Lamb Tissue
Subcutaneous ovine fat from the loin was obtained from The Ohio State University Meat Laboratory, Columbus, Ohio (one-week post-mortem). The fat was placed in meat trays, wrapped with Resinite stretch meat film (Burdon Chemical, Griffin, GA) and stored at 2 ± 2oC prior to analysis. Immediately prior to analyses, 50 g of fat was coarsely ground in a Waring lab blender (Model 1120, Dynamic Corp. of America, New Hartford, CN) for 30 seconds. No rendering or homogenization procedures were employed in order to prevent heat-induced oxidation. All procedures were conducted at 2 ± 2oC.
Corn Oil
A four-year-old, aged Mazola® brand 100% pure corn oil (with 57.1% poly-unsaturated, 14.3% saturated, and 28.6% mono-unsaturated fatty acids), which was stored in a laboratory under ambient light at room temperature, was used for PV and TBA analyses.
2-Thiobarbituric Acid Assay
All reagents used were ACS reagent grade (Fisher Scientific, Fair Lawn, NJ). The lipid oxidation of each sample was determined by the TBA method described by Witte et al. (1970) as the cold extraction procedures were modified by Pensel (1990). The details of the TBA methodology are presented in Figure 1. Five grams of either coarsely ground unrendered lamb fat or corn oil was placed in a polyethylene bag. An additional empty polyethylene bag was prepared as a blank. Fifty mL of a cold (2 ± 2oC) 20% trichloroacetic acid (TCA) and 1.6% m-phosphoric acid mixture was added to each polyethylene bag and blended in a Seward Laboratory blender (Tekmar Co., Cincinnati, OH) for 2 min. Fifty ml of cold (2 ± 2oC) distilled water was added to each bag and blended for an additional 30 seconds. The slurry was filtered through Whatman No. 1 filter paper to remove cellar debris. Five ml of the filtered slurry was added to 5.0 ml of freshly prepared 0.02 M 2-thiobarbituric acid and mixed for 5 seconds. The samples were subsequently stored in the dark at room temperature for 15 hours to develop color. The absorbance of the chemical reactions was measured at 530 nm using a Spectronic® Genesys spectrophotometer (Spectronic Instruments Inc., Rochester, NY).
AOCS Peroxide Value
The PV of each sample was determined using the American Oil Chemists' Society official method (AOCS, 1998). The procedures are shown in Figure 2. The chemical reactions of the AOCS official PV are presented in the following equations [1] to [4]:
| R· + O2 + H·-------- R·OO·H | [1] | |
| 2 KI + 2 CH3COOH----2 HI + 2 CH3COO-K+ | [2] | |
| R·OO·H + 2 HI---------ROH + H2O + I2 | [3] | |
| + starch indicator | ||
| I2 + 2 Na2S2O3--------Na2S4O6 + 2 NaI | [4] | |
| Purple colorless | colorless | colorless |
Peroxides (R·OO·H) are reacted with potassium iodide (KI) in the presence of acetic acid, and liberated iodine is measured after addition of Na2S2O3 (Asakawa and Matsushita, 1980). AOCS peroxide value is based on the amount of Na2S2O3 used to change the color of the librated iodine (I2) in the aqueous phase from purple to colorless iodide (I-), which was converted into peroxide value by employing the following formula:
Where:
Spectrophotometric Peroxide Method for Peroxide Value
Spectrophotometric Scanning. The absorption maximum of an iodine solution alone and an iodine-starch complex were determined by scanning a 10 ml of 10-3 N iodine solution and a 10 ml of 10-3 N iodine with 0.5 ml of 1% starch indicator (w/ 0.3% chloroform) (Lab Chem Inc., Pittsburgh, PA) from 400 nm to 900 nm (Figure 3). A 10-ml deionized water along with 0.5 ml of 1% starch indicator solution was also prepared for spectrophotometric scanning procedures.
Linear Regression for the Standard Curve: Iodine Equation. An iodine standard curve was generated in order to convert iodine concentration to determine the peroxide value. A 0.1 N iodine solution was freshly prepared as a stock solution for dilution. Four dilutions of 5 x 10 -4, 1 x 10 -3, 2 x 10 -3, and 4 x 10 -3 N iodine were prepared from the stock solution in de-ionized water. Ten ml of each iodine solution was dispensed into assay tubes and 0.5 ml of the 1% starch indicator was added to each tube. The absorption of de-ionized water (blank) and samples prepared as described were immediately measured at 563 nm. Absorbance was plotted against iodine concentration (Figure 4A) and linear regression analyses performed employing the following equation. Iodine equation from the standard curve (with R2 = 0.99) is obtained as follows:
Measurements. The flow diagram of the SPM analysis for peroxide value is shown in Figure 2. Five grams of fat were placed into a 250 ml flask, 30 ml of acetic acid-chloroform (3:2, v/v) reagent was added, and the flask was shaken vigorously for 1 min. One half mL of saturated potassium iodide was added to a flask, mixed for 1 min. and 30-ml of de-ionized water added and mixed again for 1 min. After incubating the flask for 5 min., 10 ml of the upper aqueous solution from each flask was dispensed into a 16 mm x 120 mm borosilicate glass assay tube. If preliminary analyses indicated that the sample was highly oxidized the sample was diluted to 10 fold with de-ionized water. The procedure continued by the addition of 0.5-ml of 1% starch indicator (with 0.3% chloroform) and mixing for 5 seconds. The absorbance of samples was immediately measured at 563 nm.
The Calculation of the Spectrophotometric Peroxide Value. The determination of spectrophotometric peroxide values was based on the chemical reactions, shown in the following equations [1] to [3] and [5]:
| R· + O2 + H·-----------R·OO·H | [1] | |
| 2 KI + 2 CH3COOH----2 HI + 2 CH3COO-K+ | [2] | |
| R·OO·H + 2 HI---------ROH + H2O + I2 | [3] | |
| + Starch indicator | ||
| I2------------------------------Iodine-starch complex | [5] | |
| Purple | Blue | |
Peroxide value, determined by the SPM method, is based on measuring the absorbance 563 nm of the iodine-starch complex. The determination of the peroxide value is corresponding to the amount of I2 produced, according to equation [3], i.e., 1 mole of peroxide (R·OO·H) generates 1 mole of iodine (I2). Thus, by determining the concentration (meq.) of iodine-starch complex in 10 ml of solution from the absorbance and the iodine equation from the standard curve, PV can be directly read from the standard curve (Figure 4B).
Peroxide value can be originally calculated from the required amount of Na2S2O3 ml in [4]. However, no Na2S2O3 was used in the SPM. One mole of I2 is equal to 2 equivalents (eq.) of I2. The total volume of the aqueous layer was 48 ml (30 ml x 60% of acetic acid + 30 ml of H2O = 48 ml). A 10-ml of aqueous layer was used to measure the absorbance. Original sample weight is 5 g. Therefore, the peroxide value by spectrophotometric method is converted as the equation [7]:
| SPV = [(OD value + 0.0131) /
3477.3] x 2 meq. x (48 ml/10 ml)/5g |
[7] | |
Statistical Analyses
All analyses were performed in six replications. The coefficient of variation (CV) of the AOCS, SPM, and TBA for lamb tissue and corn oil were calculated by the following equation:
Coefficient of Variation (CV) = (s / x ) x 100%
Statistical significance of the differences between means of PV and TBA were determined by the Tukey's multiple comparison test at 95% confidence level using the SAS 7.0 for Windows (SAS, 1998).