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研究生:張源義
研究生(外文):Yuarn-Yee Chang
論文名稱:乳酸菌發酵牡蠣風味乳製品產製技術與其生理活性功能之探討
論文名稱(外文):Studies on the Production Technology and Biological Activities of Lactic Acid Bacteria Fermented Oyster-Milk Food Product
指導教授:潘崇良
指導教授(外文):Chorng-Liang Pan, Ph. D.
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
畢業學年度:93
語文別:中文
論文頁數:111
中文關鍵詞:乳酸菌牡蠣抗氧化性抗致突變性抑制血管收縮素轉換酶
外文關鍵詞:Lactic acid bacteriaOysterAntioxidationAntimutagenecityinhibitory activity against angiotensin converting enzyme
相關次數:
  • 被引用被引用:10
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  • 下載下載:183
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本文研究乳酸菌發酵牡蠣風味乳製品產製技術、探討該製品之抗氧化、抗致突變性、及抑制血管收縮素轉換酶能力等生理活性。三組混合乳酸菌種 (4%) 發酵添加 0-15% 以生鮮牡蠣經熱水萃取後所餘牡蠣肉渣與 1% 葡萄糖之 15% 脫脂奶粉復原乳於 37oC 下進行發酵試驗。實驗結果顯示15 組乳酸發酵製品中,以 Lactobacillus (Lb.) plantarum BCRC 12250 (2%) 和 Lb. plantarum BCRC 10069 (2%) 之所有組別於發酵 16 hr 後 pH 值皆可降至 4.6 以下,其中添加 5-15% 牡蠣肉渣之乳酸菌發酵牡蠣風味乳製品之 pH 值於發酵 12.5-14 hr即可達到 4.6 以下,該組樣品之可滴定酸度 0.96-1.02% 與乳酸菌菌數分別為在 9.10-9.21 log CFU/g。
三組乳酸菌發酵牡蠣風味乳製品 (FSM-OMO5、FSM-OMO10、與 FSM-OMO15) 在 DPPH 自由基之清除效應方面皆較發酵牛乳組為佳 (20.40-27.42% 與 16.99%);在抑制血紅素催化亞麻油酸自氧化能力方面,三組牡蠣風味乳製品呈現 19.32-23.30% 之抑制能力;而在螯合亞鐵離子方面,三組牡蠣風味乳製品皆可達 80% 以上之螯合能力,且隨牡蠣肉渣添加量增加至 15% 而有較高之螯合能力表現 (91.16%)。
對於直接型致突變劑 4NQO 所誘導 Sal. typhimurium TA 98 與 TA 100 突變之抑制率分別為 29.93-36.25% 與 56.41-64.76%;而對於間接型致突變劑 B[a]P 所誘導 TA 98 與 TA 100 突變的抑制效果則分別呈現 42.76-57.46% 與 69.56-75.22% 之抑制率。
於 24 hr 發酵期間三組牡蠣風味乳製品胜肽含量由 0.60-1.09 mg/mL 增加至 0.85-1.65 mg/mL,而對抑制血管收縮素轉換酶 (angiotensin converting enzyme, ACE) 能力由 52.69-54.18% 上升至 81.59-83.10%,皆較對照之發酵牛乳組 (42.38%/77.89%) 有較佳之抑制活性。利用膠體過濾層析法 (gel permeation chromatograph, GPC) 發酵牛乳與牡蠣風味乳製品 (FSM-OMO15) 劃分可得 664-757 Da 與 1,198-1,366 Da 二群胜肽,在 ACE-I 活性測試中,牡蠣風味乳製品分別為 86.1% 與 78.2%,發酵牛乳組 (對照組) 為85.7% 與 78.8%,統計學上二組發酵產物於相同分子量範圍胜肽收集物之 ACE-I 並未呈現顯著差異。
The production technology of lactic acid bacteria (LAB) fermented skim milk-oyster meat offal (FSM-OMO), and their antioxidant properties, antimutagenicity and inhibitory activity against angiotensin converting enzyme (ACE) were studied. Three groups of lactic acid bacteria fermented with 0-15% oyster meat offal, 1% glucose, and 15% reconstituted skim milk at 37oC. Overall, the time taken to below pH 4.6 was 16 hr for all the 15 lactic acid fermented products by strains Lactobacillus (Lb.) plantarum BCRC 12250 and Lb. plantarum BCRC 10069. Conversely, the fermentation time decreased to 12.5-14 hr for LAB FSM15-OMO supplemented with 5-15% oyster meat offal, the titratable acidity value and LAB counts ranged 0.96%-1.02% and 9.10-9.21 log CFU/g, respectively.
