Immunomodulatory and Antioxidative Activity of Lactic Acid Fermented Defatted Sesame Meal

芝麻粕為榨油後的廢棄物，成分主要以蛋白質為主，常作為動物飼料或是肥料來利用。而芝麻粕中的木酚素 (lignan)，已經證實具許多生理活性，研究指出芝麻粕中以sesaminol triglucoside (STG) 為主要成份，但體外實驗中其抗氧化力的效果並不顯著，不過經過代謝或水解卻可增加其抗氧化力，乳酸菌具有β-glucosidase，可將配糖體化合物進行去糖基，同時具有對人體健康有益的功效，因此本研究利用Lactobacillus plantarum BCRC 11697、Lactobacillus delbrueckii subsp. bulgaricus BCRC 10696、Lactobacillus acidophilus BCRC 14079、Lactobacillus rhamnosus GG ATCC 53013、Streptococcus thermophilus BCRC 14087、Lactobacillus casei 01和Lactococcus lactis 等常見的七株乳酸菌進行發酵並評估其生理活性。本實驗中分析乳酸菌在芝麻粕的生長情形，進一步評估芝麻粕發酵液的水萃物和95%乙醇萃出物之抗氧化力和免疫調節活性。果顯示 L. delbrueckii subsp. bulgaricus BCRC 10696和 L. casei 01表現較高的菌數、較低的pH值以及較高的可滴定酸，同時去除STG的醣基能力也較佳。在抗氧化方面，以乙醇萃取物表現較好，其中以 L. delbrueckii subsp. bulgaricus BCRC 10696的乙醇萃取物其DPPH的 EC50 (160.6 ± 6.50 μg /mL)、TEAC (0.34 ± 0.02 mM Trolox equiv.)、ORAC (1.06 ± 0.02 mM Trolox equiv.) 等抗氧化力，以及總酚含量上都優於其他菌種。 調節免疫功能方面，顯示水萃物具有較佳的促免疫效果，其中又以 S. thermophilus BCRC 14087對於增加巨噬細胞 RAW 264.7分泌 TNF-α 最顯著；而乙醇萃取物則對降發炎有較好的效果，其中以 L. delbrueckii subsp. bulgaricus BCRC 10696效果最好，在100 μg/mL下可以分別降低TNF-α約31.25 %以及IL-6約36.5%的分泌量。進一步以芝麻粕乙醇萃取物來進行發酵來確認效果的來源，結果顯示抗氧化增加的主要來源可能是STG經去醣基後造成的；而降發炎的效果可能不是由脫脂芝麻粕中的木酚素或是梅納反應的物質所主導，推測可能是其他小分子經代謝後表現的效果。Sesame meal obtained from oil extraction of sesame. The main composition is protein and was commonly used as feed ingredients or compost for domestic animals. Sesame meal contains lignan compounds which have been proven to exert many biological activties. Studies indicated that sesaminol triglucoside in sesame seeds are the major lignans that display biological activities. The antioxidative activities of sesaminol triglucoside were weak in vitro, but they have been reported to suppress oxidative stress after being metabolized or hydrolized in vivo. Lactic acid bacteria (LAB) have high β-glucosidase activity which can hydrolyze the glycosides attached to the lignans. Moreover, LAB also exhibit several bio-functions. In this study, the sesame meal were first prepared by cultured with lactic acid bacteria such as Lactobacillus plantarum BCRC 11697, Lactobacillus delbrueckii subsp. bulgaricus BCRC 10696, Lactobacillus acidophilus BCRC 14079, Lactobacillus rhamnosus GG ATCC 53103, Streptococcus thermophilus BCRC 14087, Lactobacillus casei 01 and Lactococcus lactis, which to enhance the function of sesame meal. The growths of various LAB in sesame meal were observed. The antioxidative and immunomodulatory activity of water extract and ethanol extract of lactic acid bacteria fermented defatted sesame meal were further determined.esults showed that L.delbrueckii subsp. bulgaricus BCRC 10696 and L. casei 01 have higher cell counts, lower pH, higher titratable acidity, and higher deglycosilation activity. Generally, ethanol extract exhibited stronger antioxidant actvity than water extracts. Additionally,the ethanol extract of L. delbrueckii subsp. bulgaricus BCRC 10696 showed the strongest antioxidative of EC50 for DPPH(160.6 ± 6.50 μg /mL), TEAC(0.34± 0.02 mM Trolox equiv.), ORAC(1.06 ± 0.02 mM Trolox equiv.), and also contains the highest concentration of total polyphenols.ater extracts showed the highest immune promotion activity in immunoregulation assays. S. thermophilus BCRC 14087 exhibited the highest activity which increased the secretion of TNF-α. However, 95% ethanol extracts of L. delbrueckii subsp. bulgaricus BCRC 10696 exerted strongest anti-inflammation activity, with , of which under 100 μg/mL, TNF-α was decreased by 31.25 %, and IL-6 decreased by 36.5 %.o further confirm the results, a sesame meal extract was used. Results indicated that the increased of antioxidative activity in fermented sesame meal were due to deglycosylated of sesaminol triglucoside. In addition, the results illustrated that small molecules are responsible for the immunoregulatory effect other than the Maillard reaction products or sesame lignans.