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Animal Feed Science and Technology 178 (2012) 27– 39

Contents lists available at SciVerse ScienceDirect

Animal Feed Science and Technology

journal homepage: www.elsevier.com/locate/anifeedsci

ntegration of extruded linseed into dairy sheep diets: Effects on milkomposition and quality and sensorial properties of Pecorino cheese

. Mughetti a, F. Sinesiob, G. Acutia, C. Antoninia, E. Monetab, M. Peparaiob,. Trabalza-Marinuccia,∗

Dipartimento di Patologia, Diagnostica e Clinica Veterinaria, Università degli Studi di Perugia, Via S. Costanzo 4, 06126, Perugia, ItalyINRAN – Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione, via Ardeatina 546, 00178, Roma, Italy

r t i c l e i n f o

rticle history:eceived 4 March 2011eceived in revised form 24 August 2012ccepted 21 September 2012

eywords:xtruded linseedheepecorino cheeserganoleptic properties

a b s t r a c t

The objective of this work was to study the influence of including extruded linseed at differ-ent supplementation levels in dairy sheep feed on the chemical, organoleptic and nutritionalquality of milk and Pecorino cheese. Three hundred and thirty multiparous Sarda ewes,three weeks before their expected date of parturition, were divided into three groups of110 animals each. Ewes were fed one of three different concentrates: a control concentrate(CTR) without linseed and two concentrates supplemented with different levels (100 and200 g/kg as fed; EL-10 and EL-20, respectively) of extruded linseed. The experimental con-centrates were fed to the ewes during late pregnancy (400 g per head per day) and earlylactation (60 days after parturition; 800 g per head per day). All animals had unlimitedaccess to pasture and hay. Milk production was recorded, and milk samples were collectedfor the analysis of chemical composition and clotting properties. Cheeses were made withbulk milk from the three groups using a traditional cheese-making technique. After 60 daysof ripening, the chemical composition and organoleptic properties of the cheeses were ana-lysed using a Panel test. Milk yield and all major milk components, except milk fat yield,were both linearly (0.01<P<0.001) and quadratically (0.05<P<0.001) related to the level oflinseed in the diet. Except for the urea content, which decreased at a decreasing rate (lin-ear P<0.001 and quadratic P<0.05), milk components increased at an increased rate withincreasing EL supplementation. The fatty acid composition of milk and cheese was affectedby dietary linseed supplementation. Milk fat of groups receiving feed that included extrudedlinseed showed higher levels of monounsaturated fatty acids and lower levels of saturatedfatty acids. Linseed administration linearly increased milk polyunsaturated fatty acid con-tent (P<0.001). The C18:3 n-3 milk content increased by 36 and 87% (P<0.001) for the EL-10and EL-20 groups, respectively. The higher content of total n-3 fatty acids in milk causeda linear decrease of the n-6/n-3 fatty acid ratio in the EL groups (P<0.001). Dietary treat-ments affected the chemical composition of cheese; the increase in the level of extruded

linseed was negatively correlated with the moisture content of cheeses. Modifications tothe fatty acid profiles of the cheeses were similar to those observed for milk. Sensory prop-erties of cheese were not negatively affected by dietary treatments. Cheeses produced fromgroups fed diets with added linseed had higher scores for overall and ripe cheese flavour

Abbreviations: ADF, acid detergent fibre; ALA, alpha-linolenic acid; CLA, conjugated linoleic acid; CTR, control concentrate with no added extrudedinseed; DHA, docosaesaenoic acid; DM, dry matter; EL, extruded linseed; EL-10, concentrate with 100 g/kg of extruded linseed; EL-20, concentrate with00 g/kg of extruded linseed; EPA, eicosapentaenoic acid; FA, fatty acids; NDF, neutral detergent fibre; PCA, principal component analysis; PCs, principalomponents; PUFA, polyunsaturated fatty acids.∗ Corresponding author. Tel.: +39 075 585 7707; fax: +39 075 585 7764.

E-mail address: massimo.trabalza@unipg.it (M. Trabalza-Marinucci).

377-8401/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.anifeedsci.2012.09.005

28 L. Mughetti et al. / Animal Feed Science and Technology 178 (2012) 27– 39

and texture properties. Treated cheeses had no off-flavours and were characterised by amore marked grainy texture. The present work indicated that a concentrate containing100 g/kg of extruded linseed can be used to increase the overall quality of Pecorino cheesewithout negative effects on its typical sensorial and organoleptic characteristics or on theproductive performance of the animals.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Linseed (Linum usitatissimum) is a rich source of C18:3 n-3, which makes up 180 g/kg of the total seed weight and 530 g/kgof the total fatty acids (Mustafa et al., 2003).

The benefits of linseed administration on the acidic composition of animal products are well known (Luna et al., 2008,2005; Zhang et al., 2006a,b). Sheep and cows fed with extruded linseed (EL) show increased milk polyunsaturated fatty acids(PUFA) content, particularly n-3 fatty acids (FA), trans-11 C18:1 FA, and total conjugated linoleic acid (CLA) concentrations,and reduced ratios of n-6 to n-3 FA (Mele et al., 2011; Fuentes et al., 2008).

The enrichment of dairy products with n-3 FA could potentially result in niche marketing opportunities for milk andcheese producers. European Union regulations (1924/2006 CE and 116/2010 CE) claim that a food can be considered to be asource of omega-3 fatty acids if it contains at least 0.30 g of 18:3 n-3 per 100 g or at least a total of 40 mg of eicosapentaenoicacid and docosahexaenoic acid per 100 g.

Oilseed enrichment of sheep diet could lead to the development of natural and consumer-acceptable systems and theproduction of high quality dairy foods with enhanced healthful properties. However, the higher costs that characterise theproducts enriched with healthy fatty acids must also be justified by sensorial qualities that are equal or greater than thoseof traditional products.

Few studies have investigated the effects of linseed supplementation on the organoleptic properties of sheep cheese.Previous descriptive sensory analyses of dairy products high in polyunsaturated fatty acids did not provide definitive results.However, extensive trials on the modification of fat composition indicate that increasing the concentration of unsaturatedfatty acids promotes undesirable processes of lipolysis and oxidation, leading to degradation of fat and unacceptable changesin the colour and flavour of milk and dairy products (Campbell et al., 2003).