DPPH radical scavenging effect of LAB FSM-OMO5, FSM-OMO10, and FSM-OMO15 were higher than FSM, 20.40-27.42% versus 16.99%. In the inhibition effect of the hemoglobin-catalyzed peroxidation of linoleic acid, the inhibitory effects of 3 LAB FSM-OMO were exhibited from 19.32% to 23.30%. As to the ability of chelating on ferrous ion, the results indicated that 3 LAB FSM-OMO performed > 80% chelating effect, and following the addition of 15% oyster meat offal to FSM, which performed the highest chelating ability (91.16%).
The inhibition of 3 LAB FSM-OMO against the mutagenicity induced by direct-acting mutagen 4NQO evaluated by Sal. typhimurium TA 98 and TA 100 were 29.93-36.25% and 56.41-64.76%, respectively. The inhibition against the mutagenicity induced by indirect-acting mutagen B[a]P evaluated by Sal. typhimurium TA 98 and TA 100 were 42.76-57.46% and 69.56-75.22%, respectively. The antimutagenicity ability of exhibited by B[a]P was better than that of 4NQO.
The peptide contents increased from 0.60-1.09 mg/mL to 0.85-1.65 mg/mL and the inhibitory activity againsted ACE increased from 52.69-54.18% to 81.59-83.10% of 3 LAB FSM-OMO during fermentation (0-24 hr). The result showed that 3 LAB FSM-OMO performed the better ACE inhibitory activity than that of FSM. The FSM and FSM-OMO15 were fractionated by gel permeation chromatograph, two groups of peptide fractions have the molecular weights in the ranges of 664-757 Da and 1,198-1,366 Da. These fractions were tested for ACE inhibitory activity, the results show that ACE-I of FSM-OMO15 exhibited 86.1% and 78.2%, and FSM exhibited 85.7% and 78.8% in these two peptide fractions, respectively. There is no difference between the ACE-I on the same molecular weight peptide fraction of these two fermented products in statistics.
目 錄

摘要 i
Abstract iii
目錄 v
表目錄 x
圖目錄 xii
壹、前言 1
貳、文獻整理 3
一、牡蠣簡介 3
1. 牡蠣養殖概況 3
2. 牡蠣之生態 3
3. 牡蠣之消費與加工概況 4
4. 牡蠣之營養價值 5
二、乳酸菌簡介 6
1. 乳酸菌之定義與分類 6
2. 乳酸菌之功效 7
(1) 抑制致病菌維持腸道內菌叢之平衡 7
(2) 緩和乳糖不耐症 8
(3) 增加營養價值 8
(4) 降低血清中膽固醇 8
(5) 抗癌性 9
三、乳酸菌及其發酵製品之抗氧化性 10
1. 自由基與活性氧 10
2. 抗氧化劑的種類及作用機制 11
