Health-conscious consumers are demanding nutritious food with beneficial features from a human health perspective. The major issue in relation to milk consumption is the milk fatty acid (FA) profile, mainly represented by saturated fatty acids, which account for 62–70% of the total milk fatty acids. However, milk and dairy products are characterised also by the presence of rumenic acid (RA) and its precursor vaccenic acid (VA), which have potential health benefits, including anticarcinogenic properties in experimental animals (Hughes & Dhiman, Reference Hughes and Dhiman2002).
Milk fat composition can be modulated by supplementing the diets of cows with unsatured lipids to meet consumers’ demands. The main sources of polyunsaturated fatty acids (PUFA) are oilseed lipids, such as flaxseed (Glasser et al. Reference Glasser, Ferlay and Chilliard2008), that can be fed to dairy animals to modify the milk fatty acid profile of milk for human consumption (Caroprese et al. Reference Caroprese, Marzano, Marino, Gliatta, Muscio and Sevi2010). In previous experiments whole flaxseed supplementation of dairy cows at about 7% of daily DM intakes succeeded in increasing PUFA in milk. However, oilseed supplementations, and in particular flaxseed, have a negative impact on management costs of dairy farms, thus discouraging farmers in their utilisation. As a consequence, the study of the reduction of the amount of flaxseed administration in the cows’ diet in order to reduce costs and to obtain improvement on milk yield and composition is required.
In Italy, about 80% of dairy farms produce milk from Friesian cows both for direct consumption and for cheese production. In addition, Jersey milk is used to increase cheese yield production and meet market demand in terms of suitability for cheese making. The use of Jersey milk for Cheddar cheese making was found to lead to an improvement in profit for the cheese makers, especially at higher inclusion rates (Bland et al. Reference Bland, Bailey, Grandison and Fagan2015). However, Friesian and Jersey cows showed differences in the composition of milk fat when increasing dietary intake of calcium salts of palm fatty acid distillate were tested (Beaulieu & Palmquist, Reference Beaulieu and Palmquist1995).
Milk coagulating properties can be influenced by composition and structural organisation of proteins and fat milk (Logan et al. Reference Logan, Auldist, Greenwood and Day2014). Flaxseed administration to lactating ewes, besides changing milk fatty acid composition, succeeded in enhancing milk coagulating properties, probably as a result of increasing fat and casein content (Caroprese et al. Reference Caroprese, Albenzio, Bruno, Fedele, Santillo and Sevi2011). Timmen & Patton (Reference Timmen and Patton1988) affirm that milk fat globule (MFG) size could be influenced by differences in fatty acids composition. Both MFG and casein micelle (CM) size can be influenced by genetics variations (Bijl et al. Reference Bijl, de Vries, van Valenberg, Huppertz and van Hooijdonk2014; Logan et al. Reference Logan, Auldist, Greenwood and Day2014), and feed (Devold et al. Reference Devold, Brovold, Langsrud and Vegarud2000; Couvreur & Hurtaud, Reference Couvreur and Hurtaud2007). Our hypothesis is that flaxseed administration could have an effect on milk coagulation properties, thus influencing its cheese-making ability, by altering the milk fatty acid composition. The objectives of the present experiment were to: (i) evaluate the effects of flaxseed administration on fatty acid profile of milk from Friesian and Jersey cows, and (ii) evaluate the change in milk coagulation properties in Friesian and Jersey cows as affected by flaxseed administration.
Materials and method
Animals, experiment al design, and dietary treatments
The experiment was conducted in a farm located in Gravina in Puglia (BA), Apulia, Southern Italy (latitude: 40°49′14″52 N and longitude 16°25′24″96 E). A 30 d trial was performed from May to June of 2014 with 20 Italian Friesian cows and 20 Jersey cows divided into 2 groups of 10 animals which were balanced according to days in milk (112·31 ± 5·15 d). Experimental diets were (1) a conventional diet (CON) administrated as unifeed (2) a diet containing 0·5 kg/d of whole flaxseed (FS) in substitution of an equal amount of cotton seed administrated in the unifeed. Formulation of experimental diets and chemical composition of the diets are shown in Table 1. Cows were housed in tie stalls and individually fed; water was available ad libitum. The diets were fed twice daily and each group of cows was fed separately.
Table 1. Chemical composition, and NEL of the experimental diets (DM basis)
† Sunflower, corn gluten feed, sugar beet pulp.
