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第 2 筆
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論文名稱: | 鋸齒麒麟菜乳酸發酵產品之生產與抗氧化特性之探討
| Study on Production and Antioxidative Properties of Lactic Acid Bactria Fermented Ecuheuma serra Product
研究生: | 游欣儀
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: | T97420014
畢業學年度: | 100
語文別: | 中文
關鍵字: | 麒麟菜 Eucheuma serra
| 乳酸菌 lactic acid bacteria
全文影像連結: | | (本論文20150730對校內公開)
| 論文全文(pdf)

論文頁數: | 112
摘要: | 本研究探討麒麟菜 (Eucheuma serra) 多醣水溶液以酵素水解，經乳酸菌發酵後，測試該製品抗氧化能力、抗日本腦炎病毒及儲存安定性。將 3% (w/v) 麒麟菜熱萃多醣水溶液以 5unit/mL cellulase、 A. salmonicida MAEF108 及 P. vesicularis MA103 所產生粗酵素液，在 40℃下水解 24 hr，這 4 組水解液分為添加及不添加 1% glucose組，分別接種 2 組乳酸菌酛 (A): Lb. plantarum BCRC 12250 (2.5%,v/v) + BCRC 10069 (2.5%,v/v) 及 (B) Lb. bulgaricus BCRC 10696 (2.5%,v/v) + Strep. thermophilus BCRC 12268 (2.5%,v/v)，共得 16 組乳酸發酵製備液。在 37℃ 下進行發酵 8 hr 後 pH 值皆達發酵終 4.6 以下， pH 介於 3.45-4.47 之間。添加 1% glucose 組之 pH 值在發酵 6 hr 後，均達發酵終點，乳酸菌數量介於 7.43-8.28 CFU/mL，未添加之 8 組發酵液經發酵 8hr 達發酵終點，乳酸菌量為 6.78-7.85 log CFU/mL。
| DPPH 清除自由基能力，麒麟菜多醣液並不顯著，而 16 組發酵產品則有提升結果 (5.33%-25.71%)，其中以 cellulase 水解所得之水解液，以未加 1% glucose 組效果最好分別為 25.77% 及 25.85%。螯合亞鐵離子能力以 MAEF 108 水解所的之水解液未加 1% glucose 最好，分別為 70.39% 及 73.70% 。Euc 還原力介於 6.42 ± 0.02 到 8.28 ± 0.08 ppm ascorbic acid 之間，16 組麒麟菜發酵產品還原力介於 22.31 ± 1.11 到 27.11 ± 1.00 ppm ascorbic acid 之間。由此可知未加 1% glucose 乳酸發酵產品有抗氧化活性。
| 以 BHK-21 細胞測試抗日本腦炎病毒 (Japanese Encephalitis Virus, JEV)，實驗結果指出 Euc 對於抗 JEV 之 BHK-21 細胞並無毒性。在抑制病毒對宿主細胞吸附及抑制病毒繁殖反應皆未有效果。
| 乳酸發酵產品在 4℃ 儲藏 14 天，加 1% glucose 發酵產品之 pH 值在 3.15-3.51 之間，乳酸菌數及好氣性生菌數分別為 6.14-6.84 log CFU/mL 及 6.00-6.87 log CFU/mL。未加 1% glucose 組之 pH 值為 3.55-4.14 之間，乳酸菌數及好氣性生菌數分別在於 8.87-7.41 log CFU/mL 及 8.75-6.71 log CFU/mL，由此可知加 1% glucose 進行乳酸發酵可能讓乳酸菌快速利用醣類而使得 pH 值快速下降達到發酵終點，但因 pH 下降而可能抑制乳酸菌生長。隨儲存天數的增加乳酸菌發酵麒麟菜產品之可滴定酸度及還原醣含量也隨之提升，而黴菌和酵母菌及大腸桿菌群於儲藏期間均未被檢出。
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| This study investigates Eucheuma (Eucheuma serra, Euc) polysaccharide solution of enzyme hydrolysis. After it goes lactic acid fermentation, it starts to test the product’s stability, antioxidant capacity, anti-Japanese encephalitis virus, and storage stability. Use 3% (w/v) heat extracts of Eucheuma polysaccharide aqueous solution by 5unit/mL cellulase, A. salmonicida MAEF108 and P. vesicularis MA103 produced the crude enzyme, hydrolysis under 40oC degraded for 24 hr. These 4 hydrolyzates groups contain addition 1% glucose and non-addition 1% glucose. Other two groups of lactic acid bacteria starters are:  (A): Lb. plantarum BCRC 12250 (2.5%, v/v) + BCRC 10069 (2.5%, v/v) and ( B) Lb. bulgaricus BCRC 10696 (2.5%, v/v) + Strep thermophilus BCRC 12268 (2.5%, v / v), and the results come to 16 groups of lactic acid fermentation liquid. The pH ranged from 3.45-4.47 was fermented for 8 hr at 37oC, and its fermentation is controlled at 