer milk (0·58 vs. 1·12). This significant increase is probably due to the extensive rumen biohydrogenation of pasture LA and ALA, with simultaneous TVA production.
Important ratios, indices and groups of FAs
Spring and summer milk was characterised by a high concentration of MUFA (mainly oleic acid) and PUFA. The high concentration of PUFA in fresh grass consumed by the animals, increase the PUFAs in milk, thus significantly improving the PUFA/SFA ratio in milk. Furthermore, the biologically important n-6/n-3 ratio, was also significantly ameliorated, thus being closer to the optimum level (1 : 1) suggested for human diet (Simopoulos, Reference Simopoulos2010).
In contrast to medium chain saturated fatty acids (C6-C10), the fatty acids C12 : 0, C14 : 0, and C16 : 0 exhibit a cholesterol elevating action with C14 : 0 having four times more cholesterol raising potential. Apart from their atherogenic action, C14 : 0, C16 : 0 along with C18 : 0 possess also a thrombogenic potential (Ulbricht & Southgate, Reference Ulbricht and Southgate1991). Taking into consideration the above factors, Ulbricht & Southgate (Reference Ulbricht and Southgate1991) proposed the atherogenicity and thrombogenicity indices as a measure of estimating the atherogenic and thombogenic potential of the dietary fats. Fats with higher A.I. and T.I. values are considered to be more detrimental to human health. The pasture based management, implemented during spring and summer caused a decrease in A.I. and T.I. in milk. This result could be explained by: (a) the inhibitory action of pasture-derived UFAs, upon the short- and medium chain fatty acid de novo synthesis and (b) the increase concentration of PUFAs in spring and especially summer milk.
Terpene composition of ewe milk
Terpenes are plant secondary metabolic products, built up from isoprene units. In general, these secondary metabolites are detected mostly in dicotyledons (Mariaca et al. Reference Mariaca, Berger, Gauch, Imhof, Jeangros and Bosset1997). Species belonging to the families Asteraceae and Lamiaceae have been reported to be very rich in terpenes (Serrano et al. Reference Serrano, Cornu, Kondjoyan, Micol and Figueredo2007). Terpene content and profile in ruminant milk is influenced by feed and especially by the botanical composition of the pasture been grazed by the animals (Tornambe et al. Reference Tornambe, Cornu, Pradel, Kondjoyan, Carnat, Petit and Martin2006; De Noni & Battelli, Reference De Noni and Battelli2008; Abilleira et al. Reference Abilleira, De Renobales, Nájera, Virto, Ruiz de Gordoa, Pérez-Elortondo, Albisu and Barron2010).
Similarly to fatty acids, the seasonal changes in the botanical composition of the pasturelands, affect the profile and content of milk terpene constituents. This was clearly revealed by our results, as the terpene profile of winter milk was very different from spring and summer milk.
The terpene profile of milk samples, in all three seasons, showed the high presence of the monoterpenes: a-pinene, b-pinene and D-limonene. These terpenes are considered common in other studies (Viallon et al. Reference Viallon, Verdier-Metz, Denoyer, Pradel, Coulon and Berdagué1999; Fernandez et al. Reference Fernandez, Astier, Rock, Coulon and Berdagué2003). Their concentrations in milk: for the a-pinene were higher during spring and summer than the winder months, for the b-pinene was higher in summer and for D-limonene there are significant differences between seasons, but the highest rate (2·40) was found in winter (Table 3).
The limited supply of fresh grass and the supplementary feeding to the ewes during winter did not offer significant concentrations of the aforementioned monoterpenes. While, during grazing periods (spring and summer), the animals consumed a diversity of botanical species, mostly dicotyledons, thereby increasing the number of monoterpenes detected in milk.
The most characteristic sesquiterpenes detected in milk, especially during spring and summer, were β-caryophyllene and α-caryophyllene. Even though their concentrations were not significantly changed, their rFOA was gradually increased from winter to summer. B-caryophyllene has been proposed as a possible biochemical marker and discrimination tool between animals raised on pasture from those consuming concentrates (Prache et al. Reference Prache, Cornu, Berdague and Priolo2005). According to our results, a-caryophyllene could also serve as a potential biomarker of feed-type indication, as it was detected in a higher frequency in summer than in winter milk.
The presence of some new sesquiterpenes in milk samples during summer such as: α-muurolene, γ-muurolene, D-germacrene, a-amorphene, α-elemene, β-panasinsene, isodelene, β-cadinene, γ-cadinene, δ-cadinene, and valencene, indicates that the animals had access to new botanical species.
Conclusion
This study revealed the effect of seasonality on the FA composition of ewe milk. The significant variability was mostly attributed to the diet. Specifically, the pasture-based diet during the months of spring and especially of summer resulted to the amelioration of important ratios, indices and groups of FAs in sheep milk. Furthermore, pasture-feeding mainly in seasons of spring and summer enhanced the concentration of terpene molecules in sheep milk.
This work was funded by the National programme PINDOS.
