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第 29 筆
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論文名稱: | 乳酸菌發酵吳郭魚保健食品產製技術及生理活性之探討
| Studies on the Health Food Production Technology and Biological Activities of Lactic acid Bacteria Fermented Tilapia Meat Product
研究生: | 陳玉真 Hu-chen chen
指導教授: | 潘崇良 Chorng-Liang Pan
學位類別: | 碩士
學校名稱: | 國立臺灣海洋大學
系所名稱: | 食品科學系
學號: | M91320050
畢業學年度: | 92
語文別: | 中文
關鍵字: | 乳酸菌 lactic acid bacteria
| 吳郭魚 tilapia
| 生理活性 biological activity
全文點閱次數: | 12

全文影像連結:

論文全文(pdf)

論文頁數: | 145
摘要: | 摘要
|
| 將乳酸菌 Pediococcus (Ped.) pentosaceus MFL、MFS、Lactobacillus (Lb.) plantarum BCRC12250 混合乳酸菌為菌酛及未添加菌種 (Non-starter) 添加 4% Sucrose、1% Glucose、2% NaCl、及 50% Distilled water，利用吳郭魚肉漿為發酵基質。以乳酸菌做為菌種發酵吳郭魚食品能使其在發酵 12 hr 後，pH 值急速下降至 4.6 以下，而縮短發酵時間至 12 hr 且能成為發酵吳郭魚食品中之優勢菌種，可抑制吳郭魚食品中雜菌生長。揮發性鹽基態氮含量變化，以添加乳酸菌酛組其揮發性鹽基態氮值具有緩慢增加。添加乳酸菌菌酛實驗組之發酵 24 hr 後，膠強度 (Gel strength) 為 621.08-742.74 cm x g，而未添加菌酛之膠強度為 429.48 cm x g，因此添加菌種發酵組具有增加膠強度能力。添加菌酛與未添加組之間，其 Hunter a、b 值無明顯之差異，但添加菌酛之實驗組之白度則有明顯改善 (由51.71增加至58.34-61.98)。SDS-PAGE 蛋白質電泳分析結果發現發酵製品在 12 hr 發酵後水溶性和鹽溶性肌肉蛋白質均被降解，並且游離胺酸含量均有提高之效果。吳郭魚發酵食品之冷水萃取物 (50 mg/mL) 之抗氧化能力方面，在清除 DPPH 自由基能力為 40.12-94.93□，其中以 Ped. pentosaceus MFL 與 MFS 之組合具有較佳表現 (94.93□)。在螯合亞鐵能力分別有 68.17-98.86□ 之螯合能力表現，抑制血紅素催化亞麻油酸自氧化能力，以 Ped. pentosaceus MFL 與 MFS 之組合菌種發酵吳郭魚食品表現可達到 81.44% 較為顯著，在還原力表現方面，以添加乳酸菌菌酛組合之還原力具較佳表現為 A700nm = 0.85-0.92。4 組乳酸菌發酵吳郭魚之冷水萃取物對於 4NQO 和 B[a]P 所誘導 S. typhimurium TA100 突變之抑制率分別為 12-26% 與 28-55%。其中又以對 B[a]P 的抗致突變能力要對 4NQO 者為佳。吳郭魚及未添加菌種發酵吳郭魚之冷水萃取物對 4NQO 及 B[a]P 的抗致突變能力並不顯著，其中 Non-starter 組之抑制突變率皆在 15% 以下。4 組乳酸菌發酵吳郭魚食品之冷水萃取物與吳郭魚及未添加菌種組相比較顯現於對於免疫細胞株 HB4C5 及 THP-1 之促進生長能力。4 組添加乳酸種菌發酵之吳郭魚食品組 (IC50 = 0.06-29.47 mg/mL) 對抑制 ACE 活性均較未添加菌種組 (IC50 = 16.70 mg/mL) 顯著，其中以菌種 MFL+MFS+12250 組發酵吳郭魚食品之抑制能力較高可達至IC50 = 0.06 mg/mL。
|
| Abstract
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| Tilapia were fermented with 4% Sucrose, 1% Glucose, 2% NaCl, 50% Distilled water, and lactic acid bacteria (LAB) strains of Pediococcus (Ped.) pentosaceus MFL, Ped. pentosaceus MFS, Lactobacillus (Lb.) plantarum BCRC12250, or their combination, and non-starter.  LAB were as starters in tilapia meat paste.  After 12 hr fermentation, the pH value rapidly declined to under 4.6 and cut down fermented time to 12 hr.  The LAB growth rapidly, became predominant during fermented tilapia meat paste period and the growth of other microorganisms was substantially inhibited.  