Pecorino cheese is the most important sheep cheese in Italy. To our knowledge, there are no published studies examiningthe effects of dietary supplementation with EL on the sensorial properties of this milk product. The objective of the presentstudy was to evaluate the effects of different levels of linseed supplementation in the diet on the chemical composition ofmilk and on the chemical and organoleptic properties of Pecorino cheese.

2. Materials and methods

2.1. Animals, experimental design, and diets

The present study was carried out in accordance with the guidelines of the Animal Welfare Committee at the Universityof Perugia, Italy.

The experiment was carried out using 330 Sardinian pluriparous sheep. All ewes were housed in a stable from 4.00 pmto 8.00 am and had unlimited access to pasture for the rest of the day.

Three weeks before the expected date of parturition, the ewes were randomly divided into three groups of equal size,balanced for body weight (45.1 ± 1.2 kg) and body condition score (2.29 ± 0.02; Russell et al., 1969), and fed isoenergeticand isonitrogenous concentrates. Three concentrates were formulated: a control concentrate (CTR), without EL, and twoexperimental concentrates which contained, respectively, 100 g/kg (EL-10) and 200 g/kg (EL-20) of ground and EL. Extrusionof linseed, ground to pass a 4 mm screen, was performed in a single screw extruder (Berga, Treviso, Italy) with a through-put of 1600 kg/h (barrel length: 3.2 m; die diameter: 7 mm; screw speed: 300 rpm; temperature at the end of the barrel:130–138 ◦C; duration: 1 min). After extrusion the product was dried in a counter flow cooler for 12 min. Experimental dietswere administered from three weeks before the expected date of parturition to 60 days post partum. Animals were fed 400to 800 g per head per day of concentrate (during late pregnancy and early lactation, respectively), which was administeredin two equal portions during milking. Alfalfa hay was provided in box feeders ad libitum. Concentrate represented 253 and290 g/kg of total estimated dry matter (DM) intake during late pregnancy and early lactation, respectively.

After lamb weaning, at 40 days of age, ewes were machine-milked twice a day. From days 40 to 60, milk yield was recordeddaily. Pooled morning and evening milk samples from each group were collected every two days in order to analyse thechemical composition and somatic cell count by the infrared method, using MilkoScan 4000 (Foss Electric, Hillerød, Denmark;Biggs, 1978), and to analyse clotting properties such as renneting time, curd consistency and rate of firming (Zannoni and

Annibaldi, 1981; Annibaldi et al., 1977). Milk samples were also stored at −20 ◦C for lipid profile analysis.

A traditional cheese-making technique was used to make Pecorino cheese from bulk milk samples collected from thethree groups at the end of the experimental period. Briefly, 50 L of raw milk from each batch was heated to 38 ◦C, and liquidcalf rennet was added to curdle the milk. After the milk had clotted (after approximately 18 min), the curd was cut to the

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L. Mughetti et al. / Animal Feed Science and Technology 178 (2012) 27– 39 29

ize of little maize grain. The curd was cooked at 42 ◦C for 5 min and, after being removed from the vat, was pressed into4 cm diameter, cylindrical, perforated moulds in order to drain the whey. Cheeses (10 per experimental group) were salted

n brine for 24 h and then transferred to a non-conditioned ripening room where they remained at a temperature of 11 ± 1 ◦Cnd a relative humidity of 73–75% for 60 days.

.2. Recordings, sampling, and analytical procedures

The chemical compositions and lipid profiles of all cheeses were analysed at the end of the ripening period. Cheeses werenalysed for moisture content by AOAC (2000) method 934.01 and for ash content by AOAC (1990) method 942.05. Theat content of cheese samples was determined using the Gerber method, and the total protein content was determined by

easuring the total nitrogen, according to AOAC (1990) Kjeldahl method 976.06, and converting this value to the proteinontent by multiplying by 6.38.

During the trial, samples of pasture, alfalfa hay and concentrates were collected and analysed for chemical composition.M was evaluated following AOAC (2000) method 934.01, and pasture samples prior to DM determination were dried at0 ◦C for 24 h in a forced air oven. Crude protein, crude fat and ash were determined according to AOAC methods 976.06,20.39 and 942.05, respectively (AOAC, 1990). The methods of Van Soest et al. (1991) were used in the analyses of NDF (notssayed with a heat stable amylase), ADF and lignin (sa). Sodium sulphite was used in the NDF procedure, and both NDF andDF were expressed inclusive of ash.

Calcium and phosphorous concentrations were determined following AOAC methods 985.35 (Julshamn et al., 1998) and65.17 (AOAC, 1996), respectively.

Fatty acids in milk samples were extracted according to the Röse-Gottlieb method (AOAC, 1990) modified by Secchiarit al. (2003). Fatty acid extractions in cheese and feed samples were performed according to the method of Folch et al. (1957).

Methylation of milk, cheese and feed fat was conducted using the sodium methoxide methylation procedure (Cecchi et al.,985). Samples of 50–100 mg were accurately weighed into screw-capped centrifuge tubes. Then, 0.5 mL of a 0.3 M NaOMeolution in methanol were added, and the vial was closed, shaken for 1 min, and left to rest at room temperature for 2 min.or neutralization purposes, 0.1 mL of a 0.5 M H2SO4 solution were added and the mixture was shaken for a few seconds.hen, 1.5 mL of distilled water was added, shaken for 10 s, and centrifuged. The organic layer was separated, evaporated toryness under nitrogen, and redissolved in 2 mL of diethyl ether. Finally, 0.8, 0.5 and 1 �L from the organic layer of milk,heese and feed samples, respectively, were injected directly onto the gas-liquid chromatograph. Nonadecanoic acid (C19:0)Sigma–Aldrich, St. Louis, MO, USA) was used as an internal standard.

Fatty acid methyl esters were quantified using a gas-chromatograph Perkin-Elmer-8410 (PerkinElmer, Norwalk, CT,SA) fitted with a fused silica capillary column [SPTM-2380, 100 m × 0.25 mm (internal diameter) with 0.2 �m film thick-ess; Supelco, Inc., Bellefonte, PA, USA]. Hydrogen was used as the carrier gas with a pressure of 120 kPa. The injector andetector temperature was 250 ◦C. Gas-chromatography conditions were as follows: an initial oven temperature of 70 ◦C wasaintained for 1 min, then ramped up at a rate of 5 ◦C/min to a final temperature of 100 ◦C and maintained for 2 min; the

emperature was then increased to 175 ◦C at 10 ◦C/min and held at 175 ◦C for 28 min; the temperature was ramped up to25 ◦C at a rate of 5 ◦C/min and maintained for 25 min. The split ratio was 40:1. Fatty acid peaks were identified by comparinghe retention times of sample peaks with those of the standard mixture (37 Component FAME Mix, SupelcoTM, Bellefonte,A, USA).

