- 學位論文
蛋殼膜膠原蛋白水解液、蛋黃水解液及蛋殼粉對預防骨質疏鬆機能性之評估
Evaluation of functional properties of egg-derived products on bone cell function
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摘要
全世界之液蛋廠、餐廳及蛋品加工廠，每年會產生六百五十五萬噸之廢棄蛋殼，目前廢棄蛋殼之應用，主要以作為廢棄垃圾丟棄或加入鳥禽類飼料中，添加其鈣質營養量，其利用性值得深入探討 (Suguro et al., 2000)。最早於1962，Baker及Balch發現將蛋殼溶於酸性溶液中，其粗萃取物中發現含有大量羥脯胺酸，最後鑑定確定為第I及V型膠原蛋白。而目前已有相關研究指出利用不同來源之膠原蛋白 (牛皮、雞、魚與豬) 進行水解後，進行骨質疏鬆相關之細胞與動物實驗，結果發現不論在細胞或動物實驗方面，與控制組相比皆有正面之效果。 骨質疏鬆症 (Osteoporosis) 為一種常見的全身性骨骼代謝異常的疾病，主要是因為在骨骼的代謝過程中骨形成作用與骨吸收作用之間不平衡所導致，使骨密度減少進而造成骨質中鈣質的流失，導致骨骼支撐力降低，所以骨骼容易碎裂 (Fanny et al., 2010)。 本篇研究主要探討蛋殼粉、蛋殼膜膠原蛋白水解液以及蛋黃水解液在骨母細胞上之影響，實驗材料主要利用蛋殼膜萃取膠原蛋白，而後利用1%之胃蛋白酶進行酵素水解 ; 蛋黃粉部分則利用胃蛋白酶及胰蛋白酶兩種酵素進行水解試驗，兩者試驗皆於37℃下，水解時間0-10小時。再將此三種不同水解液進行胜肽含量測試。選出最佳胜肽含量後，額外添加類骨母細胞 (MG-63) 中進行共培養。而後進行細胞增生試驗、鹼性磷酸酶活性測試、骨礦化節點分析等功能評估。 實驗結果顯示，添加於MG-63類骨母細胞進行培養後，其中以經胰蛋白酶水解6小時之蛋黃水解液以及經胃蛋白酶水解8小時之蛋殼膜膠原蛋白水解液不論在細胞增生與鹼性磷酸酶活性都有顯著差異。而在細胞增生之結果顯示，當水解液濃度高於200 μg/mL時，對於MG-63 之增生有抑制之效果。因此，不論蛋殼膜膠原蛋白水解液或蛋黃水解液與MG-63細胞進行培養時，若要有效刺激類骨母細胞生長，其濃度在0-100 μg/mL與控制組相比有顯著之效果。本實驗結果也證實經胃蛋白酶水解8小時之蛋殼膜膠原蛋白水解液與經胰蛋白酶水解6小時之蛋黃水解液在鹼性磷酸酶試驗與控制組相比有顯著之效果，其又以額外添加0.1%蛋殼粉效果更為顯著。而在茜紅素染色試驗方面，同時添加經胃蛋白酶水解8小時之蛋殼膜膠原蛋白水解液與經胰蛋白酶水解6小時與0.1%蛋殼粉之處理組時，與控制組相比其礦化效果更為顯著。因此我們可以知道就述實驗條件下，會增加骨細胞中之鹼性磷酸酶活性與骨礦化效果，進而促進類骨母細胞之生長。 綜合上述，膠原蛋白水解液對刺激類骨母細胞其生長確實有一定的效果，未來在骨骼發展及預防骨質疏鬆上深具潛力。
並列摘要
Abstract Eggshell is a major byproduct of liquid egg industry and consists of collagen I, V and X in its membrane. Some studies suggest that hydrolyzed collagen-enriched diet improves bone collagen metabolism and BMD (bone mineral density). Using osteoblast-like MG-63 cells as a model system capable of partial osteoblastic differentiation, the effect of purified collagen on growth and morphological change were examined. The objective of this study was to investigate the effect of eggshell powder, collagen hydrolysate and egg yolk hydrolysate on bone cell function. The samples were prepared by hydrolyzing eggshell membrane collagen and egg yolk using pepsin and trypsin, at 37℃ for 1 to 10 hours. The hydrolysates of eggshell membrane collagen, egg yolk and eggshell powder were co-incubated with MG-63 cells then evaluated by the assays of proliferation, osteoblast differentiation and matrix mineralization using MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide), alkaline phosphatase (ALP) activity and alizarin red staining, respectively. The results obtained from MG-63 cells in vitro test demonstrated that the egg yolk hydrolyzed by trypsin for 6 hours (ET6) and the collagen hydrolyzed by pepsin for 8 hours (CP8) were able to stimulate cell growth and ALP activity. The results of MTT assay indicated that the growth of MG-63 was inhibited by the samples at concentration higher than 200 μg/ml. In contrast, the proliferation of MG-63 was only stimulated at low concentration (0-100 μg/ ml) of hydrolyzed egg yolk and collagen. The result also demonstrated that the CP8 and ET6 revealed a highest ALP activity, especially the hydrolysates of collagen and egg yolk to added 0.1% eggshell powder. Furthermore, the alizarin red staining indicated that the mineralization was slightly enhanced when stimulated by the sample prepared by incorporating ET6 within CP8 and eggshell powder. It is concluded that the ET6、CP8 and 0.1% of eggshell powder can increase significantly osteoblast activity, which acts on bone remodeling. Key Words: Osteoblast, Eggshell Membranes, Collagen Hydrolysate, Egg Yolk Hydrolysate, Eggshell Powder
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