Research On Production Of 1,3-diacylglycerol From Milk Fat And Transport Pathway In Vivo

Mike fat is a kind of oil which rich in polyunsaturated fatty acids,most of the fatty acids presence in the form of triglyceride.Excessive intake of triglycerides will have adverse effects on human health.It can easily cause a series of cardio-cerebrovascular diseases,such as hypertension.The current study showed that,the mixed oil which contain a certain proportion of 1,3-Diacylglycerol(1,3-DAG)has hypoglycemia activity.On the basis of testing 1,3-Diacylglycerol content in milk fat by HPLC,this paper was developed a process for preparing modified milk fat which rich in 1,3-Diacylglycerol,under the use of Novozyme 435 catalyzed glycerolysis reaction in solvent-free system.To explore the effect of modified milk fat on the expression of genes related to metabolism in the small intestinal absorption transport pathway by Caco-2 cell model,and verify the modified functional dairy product.Using a method of testing 1,3-Diacylglycerol content in mixed ester.The 1,3-Diacylglycerol was separated and detected by HPLC with differential refractive index detector.Result showed that,the different glycerin esters in the sample can be effectively separated under the condition of : N-hexane,Isopropanol and methane acid(20:3:1)as mobile phase,sample volume 10 ul,temperature of column temperature was 35?,lasting28 min.Quantitation by normalization method,the RSD of repetitive experiments was between 0.468%~1.462%,the RSD of stability test was less than 2%,and the recovery rate was between 97.4%~101.3%.This method is efficient and accurate,and suitable for test and analysis of 1,3-Diacylglycerol in mixed ester.Developed a process for preparing modified milk fat which rich in 1,3-Diacylglycerol,under the use of Novozyme 435 catalyzed glycerolysis reaction in solvent-free system.Novozyme 435 was selected from 3 kinds of lipase as catalyst.Study on the effect of4factors(reaction time,reaction temperature,enzyme addition and the molar ratio of glycerol)on the yield of 1,3-Diacylglycerol.The single factor test results were optimizedand verified by Box-Behnken test design.Under the condition of 12.2h reaction time,6%enzyme addition,the reaction temperature was 54.6 ?,the moral ratio of methanol and oil was 3.6:1,glycerol solution milk fat products after the reaction of 1,3-diacylglycerol content up to 48.72%.The content of 1,3-Diacylglycerol in milk fat products was 48.72%after glycerolysis.The related physicochemical indexes of modified milk fat all corresponding to the relevant national quality and hygiene standards.To explore the effect of modified milk fat on the expression of genes related to metabolism in the small intestinal absorption transport pathway by Caco-2 cell model.The stability test showed that the milk fat digestion liquid and can both stably exist in the 12 h in the cell culture medium before and after modified.The MTT cytotoxicity test showed that,the IC50 value of modified milk to Caco-2 cell was 1560 M,while the IC50 value of normal milk is 1617 M.The cell test was carried out under the safe dose level.Take 21 day cells,respectively;add normal milk(TG),modified milk(DAG)and culture medium(CK)co-culture in 6 well plates.After 4h,cells was collected after 4h,and measured the expression of related genes(FABP1?MTP?DGAT1?PPAR?)in lipid metabolism by real-time fluorescence quantitative PCR.The results showed that,the DAG group and TG group could significantly increase the expression of 4 genes.FABP gene expression was significantly increased in DAG group compared with TG group,meanwhile significantly reduced the expression of MTP and DGAT1(P<0.01).There was no significant difference in the expression of PPAR gene between the two groups.There was a difference between modified milk and normal milk in intestinal metabolism,only a small part of modified milk was in the form of chyle through 2-single Gan ester pathway during absorption and transportation.Combined with the early SD rat model research of our subject group,we speculated that the modified milk was aborted and transported by3-phosphoglyceraldehyde pathway.