Five cheeses per group were used to determine the sensory profile, assessed by a trained sensory panel. Two of theseheeses were used in pre-testing sessions to generate attributes and to standardise the panel’s definitions of the attributes.he other three cheeses were used for test evaluations, using two replicates for each cheese. The descriptive analysis of theheeses was replicated twice (Lawless and Heymann, 1998).

The panel was established before the present study and was composed of nine judges who had several years of experiencen sensory evaluation and were already trained to evaluate cheeses. The judges evaluated the samples in a monadic sequential

ay, scoring the attributes on a continuous unstructured line intensity scale ranging from 0 to 9 and anchored at both endsith extremes for each attribute. Evaluations were carried out in relation to 16 attributes: appearance (two attributes), odour

three attributes), flavour (six attributes) and texture (five attributes) according to the ISO 13299 criteria (ISO, 2003). Allssessments were conducted in a sensory laboratory in individual booths designed according to the international standard,SO 8589 (ISO, 2007). Cheeses were cut in slices of 1 cm, coded with three-digit random numbers, and served at 16 ± 1 ◦C in aalanced order across judges. Data collection was performed with FIZZ software (Biosystemes, Couternon, France) through

computer network.

.3. Statistical analyses

Milk yield, chemical and fatty acid composition of bulk milk and cheese and the clotting properties of cheese werenalysed using an ANOVA in the GLM (General Linear Model) procedure of SAS (2001). As for the models used for milk yield

nd composition, sampling time was not included because it was found to be not significant (P>0.05).

The final model included the fixed effect of dietary treatment and the residual error as:

Yij = � + Di + εij

30 L. Mughetti et al. / Animal Feed Science and Technology 178 (2012) 27– 39

Table 1Chemical composition and metabolisable energy content of the forages (g/kg, unless otherwise stated).

Forages

Alfalfa hay Mixed pasturea

Dry matter 919.0 191.8Crude protein 124.3 20.6Crude fat 17.7 2.0Ash 74.6 12.9NDFb 444.9 50.4ADFc 326.2 22.8Lignin (sa)d 48.1 3.4Calcium 10.1 0.7Phosphorus 5.2 0.4Metabolisable energy (Mcal/kg) 1.716 0.418

a Botanical composition of the pasture was as follows: 750 g/kg Graminaceae (Lolium spp., Festuca ovina L., Bromus spp., Agrostis spp.), 110 g/kg Leguminosae(Trifolium repens L., Vicia spp,), 140 g/kg others: Compositae, Umbelliferae, Plantagineae, Scrophulariaceae and Ranuncolaceae.

b Neutral detergent fibre.c Acid detergent fibre.d Lignin determined by solubilisation of cellulose with sulphuric acid.

where � is the overall mean; Di is the dietary treatment and εij is the residual error.Orthogonal contrasts were used to test the linear and quadratic response to the increase of the level of EL. Data were

reported as least squares means ± standard error (SEM).Sensory data were checked for panel consistency over replicate evaluations. Subsequently, a two-way analysis of variance

was applied to each sensory attribute with dietary treatment groups and cheese samples included as fixed effects. The modelused was:

Yijk = � + Di + Cj + εijk

where � is the overall mean; Di is the dietary treatment; Cj is the cheese sample and εijk is the residual error. Attributeswere analysed using a Duncan Multiple Range test (at P<0.05 or P<0.001 significance levels) to estimate the differencebetween diet groups. Univariate analyses were performed using XL-Stat (Addinsoft). The data were then averaged acrossjudges and replicates. The information carried by the original variables was projected onto a smaller number of underlying“latent” variables called Principal Components (PCs). Principal component analysis (PCA) was performed on the sensory data(covariance matrix of the panel sample means) using Unscrumbler (CAMO Process AS, Oslo, Norway).

3. Results

The average chemical composition of pasture, alfalfa hay and concentrates are reported in Tables 1–3.Milk yield and all major milk components, except milk fat yield, were both linearly (0.01<P<0.001) and quadratically

(0.05<P<0.001) related to the level of EL in the diet. Milk yield increased by 3% and decreased by 11% with the lowest and thehighest EL level, respectively. Except for the urea content, which decreased at a decreasing rate (linear P<0.001 and quadraticP<0.05), milk components increased at an increased rate with increasing EL supplementation (Table 4).

Due to the milk yield response to the dietary treatment, the daily production of all major milk constituents (protein,casein, fat and lactose) was linearly (P<0.01) and quadratically (P<0.01) related to the EL intake, with an increase at thelower EL addition level and a sharp decrease at the higher EL dietary supplementation.

Overall clotting properties of ewe milk tended to be positively affected by EL administration (Table 4). In particular, alinear (P<0.001) decrease in renneting time was observed with increasing levels of EL included in the diet.

The fatty acid composition of milk fat was affected by the dietary EL supplementation. The concentrations of mostshort-chain and medium-chain FA decreased at a constant rate with increasing EL supplementation (Table 5).

The concentrations of FA C18:0, C18:1 and C18:2 in the milk fat increased at a decreasing rate (linear P<0.001 and quadratic0.05<P<0.001) as the level of EL increased (Table 5).

EL supplementation increased linearly and quadratically (P<0.001) the content of n-3 FA in milk (Table 5). The concentra-tions of C18:3 n-3 in milk increased by 36 and 87% for the EL-10 and EL-20 treatments, respectively. The eicosapentaenoicand docosaesaenoic acid levels in milk increased at a decreasing rate as EL intake increased. (Table 5). Milk fat of the ELgroups had lower concentrations of saturated FA (linear decrease, P<0.001) and higher levels of monounsaturated FA [linear(P<0.001) and quadratic (P<0.05) increase] and total PUFA (quadratic increase, P<0.001).

The total concentration of both n-3 and n-6 FA increased and was linearly (P<0.001) and quadratically (P<0.001) affected

as EL intake increased. However, the higher level of total n-3 FA in the milk of the groups receiving EL resulted in a linearand quadratic (P<0.001) reduction of the proportion of n-6/n-3.

