臺灣博碩士論文加值系統

乳清為乾酪製造時產生的副產物，其所衍生之生物活性肽 (bioactive peptide) 已被廣泛研究。目前製備乳源生物活性肽主要分為以蛋白酵素、消化酵素水解與微生物發酵。因此本研究擬利用本實驗室自克弗爾粒 (kefir grain) 中篩選出一株可分解牛乳蛋白之乳酸菌 Lactobacillus kefiranofaciens M1 (M1) 搭配蛋白酵素 (中性蛋白酶及鹼性蛋白酶) 與消化酵素 (胃蛋白酶) 水解乳清，並探討其免疫、抗氧化與抗高血壓之機能性，期能製備出具機能性水解乳清蛋白液。
分析其水解過程中的 pH 值、水分與肽濃度，顯示濃縮乳清蛋白水解液各組在水解 12 小時有最高的肽含量，新鮮製備乳清水解液各組則在水解 4 小時有最高的肽含量，且各組中以鹼性蛋白酶組之水解率最高。在免疫分析上，將乳清水解發酵液與小鼠巨噬細胞株 (RAW 264.7) 及 BALB/c 小鼠脾臟細胞進行體外 (in vitro) 共同培養試驗，發現各水解組中以鹼性蛋白酶組具顯著誘導腫瘤壞死因子-α (tumor necrosis factor (TNF)-α) 與介白素-12 (interleukin (IL)-12) 之效果。進一步試驗發現，將水解液與 M1 共同發酵後，其誘導效果更甚酵素水解組，但卻低於純以 M1 發酵乳清組 (p<0.05)。於抗過敏試驗中證實，乳清發酵液可顯著誘導致敏脾臟細胞分泌 IL-12 並抑制其分泌 IL-4 細胞激素，使其趨向 Th1 環境。經有效成分特性分析得知，發酵乳清之免疫機能性主要受 Lb. kefiranofaciens M1上之肽聚醣 (peptidoglycan) 調控。乳清水解液在抗氧化活性之研究上，大部分樣品在不同檢測系統可表現不同程度的抗氧化能力。在清除 2,2-diphenyl-1-picrylhydrazyl (DPPH) 自由基的能力上，以鮮乳清組之清除率高於濃縮乳清組，且以中性蛋白酶水解鮮乳清可得最高清除率。螯合銅離子能力則以胃蛋白酶水解組最佳，但乳清蛋白水解液各組在亞鐵離子螯合力上皆不佳。在探討乳清水解液之抗高血壓效果上，以鹼性蛋白酶水解濃縮乳清蛋白組具有最高之血管收縮素轉化酶 (angiotensin I-converting enzyme, ACE) 抑制率，將其以分子篩區分分子量大小並做活性試驗，得知水解液之肽片段分子量越小對 ACE 之抑制效果越佳，將其進一步以管柱純化後可得分子量約在 262.37 Da 之 ACE 抑制肽。
綜合上述結果得知，本實驗將乳清蛋白經不同酵素與微生物水解，可成功製備出具抗過敏、抗氧化與抗高血壓等不同機能性之水解液。未來則須經進一步純化提升其效果。

