| Conjugated linoleic acid (CLA) is a technical term for a mixture of positional (6-8,7-9,9-11,11-13,13-15) and geometric (cis,cis; cis,trans; trans,cis; trans,trans) isomers of linoleic acid that contain conjugated double bonds. The major source of naturel CLA is milk and meat from ruminant. Milk fat is a very common and good source for natural CLA. On average, per gram of milk fat contains 10mg CLA, most of which is biological activity isomer 9c,11t-CLA. CLA have many positive biological activities, and the different isomers show different bioactivities. For instance, based on testing in animal models,9c,11t-CLA showed the function of anti-carcinogenesis, anti-atherosclerosis and immunomodulation.10t,12c-CLA could reduce the body fat, and it also is a potent inhibitor of milk-fat synthesis. Considering the use of CLA for medicinal and nutraceutical purposes, a safe isomer-selective process is required. Therefore, it is necessary to establish the reliable methods for analysis of CLA isomer.In the present study, conditions of milk fat extraction, sample pretreatment and HPLC and MEKC analysis were optimized. The major CLA isomers from milk samples were separated and tested by silver-ion high performance liquid chromatography (Ag+-HPLC) and micellar electrokinetic chromatography (MEKC). The main results were as follows:1. Different solvent combinations were evaluated for the extraction of milk fat. In comparison with others, hexane/isopropanol with the assistance of ultrasound displayed better extraction ability.2. Methylation of CLA with NaOCH3-CH3OH in mild conditions showed favorable effect. Box-Behnken design was applied to find the optimum methylation conditions. The results showed that the optimum conditions for the methylation of CLA were as follows:8.56 mL of 0.5 M sodium methoxide-methanol, reaction temperature 24.4℃, reaction time 12.8 min.3. Ag+-HPLC has been shown to be effective in the resolution of the major CLA isomers 9c,11t-CLA and 10t,12c-CLA, which were known as their beneficial biological activities. In the concentration of standard isomers from 7.812μg/mL to 500μg/mL, there was a good linear relationship between the peak area and injection concentration.4. The milk treated with heat, including pasteurization and ultra-high temperature instantaneous sterilization (UHT), could reduce the content of CLA significantly. The varations of CLA content in milk was investigated during storage by using Ag+-HPLC. With the increase in preservation time, there was sharp decline in CLA levels in all the samples. During shelf life,9c,11t-CLA content in row milk decreased by 9%,9c,11t-CLA in pasteurized milk decreased by 16%, and in UHT milk decreased by 8%. Season was another important influencing factor. The result showed that the CLA content in summer milk was significantly higher than other seasons. Additionally, there was a significantly difference in the 9c, 11t-CLA content among the cow milk, goat milk and buffalo milk (P<0.001). The CLA content of milk from different companies showed statistically difference (P< 0.001).5. Under the optimum conditions,80 mM borate (pH 9.0),54 mM sodium dodecyl sulphate,4%(w/v)β-cyclodextrin,8M urea,4%(v/v) ethanol,25 kV and 20℃, the major CLA isomers 9c,11t-CLA and 10t,12c-CLA in milk could be separated by MEKC effectively within 15 min. The result indicated that there was a significant difference in the total CLA content among raw milks, pasteurized milk, UHT milk (P<0.001). However, the content of 10t,12c-CLA from all samples was almost similar (about 3%). The result also showed there was a significant difference in the total CLA content and 10t,12c-CLA content among the cow milk, goat milk and buffalo milk (P<0.05). The total content of CLA in these samples was 9.220mg/g milk fat in the cow milk,2.972 mg/g milk fat in the buffalo milk and 6.054 mg/g milk fat in the goat milk.
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