Fatty acid profiles of cheeses were generally similar to those of milk, suggesting that processing of milk into cheese didnot alter its fatty acid composition, with the exception of FA present in only trace amounts. The concentrations of FA C4:0,

L. Mughetti et al. / Animal Feed Science and Technology 178 (2012) 27– 39 31

Table 2Ingredients, chemical composition and metabolisable energy content of the experimental concentrates.

Concentrates

CTRa EL-10b EL-20c

Ingredients (g/kg)Soybean meal 188.2 124.0 60.8Corn meal 335.9 157.0 20.0Barley grain 50.0 50.0 20.0Wheat bran 162.6 303.1 578.2Wheat flour shorts 200.0 200.0 57.4Extruded linseed – 100.0 200.0Molasses 20.0 20.0 20.0Calcium carbonate 16.9 19.7 21.6Sodium bicarbonato 5.0 5.0 5.0Bicalcic phosphate 9.4 4.2 -Sodium chloride 5.0 5.0 5.0Magnesium oxide 2.0 2.0 2.0Vitamin and mineral premixd 5.0 5.0 5.0Bonding agent – 5.0 5.0

Chemical composition (g/kg)Dry matter 919.2 922.8 933.6Crude protein 176.5 176.9 180.1Crude fat 33.4 77.1 124.5Ash 69.0 74.5 82.1NDFe 170.3 206.0 250.0ADFf 48.4 60.4 75.4Lignin (sa)g 11.2 14.6 23.1Calcium 12.8 13.4 14.0Phosphorus 3.4 4.0 4.1Metabolisable energy (Mcal/kg) 3.230 3.280 3.312

a Control concentrate with no added extruded linseed.b Concentrate with 100 g/kg of extruded linseed.c Concentrate with 200 g/kg of extruded linseed.d Mineral and vitamin premix supplied (per kg of final diet): Co, 0.30 mg (2CoCO3; 3Co(OH)2 H2O); Zn, 50.00 mg (ZnO); Fe, 15.00 mg (FeCO3); Mn, 30.00 mg

(MnO); Se, 0.60 mg (Na2SeO3); I, 1.00 mg (Ca(IO3)2); vitamin A, 50,000 IU (retinylacetate); cholecalciferol, 3000 IU; vitamin E, 50.00 mg (�-tocopherolacetate).

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e Neutral detergent fibre.f Acid detergent fibre.g Lignin determined by solubilisation of cellulose with sulphuric acid.

6:0, C8:0 and C10:0 linearly (P<0.001) decreased with increasing EL in the diet. With a few exceptions, the remaining FAere linearly and quadratically (0.001<P<0.05) related to the level of dietary EL (Table 6).

Cheese chemical composition was affected by dietary treatment (Table 7). As the level of linseed included in the dietncreased, the moisture content decreased linearly (P<0.01) and quadratically (P<0.05). A parallel quadratic increase in lipidP<0.01) and protein (P<0.05) content was observed, although these differences tended to fade when expressed on a DMasis.

After 60 days of ripening, the average cheese yield value was 136 g/kg of milk. The adjusted cheese yields (expressed as gf 37 g/100 g moisture cheese per 100 g of milk) were 13.0, 14.9 and 14.3 for the CTR, EL-10 and EL-20 groups, respectively.

ue to the higher moisture content, the weight loss of cheese was greater in the groups receiving linseed (P<0.05).

Most of the sensory attributes were affected by dietary treatments. However, the descriptors of taste “salty”, of highntensity, and “acid” and “bitter”, of mild intensity, did not change between treatments. Within each dietary treatment,

able 3ipid profile of concentrates (g/100 g of fatty acids).

Concentrates

CTRa EL-10b EL-20c

C16:0 15.32 10.43 9.71C18:0 2.00 3.54 3.93C18:1 n-9 23.03 19.71 19.26C18:2 n-6 54.08 26.22 21.86C18:3 n-3 3.49 38.91 44.00Others 2.08 1.19 1.24

a Control concentrate with no added extruded linseed.b Concentrate with 100 g/kg of extruded linseed.c Concentrate with 200 g/kg of extruded linseed.

32 L. Mughetti et al. / Animal Feed Science and Technology 178 (2012) 27– 39

Table 4Effects of linseed supplementation on yield, composition and clotting properties of milk.

Dietary treatment P value SEM

CTRa EL-10b EL-20c Ld Qe

Milk yield (g/d) 1362.25 1403.67 1217.99 <0.001 <0.001 20.49Casein (g/kg) 37.41 37.41 38.30 <0.001 <0.001 0.14Casein (g/d) 50.94 52.52 46.56 <0.01 <0.01 0.78Fat (g/kg) 59.07 60.66 61.02 <0.001 <0.001 0.70Fat (g/d) 80.40 85.15 74.19 NS <0.01 1.49Lactose (g/kg) 47.65 47.87 48.62 <0.001 <0.001 0.13Lactose (g/d) 64.89 67.19 59.10 <0.01 <0.01 0.99Protein (g/kg) 48.08 48.08 48.99 <0.001 <0.001 0.12Protein (g/d) 65.47 67.48 59.56 <0.01 <0.01 0.99Urea (mg/dl) 45.77 40.08 36.45 <0.001 <0.05 1.08rf (min) 18.88 17.74 17.01 <0.001 NS 0.40a30

g (mm) 40.21 42.57 42.37 NS NS 1.67k20

h (min) 1.71 1.57 1.51 <0.001 <0.001 0.08

SEM, pooled standard error of the mean.a Control concentrate with no added extruded linseed.b Concentrate with 100 g/kg of extruded linseed.c Concentrate with 200 g/kg of extruded linseed.d Probability of the linear component of variance.e Probability of the quadratic component of variance.f Renneting time.

g Curd consistency.h Rate of firming.

differences among cheeses were observed for “sheep milk odour” (P<0.001), “toughness” (P<0.01) and “graininess” (P<0.001)(Table 8).

The CTR samples were perceived as being moister and less firm, and they showed a heterogeneous colour (bi-coloured).Moreover, they had a less grainy (smooth) texture and a less pronounced flavour. PCA was performed on three cheesesamples in each group, and the data were averaged over individual judges and replicates. Fig. 1 shows a multidimensionalmap of the cheese samples based on the sensory attributes.

