Combi 514r

The fatty acids composition of each bovine muscle was determined by using a
slightly modified method described by O’Fallon et al. [21 (link)]. For the separation of fatty acid
methyl esters, 1 g of meat sample from each treatment was mixed with 0.7 mL of
10N KOH and 6.3 mL of methanol, placed in a constant temperature water bath at
55°C. Heated for 1 h and 30 min while vigorously shaken once every 30
min. After cooling for 1-2 min in cold water, 0.58 mL of (24N)
H₂SO₄ was added, and the mixture was again heated in a
water bath at 55°C for 1 h and 30 min and vigorously shaken again once
every 30 min. At the end of heating in a water bath, 3 mL of hexane was added to
samples and centrifuged (Combi-514R, Hanil scientific) at 3,000 rpm for 5 min.
After immersing in a vial using a Pasteur pipette, the fatty acid analysis was
performed using the Gas Chromatograph-Flame Ionization Detector (FID) (7890
series, Agilent, Santa Clara, CA, USA) under the following conditions. The
injector was split mode with a split ratio of 25:1, temperature was
250°C, the detector was FID. High purity air, high purity H2, and high
purity He was used as the carrier gas. The flow rate was 40 mL/min for
H₂ and 400 mL/min for air. HP-88 column (60 m × 250
μm × 0.2 mm) was used for the analysis. Fatty acids composition is
expressed as a percent of meat.

Each cheese sample was added to twice its volume of methanol (cheese:methanol
ratio of 1:2) and kept for 1 h at 30° in a shaking incubator (SI-900R,
Jeio Tech, Kimpo, Korea), centrifuged at 1,900×g for 10 min (Combi-514R,
Hanil Co., Ltd., Seoul, Korea), and passed through Whatman No. 2 filter paper.
The filtrates were used as samples for the analysis of antioxidant activity.
Radical-scavenging activity was determined by a
2,2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS; Sigma, St.
Louis, MO, USA) assay. We mixed 14 mM ABTS and 5 mM potassium persulfate in 0.1
M potassium phosphate buffer (pH 7.4) in a 1:1 ratio and incubated them for 16 h
in a dark room at 25°C. The mixture was diluted with 0.1 M potassium
phosphate buffer (pH 7.4) until the absorbance at 734 nm wavelength reached
0.7±0.02 on a spectrophotometer (X-ma 3200, Human Co., Ltd., Seoul,
Korea). A 20 μL sample was then added to 980 μL of the above
solution, and the mixture was incubated for 5 min in 37°C. Absorbance was
measured at 734 nm. The antioxidant activity was calculated as follows:
A_c: absorbance values of the negative control
A_s: absorbance values of an experimental sample