‡ Lin Tech (Tecnozoo srl, Torreselle di Piombino Dese, Italy).
§ Ca salts of fish oil, NaHCO3, NaCl.
¶ Calculated according to NRC (2001).
Feed sampling and analysis
The chemical composition of diets was determined by standard procedures (AOAC, 1990). The crude fibre and fibre fractions were determined by Fiber Cap (FC 221, FOSS). A representative sample of feed was collected for fatty acid (FA) analysis of feed according to O'Fallon et al. (Reference O'Fallon, Busboom, Nelson and Gaskins2007). The fatty acid methyl esters (FAME) were analysed on a Agilent 6890N gas chromatograph. Separation of the FAME was performed using a DB 23 fused-silica capillary column 60 m × 0·25 mm (i.d.) with 0·25 µm film thickness. Operating conditions were: helium flow rate of 1·2 ml/min; FID detector at 250 °C; a split-splitless injector at 240 °C and an injection volume of 1 µl with a split ratio 1 : 50. The initial column temperature was set at 60 °C, increased to 180 °C at 25 °C/min and finally increase to 230 °C at 6 °C/min and held for 15 min. Individual FAME's peaks were identified using standards from Matreya (Matreya, 2178 High Tech Road, State College, PA 16803 USA). Each fatty acid was reported as a percentage of FAME (NRC, 2001).
Milk sampling and analysis
Cows were milked twice daily (07 : 00 and 14 : 30) using pipeline milking machines. Milk yield was recorded daily by means of graduated measuring cylinders attached to individual milking units. At the beginning, at day 15 and then at day 30 of the experiment milk samples consisting of proportional volumes of morning and evening milk, were individually collected in 200 ml sterile plastic containers after cleaning and disinfection of teats (70% ethyl alcohol) and discharging the first streams of foremilk. Milk samples were carried in our laboratory by means of transport tankers at 4 °C. One aliquot was stored at −20 °C for fatty acid analysis. Fresh samples were used for chemical analysis as following: pH (GLP 21 Crison, Spain), total protein, casein, fat and lactose content using an infra red spectrophotometer (Milko Scan 133B; Foss Electric, Hillerød, Denmark) according to the International Dairy Federation standard (IDF, 1990); evaluation of the milk coagulation properties (rennet coagulation time, time to clot firmness, and clot firmness after 30 min) measured by a Foss Electric Formagraph (Foss Electric, Hillerød, Denmark). At day 30 of the experiment bulk milk from each experimental group was collected and taken to our laboratory by means of transport tankers at 4 °C to perform electrophoresis analysis.
For the analysis of milk fatty acids, milk fat was extracted according to the procedure of Luna et al. (Reference Luna, Juárez and de la Fuente2005) and trans-esterification of fatty acids according to ISO-IDF (2002) procedures. Briefly, fatty acid methyl esters were separated and measured using a gas chromatograph (Agilent 6890N) equipped with CP-Sil 88 fused-silica capillary column (100 m × 0·25 mm i.d. with 0·25-μm film thickness). Operating conditions were a helium flow rate of 1 ml/min, a flame-ionisation detector at 260 °C, a split-splitless injector at 260 °C, and an injection volume of 1 µl with a split ratio 1 : 50. The temperature program of the column was set at 100 °C with a subsequent increase to 240 °C at 3·5 °C/min and held for 15 min. Fatty acids were identified by comparing their retention times with the fatty acid methyl standards (FIM-FAME-7-Mix, Matreya LLC, Pleasant Gap PA), with the addition of C18 : 1–11t, C18 : 2–9c, 11t, C18 : 2–9c, 11c, C18 : 2–9t, 11t, and C18 : 2–10t, 12c (Matreya LLC, Pleasant Gap PA). Data were collected and integrated using Agilent Chemstation Software rev. B.04·03. Fatty acids were reported as grams per 100 g of FA. Saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and PUFA were calculated. Atherogenic (AI) and thrombogenic (TI) indices were calculated according to Ulbricht and Southgate (Reference Ulbricht and Southgate1991) formula.
