Dairy Sheep Symposium - the Department of Animal Sciences ...

ing, the cheeses were brined for 18 h at 5°C. Polycoat (RV XP164, Natural Purity, Inc. Minneapolis,
MN) was applied to cheese surfaces and cheeses were aged at 7°C in an 85% humiditycontrolled
room. At the time of sampling, a representative wedge was cut from the Manchego
wheel and ground for compositional analysis.
Analytical Methods
All compositional analyses were carried out on the cheeses in duplicate. Pasteurized milk
samples were analyzed for total solids (Green and Park, 1980), fat by Mojonnier (AOAC, 1995),
protein (total percentage N × 6.35) by Kjeldahl (AOAC, 1995) and casein (AOAC, 1995). Nonprotein
nitrogen (NPN) of the milks was also measured using the method described by Johnson
et al. (2001). Cheeses were analyzed for moisture by vacuum oven (Vanderwarn, 1989), fat by
Mojonnier (AOAC, 1995), pH by the quinhydrone method (Marshall, 1992), salt by chloride
electrode (model 926; Corning Glass Works, Medfield, MA; Johnson and Olson, 1985) and
protein by Kjeldahl (AOAC, 1995). Proteolysis was monitored during ripening by measuring the
amount of 12% TCA soluble nitrogen at 3 d, 1, 3, 6 and 9 mo (AOAC, 1995).
Extraction of Free Fatty Acids
Individual free fatty acids (FFA) were determined using a modified version of the procedure
of Ha and Lindsay (1991) at 1, 3, 6 and 9 months. Approximately 5 g of finely grated cheese,
0.4 ml of diethyl ether containing 500 μg of nonanoic acid (internal standard), 15 ml of diethyl
ether and 0.5 ml of 5.5 N H 2 SO 4 were mixed in a Qorpak tube (Fisher Scientific, Chicago, Il).
The mixture was blended thoroughly with an homogeniser (Ultra-Turrex ® T25 basic, IKA ®
Works, Inc., Wilmington, NC) fitted with the dispersing tool 25 (S25N 10G) at a speed of 13,000
min -1 for 2 min. The dispersing tool was rinsed twice with 10 ml of hexane. Each rinsed solution
was combined with the sample extract. To remove water, 12.5 g of anhydrous Na 2 SO 4 was added
to each tube and vortexed thoroughly. The mixture was then centrifuged in a padded cup of
Babcock centrifuge for 5 min and the supernatant was then used for isolation of FFA.
Alumina columns were prepared by filling empty 20 ml BondElut columns (Varian Inc.,
Walnut Creek, CA) with 5 g of deactivated alumina and the columns were placed onto the
vacuum manifold unit (Supelco, Sigma-Aldrich, St. Louis, MO). Deactivation of the alumina
was carried out as described by Ha and Lindsay (1991). The pooled ether/hexane extract was
passed through the alumina column twice. The column was then washed twice with 10 ml of
hexane:diethyl ether (1:1, v/v) each to remove any neutral lipids. The alumina, with the
adsorbed FFA, was dried under vacuum for a few seconds and transferred to a screw-capped
tube. 5 ml of 6% formic acid in Na 2 SO 4 -dried diisopropyl ether (v/v) were added to the alumina
and mixed thoroughly. Samples were then centrifuged at 2,000 x g for 5 min and the supernatant
was then carefully transferred to a screw-capped tube containing 2.5 g of Na 2 SO 4 to remove any
water. The contents were mixed thoroughly and centrifuged again for 5 min. Supernatant were
carefully transferred to screw-capped graduated glass centrifuge tubes (17 × 117 mm, Heavyduty
Kimax Brand, Fisher Scientific, Inc., Chicago, IL) and concentrated to 500 μL using a
nitrogen stream in a vacuum manifold system with the drying unit attached to it (Supelco,
Sigma-Aldrich, St. Louis, MO). Three separate extractions were carried out per sample.
Gas Chromatography
The underivatized sample (1.0 μL) was then separated using a fused silica capillary column
(DB-FFAP; 30 m × 0.25 mm i.d.; 0.25 μm film thickness; J & W Scientific, Folsom, CA) in an
Agilent model 6890 Series gas chromatograph (Agilent Technologies, Inc., Wilmington, DE)