| Summary: | 碩士 === 靜宜大學 === 食品營養學系 === 100 === It is known that the biopolymers (mainly glycoproteins) in hot water extracts of Chlorella sp. show in vitro and in vivo immunostimulatory activities. The immunostimulatory activities may be influenced by coexisting starch in the extract and processing conditions during extraction or purification of the active glycoproteins involved. Therefore, the aim of this study was to investigate the effects of de-starching treatment, thermal treatment, and the variety and concentration of stabilizing agents on the surface hydrophobicity and in vitro immunostimulatory activity in murine macrophage RAW 264.7 model of Chlorella glycoproteins. The samples studied included the macromolecular fraction WS-F1 and its destarched fraction WS-ds-F1 (both molecular weights 10 kDa). The WS-F1 was prepared by extraction with water at 90oC for 1 hr on Chlorella sorokiniana powder, following by ultrafitration with a membrane of molecular-weight cut-off 10 kDa. The WS-ds-F1 was yielded as the above procedure but with an additional degradation with thermally resistant -amylase during extraction. The resultant WS-F1 yielded 8.5% (w/w) and contained a total protein content of 55% (w/w) and sugar composition of mainly glucose (58 mol% based on detectable carbohydrates), followed by ribose (18 mol%) and galactose (14 mol%). In contrast to the WS-F1, the destarched WS-ds-F1 showed a similar yield and total protein content, sugar composition of a higher molar percentage in glucose and galactose and less in ribose, and a greater in vitro immumostimulatory activity. The Ws-ds-F1 was accordingly used for the stabilizing formulation study. The stabilizers employed included two sugars (trehalose and mannitol; WS-ds-F1 protein: sugar ratio = 1: 0.5 1: 6 (w/w)) and eight salts (NH4Cl, NaCl, KCl, CaCl2, Na2SO4, KH2PO4, NaH2PO4 and Na2HPO4; 0.011 M). The ANS fluorescence probe was applied to detect the surface hydrophobicity change of proteins in stabilized WS-ds-F1 formulation solutions. The results illustrated that, among the stabilized formulations, the formulations with 0.5-fold weight of trehalose (i.e. WS-ds-F1 protein: trehalose = 1:0.5 (w/w)), 1-fold weight of mannitol (i.e. WS-ds-F1 protein: mannitol = 1:1 (w/w)), 10 mM NaCl, and 10 mM Na2SO4 exhibited a significantly lower ANS characteristic fluorescence (at an emission wavelength of 480 nm) than the control without stabilizers, showing the best stabilizing effect for WS-ds-F1 protein. The immunostimulatory activities of these four formulations in RAW 264.7 cell model indicated that most of samples with Chlorella glycoproteins at low concentrations displayed promoting effects on RAW 264.7 viability, especially the mannitol-WS-ds-F1 formulation at 5 g/mL that increased effectively the cell viability to 233%. All samples containing Chlorella glycoproteins stimulated a significant amount of NO secreted from RAW 264.7 cells. The effectiveness of stimulating NO secretion followed the order of: stabilized WS-ds-F1 formulations > WS-F1 and WS-ds-F1 alone > related sugars. The trehalose-WS-ds-F1 formulation showed the highest effect on increasing NO level (e.g. 3.44 M NO at 100 g/mL formuation). At 10100 g/mL, WS-F1, WS-ds-F1, and its stabilized formulations all promoted effectively the cytokines TNF- and IL-6 secreted from RAW 264.7 cells generally in a dose dependence. All WS-ds-F1 samples at 50100 g/mL caused a similar TNF- level but significantly higher IL-6 level than those did by 1 g/mL lipopolysaccharide. Conclusively, the in vitro immunostimulatory activity in RAW 264.7 of WS-ds-F1 was greater than that of WS-F1. Among the stabilizers examined for WS-ds-F1, trehalose, mannitol, NaCl, and Na2SO4 were the better stabilizers with negligible effects on the immunostimulatory activity of WS-ds-F1.
|
|---|