4.6 below. After 6 hours, the group which is added 1% glucose that its pH range reach to the final fermentation. On the contrary, the 8 groups of fermentation liquid which are not added 1% glucose that ferment after 8 hours, the lactic acid bacteria range fall from 6.78 to 7.85 log CFU/mL, and the group which is added 1% glucose that its lactic acid bacteria range fall from 7.43-8.28 log CFU/mL. DPPH radical scavenging capacity, and the Eucheuma polysaccharide are not obviously, but 16 groups of lactic acid fermentation liquid products enhance the results from 5.33%-25.71%. The hydrolyzate of cellulase hydrolysis, and the group which is not added 1% glucose are the best results. Their ranges are 25.77% and 25.85%. The best capacity of the chelating Fe 2+ ion is the MAEF 108 hydrolysis of the hydrolyzate which is not added 1% glucose, and the percentages are 70.39% and 73.70%. The Euc reducing power range falls to 6.42 ± 0.02 8.28 ± 0.08 ppm of ascorbic acid in between. These 16 groups of lactic acid fermentation liquid products range fall between 22.31 ± 1.11 to 27.11 ± 1.00 ppm, ascorbic acid. Thus, the group which is not added 1% glucose lactic acid fermentation products that contain antioxidant avtivity. Taking BHK-21 cells test against Japanese Encephalitis virus as an example, and the experiment points out that the Euc against the anti-JEV BHK-21 cells which does not have any toxic reaction. Presenting results in the inhibition of viral adsorption to host cells and inhibit virus propagation do not show any reactions. Take 16 groups of lactic acid fermentation products and store them in 4oC for 14 days, the fermentation products which are added 1% glucose, the pH value is 3.15-3.51 in between. The lactic acid bacteria and aerobic health bacterial are 6.14-6.84 log CFU/mL and 6.00-6.87 log CFU/ml. The not added 1% glucose, the pH value is 3.55-4.14 in between. The lactic acid bacteria and aerobic health bacterial are 8.87-7.41 log CFU/mL and 8.75-6.71 log CFU/ml. In conclusion, adding 1% glucose to lactic acid fermentation may lead to the lactobacillus of carbohydrates fast used and make the pH value decline rapidly in order to reach the final fermentation. However, the fall in pH may inhibit the growth of lactic acid bacteria. Acidity and reducing sugar content are increasing with the increase in the number of storage days, then molds, yeasts, and coliforms will not be able detected during the storage time.
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 (Abstract)    xiii
| 壹、前言    1
| 貳、文獻整理    3
| 一、藻類    3
| 二、麒麟菜 (Eucheuma serra)    4
| 2-1. 分佈和形態    4
| 2-2. 成分    5
| 2-3. 利用價值    5
| 三、海藻多醣    6
| 3-1. 海藻多醣定義與特性    6
| 3-2. 海藻多醣萃取方法    7
| 3-3. 海藻多醣之應用    7
| 3-4. 海藻多醣硫酸化應用    8
| 四、海藻寡醣    9
| 4-1. 海藻寡醣萃取方法    9
| 4-2. 海藻寡醣之生理活性    10
| 4-2-1. 免疫性    10
| 4-2-2. 益菌性    10