Three PCs explained 0.87 of the overall variance in the data (Fig. 1). PC1 (0.60 of variance) separated CTR cheese samples(CTR a, CTR b and CTR c) from the linseed added groups. Cheeses from the EL-10 and EL-20 groups were distinguished by theirhigher scores for flavour and texture. The CTR cheese samples, located on the negative semi-axis of PC1, were described asbeing more moist and having a more intense “sheep milk odour”. PC2 explained 0.16 of the variation in sensory properties(ordinate of the Fig. 1a), whereas PC3 explained 0.11 of the variation (ordinate of the Fig. 1b). PC2 and PC3 were mainlyrelated to intra-group differences in colour (uniformity) and “holes”. The contribution of “bitter” and “acid” tastes to PC2,and of “salty” and “greasy” properties to PC3 were only apparent because differences in taste were not significant.

4. Discussion

The lower milk yield observed with the higher EL intake may have occurred due to the higher lipid content of the diet(64 g/sheep/day derived from the linseed supplementation). Higher PUFA intake may reduce the digestibility of the diet,particularly that of structural carbohydrates (Ikwuegbu and Sutton, 1982; Jenkins and Palmquist, 1984).

Moallem (2009) supplemented the diet of dairy cattle with 40 g/kg EL and observed a higher milk yield (+2.7%), confirmingobservations of previous authors who used fat supplements in the diet (Jonson et al., 2002; Chiofalo et al., 2004). However,these results are contrary to those of other authors who observed a reduced milk yield (Petit et al., 2005) or no variation inmilk yield (Kitessa et al., 2003) using either untreated whole linseed (Kennelly, 1996; Mustafa et al., 2003) or EL (Gonthieret al., 2005).

Moderate levels of fat inclusion in the diet may increase milk yield by improving feed efficiency (Sarrazin et al., 2004),while higher inclusion levels may reduce feed intake (Petit et al., 2005) and milk yield by depressing ruminal function(Gonthier et al., 2005).

Results regarding the effects of fat supplementation on the milk fat content of ewes were inconsistent. The scientificliterature shows a reduction (Chiofalo et al., 2004; Luna et al., 2005), an increase (Horton et al., 1992) or no difference(Kitessa et al., 2003) in the milk fat content.

The reduction of fat levels is explained by the inhibiting effect of high concentrations of PUFA in the diet on the de novosynthesis of FA. Currently there are several recognised mechanisms that could explain this effect. Palmquist et al. (1993)

argue that the reduction of the de novo synthesis of FA in the udder is largely due to an increased uptake and subsequentsecretion of dietary- and ruminally-derived FA. These FA compete for esterification with short-chain FA synthesised in themammary gland, which causes negative feedback on lipogenic enzymes.

L. Mughetti et al. / Animal Feed Science and Technology 178 (2012) 27– 39 33

Table 5Effects of linseed supplementation on milk fatty acid composition (g/100 g of fatty acids).

Dietary treatment P value SEM

CTRa EL-10b EL-20c Ld Qe

C 4:0 5.88 5.61 5.42 <0.001 NS 0.37C 6:0 3.65 2.94 2.28 <0.001 NS 0.21C 8:0 2.83 2.05 1.51 <0.001 NS 0.15C 10:0 6.72 4.85 3.62 <0.001 NS 0.28C 11:0 0.15 0.11 0.08 <0.001 <0.001 0.04C 12:0 3.51 2.83 2.37 <0.001 NS 0.09C 13:0 0.08 0.09 0.09 <0.001 <0.001 0.02C 14:0 10.26 9.48 8.29 <0.001 NS 0.13C 14:1 0.16 0.16 0.13 <0.001 <0.001 0.03C 15:0 1.04 0.91 0.94 <0.001 <0.001 0.02C 16:0 24.27 23.99 22.21 <0.001 NS 0.30C 16:1 0.95 0.77 0.74 <0.001 <0.001 0.05C 17:0 0.58 0.56 0.55 <0.001 <0.001 0.02C 17:1 0.14 0.17 0.12 <0.001 <0.001 0.02C 18:0 11.29 12.59 13.79 NS <0.05 0.48C 18:1 t 3.05 4.42 7.80 <0.001 <0.05 0.29C 18:1 c 19.09 20.69 21.30 NS <0.05 0.60C 18:2 t n-6 0.54 0.73 0.79 <0.001 <0.001 0.03C 18:2 c n-6 2.16 2.41 2.42 <0.001 <0.001 0.05C 20:0 0.32 0.27 0.29 <0.001 <0.001 0.02C 18:3 n-6 0.03 0.03 0.04 <0.001 <0.001 0.01C 20:1 1.30 1.87 2.23 <0.001 <0.001 0.07C 18:3 n-3 1.21 1.65 2.26 <0.001 <0.001 0.07C 21:0 0.08 0.07 0.08 <0.001 <0.001 0.01C 20:2 n-6 0.13 0.03 0.04 <0.001 <0.001 0.04C 22:0 0.14 0.12 0.13 <0.001 <0.001 0.01C 20:3 n-6 0.02 0.03 0.01 <0.001 <0.001 0.01C 22:1 0.01 0.01 0.04 <0.001 <0.001 0.01C 20:3 n-3 0.07 0.06 0.04 <0.001 <0.001 0.02C 20:4 n-6 0.10 0.07 0.06 <0.001 <0.001 0.03C 23:0 0.08 0.08 0.07 <0.001 <0.001 0.02C 22:2 n-6 0.05 0.06 0.04 <0.001 <0.001 0.01C 24:0 0.05 0.10 0.07 <0.001 <0.001 0.01C 20:5 n-3 0.05 0.06 0.06 <0.001 <0.001 0.02C 24:1 0.01 0.03 0.00 <0.001 <0.001 0.01C 22:6 n-3 0.05 0.09 0.10 <0.001 <0.001 0.02SFAf 70.92 66.65 61.79 <0.001 NS 0.81MUFAg 24.70 28.13 32.36 <0.001 <0.05 0.74PUFAh 4.39 5.22 5.85 NS <0.001 0.17n-6 3.02 3.36 3.39 <0.001 <0.001 0.10n-3 1.37 1.85 2.46 <0.001 <0.001 0.09n-6/n-3 2.22 1.83 1.40 <0.001 <0.001 0.06

SEM, pooled standard error of the mean.a Control concentrate with no added extruded linseed.b Concentrate with 100 g/kg of extruded linseed.c Concentrate with 200 g/kg of extruded linseed.d Probability of the linear component of variance.e Probability of the quadratic component of variance.f Saturated fatty acids.g Monounsaturated fatty acids.