VBN of sample fermented with LAB were slow increased after 24 hr fermentation.  The gel strength of the 24 hr fermented tilapia meat paste as LAB was used the starters was 621.08-742.74 cm x g and the gel strength obtained from the 24 hr fermented tilapia meat paste without LAB was 429.48 cm x g.  These results may suggest that fermented tilapia meat paste with the addition of LAB can enhance the gel strength ability.  Between fermented tilapia meat paste with/without the addition of LAB, its Hunter a and b value had no significant difference.  But the whiteness of samples with the LAB fermented tilapia meat paste were significantly higher than without starter (whiteness: from 51.71 increased to 58.34 and 61.98).  The results of SDS-PAGE of LAB fermented tilapia meat paste suggested that most of water- and salt-soluble muscle protein were rapidly degraded after 12 hr fermentation and increased in amounts of free amino acid.  In the evaluation on the antioxidation effect of the cold water extracts of fermented tilapia food (50 mg/mL), the ability for scavenging DPPH free radical was examined to perform 40-94%.  Scavenging DPPH free radical of the cold water extracts of fermented tilapia food by Ped. pentosaceus MFL and MFS starters demonstrated 94% antioxidative activity.  The results indicated that the cold water extracts of five tilapia fermented food performed 68-98% chelating effect by assaying the ability of chelating on Fe (II) ion.  The antioxidative effect was measured via the inhibition of hemoglobin-induced linoleic acid oxidation. The cold water extracts of fermented tilapia food by using Ped. pentosaceus MFL and MFS starters significantly obtained 81% antioxidative activity.  Reducing power of the cold water extracts of fermented tilapia food with LAB were performed well to A700nm = 0.85-0.92.  Four groups of the cold water extracts of fermented have good performance on inhibiting S. typhimurium TA100 mutation induced by 4NQO or B[a]P, the antimutagenicity observed were 12-26% and 28-55%, respectively.  The antimutagenicity ability of exhibited by B[a]P was better than 4NQO.  The antimutagenicity ability of the cold water extracts of tilapia and without starter fermented were not so pronounced, especially for without starter group the mutation inhibition capability was below 15%.  