Electrophoresis analysis of caseins
Polyacrilamide gel isoelectric focusing
Casein fraction from bulk milk of Friesian and Jersey cows, fed in CON and FS condition, was prepared according to the procedure of Aschaffenburg & Drewry (Reference Aschaffenburg and Drewry1959). Wet caseins were freeze-dried and stored at −20 °C until electrophoresis analysis. Isoelectric focusing was performed on a thin layer polyacrylamide gel (0·25 × 120 × 100 mm3) in a pH gradient 2·5 ÷ 6·5 obtained by mixing pharmalyte 2·5–5·0, 4·0–6·5 and 5·0–6·0 in the proportion 42, 42 and 16% (v/v), respectively. Electrophoretic conditions were: 1000 Vmax, 4 mA max and 20W for 700 V/h in the pre-focalisation phase and 1500 Vmax, 4 mA max and 20W for 4490 V/h.
Two dimensional gel electrophoresis
Each sample (0·10 g of wet casein) was dissolved in 1·0 ml of 9 M urea to obtain a sample solution. An aliquot of sample solution (100 µl) was mixed with 150 µl of DeStreak and 100 mm DTT to obtain 250 µl of rehydration solution. The immobilised pH linear gradient (IPG) 13-cm dry strips in pH 3–10 (Amersham Biosciences, Melbourne, VIC, Australia) were placed in the 250 µl of rehydration solution, covered with the cover fluid (Amersham Biosciences, Melbourne, VIC, Australia) and kept for a minimum of 8 h at room temperature. Rehydrated IPG strips were submitted to the first dimension using an Ettan IPGphor 3 (GE-Healthcare Bio-Sciences) electrophoresis unit (Amersham Biosciences). Electrophoretic separation was run in 4 steps: 500 V/h followed by 1000 V/h, a gradient to 8000 V over 90 min, and 8000 V for 3 h for a total of 36000 V/h. After the IPG electrophoresis, the strips were equilibrated, first in a solution containing 1·5 M Tris-HCl, pH 6·8, 6 M urea, 30% glycerol (w/v), 2% SDS, 64 mm DTT, and traces of bromophenol blue, for 15 min, and then, in a solution containing 1·5 M Tris-HCl, pH 6·8, 6 M urea, 30% glycerol (w/v), 2% SDS, 135 mm iodoacetamide, and traces of bromophenol blue, for further 15 min. The equilibrated IPG dry strips were loaded on top of the 8–18% SDS-PAGE gradient polyacrylamide gel (1·5 × 18 × 16 mm3), embedded with 1% agarose. The electrophoresis was carried out in an Hoefer SE 600 Series Ruby Standard Vertical Electrophoresis Unit (GE-Healthcare Bio-Sciences) at a constant voltage of 100 V, at 30 mA/gel, for 16 h at 15 °C. Gels were stained with 0·25% w/v CBB overnight and destained in water. Images were captured on an Image Master Scanner (GE-Healthcare Bio-Sciences) in transmission mode, at a resolution of 300 dpi.
Statistical analysis
Data were processed using ANOVA with the REPEATED statement in PROC MIXED with CV (variance components) as covariance structure of SAS (SAS, 2013). Diet, time of sampling, breed, and their interactions were Fixed factors. Animal was a Random factor nested in the treatment. When significant differences between means were found (P < 0·05), a Tukey post-hoc test was performed to adjust tests in multiple comparison.
Results and discussion
Milk yield and milk composition
Average of the three sampling times of the milk yield and composition of cows fed control diet (CON) and flaxseed (FS Table 2) is shown in Table 2. Milk production was influenced only by breed; Friesian cows showed higher milk production than Jersey cows (P < 0·001). In previous experiments FS supplementation to Friesian cows increased milk yield (Caroprese et al. Reference Caroprese, Marzano, Marino, Gliatta, Muscio and Sevi2010, Reference Caroprese, Sevi, Marino, Santillo, Tateo and Albenzio2013); in the present experiment the absence of apparent effects of FS administration on milk yield, however, was an expected result based on the reduction of FS supplemented by about three-fold. In the present experiment protein and casein yield, and milk composition were significantly influenced only by breed. It is well known that Jersey cows yield lower milk than Friesian cows with higher fat content than milk from Friesian cattle (4·1–4·9 vs. 3·3–4·1%, respectively) (Buchanan et al. Reference Buchanan, Fitzsimmons, Van Kessel, Thue, Winkelman-Sim and Schmutz2002). Differences in milk yield and composition between Jersey and Friesian milk can be ascribed both to differences in genetics and to different capability of utilising dietary fibre and N between Jersey and Friesian as reported in Aikman et al. (Reference Aikman, Reynolds and Beever2008). Those authors reported that despite the faster rate of passage through the gastrointestinal tract, the digestibility of dietary components by Jersey cows was similar or higher than Friesian as a result of increased eating and rumination time per kilogram of DM consumed.
