| The triglycerides are95%-98%of total fat in breast milk, the content of palmitic acid in total fatty acids of human milk is about24%-26%, SN-2palmitic acid is50%-70%of total palmitic acid, while content of palmitic acid in milk is26%-35%, SN-2palmitic acid is only32%-40%in palmitic acid。Palmitic acid of different position in triglyceride directly affect the physiological function of palmitic acid in the human body’s. The palmitic acid at the Sn-2position of triglycerides can promote the fatty acid absorption in human’s body. Especially for infants and young children, the palmitic acid at the Sn-2position of triglycerides can prevent the loss of calcium, magnesium and other bone minerals, and reduce stool hardness. In recent years, more and more research breast milk of formula which have the same proportion of Sn-2fatty acids with human milk. Therefore, the detection methods of the palmitic acid at the Sn-2position of triglycerides is very important. A quick and sensitive method was developed for the determination of sn-2fatty acid composition in milk fat. Specific content and results are as follows:1. The known content and composition of the standard triglycerides were analyzed by gas chromatography which were used the method of GB5413.27-2010to esterify the glyceride.And The text select the column which detect triglycerides. The text adjust the conditions of gas chromatography instruments to determine the conditions of the separation of the fatty acids,such as split ratio and temperature-programmed gas chromatography. The optimize conditions were as follows:The GC column was DB-23column; The carrier gas was high purity nitrogen (≥99.99%);The flow of carrier gas was1.0mL/min; The ex-column pressure was17.869psi; The inlet heater temperature was270℃; The detector temperature was250℃; The injection volume was1μL;The optimal temperature-programmed from160℃to215℃at5°C/min, held for2min at160℃and held for2min at215℃; The time of analysis was15min; The split ratio was set to30:1.2. Specific pancreatic lipase hydrolysis standard triglycerides. Gas Chromatograph detection hydrolysis extent of triglyceride. The optimize conditions were as follows:The amount of milk sample was30mg. The amount of pancreatic lipase which was used to hydrolyze triglycerides was30mg. The reaction time of hydrolysis was2min. Amino solid-phase extraction column was used to separate hydrolyzates, and the monoglycerides was obtained.And the eluents were hexane and ethyl acetate mixed solution and dichloromethane and methanol mixed solution which were different ratios. The test determine the ratio of eluent and the times of elution. The optimize conditions were as follows:Eluent was hexane and ethyl acetate mixed solution (85:15,v:v) and dichloromethane and methanol mixed solution(2:l,v:v). The times of elution was four and the elution times were four times with2mL each time. The method of acetyl chloride and methanol and the potassium hydroxide and methanol were selected to esterified the glyceride. The2-position fat acid composition were determined by GC of the fat acid methyl esters. And the optimize method of the fat acid methyl esters was the method of acetyl chloride and methanol.3. Specific pancreatic lipase hydrolysis standard triglycerides. And Compare recovery rate of SPE and TLC, the results showed that the recovery rate of the method with the amino solid-phase extraction was93.5%-95%, and the recovery rate of the method with the TLC was81.5%-87.25%. So the SPE was simpler and easier to use to separate.4. Rose-Gettlieb method and Chloroform-methanol method are used to extract milk fat. And the fat extraction rates of two methods are compared and choose a better way to extract the milk fat.The result are3.22%-3.77%and1.67%-2.33%,respectively. So Rose-Gettlieb method was used to extract the milk fat,and the new method was used in milk fat. And the results showed that the palmitic acid of milk fat was33.19%, and the palmitic acid at the Sn-2position of triglycerides was38.15%. The method recoveries were89.88%-92%. |
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