The growth-promotion ability of four groups of the cold water extracts of fermented tilapia food with LAB to immuno-cell line, HB4C5 and THP-1, showed significant difference to the cold water extracts of tilapia and without starters.  The ACE inhibiting activity ability of four groups from the cold water extracts of fermented with LAB was the better to perform (IC50 = 0.06-29.47 mg/mL) than without starters (IC50 = 16.70 mg/mL), especially for the cold water extracts by using Ped. pentosaceus MFL and MFS can reach IC50 = 0.06 mg/mL
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論文目次: | 目 錄
| 目    錄    i
| 表 目 錄    vi
| 圖 目 錄    viii
| 中文摘要    x
| 英文摘要    xii
| 一、前言    1
| 二、文獻整理    3
| I.    乳酸菌之特性、利用及功能性    3
| II.    乳酸菌之代謝產物對於病原菌之抑制作用    5
| III.    乳酸菌發酵對食品風味及蛋白酵素系統之影響    6
| IV.    乳酸菌之抗氧化特性    8
| IV-1.    自由基與活性氧之形成    9
| IV-2.    氧化傷害    9
| IV-3.    抗氧化物的種類    10
| (1)    自由基終止型    10
| (2)    還原劑或氧清除劑    10
| (3)    單重態氧抑制劑    11
| (4)    金屬螯合劑    11
| (5)    抗氧化酵素    11
| IV-4.    乳酸菌之抗氧化    11
| V.    乳酸菌及其發酵製品之抗致突變性研究    12
| VI.    抗致突變物之作用機制    14
| VI-1.    去致突變物    14
| (1)    防止致突變物生成之抑制劑    14
| (2)    降低致突變物造成DNA傷害之阻斷劑    14
| a. 致原致突變物之不活化作用    15
| b. 對代謝活化酵素系統之抑制作用    15
| c. 對原致突變物代謝活化物之不活化作用    15
| d. 促進解毒代謝系統    15
| (3)    抗氧化劑    15
| VI-2.    生物抗致突變    16
| (1)    作用於 DNA 修補之化合物    16
| (2)    抑制腫瘤促進與增殖作用之抑制劑    16
| VII.    抗致突變性之檢測方法    16
| VIII.    發酵食品降血壓功效    18
| IX.    免疫活性物質之評估方法    21
| IX-1.    免疫細胞之活化    21
| (1)    T 細胞之活化    21
| (2)    自然殺手細胞之活化    22
| (3)    吞噬細胞之活化    22
| IX-2.    免疫活性因子之分析方法    22
| (1)    細胞存活率 (MTT 還原分析法)    22
| (2)    毒胞活性一氧化氮 (NO) 產生量分析法    23
| (3)    吞噬球呼吸爆 (Respiratory burst) NBT 還原
| 分析法    23
| 三、材料與方法    24
| I.    實驗步驟    24
| II.    實驗材料    26
| II-1.    吳郭魚原料    26
| II-2.    試驗菌株及細胞株    26
| II-3.    培養基及藥品    26
| II-4.    儀器設備    30
| III.    實驗方法    32
| III-1.    魚肉漿發酵    32
| III-1-1.    乳酸菌之保存、活化及生長測試    32
| III-1-2.    乳酸菌發酵菌酛的製備    33
| III-1-3.    乳酸菌發酵吳郭魚肉漿之製備    33
| III-2.    發酵期間生菌數及 pH 值的變化    33
| III-2-1.    主要微生物菌相之計數    33
| III-2-2.    pH value 之測定    34
| III-3.    樣品之一般成份分析    34
| (1)    水分含量    34
| (2)    灰份含量    35
| (3)    粗脂肪量    35
| (4)    粗蛋白含量之測定    35
| (5)    醣量的測定    36
| III-4.    品質檢測    36
| III-4-1.    揮發性鹽基態氮 (VBN) 含量之測定    36
| III-4-2.    Hunter L、a、b 值之測定    36
| III-4-3.    膠強度 (Gel strength) 之測定    37
| IV.    乳酸菌發酵對魚肉蛋白質之影響    37
| IV-1.    游離胺基酸 (Free amino acid, FAA) 分析    37
| IV-1-1.    製備 TCA 抽出物    37
| IV-1-2.    分析游離胺基酸    37
| IV-2.    電泳分析    37
