| Summary: | 碩士 === 國立中興大學 === 動物科學系所 === 107 === Milk fat globule membrane (MFGM) is a three-layered structure that surrounding triglycerides in milk. It composes of kinds of phospholipids with many membrane proteins embed on it. Researches show that the phospholipid of MFGM improves the neuromuscular development in newborns, and lactadherin, one of the membrane proteins, stimulates engulfment of apoptotic cells, accelerates the intestinal development, and has anti-inflammatory effects. Nevertheless, the method of MFGM isolation hasn’t been unified, so the biological functions might be different. On the hand, the researches of MFGM mainly focuses on bovine MFGM, the composition and the biological functions of goat MFGM are not clear yet. Thus, the aim of this study is to investigate the differences of the compositions between bovine and goat MFGM, and evaluate the effects on wound healing of intestinal epithelial cell.
Firstly, cream was separated from raw milk and was further churned into buttermilk, and then freeze-dried to get the buttermilk powder. Hydroxyapatite (HA) or sodium citrate (SC) was added to reconstituted buttermilk for dissociating casein micelles, following by isolating MFGM isolates through microfiltration. Crude protein, phospholipid, total carbohydrate contents and protein profile were analyzed. The results showed that, hydroxyapatite pretreatment could successfully remove the casein micelle, especially almost removed all of the casein micelle in goat buttermilk, and the content of phospholipid of MFGM isolates were more than those added sodium citrate. In summary, hydroxyapatite could effective removed the casein micelle to increase the purity of MFGM isolates.
The wound healing effects were evaluated by using intestinal epithelial cell model in vitro, while caco-2 cell line was used to co-incubate with MFGM isolates from different milk origin, including bovine milk, goat milk or commercial bovine buttermilk. The caco-2 cells were co-incubated with MFGM isolates for 24 hours, following that, cell viability, cell proliferation, cell migration, and the content of cytokines were analyzed. The results showed that, the content of inflammation related cytokines CCL20 and IL-8 weren’t stimulated by MFGM isolates, and both 0.01 μg/mL and 0.1 μg/mL MFGM isolates could increase cell viability and cell proliferation, especially 0.1 μg/mL MFGM isolates could significantly improve caco-2 cell migration area. Next, 1 μg/mL LPS, 100 ng/mL TNF-α, and 20 ng/mL IFN-γ were added to caco-2 cell medium and incubated for 24 hours for stimulating cell apotosis. The effects of MFGM isolates on wound healing and inflammation induced apotosis were analyzed by using cell viability, cell proliferation, the content of cytokines, and the permeability of caco-2 cell monolayer. The results showed that, MFGM isolates could decrease the inflammatory mediator-induced cell death and increase the proliferation. On the other hand, MFGM isolates also decrease the content of CCL20 and IL-8 under inflammation status. Otherwise, MFGM could improve the loss of permeability in the inflammatory mediator-induced caco-2 cell monolayer. The underlying mechanism was evaluated while MFGMs were found to combine with epidermal growth factor receptor (EGFR) and further stimulated the downstream reaction. In summary, MFGM isolates could improve the growth of caco-2 cell, and assisted wound healing after inflammatory mediator-induced inflammation. In conclusion, hydroxyapatite has the ability to remove the casein micelle to increase the purity of MFGM isolates, and improve the growth and wound healing of intestinal epithelial cell.
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