Table 2. Least Square Means ± sem of milk yield, composition of Friesian and Jersey cows fed control diet or flaxseed (CON and FS)
NS, not significant.
*, P < 0·05; **, P < 0·01, ***, P < 0·001.
Milk fatty acid composition
Table 3 shows the average of each milk fatty acid at the three sampling times. Milk C10 : 0 and C12 : 0 content was influenced by diet (P < 0·001 for C10 : 0 and P < 0·01 for C12 : 0,) with higher contents measured in milk from CON than in milk from FS diet. Both C14 : 0 and C16 : 0 milk contents were influenced by breed (P < 0·05), and diet (P < 0·01) displaying higher contents on average in Jersey than in Friesian milk. These results are in agreement with previous reports (Drackley et al. Reference Drackley, Overton and Douglas2001), in which C16 : 0 content was higher for Jersey milk than for Friesian milk; however, in Drackley et al. (Reference Drackley, Overton and Douglas2001) experiment fat supplementation did not influence C16 : 0 content of milk. The content of C18 : 0 was significantly influenced by breed and by the interaction breed x diet (P < 0·001 and P < 0·01, respectively). The C18 : 0 content was higher in Jersey than in Friesian milk, and in FS milk from Jersey than from Friesian in agreement with previous findings (Mustafa et al. Reference Mustafa, Chouinard and Christensen2003; Loor et al. Reference Loor, Ferlay, Ollier, Doreau and Chilliard2005, Caroprese et al. Reference Caroprese, Marzano, Marino, Gliatta, Muscio and Sevi2010). C18 : 1trans-11 showed higher values in Friesian than in Jersey milk (P < 0·001), and in FS than in CON milk (P < 0·01). In addition, a significant interaction breed x diet was found, with higher content of C18 : 1trans-11 measured in milk from FS Friesian cows than from FS Jersey cows (P < 0·01). As reported by Bell et al. (Reference Bell, Griinari and Kennelly2006) the synthesis of C18 : 1trans-11 is influenced by the accumulation of trans C18 : 1 in the rumen. It could be argued that Friesian and Jersey cows are characterised by differences in ruminal biohydrogenation. Whole flaxseed administration, however, in Friesian breed succeeded in enhancing milk content of C18 : 1trans-11, which is considered the main health promoting fatty acid in milk together with CLA (Bauman & Lock, Reference Bauman, Lock, Fox and McSweeney2006). The increase of C18 : 1 isomers can be the result of a partial biohydrogenation in the rumen due to increasing of linolenic acid with flaxseed diet, and of the desaturation of C18 : 0 in the mammary gland (Kennelly, Reference Kennelly2006). The evolution of C18 : 3 n-3 in milk from Friesian and Jersey cows fed control diet or flaxseed during time is shown in Fig. 1. In general, significant differences were found in C18 : 3 n-3 content, which was influenced by breed (P < 0·01) and diet (P < 0·001). No differences at the beginning and at 15 d were found; whereas, at 30 d of the experiment FS milk from both Friesian and Jersey cows displayed significantly higher contents than CON milk from both Friesian and Jersey cows. On average, the content of C18 : 3 n-3 was higher in Friesian milk than in Jersey milk, according to Drackley et al. (Reference Drackley, Overton and Douglas2001). However, in the present experiment, flaxseed administration resulted in an enhancement of C18 : 3 n-3 content to such an extent that no differences between supplemented Friesian and Jersey cows were found at the end of the experiment. This is in agreement with Palladino et al. (Reference Palladino, Buckley, Prendiville, Murphy, Callan and Kenny2010).
Fig. 1. Least Squares means ± sem of C18 : 3 n-3 content measured in milk from Friesian and Jersey cows fed control diet or flaxseed (CON and FS) at 0, 15, and 30 d of the experiment. a,b,c Values with different superscripts differ between feeding treatments within a sampling day (P < 0·05).
Table 3. Least Square Means ± sem of fatty acid composition (g/100 g of total fatty acids) of milk from cows fed control diet or flaxseed (CON and FS)
NS, not significant.
*, P < 0·05; **, P < 0·01; ***, P < 0·001.