Whey is a byproduct of cheese manufacture resulting from the coagulation. Bioactive peptides derived from whey protein are widely investigated and their amino acid sequences have been identified. Fermentation by microorganisms and enzymatic hydrolysis with digestive enzymes and protease result in their release. Thus, the objective of this study was to investigate the effects of different hydrolytic reactions (alcalase, neatrase, pepsin, Lb. kefiranofaciens M1 (M1)) on in vitro biological properties, such as antihypertensive, antioxidative and immunomodulatory reaction of whey protein.
Immunomodulatory results demonstrated that whey protein hydrolysates (WPHs) from alcalase could significantly induce the production of TNF-α and IL-12 in murine macrophage cell line (RAW 264.7) and murine splenocytes. Further evaluating the effects of the WPHs obtained from combination of alcalase hydrolysis and Lb. kefiranofaciens M1 fermentation on cytokine production displayed that the secretion of cytokines induced by WPHs from Lb. kefiranofaciens M1 and combination group significantly higher than WPHs from alcalase only and whey control on both cells. In addition, Th2-polarized splenocytes revealed that milk whey fermented by Lb. kefiranofaciens M1 had IL-12 inducing and IL-4 repressing activities. These results suggest that the whey protein hydrolysates from Lb. kefiranofaciens M1 may be able to direct the Th1/Th2 balance toward Th1. The putative immunomodulin in the whey protein hydrolysates from Lb. kefiranofaciens M1 might be peptidoglycans in Lb. kefiranofaciens M1. Antioxidant results indicated that WPHs obtained from neutrase in fresh whey exhibited the highest DPPH radical-scavenging activity compared with other treatments. In addition, evaluation of chelating ion ability showed that WPHs hydrolyzed by pepsin had the highest ability of Cu2+ chelation. In angiotensin I-converting enzyme (ACE) inhibitory activity, WPHs obtained from neutrase in WPC exhibited the highest ACE inhibitory activity. Further purification by microfiltration and size exclusion chromatography, one peptide with molecular mass 262.37 Da showed the highest ACE inhibitory activity. The sequence of this peptide will be determined in the near future.
In conclusion, we successfully manufactured whey protein hydrolysates which contain antihypertention, antioxidation and immunomodulation properties by different hydrolytic reactions. In the future, the animal tests will be conducted to verify the in vitro biological effects of whey protein hydrolysates and the purified biological peptides of whey protein will be further identified to boost the biological effects.

口試委員會審定書
誌謝
目錄 I
圖目錄 V
中文摘要 i
英文摘要 iii
緒言 v
壹、 文獻檢討 1
一、 乳清蛋白簡介 1
(一) 乳清蛋白 1
(二) 乳清蛋白之組成 1
二、 生物活性肽簡介 6
(一) 肽對於生物體之重要性 6
(二) 生物活性肽之製備 6
(三) 乳清蛋白源生物活性肽之機能性 9
貳、 材料與方法 41
第一部分：乳清蛋白水解液製備及基本成分 41
第二部分：乳清蛋白水解液之免疫機能性 47
第三部分：乳清蛋白水解液之抗氧化機能性 54
第四部分：乳清蛋白水解液之抗高血壓機能性 57
參、 結果與討論 63
第一部份：乳清蛋白水解液製備及基本成分 63
一、 乳清蛋白水解液製備 63
(一) 基本組成分 63
(二) 蛋白水解酵素最佳作用溫度試驗 64
(三) 乳清蛋白水解液之 pH 值與酸度變化 65
(四) 乳清蛋白水解液之肽濃度變化 66
二、 乳清發酵液製備 72
(一) 乳清蛋白液中Lb. kefiranofaciens M1菌株之生長 72
(二) 乳清蛋白發酵液之pH值與酸度 72
(三) 乳清蛋白發酵液之肽濃度變化 73
第二部份：乳清蛋白水解液之免疫機能性分析 78
一、 乳清水解液誘導免疫細胞分泌細胞激素之影響 78
(一) 乳清水解液對 RAW 264.7細胞株上之影響 78
(二) 乳清水解液對脾臟細胞之影響 78
二、 乳清發酵液誘導免疫細胞分泌細胞激素之影響 84
(一) 乳清發酵液對 RAW 264.7 細胞株之影響 84
(二) 乳清發酵液在脾臟細胞上之影響 84
三、 乳清發酵液抗過敏試驗 89
四、 有效成分特性分析 91
(一) 確認有效物質 91
(二) 確認有效部位 91
第三部份：乳清蛋白水解液之抗氧化機能性分析 97
一、 乳清蛋白水解液清除 DPPH自由基之能力 97
二、 乳清蛋白水解液螯合亞鐵離子能力 100
三、 乳清蛋白水解液螯合銅離子能力 102
第四部份：乳清蛋白水解液之抗高血壓機能性分析 104
一、 乳清蛋白水解液抑制 ACE 能力 104
二、 鹼性蛋白酶水解液活性位置區分 106
三、 離子子交換樹脂純化 108
四、 乳清蛋白源 ACE 抑制肽分子量 111
肆、 結論 114
伍、 參考文獻 116
陸、 作者小傳 136

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