Hi

M

isota

h Polyunsaturated fatty acids.

Another mechanism that could explain the inhibitory effect of PUFA was described by Morales et al. (2000) and Loor anderbein (2003). This proposed mechanism is based on the direct inhibition of de novo synthesis of FA, which involves the

ntermediate products of biohydrogenation in the rumen, such as C18:1 trans-11 and C18:2 trans-10, cis-12.No detrimental effects of linseed administration on the milk fat content were observed (Khorasani and Kennelly, 1994;

ustafa et al., 2003), even though the linseed included in the diet is rich in PUFA and, in particular, in C18:3 n-3.Other important aspects to consider, in addition to the degree of unsaturation of the dietary FA, are their degradability

n the rumen and the physical form in which they are administered. Whole seeds release the FA into the rumen morelowly compared to oil administration; therefore, the risk of biohydrogenation is reduced. Moreover, in comparison to otherilseeds, linseed is characterised by a lower rate of ruminal degradation (Dhiman et al., 2000; Secchiari et al., 2003) and, in

he present study, it was extruded. Heat treatments reduce the rumen degradability of lipids due to their encapsulation in

denatured protein matrix (Chouinard et al., 1997).

34 L. Mughetti et al. / Animal Feed Science and Technology 178 (2012) 27– 39

Table 6Effects of linseed supplementation on cheese fatty acid composition (g/100 g of fatty acids).

Dietary treatment P value SEM

CTRa EL-10b EL-20c Ld Qe

C 4:0 7.00 5.91 5.99 <0.01 NS 0.53C 6:0 3.83 3.00 2.89 <0.001 NS 0.24C 8:0 2.69 2.05 2.04 <0.001 NS 0.13C 10:0 6.16 4.90 4.87 <0.001 NS 0.17C 11:0 0.14 0.04 0.05 <0.001 <0.001 0.03C 12:0 3.28 3.00 3.06 <0.001 <0.001 0.02C 13:0 0.05 0.09 0.08 <0.001 <0.001 0.01C 14:0 9.98 10.19 10.11 <0.001 <0.01 0.13C 14:1 0.15 0.29 0.15 <0.001 <0.001 0.05C 15:0 1.00 0.95 1.11 <0.001 <0.001 0.02C 16:0 24.22 24.17 23.29 <0.01 NS 0.41C 16:1 0.89 0.87 0.84 <0.001 <0.001 0.02C 17:0 0.56 0.51 0.56 <0.001 <0.001 0.01C 17:1 0.14 0.11 0.12 <0.001 <0.001 0.004C 18:0 10.26 8.89 9.15 <0.01 NS 0.40C 18:1 t 3.72 5.67 6.56 <0.01 <0.001 0.13C 18:1 c 20.26 21.60 20.17 NS <0.01 0.48C 18:2 t n-6 0.52 0.72 0.94 <0.001 <0.001 0.02C 18:2 c n-6 2.11 2.41 2.57 <0.001 <0.001 0.04C 20:0 0.26 0.20 0.24 <0.001 <0.001 0.01C 18:3 n-6 0.02 0.04 0.04 <0.001 <0.001 0.01C 20:1 1.16 1.96 2.54 <0.001 <0.001 0.02C 18:3 n-3 1.18 1.84 2.02 <0.001 <0.001 0.03C 21:0 0.06 0.06 0.07 <0.001 <0.001 0.003C 20:2 n-6 0.02 0.03 0.02 <0.001 <0.001 0.01C 22:0 0.11 0.09 0.11 <0.001 <0.001 0.003C 20:3 n-6 0.00 0.02 0.01 <0.001 <0.001 0.01C 22:1 0.00 0.00 0.02 <0.001 <0.001 0.01C 20:3 n-3 0.00 0.00 0.06 <0.001 <0.001 0.01C 20:4 n-6 0.11 0.13 0.06 <0.001 <0.001 0.02C 23:0 0.06 0.05 0.06 <0.001 <0.001 0.01C 22:2 n-6 0.01 0.04 0.01 <0.001 <0.001 0.01C 24:0 0.02 0.08 0.07 <0.001 <0.001 0.01C 20:5 n-3 0.02 0.04 0.04 <0.001 <0.001 0.01C 24:1 0.00 0.02 0.03 <0.001 <0.001 0.01C 22:6 n-3 0.03 0.05 0.06 <0.001 <0.001 0.02SFAf 69.67 64.17 63.73 <0.001 <0.01 0.75MUFAg 26.31 30.52 30.43 <0.05 <0.01 0.65PUFAh 4.02 5.31 5.84 NS <0.001 0.12n-6 2.79 3.38 3.65 <0.001 <0.001 0.07n-3 1.22 1.92 2.19 <0.001 <0.001 0.05n-6/n-3 2.29 1.76 1.67 <0.001 <0.001 0.02

SEM, pooled standard error of the mean.a Control concentrate with no added extruded linseed.b Concentrate with 100 g/kg of extruded linseed.c Concentrate with 200 g/kg of extruded linseed.d Probability of the linear component of variance.e Probability of the quadratic component of variance.f Saturated fatty acids.

g Monounsaturated fatty acids.h Polyunsaturated fatty acids.

The quantity of protein contained in milk may change in different ways following the administration of oilseeds. Fuenteset al. (2008) used EL in lactating dairy cows and observed an increase in the concentration of milk protein. This increase wasexplained by the reduction of milk yield (the amount of protein excreted was similar to that of the control group). A similarmechanism was likely at work in the present study.

The use of fish or linseed meal is often associated with an increased concentration of milk protein (Gonzalez et al., 1982),probably because of the low rumen degradability and the high intestinal digestibility of these foods (Allison and Garnsworthy,2002), which causes an increased availability of amino acids for protein synthesis in milk.

Other studies have indicated a decrease (Chiofalo et al., 2004; Horton et al., 1992; Gonthier et al., 2005) or no changesto the protein content of sheep (Zhang et al., 2006a; Luna et al., 2005) or cow milk (Mustafa et al., 2003) following linseed

administration.

Sometimes, a reduction in the proportion of casein, with no reduction of total protein content, can occur in sheep’s milk,as observed after the administration of calcium soap of FA with or without raw sunflower seeds (Osuna et al., 1998).

L. Mughetti et al. / Animal Feed Science and Technology 178 (2012) 27– 39 35

Table 7Effects of linseed supplementation on chemical composition of cheese after 60 days of ripening.

