| Milk fat is dispersed in milk in a form of sphere,which is called milk fat globule(MFG).It is wrapped by a membrane structure containing lipids,proteins and phospholipids,which is known as milk fat globule membrane(MFGM).The composition and content of proteins on milk fat globule membrane are affected by species,lactation period and nutrition.In this study,conjugated linoleic acid(CLA)was used to feed normal lactating Holstein cows.The milk composition and milk fat globule size was measured and analyzed.MFGM protein was extracted for proteomic analysis,and the effect of milk fat globule membrane protein on the formation of lipid droplets in breast epithelial cells was studied through experiments of cells,so as to lay a foundation for understanding the regulatory role of MFGM protein composition in the process of milk fat globule size.(1)Experiment of animal: Firstly,16 Holstein cows with similar health conditions were selected for the experiment.The control group(CON group)was fed normally.Each cow in the experimental group(CLA group)was fed with 350 g/d CLA for 10 days.The production performance,the milk composition of cows was measured and analyzed on days 0,2,4,6,8,9 and 10 respectively.The size of milk fat globules was measured by Mastersizer 3000.And the milk was collected on 10 th day for proteomics analysis,and was verified by Western blot.The results showed that with an increasing of feeding days of CLA,the milk yield,the milk protein and the lactose content of the two groups had no significant effect.The milk fat content,the MFG size D[3,2],D[4,3],and the MFG size parameters DV(10),DV(50),DV(90)and DV(100)all decreased significantly with an increasing of feeding days(P<0.05).The number of MFGs was counted by dilution counting method.It was found that the number of MFGs also decreased significantly with the increasing of feeding time of CLA(P<0.05).The MFGM protein was extracted from the the control group and the experimental group for 10 th day.After SDS-PAGE polyacrylamide gel electrophoresis,the MFGM protein was successfully extracted.The two kinds of milk were detected and analyzed by i TRAQ proteomics quantitative technique.The composition and expression profiles of the milk fat globule membrane protein were obtained,and the differential proteins were screened.GO and KEGG enrichment analysis were performed to obtain differential expression of membrane proteins,and FABP3 was found in the CLA group compared with the control group.XDH and PLIN2 decrease by 0.89,0.82 and 0.77 times respectively(P<0.05),while MUC15,VLDLR and MFG-E8 increase by 1.59,1.36 and 1.17 times respectively(P<0.05).Then BTN,XDH and PLIN2 were selected for Western blot to verify the protein expression.The results were similar to those of proteomics,indicating that the composition of MFGM protein plays a regulatory role in the size of milk fat globules.(2)Experiment of cell: cow mammary epithelial cells MAC-T were cultured and treated as follows: LA + CON-MFGM group(100 μM linoleic acid + 100 μg/m L CON-MFGM protein),LA + CLA-MFGM group(100 μM linoleic acid + 100 μg/m L CLA-MFGM protein).The treated cells were stained with oil red O and the diameter and proportion of lipid droplets were measured,the results showed that the lipid droplet diameter of MFGM protein treated breast epithelial cells in CLA group increased to 1.33 μm compared with 1.07 μm in CON group.It was found that MFGM protein treatment in CLA group reduced the proportion of small lipid droplets(size<1 μm)and increased the proportion of large lipid droplets(size>3 μm). |
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