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研究生:劉凱崴
論文名稱:山藥酸乳酪之發酵技術與抗氧化性及抗致突變性之探討
論文名稱(外文):Studies on the Fermentation Technology and the Characteristics of Antioxidation and Antimutagenicity of Yam Yogurt
指導教授:潘崇良
學位類別:碩士
校院名稱:國立海洋大學
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
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:118
中文關鍵詞:山藥酸乳酪抗氧化性抗致突變性
外文關鍵詞:YamYogurtAntioxidationAntimutagenicity
相關次數:
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山藥為中國傳統中藥材及養生藥膳重要原料之一,而乳酸菌食品則是長久以來與人類飲食生活關係密切,發酵酸乳酪即是其中一項。本研究即嘗試將山藥結合酸乳酪開發成為保健食品,但在觀察初期以 Lactobacillus (Lb.) plantarum CCRC 10069 (1%) + CCRC 12250 (2%) 發酵添加 10% 山藥所製成的酸乳酪時發現,於發酵及貯藏的過程中呈現離水現象及澱粉沉積等問題待解決。故在降低山藥酸乳酪離水現象的探討中,分別將不同菌種組合及接種比例之 4 株產 extracellular adhesive substance (EAS) 乳酸菌,接種於額外添加 0-6% 脫脂奶粉之山藥脫脂牛乳中進行發酵。在以六組產 EAS 乳酸菌發酵的山藥酸乳酪中,離水現象皆較不產 EAS 乳酸菌之控制組產品為低,其中以 Lb. plantarum CCRC 10069 (2%) + Lc. lactis CCRC 12315 (3%) 發酵額外添加 6% 脫脂奶粉之山藥酸乳酪於 3 天 4oC 貯藏後,離水現象可由原來 43% 降至 30%。
1% 山藥水煮液以 cellulase R-10 (E/S = 3% 或 6%) 分別降解 30 hr 和 21 hr 後失活,再使用 50 unit -amylase 降解 2 hr,可分別得到 14.78 及 16.57 mg/mL 之還原醣濃度。利用膠體過濾層析法分析山藥水解液中醣類與蛋白質的成分變化,推測山藥水煮液經多醣分解酵素降解後,其主要成份可能為醣蛋白。山藥先經多醣降解酵素分解後可以解決山藥酸乳酪中大部分澱粉沉積的問題。將 2% 山藥添加於 6% 脫脂奶粉之脫脂牛乳中,以 Lb. plantarum CCRC 10069 (2%) + Lc. lactis CCRC 12315 (3%) 進行發酵試驗,添加 2% 山藥之酸乳酪可於 37oC 下發酵 18 hr 後 pH 值即達到 4.6 以下。山藥先經酵素降解後所製成之酸乳酪,其 pH 值則會隨著發酵時間增加而降低,其中分別以 E/S = 3% 或 6% cellulase R-10 降解後再以 50 unit -amylase 分解之山藥水煮液於發酵 12 hr 後所產酸乳酪 pH 值可至 4.5。在發酵 12 hr 後山藥 (2%) 酸乳酪組可滴定酸度較控制組增加量為多 (0.93% vs. 0.80%),而先經酵素降解山藥水煮液的組別,則是有更高的可滴定酸度 (1.03-1.41%),mold/yeast count 及 coliforms count (CFU/g) 在發酵期間 (48 hr) 內則均未被檢測出。於 4oC 下貯藏 14 天後,山藥酸乳酪其乳酸菌活菌數仍維持在 108 CFU/g 以上。
山藥酸乳酪冷水或熱水萃取物 (50 mg/mL) 分別有 15-19% 與16-18% 清除 DPPH 自由基能力。抑制亞麻油酸產生氫過氧化物方面,山藥酸乳酪冷水或熱水萃取物較未添加山藥之酸乳酪萃取物為佳。而在螯合亞鐵離子能力方面,山藥冷水或熱水萃取物 (10 mg/mL) 以及經過多醣分解酵素降解之山藥水解液 (10 mg/mL),均較山藥酸乳酪冷水或熱水萃取物表現為強 (28-29%/10-18%)。
4 組山藥酸乳酪與其對應之 1% 凍乾粉末復水溶液對於 4NQO 和 B[a]P 所誘導 S. typhimurium TA100 突變之抑制率分別為 58-72% 與 52-59% 以及 71-81% 與 54-81%。其中又以對 B[a]P 的抗致突變能力要較對 4NQO 者為佳。山藥冷水或熱水萃取物對 4NQO 及 B[a]P 的抗致突變能力並不顯著,其中山藥熱水萃取物之抑制突變率皆在 1% 以下。而山藥經多醣分解酵素降解的 3 種水解液對 4NQO 及 B[a]P 致突變的抑制效果則分別自 1-5% 與 1-9% 提升至 35-65% 與 31-49%。
Yam is one of important material of traditional Chinese herbs and health-keeping medicines. Products of lactic acid bacteria have a very close relationship with our diet for a long time and yogurt is one of them. This study aims to integrate yam into yogurt to develop a new health food. In the early progressing research work, 10% yam yogurt fermented with Lactobacillus (Lb.) plantarum CCRC 10069 (1%) and CCRC 12250 (2%) presented the wheying off phenomenon and the precipitation of yam starch particle during the fermentation and storage periods. To reducing syneresis, four extracelluar adhesive substance (EAS) producing lactic acid bacteria (EAS+ lactic) were inoculated into the original yam yogurt base with an extra 0-6% skim milk powder addition, and the EAS producing (EAS+) starter combinations were also employed. A significant decrease in syneresis has been observed in six groups with EAS+ lactic starters while compared to the control group. The largest reduction of syneresis (from 43% to 30%) has been observed from the group containing combinations of Lb. plantarum CCRC 10069 (2%) and Lc. lactis CCRC 12315 (3%) with 6% skim milk powder addition in the yam yogurt, which has been stored at 4oC for three days.