Dietary treatment P value SEM

CTRa EL-10b EL-20c Ld Qe

Moisture (g/kg) 385.1 335.6 351.1 <0.01 <0.05 8.7Protein (g/kg) 228.2 252.3 247.8 NS <0.05 5.5Protein (g/kg DM) 371.0 379.6 381.8 NS <0.05 3.5Fat (g/kg) 286.5 306.5 311.0 NS <0.01 4.3Fat (g/kg DM) 465.8 461.3 479.2 <0.001 NS 1.3Ash (g/kg) 67.7 71.8 69.9 <0.001 <0.01 1.3Ash (g/kg DM) 110.1 108.0 107.7 <0.001 <0.05 1.0

SEM, pooled standard error of the mean.a Control concentrate with no added extruded linseed.b Concentrate with 100 g/kg of extruded linseed.c Concentrate with 200 g/kg of extruded linseed.d Probability of the linear component of variance.e Probability of the quadratic component of variance.

Table 8Effect of dietary treatment on the organoleptic properties of cheeses.

Attributes Dietary treatment (DT) F-values

DT Replicate (R) DT × R

CTRa EL-10b EL-20c SEM dfd = 2 df = 2 df = 4

Colour homogeneity 4.0C 4.7B 6.7A 0.13 99.7*** 0.9 1.9Holes 4.8B 5.8A 4.4C 0.09 28.0*** 1.4 0.7Overall Odour 6.3b 6.8a 6.7a 0.07 3.8* 1.3 0.6Sheep milk odour 4.5A 3.6B 2.6C 0.11 64.3*** 12.0*** 3.3*Ripe cheese odour 4.2C 5.4B 5.7A 0.09 43.8*** 3.0 0.4Salty 7.1 6.9 7.0 0.07 0.3 1.2 0.7Acid 2.3 2.5 2.2 0.06 1.4 2.3 3.5*Bitter 1.7 1.7 1.6 0.06 0.5 0.7 1.0Overall flavour 6.5B 7.0A 7.1A 0.08 5.6** 1.5 0.9Ripe cheese flavour 5.3B 6.3A 6.4A 0.08 20.9*** 0.5 0.2Sharpness 2.0C 2.8B 3.3A 0.09 21.8*** 1.9 1.5Thoughness 4.3B 6.1A 6.2A 0.11 80.6*** 7.3** 0.3Graininess 3.4C 6.1B 6.5A 0.15 241.9*** 14.0*** 0.3Screeching 1.1B 1.6A 1.8A 0.08 8.3*** 2.3 3.1*Moisture 5.2A 3.0B 2.6C 0.13 181.2*** 3.0 0.8Greasiness 4.7a 4.3b 4.4ab 0.07 3.4* 2.8 0.2

SEM, pooled standard error of the mean. Means in the same row with different superscripts are significantly different (a, b = P<0.05; A, B, C = P<0.01; A,B = P<0.001). *P<0,05; ** P<0,01; ***P<0,001.

a Control concentrate with no added extruded linseed.

fit

tcft2f

ucn1

R

b Concentrate with 100 g/kg of extruded linseed.c Concentrate with 200 g/kg of extruded linseed.d Degrees of freedom.

There are two mechanisms that could explain these changes. The first is the limited availability of amino acids that resultsrom increased milk production stimulated by the presence of lipids in the diet (Gaynor et al., 1994). Another hypothesisnvolves the modification of insulin or glucose metabolism in the mammary gland after fat supplement administration inhe ruminant diet (Dhiman et al., 1995).

The variability of the effects reported in the literature for linseed administration can be explained with the differentechnological treatments of seeds. In two studies in which linseeds were treated with formaldehyde, a higher milk proteinontent was observed (Petit et al., 2001; Petit, 2003), indicating that the addition of formaldehyde can protect linseed proteinrom ruminal degradation. Heat treatments can modify where proteins contained in the seeds are digested, causing proteinso be digested in the small intestine rather than in the rumen, without modifying the whole tract digestibility (Gonthier et al.,004). However, Gonthier et al. (2004) indicated that extrusion is the least effective heat treatment in protecting oilseedsrom ruminal degradation.

Our results regarding the urea content of milk contrasted with those of Moallem (2009), who observed an increment of therea concentration after administration of EL in the diet. In sheep, there is a strong correlation between milk urea nitrogenoncentration and the amount of protein in the diet. If the dietary protein concentration increases, a reduced efficiency of

itrogen utilisation for the synthesis of milk proteins is observed, which results in a greater loss of nitrogen (Cannas et al.,998).

An extreme variability in the physical and chemical parameters of milk, particularly sheep milk (Pulina et al., 2006;eynolds et al., 2006), is sometimes observed after the administration of lipid supplements. This is mainly due to the

36 L. Mughetti et al. / Animal Feed Science and Technology 178 (2012) 27– 39

Fig. 1. PCA. Bi-plot PC1vs PC2 (a) and PC1vs PC3 (b) for all 16 sensory attributes of all dietary treatments. CTR = Control concentrate with no added extrudedlinseed. EL-10 = Concentrate with 100 g/kg of extruded linseed. EL-20 = Concentrate with 200 g/kg of extruded linseed. Letters (a, b, c) indicate replicateswithin experimental groups of cheese.

fa

tr

oeo(

Td1

e

cSn

m

aic

aatl

osse

sptcwccfo

B(vo

ta

Mio

l

L. Mughetti et al. / Animal Feed Science and Technology 178 (2012) 27– 39 37

act that scientific reports are characterised by differences in the dose, physical form and composition of the supplementsdministered, which could affect the degree of availability of fats in the rumen and their impact on microbial fermentations.

In addition, it cannot be excluded that the higher NDF and ADF content, and the lower non-fiber carbohydrate content ofhe EL-enriched concentrates might have negatively affected the milk yield. However, it was estimated that the concentrateepresented only a minor component of the daily ration (approximately 290 g/kg total DM intake).

With respect to the FA composition of milk and cheese, a reduction of short and medium chain FA synthesis is commonlybserved after the administration of lipid supplements, especially if they are rich in PUFA (Goodridge et al., 2001; Mustafat al., 2003; Sarrazin et al., 2004; Zhang et al., 2006b). Including high amounts of PUFA in the diet can lead to the accumulationf trans FA (C18:1 trans-11, C18:2 trans-10, cis-12), known for their inhibitory effect on the de novo synthesis of milk FABauman and Griinari, 2001).