| 4-2-3. 抗氧化性    11
| 4-2-4. 抗凝血    14
| 4-2-5. 抗病毒    15
| 4-2-6. 抗致突變    16
| 4-2-7. 抑制血管收縮素轉化酵素    18
| 五、洋菜酶 (Agarose)    18
| 5-1. 洋菜酶    18
| 5-2. 洋菜生產菌株及其分離來源    19
| 六、乳酸菌簡介    20
| 6-1. 乳酸菌之定義    20
| 6-2. 益生菌    22
| 6-3. 乳酸菌之應用與生理功能    22
| 6-3-1    維持腸道正常功能    22
| 6-3-2    提升免疫功能    23
| 6-3-3    抗癌基制    23
| 6-3-4    抗菌作用    23
| 6-3-5    預防心血管疾病    24
| 6-3-6    緩和乳糖不耐症    24
| 七、麒麟菜之抗氧化特性    24
| 7-1. 自由基 (Free radicals) 與活性氧之形成    24
| 7-2. 氧化傷害與疾病之相關性    25
| 7-3. 抗氧化劑種類    27
| 7-3-1    自由基終止型 (Free radical terminator)    27
| 7-3-2    還原劑或氧清除劑 (Reducing agent or
| oxygen scavengers)    27
| 7-3-3    單重態氧抑制劑 (Singlet oxygen inhibitor)    28
| 7-3-4    金屬螯合劑 (Chelating agent)    28
| 7-3-5    抗氧化酵素 (Antioxidative enzyme)    28
| 7-4. 抗氧化活性測試原理    29
| 7-4-1  DPPH (1,1-diphenyl-2-picrylhydrazy)l 自由基
| 清除力指標與其原理    29
| 7-4-2    亞鐵離子螯合能力測定指標及其原理    29
| 7-4-3    還原力測定指標及其原理    29
| 7-4-4    總酚量測定指標及其原理    29
| 參、實驗設計    31
| 肆、實驗材料與方法    32
| 一、實驗材料    32
| 1-1. 原料    32
| 1-2. 實驗菌株    32
| 1-2-1    產洋菜酶海洋菌株    32
| 1-2-2    乳酸菌株    32
| 1-2-3    實驗細胞株    32
| 1-3. 試驗藥品    32
| 1-3-1    藥品    32
| 1-3-2    培養基組成    34
| 1-3-3    DNS 溶液    35
| 1-4. 儀器設備    35
| 二、實驗方法    36
| 2-1. 麒麟菜一般成分分析    36
| 2-1-1    水分含量    36
| 2-1-2    灰份含量    36
| 2-1-3    粗蛋白含量    36
| 2-1-4    粗脂肪含量    37
| 2-2. 產洋菜酶海洋菌株之保存與活化    37
| 2-2-1    產洋菜酶海洋菌株保存    37
| 2-2-2    Agarase 粗酵素活性測定    37
| 2-3. 乳酸菌菌株之保存與活化    37
| 2-3-1    乳酸菌菌株保存    37
| 2-3-2    乳酸菌菌株活化    38
| 2-4. Agarase 粗酵液之生產    38
| 2-4-1    Agarase 粗酵液之生產    38
| 2-4-2    Agarase 粗酵素液活性測定    38
| 2-5. 麒麟菜發酵液之製備及分析    39
| 2-5-1    麒麟菜熱萃多醣液之製備    39
| 2-5-2    以多醣分解酵素降解麒麟菜熱萃取液    39
| 2-5-3    還原醣含量測定    39
| 2-5-4    麒麟菜多醣水溶液及其水解液經 Sephadex  膠體過濾層析之測定    40
| 2-5-5    薄層色層分析    40
| 2-5-6    高效能液相層析    41
| 2-6. 麒麟菜發酵液製備    41
| 2-6-1    pH 值之測定    41
| 2-6-2    可滴定酸度測定    41
| 2-6-3    微生物品質測定    42
| 2-6-4    還原醣量測定    42
| 2-6-5    儲藏試驗    42
| 2-7. 麒麟菜多醣水溶液及乳酸發酵液之抗氧化活性    42
| 2-7-1    清除DPPH 自由基能力測定    42
| 2-7-2    亞鐵離子螯合能力之測定    43
| 2-7-3    還原力測定    43
| 2-7-4    總酚含量測定    43
| 2-8. 抗病毒測試    44
| 2-8-1    細胞培養    44
| 2-8-2    細胞繼代培養    44
| 2-8-3    細胞存活率 (MTT assay)    44
| 2-8-4  抗日本腦炎病毒試驗    45
| 2-8-4-1病毒對宿主細胞吸附及抑制繁殖反應    45
| 伍、結果討論    47
| 一、麒麟菜一般成分分析    47
| 二、多醣分解酵素降解麒麟菜熱水萃取液    47
| 2-1.  菌株活化及粗酵素液誘導    47
| 2-2.  還原醣含量變化    48
| 2-3.  膠體過濾層析分析    49
| 2-4.  薄層色層分析    49
| 2-5.  高效能液相層析分析    50
| 三、多醣分解酵素降解麒麟菜多醣水溶液發酵實驗結果    51
| 四、麒麟菜乳酸發酵液抗氧化之探討    52
| 4-1.  清除 DPPH 自由基能力    52
| 4-2.  螯合亞鐵離子能力    53
| 4-3.  還原力    54
| 4-4.  總酚含量    54
| 五、麒麟菜乳酸發酵液抗日本腦炎細胞實驗評估    55
| 5-1.  對倉鼠腎臟細胞 BHK-21 存活率及抗病毒之影響    55
| 六、麒麟菜乳酸發酵液之儲存試驗    56
| 6-1.  pH 值變化    56
| 6-2.  可滴定酸度變化    56
| 6-3.  還原醣變化    57
| 6-4.  乳酸菌含量變化    57
| 陸、結論    59
| 柒、參考文獻    61
|
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2012 年 11 月 19 日 8:54