| IV-2-1.    製備水溶性蛋白    37
| IV-2-2.    製備鹽溶性蛋白    38
| IV-2-3.    電泳分析    38
| V.    吳郭魚及發酵吳郭魚食品之抗氧化性之探討    38
| V-1.    吳郭魚及發酵吳郭魚食品冷水萃取物之製備    38
| V-2.    清除 □,□-diphenyl-□-picrylhydrazyl (DPPH) 自由基
| 能力之測定    38
| V-3.    抑制血紅素催化亞麻油酸自氧化測定    39
| V-4.    螯合亞鐵離子能力之測定    39
| V-5.    還原力測定    39
| VI.    安氏試驗法    40
| VI-1.    試驗菌株之保存    40
| VI-2.    試驗菌株之活化    40
| VI-3.    S. typhimurium TA100 基因型態之確認    40
| VI-3-1.    組織胺需求性 (Histidine requirement) 之
| 確認    40
| VI-3-2.    rfa 突變之測試    41
| VI-3-3.    uvr B 突變之測試    42
| VI-3-4.    R-factor 之測試    42
| VI-4.    毒性試驗    43
| VI-5.    致突變試驗    43
| VI-6.    抗致突變試驗    43
| VII.    MTT 細胞存活率分析    44
| VII-1.    培養動物細胞株    44
| VII-2.    培養基使用條件    45
| VII-3.    細胞存活率 (MTT assay)    45
| VIII.    發酵吳郭魚肉漿對 ACE 活性之測定    45
| VIII-1.    可溶性蛋白質含量的測定    45
| VIII-2.    胜肽含量之測定    46
| VIII-3.    對 ACE 抑制能力的測定    46
| VIII-4.    IC50 值的測定    47
| VIII-5.    Sephadex 膠體過濾    47
| IX.    統計分析    48
| 四、結果與討論    49
| I.    吳郭魚及吳郭魚發酵食品之一般成分    49
| II.    發酵期間試驗    49
| II-1.    發酵期間 pH 值、可滴定酸度、VBN 值及主要
| 微生物菌相變化    49
| II-2.    發酵期間之膠強度與白度之變化    51
| II-3.    發酵期間蛋白質水解之變化    52
| III.    貯藏試驗    54
| III-1.    pH值、可滴定酸度、及微生物含量變化    54
| III-2.    膠強度與白度之變化    56
| IV.    吳郭魚及乳酸菌發酵吳郭魚食品抗氧化活性之
| 探討    57
| IV-1.    清除 DPPH 自由基之能力    57
| IV-2.    螯合亞鐵離子之測定    58
| IV-3.    抑制血紅素 (Hemoglobin) 催化亞麻油酸自氧化能力
| 測定    59
| IV-4.    還原力測定    60
| V.    吳郭魚及乳酸菌發酵吳郭魚食品抗致突變性之
| 探討    61
| V-1.    S. typhimurium TA100 試驗菌株基因型態之確認    61
| V-2.    毒性試驗    62
| V-3.    致突變性試驗    62
| V-4.    抗致突變性試驗    62
| VI.    吳郭魚及乳酸菌發酵吳郭魚食品對免疫細胞株
| HB4C5 與 THP-1 之細胞存活率影響    64
| VII.    吳郭魚及乳酸菌發酵吳郭魚食品之抑制血管縮收
| 素轉換酶 (Angiotensin converting enzyme, ACE)
| 活性測定    65
| VII-1.    可溶性蛋白質與胜肽含量    65
| VII-2.        吳郭魚及乳酸菌發酵吳郭魚食品冷水萃取物對
| ACE 之IC50 值    66
| VII-3.        吳郭魚及乳酸菌發酵發酵食品劃分物與抑制 ACE
| 活性的關係    68
| 五、結論    71
| 六、參考文獻    73
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|
| 表 目 錄
|
| 表一、    吳郭魚及發酵吳郭魚肉漿之一般成份組成    92
| 表二、    乳酸菌發酵吳郭魚肉漿在 37oC 發酵過程中 pH 值及
| 好氣性總生菌、乳酸菌、腸內細菌菌科、葡萄球菌屬、
| 和假單胞菌屬之菌數變化    93
| 表三、    乳酸菌發酵吳郭魚肉漿在 37oC 發酵過程中 VBN 值之
| 變化    94
| 表四、    吳郭魚肉漿於發酵期間膠強度 (Gel strength) 之變化    95
| 表五、    乳酸發酵吳郭魚肉漿在 37oC 發酵過程中 Hunter L、a、
| b 值及白度之變化    96
| 表六、    乳酸菌發酵吳郭魚肉漿發酵 24 hr 後之游離胺基酸組成    97
| 表七、    吳郭魚及乳酸菌發酵食品冷水萃取物對 DPPH 自由基之
| 清除效應    98
| 表八、    吳郭魚及乳酸菌發酵食品冷水萃取物對螯合亞鐵離子之
| 效應    99
| 表九、    吳郭魚及乳酸菌發酵食品冷水萃取物對抑制血紅素催化
| 亞麻油酸自氧化之能力    100