Among the CLA isomers in milk CLA cis-9trans-11 was influenced by breed with higher contents in Friesian than in Jersey milk (P < 0·001). Nevertheless, even if no effects of diet on milk CLA content was measured, the diet led to an increase in both C18 : 3 n-3 and in C18 : 1trans-11 in Friesian milk, suggesting that reducing the amount of flaxseed administration can still enrich milk fatty acid profile. In addition, our results confirmed that significant genetic variation exists for the concentration of several fatty acids in milk, and this is most likely a function of differential expression of genes coding for enzymes catalysing FA synthesis within the mammary gland (Soyeurt & Gengler, Reference Soyeurt and Gengler2008; Mele et al. Reference Mele, Dal Zotto, Cassandro, Conte, Serra, Buccioni, Bittante and Secchiari2009). SFA, MUFA, and PUFA were influenced by breed (P < 0·001); on average milk from Jersey cows showed higher values of SFA, and lower of MUFA, and PUFA than milk from Friesian cows. In general, flaxseed administration resulted in a reduction of SFA (P < 0·001) and an increase in MUFA (P < 0·001) content. Furthermore, an effect of the interaction breed x diet was found for both SFA and MUFA (P < 0·05); milk from FS group displayed lower SFA and higher MUFA than CON milk both in Friesian and in Jersey milk. Furthermore, the diet supplemented with flaxseed resulted in increased content of n-3 FA in milk. The Atherogenic and Trombogenic indexes were influenced by breed (P < 0·001) and diet (P < 0·001) with lower values in flaxseed group especially in Friesian cattle. Milk from FS cows was characterised by reduced Atherogenic and Trombogenic indexes, according to previous findings in Caroprese et al. (Reference Caroprese, Marzano, Marino, Gliatta, Muscio and Sevi2010), suggesting that, even at low level of supplementation, flaxseed in the diet of dairy cows yields milk with healthier features from a human perspective.
Milk coagulation properties and Electrophoresis analyses
Milk coagulating properties shown in Fig. 2 were significantly influenced by diet (P < 0·01 for coagulation time; P < 0·001 time to clot firmness and clot firmness after 30 min). Jersey cows showed lower values of coagulation time, and time to clot firmness, and higher values of clot firmness at 30 min than Friesian cows. Breed comparison revealed that milk from Jersey cows generally exhibited superior coagulation properties when compared with milk from Friesian cows (Jensen et al. Reference Jensen, Poulsen, Andersen, Hammershøj, Poulsen and Larsen2012). FS diet reduced time to clot firmness and increased clot firmness in Friesian milk; it resulted in increased clot firmness in Jersey milk. In Friesian milk time to clot firmness was affected by the interaction diet x breed, showing a significant reduction in flaxseed when compared to the control group.
Fig. 2. Least Squares means ± sem of coagulation time, time to clot firmness, and clot firmness after 30 min measured in milk from Friesian and Jersey cows fed control diet or flaxseed (CON and FS). a,b,cValues with different superscripts differ between feeding treatments within a sampling day (P < 0·05).
To better investigate the results of flaxseed administration on milk coagulating properties and to examine the relation between changes in fatty acid profile and casein fractions from Friesian and Jersey bulk milk, an electrophoresis study was performed. It was stated that cows carrying the αs1-B, β-B, κ-B caseins and β-LG B haplotype show the better coagulation properties (Aleandri et al. Reference Aleandri, Buttazzoni, Schneider, Caroli and Davoli1990; Jakob & Puhan, Reference Jakob and Puhan1992). Isoelectrofocusing patterns of Jersey and Friesian whole caseins (Fig. 3) showed a different genetic polymorphism. Jersey cows carried homozygous variant B at locus κ-CN, heterozygous variants A1, A2 and B at locus β-CN and heterozygous variants B and C at locus αs1-CN. Friesian cows carried heterozygous variants A and B at locus κ-CN, heterozygous variants A1, A2 and B at locus β-CN and homozygous variant B at locus αs1-CN. Friesian and Jersey milk also differed for the content of variant A1 at β-CN locus as a result of a different distribution of this variant. Beside the different αs1-CN variants found in Friesian and Jersey cows, the 2DE maps showed a different form of the αs1-CN spots in milk from cows fed flaxseed with respect to 2DE maps of milk from control cows irrespective of breed. Previous findings led to the hypothesis that a different aggregation of αs1-CN fraction occurred in bulk milk from flaxseed fed cows (Fig. 4).