Linseed is rich in C18:0, C18:1 and C18:3 FA, which can contribute to an increased concentration of C18:1 FA in milk.he C18:1 FA are derived from the partial biohydrogenation of the C18:2 and C18:3 FA that occurs in the rumen, and by theesaturation of stearic acid, which occurs in the mammary gland due to the action of the enzyme �-9-desaturase (Kennelly,996; Loor and Herbein, 2003; Dhiman et al., 2000).

High levels of C18:1 and C18:2 FA in milk have been well documented in cattle (Khorasani and Kennelly, 1994; Mustafat al., 2003) and in sheep (Zhang et al., 2006a,b; Luna et al., 2008) following linseed administration.

Another effect associated with dietary lipid supplements rich in PUFA is the increase of alpha-linolenic acid (ALA) con-entrations in milk. This effect has been demonstrated both in sheep (Kitessa et al., 2003) and in other animal species (Sanzampelayo et al., 2002; Mustafa et al., 2003; Sarrazin et al., 2004). The results obtained in the present work for the C18:3-3 concentration largely confirm the data reported in the literature (Luna et al., 2005, 2008; Zhang et al., 2006a,b).

When fresh forage is added to diet, as in the present work, the effects of linseed on the FA profile of milk can be partiallyitigated. This relates to the high ALA content of fresh forage (50–75 g/100 g of total FA; Elgersma et al., 2006).Eicosapentaenoic (EPA) and docosaesaenoic (DHA) acids are present in very small amounts in both conventional diets

nd in milk fat (<0.1 g/100 g of total methyl esters of FA), with the exception of milk obtained by including marine productsn ruminant diets (Luna et al., 2005). Results from this experiment (Tables 5 and 6) indicate a slight increase of EPA and DHAoncentrations in both milk and cheese obtained from EL supplemented groups.

Following the integration of linseed into cattle diets, Moallem (2009) noted an increase in EPA and docosapentaenoiccid (C22:5 n-6) concentrations in milk, although these essential FA were not identified in the diet administered to thenimals. Wathes et al. (2007) showed that these long-chain PUFA can be synthesised in the liver from shorter FA, throughhe desaturation and elongation activities that occurred due to different enzyme systems. Even in humans, increases in bloodevels of the n-3 long chain FA were observed in response to diets enriched with ALA (Weill et al., 2002).

The fact that the transformation of milk into cheese did not change the lipid profile is in line with the observations maden cattle by Dhiman et al. (1999) following the administration of EL, and by Baer et al. (1996) with the use of extrudedoybeans. More recently, Zhang et al. (2006a, b) enriched cheeses with CLA and ALA using milk from animals fed linseed orunflower seeds. Similar results were observed in different types of cheese made with sheep milk (Addis et al., 2005; Lunat al., 2005, 2008; Nudda et al., 2005).

Results concerning cheese yields in the present work are in line with previous reports by Zhang et al. (2006a).Data in the literature regarding cheese enriched with PUFA and CLA do not provide definitive indications as far as sen-

ory characteristics are concerned. Kraggerud et al. (2008) observed correlations between the FA composition and sensoryroperties of bovine semi-hard cheese. Variations in the firmness of cheeses were attributed to the season (winter) and tohe unsaturated FA content. Luna et al. (2005) did not observe differences in appearance, taste and acceptability in sheepheese made from animals fed raw linseed in comparison to the control. With regard to flavour and texture, lower scoresere observed in cheeses made from animals fed linseed at two months of ripening, but these differences were not signifi-

ant. At three months of ripening, these changes disappeared. Luna et al. (2008) submitted cheeses made from sheep fed aoncentrate containing whole linseed (18.5 g/kg DM) and sunflower oil (8.1 g/kg DM) to a professional panel. The authorsound no differences compared to the control for any of the sensory attributes considered (appearance, aroma, taste, texture,verall quality).

Modifications of organoleptic properties were indicated for milk and cheese enriched with CLA (Campbell et al., 2003;aer et al., 2001). These variations were able to affect consumer acceptability as well. More recently, however, Lynch et al.2005) indicated that untrained assessors were unable to distinguish differences in the flavour of milk enriched with CLA oraccenic acid. However, milk enriched with CLA can be used to produce Edam cheese with a softer structure and acceptablerganoleptic properties (Ryhänen et al., 2005).

Jones et al. (2005) observed that bovine cheeses enriched with high levels of CLA were less compact and less crumblyhan the control samples, but the flavour, aroma and the overall impression were found to be unaffected by the treatment. Inddition, higher levels of C18:1-trans and a reduction of the C18:0 and the total saturated FA concentration were observed.

Recently, Bermúdez-Aguirre and Barbosa-Cánovas (2011) observed that changes in texture of Queso Fresco, Cheddar andozzarella cheese, obtained from milk fortified with either animal or vegetable sources of omega-3, could be related to the

nteraction between the milk components (particularly fat globules, casein micelles, calcium ions), rennet and sources ofmega-3.

Results from the present study on Pecorino cheese seem to confirm observations of previous authors who ana-ysed changes in the organoleptic properties of different types of cheese. Cheeses obtained from both EL groups had no

38 L. Mughetti et al. / Animal Feed Science and Technology 178 (2012) 27– 39

off-flavours and showed a marked grainy texture (less compact than control cheese samples). However, cheese toughnesswas not related to the unsaturated FA content. Instead, cheese toughness appeared to be related to the weight loss percentageof the cheeses at 60 days of ripening.

5. Conclusions

The use of a diet supplemented with EL allows products with a nutritional value higher than that of similar conventionalproducts to be developed, at least in the experimental conditions adopted in this research. The higher n-3:n-6 fatty acidratio is considered to be healthier for the consumer.

The modifications observed in the sensorial properties of cheeses did not alter the overall acceptability of the treatedproducts.

The present work indicated that a concentrate containing 100 g/kg of EL can be used to increase the overall qualityof Pecorino cheese without negative effects on its typical sensorial and organoleptic characteristics or on the productiveperformance of the animals.

Acknowledgments

The authors would like to thank S. De Vincenzi and E. Cestola for help in laboratory analysis and D. Sanna for the technicalassistance and for care of the animals. Mignini & Petrini S.p.a. is kindly acknowledged for providing the technical specificationsof linseed extrusion. This project was sponsored by the Italian Ministry of Agriculture (MiPAAF), DM 293/7303/05.

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