(1) 自由基終止劑 (free radical terminator) 11
(2) 還原劑或氧清除劑 (reducing agent or oxygen
scavenger) 11
(3) 金屬螯合劑 (chelating agent) 12
(4) 單重態氧抑制劑 (singlet oxygen inhibitor) 12
(5) 抗氧化酵素 (antioxidative enzyme) 13
3. 乳酸菌及其發酵製品之抗氧化 13
四、乳酸菌及其發酵製品之抗致突變性 15
1. 膳食與癌症之相關性 15
2. 食物中之致突變物及致癌物 16
3. Ames test 之檢測方法 18
(1) histidine requirement 19
(2) rfa mutation 19
(3) uvrB mutation 20
(4) R-factor 20
(5) pAQ1 plasmid 20
4. 乳酸菌及其發酵製品之抗致突變性 20
五、高血壓之定義與分類 22
1. 定義 22
2. 分類 23
(1) 原發性高血壓 23
(2) 續發性高血壓 23
六、血管收縮素轉換酶 (angiotensin converting enzyme, ACE)
簡介與其酵素活性抑制物 24
1. ACE 生化特性 24
2. ACE 的主要功能 24
3. ACE 抑制物與發酵乳製品中之來源 25
參、實驗設計 29
肆、材料方法 30
一、實驗材料 30
1. 原料 30
2. 試驗菌株 30
3. 培養基 31
4. 藥品 34
5. 材料及儀器設備 36
二、實驗方法 37
1. 乳酸菌菌株之保存與活化 37
(1) 菌株之保存 37
(2) 菌株之活化 37
2. 乳酸菌發酵牡蠣風味乳製品之生產條件探討 37
(1) 乳酸菌發酵牡蠣風味乳製品的製備 37
(2) 微生物菌相測定 38
(3) 酸鹼值之測定 38
(4) 可滴定酸度之測定 38
(5) 貯藏試驗 39
(6) 離水現象之測定 39
3. 乳酸菌發酵牡蠣風味乳製品抗氧化性之測定 39
(1) 清除 DPPH 自由基能力之測定 39
(2) 抑制血紅素催化亞麻油酸自氧化之測定 40
(3) 螯合亞鐵離子能力之測定 41
4. 乳酸菌發酵牡蠣風味乳製品之抗致突變性測定 42
(1) 試驗菌株之保存與活化 42
a. 菌株 TA 98 及 TA 100 之保存 42
b. 菌株 TA 98 及 TA 100 之活化 42
(2) 試驗菌株基因型態之確認 42
a. 組胺酸需求性之確認 42
b. rfa 突變測試 43
c. uvrB 突變測試 43
d. R-factor 之測試 43
(3) 毒性試驗 43
(4) 致突變實驗 44
(5) 抗致突變實驗 45
5. 乳酸菌發酵牡蠣風味乳製品對 ACE 抑制能力
的測定 46
(1) 可溶性蛋白質含量之測定 46
(2) 胜肽含量之測定 46
(3) 對 ACE 抑制能力的測定 46
a. 藥品配製 46
b. 試驗步驟 47
(4) IC50 值之測定 48
(5) Sephadex 膠體過濾層析 48
(6) 劃分收集物對 ACE 抑制能力的測定 48
6. 統計分析 48
伍、結果與討論 50
一、以三種乳酸菌之菌種組合發酵牡蠣風味乳製品 50
1. 乳酸菌菌株之選用 50
2. 牡蠣風味乳製品發酵實驗結果 51
二、乳酸發酵牡蠣風味乳製品抗氧化性之探討 52
1. 清除 DPPH 自由基之能力 52
2. 抑制血紅素催化亞麻油酸自氧化之能力 53
3. 螯合亞鐵離子之效果 54
三、乳酸發酵牡蠣風味乳製品抗致突變性之探討 54
1. Sal. typhimurium TA 98 及 TA 100 試驗菌株
基因型態之確認 54
(1) 組胺酸需求性 (histidine requirement) 之確認 55
(2) rfa 突變 (rfa mutation) 之測試 55
(3) uvrB 突變 (uvrB mutation) 之測試 55
(4) R-factor 之測試 55
2. 毒性試驗 55
3. 致突變性試驗 56
4. 抗致突變性試驗 56
四、乳酸菌發酵牡蠣風味乳製品對 ACE 活性
之抑制效果 58
1. 發酵期間可溶性蛋白質含量與胜肽含量變化 58
2. 發酵期間胜肽含量及對 ACE 抑制能力的影響 59
3. 乳酸菌發酵牡蠣風味乳製品劃分物與抑制 ACE
活性的關係 60
五、乳酸菌發酵牡蠣風味乳製品貯藏試驗之探討 62
1. pH 值與可滴定酸度含量變化 62
2. 乳酸菌菌數變化 63
3. 離水現象 64
4. 可溶性蛋白質含量與胜肽含量 65
5. ACE 抑制力與 IC50 值之變化 65
陸、結論 69
柒、參考文獻 70
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