Fig. 3. Polyacrylamide gel isoelectric focusing in the pH range 2·5–6·5 of whole caseins from Friesian and Jersey cows fed control diet or flaxseed (CON and FS). Different genetic polymorphism was revealed in two breed.
Fig. 4. Two dimensional gel electrophoresis (2-DE) of whole caseins from Friesian and Jersey cows fed control diet or flaxseed (CON and FS). 2-DE maps were obtained by using IPG linear gradient 3·0–10·0 in first dimension and SDS-pore gradient gel 8–18% electrophoresis (PGGE) in second dimension.
The αs1-CN membranous form has been suggested to play a key role in the early steps of casein micelle biogenesis and/or casein transport during the secretory pathway, being required for the efficient export of the other caseins from the endoplasmic reticulum (ER: Chanat et al. Reference Chanat, Martin and Ollivier-Bousquet1999; Le Parc et al. Reference Le Parc, Houeto, Pigat, Chat, Léonil and Chanat2014). The lipid raft interaction of αs1-CN with the mammary epithelial cell membranes is strengthened by its aggregative properties acting through disulphide bonds (Le Parc et al. Reference Le Parc, Houeto, Pigat, Chat, Léonil and Chanat2014). A strong relation between αs1-CN and biophysical properties of milk fat globules (MFG) has been found, in particular, the genetic polymorphism at αs1-CN locus can affect both structure and composition of MFG membrane (MFGM: Cebo et al. Reference Cebo, Lopez, Henry, Beauvallet, Ménard, Bevilacqua, Bouvier, Caillat and Martin2012). Therefore, a strong relation between fat composition of MFGM and αs1-caein fraction is well documented. In the present study, cows fed flaxseed increased by 6% to about 9% the proportion of unsaturated fatty acids, respectively in Jersey and Friesian milk, compared with control milk. The alteration in the proportion of unsaturated fatty acids induced by flaxseed could have influenced the size and behaviour of milk fat globule as previously demonstrated by Briard et al. (Reference Briard, Leconte, Michel and Michalski2003); Couvreur et al. (Reference Couvreur, Hurtaud, Marnet, Faverdin and Peyraud2007) and Wiking et al. (Reference Wiking, Stagsted, Björck and Nielsen2004). Previous studies found that milk from cows fed supplemental fish meal, fish oil and sunflower oil or pasture displayed a decrease in MFG and casein micelles (CN) size (Jones et al. Reference Jones, Weiss and Palmquist2000; Avramis et al. Reference Avramis, Wang, McBride, Wright and Hill2003; Couvreur et al. Reference Couvreur, Hurtaud, Marnet, Faverdin and Peyraud2007). Therefore, the interaction of MFGM-CM influenced coagulation properties of milk (Couvreur et al. Reference Couvreur, Hurtaud, Marnet, Faverdin and Peyraud2007; Logan et al. Reference Logan, Auldist, Greenwood and Day2014). On the basis of these considerations and of the changes measured in the proportion of unsaturated fatty acids in milk from cows with flaxseed diet we could hypothesise a rearrangement of MFGM, interacting with casein micelles and responsible for self-assembling of two αs1-CN genetic variants. Further studies are needed to clarify this hypothesis by investigating the changes occurring in MFGM composition from cows with a flaxseed diet.
Conclusions
The results of the present study demonstrated that flaxseed in the diet of dairy cows can improve milk fatty acid profile with a reduction of saturated fatty acids, an increase of monounsaturated and polyunsaturated fatty acids, in particular in Friesian milk. Some differences in the content of saturated fatty acids, and monounsaturated fatty acids on milk fatty acid profile after flaxseed administration emerged between Friesian and Jersey cows.
Milk coagulating ability was affected by the flaxseed administration in both breeds, resulting in a reduced time to clot firmness and increased clot firmness, probably as a result of different aggregation of αs1-casein micelles, caused by the change in fatty acid profile of milk fat globule. Further studies are needed to elucidate the relationships between flaxseed administration and altered milk fat globule size and composition, and in milk fat globule interactions with casein micelles.
This work was financially supported by Regione Puglia – Cooperation Projects misura 124- through the project: ‘Sviluppo di prodotti lattiero-caseari ad elevata garanzia e funzionalità (Pro.Lat.Gar.Fu)’.