| 表十、    吳郭魚及乳酸菌發酵吳郭魚食品冷水萃取物之還原力    101
| 表十一、    沙門氏菌變異株 Sal. typhimurium TA100 之基因型態
| 檢測    102
| 表十二、    吳郭魚及乳酸菌發酵吳郭魚食品之冷水萃取物
| (50 mg/mL) 對 S. typhimurium TA100 之毒性    103
| 表十三、    吳郭魚及乳酸菌發酵吳郭魚食品之冷水萃取物
| (50 mg/mL) 對 S. typhimurium TA100 之致突變性    104
| 表十四、    吳郭魚及乳酸菌發酵吳郭魚食品之冷水萃取物
| (50 mg/mL) 對 4NQO 和 B[a]P 之抗致突變性    105
| 表十五、    吳郭魚及乳酸菌發酵吳郭魚食品冷水萃取物
| (150 mg/mL) 之可溶性蛋白質及胜肽含量的影響    106
| 表十六、    吳郭魚及乳酸菌發酵吳郭魚食品之冷水萃取物對
| ACE IC50 值的影響    107
| 表十七、    吳郭魚及乳酸菌發酵吳郭魚食品之冷水萃取物個別劃分收集物抑制 ACE 能力的關係    108
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|
|
| 圖 目 錄
|
| 圖一、    乳酸菌發酵吳郭魚肉漿在 37oC 發酵過程中可滴定
| 酸度 (Titrable acidity, %) 之變化    109
| 圖二、    吳郭魚肉漿混合接種乳酸菌Ped. pentosaceus MFL
| 及 MFS 後於發酵過程中水溶性蛋白質 (WSP) 與
| 鹽溶性蛋白質 (SSP) 之 SDS-PAGE 電泳圖之變化    110
| 圖三、    吳郭魚肉漿混合接種乳酸菌 Ped. pentosaceus MFL
| 及 Lb. plantarum BCRC12250 後於發酵過程中水溶性
| 蛋白質 (WSP) 與鹽溶性蛋白質 (SSP) 之 SDS-PAGE
| 電泳圖之變化    111
| 圖四、    吳郭魚肉漿混合接種乳酸菌 Ped. pentosaceus MFS 及 Lb. plantarum BCRC12250 後於發酵過程中水溶性蛋白質 (WSP) 與鹽溶性蛋白質 (SSP) 之 SDS-PAGE 電泳圖之變化    112
| 圖五、    吳郭魚肉漿混合接種乳酸菌 Ped. pentosaceus MFL、
| Lb. plantarum BCRC12250、及 Ped. pentosaceus MFS 後於發酵過程中水溶性蛋白質 (WSP) 與鹽溶性蛋白質 (SSP) 之 SDS-PAGE 電泳圖之變化    113
| 圖六、    吳郭魚肉漿混合未接種乳酸菌 (Non-starter) 後於發酵過程中水溶性蛋白質 (WSP) 與鹽溶性蛋白質 (SSP) 之 SDS-PAGE 電泳圖之變化    114
| 圖七、    乳酸菌發酵吳郭魚食品於 4oC 貯藏期間之 pH 值與可滴定酸度 (Titrable acidity) 變化    115
| 圖八、    乳酸菌發酵吳郭魚食品於 -18oC 貯藏期間之 pH 值與可滴定酸度 (Titrable acidity) 變化    116
| 圖九、    乳酸菌發酵吳郭魚食品於 4oC 貯藏期間之好氣性總生菌、乳酸菌、腸內細菌菌科、葡萄球菌屬、和假單胞菌屬之菌數變化    117
| 圖十、    乳酸菌發酵吳郭魚食品於 -18oC 貯藏期間之好氣性總生菌、乳酸菌、腸內細菌菌科、葡萄球菌屬、和假單胞菌屬之菌數變化    118
| 圖十一、    乳酸菌發酵吳郭魚食品於 4oC 貯藏期間之膠強度 (Gel strength) 之變化    119
| 圖十二、    乳酸菌發酵吳郭魚食品於 -18oC 貯藏期間之膠強度 (Gel strength) 之變化    120
| 圖十三、    乳酸菌發酵吳郭魚食品於 4oC 貯藏期間之 Hunter L、a、b 值及白度之變化    121
| 圖十四、    乳酸菌發酵吳郭魚食品於 -18oC 貯藏期間之 Hunter L、a、b 值及白度之變化    122
| 圖十五、    吳郭魚及乳酸菌發酵吳郭魚食品之冷水萃取物對細胞株 HB4C5 細胞存活率之影響    123
| 圖十六、    吳郭魚及乳酸菌發酵吳郭魚食品之冷水萃取物對細胞株 THP-1 細胞存活率之影響    124
| 圖十七、    吳郭魚及乳酸菌發酵吳郭魚食品之冷水萃取物對 ACE 抑制活性的影響    125
| 圖十八、    吳郭魚之冷水萃取物的膠體層析圖譜    126
| 圖十九、    以 Ped. pentosaceus MFL 與 MFS 發酵吳郭魚食品之冷水萃取物的膠體層析圖譜    127
| 圖二十、    以 Ped. pentosaceus MFL 與 Lb. plantarum BCRC12250 發酵吳郭魚食品之冷水萃取物的膠體層析圖譜    128
| 圖二十一、    以 Ped. pentosaceus MFS 與 Lb. plantarum BCRC12250 發酵吳郭魚食品之冷水萃取物的膠體層析圖譜    129
| 圖二十二、    以 Ped. pentosaceus MFL, MFS, 與 Lb. plantarum BCRC12250 發酵吳郭魚食品之冷水萃取物24小時的膠體層析圖譜    130
| 圖二十三、    以未添加乳酸菌發酵吳郭魚之冷水萃取物的膠體層析圖譜    131
|
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