中文摘要....................................................ibstract..................................................iii錄........................................................v目錄....................................................vii目錄.....................................................ix、前言....................................................1、文獻整理................................................2、芝麻....................................................2. 芝麻簡介.................................................2. 芝麻成分和營養價值.......................................3、益生菌..................................................9. 益生菌定義...............................................9. 益生菌的具備的特性......................................10. 益生菌的健康功效........................................11、乳酸菌.................................................17. 乳酸菌的定義和特性......................................17. 乳酸菌之分類............................................17、氧化反應及抗氧化機制...................................18. 自由基和活性氧的產生....................................18. 氧化傷害................................................18. 抗氧化作用機制..........................................19. 抗氧化活性測試原理......................................21、發炎反應...............................................23. 簡介....................................................23. 巨噬細胞................................................24. 脂多醣體 (lipopolysaccharide, LPS) 與巨噬細胞之活化.....25. 促發炎細胞激素..........................................26、實驗目的與實驗架構.....................................28、實驗目的...............................................28、實驗架構...............................................29、材料與方法.............................................31、實驗材料...............................................31. 試驗原料................................................31. 試驗用菌種..............................................31. 試驗用細胞株............................................31. 培養基..................................................31、藥品...................................................32. 發酵實驗................................................32. 抗氧化實驗..............................................32. 免疫調節實驗............................................32. 溶劑....................................................33.標準品...................................................33、儀器...................................................33. 化學抗氧化能力及免疫能力分析相關儀器設備................33. 化學分析相關儀器設備....................................34、實驗方法...............................................35.菌株之保存與活化.........................................35. 脫脂芝麻粕發酵液製備及分析..............................35. 脫脂芝麻粕發酵液水萃物及乙醇萃取物之製備................36. 水萃物和乙醇萃取物之Lignan成份分析......................37. 脫脂芝麻粕發酵液萃取物之抗氧化性測定....................37. 脫脂芝麻粕發酵液萃取物之免疫調節功能....................39. 乳酸發酵芝麻木酚素之乙醇萃取物製備......................42. 統計分析................................................43、結果與討論.............................................44、脫脂芝麻粕乳酸菌發酵過程實驗...........................44. 發酵過程中乳酸菌菌數之變化..............................44. 發酵過程中pH和可滴定酸度之變化..........................44、脫脂芝麻粕發酵液的水萃和乙醇萃取率.....................45、水萃物和乙醇萃取物之Lignan成份分析.....................45、脫脂芝麻粕發酵液水萃物和乙醇萃取物的抗氧化能力.........46. DPPH 自由基清除能力.....................................47. 總抗氧化能力 (Trolox equivalent antioxidant capacity,TEAC) ...........................................................47. 氧自由基吸收能力 (Oxygen radical absorbance capacity,ORAC) ...........................................................48. 水萃物和乙醇萃取物總酚含量..............................48、脫脂芝麻粕發酵液水萃物和乙醇萃物的免疫調節能力.........50. 細胞生存率-DNA定量分析..................................50、以乳酸發酵芝麻粕萃取物來確認抗氧化和抗發炎能力的來源...54. 乳酸發酵芝麻粕萃取物之乙醇萃取物之DPPH清除能力..........55. 乳酸發酵芝麻粕萃取物之免疫調節..........................55、參考文獻...............................................8