One percent yam boiling aqueous solutions have been hydrolyzed by cellulase R-10 (E/S = 3% or 6%) for 30 and 21 hr, respectively, and then digested by 50 unit -amylase for 2 hr. The concentration of reducing sugar of such solution was recorded as 14.78 and 16.57 mg/mL, respectively. Gel permeation chromatography was used to analyze the change of carbohydrate and protein compositions in yam aqueous solution from enzyme hydrolysis. The main component in yam boiling aqueous solutions after hydrolyzed by polysaccharide-digesting enzymes is probably glycoprotein, depending upon the results derived from GPC observation. In the following yam yogurt production, this saccharide digesting enzyme treatment was proved to solve most yam starch precipitation problem. As 2% yam yogurt base with extra 6% added skim milk powder was fermented with Lb. plantarum CCRC 10069 (2%) and Lc. lactis CCRC 12315 (3%), the results showed that the pH value of this product reached 4.6 before fermented at 37oC for 18 hr. The products of yam yogurt made with hydrolyzed yam aqueous solution mixed with skim milk, the pH value of yam yogurt declined with increasing fermentation time. The pH value of group containing treated by cellulase R-10 (3% or 6%) and 50 unit a-amylase could decrease to 4.5 in 12 hr fermentation. The titrable acidity of 2% yam add products is also higher while comparing to the control group, which as 0.93% vs. 0.80%. Titrable acidity also achieved to 1.03-1.41% in those yam yogurt groups with pre-hydrolyzed yam aqueous solution. Both mold/yeast count and coliforms count (CFU/g) have not been detected during the 48 hr fermentation time. The number of lactic acid bacteria remained above 108 CFU/g for yam yogurt stored at 4oC for 14 days.
In the evaluation on the antioxidation effect of yam yogurt, the ability for scavenging DPPH free radical was examined, and yam yogurt cold water or hot water extracts (50 mg/mL) performed a antioxidative activity as 15-19% and 16-18%, respectively. While inhibiting linoleic acid produced hydroperoxide was used to evaluate the antioxidation effect, both yam yogurt cold water and hot water extracts have better antioxidative activity than plain yogurt did. As to measure the ability of chelating on Fe (II) ion for evaluating the antioxidation effect, the results showed that yam cold water or hot water extracts (10 mg/mL) and its hydrolyzed solution (10 mg/mL) were all stronger than the yam yogurt extracts.
Both four groups of yam yogurts and their 1% yam yogurt lyophilized powder rehydrated aqueous solutions have good performance on inhibiting S. typhimurium TA100 mutation induced by 4NQO or B[a]P, the antimutagenicity observed were 58-72% and 52-59% as well as 71-81% and 54-81%, respectively. The antimutagenicity ability of exhibited by B[a]P was better than 4NQO. However, the antimutagenicity ability of yam cold water or hot water extracts were not so pronounced, especially for yam hot water extracts the mutation inhibition capability was below 1%. Although, after yam has been hydrolyzed by polysaccharide-digesting enzymes, three hydrolytic solutions showed the potentials to inhibit mutations, that induced by 4NQO and B[a]P, were from 1-5% and 1-9% to 35-65% and 31-49%, respectively.
目 錄 i
表 目 錄 vi
圖 目 錄 viii
中文摘要 xii
英文摘要 xiv
一、前言 1
二、文獻整理 3
I. 山藥之生理功能 3
I-1. 山藥簡介 3
I-2. 山藥的分佈與種原 3
I-3. 山藥之成分與營養價值 5
I-4. 山藥之利用與保健功能 6
II. 山藥之抗氧化特性 7
II-1. 自由基與活性氧之形成 7
II-2. 氧化傷害 8
II-3. 抗氧化物的種類 9
II-3-1. 自由基終止型 9
II-3-2. 還原劑或氧清除劑 9
II-3-3. 單重態氧抑制劑 9
II-3-4. 金屬螯合劑 10
II-3-5. 抗氧化酵素 10
II-4. 山藥之抗氧化性 10
III. 乳酸菌所產胞外多醣對酸乳酪質地之影響 11
III-1. Ropy 及 non-ropy 乳酸菌對酸乳酪微細結構之影響 12
III-2. 乳酸菌所產不同型態 EPS 對酸乳酪質地改變之影響 13
III-3. 乳酸菌所產 EPS 對酸乳酪品質之影響 14
IV. 乳酸菌及其發酵乳製品之抗致突變性研究 14
V. 抗致突變物之作用機制 16
V-1. 去致突變物 16
V-1-1. 防止致突變物生成之抑制劑 16
V-1-2. 降低致突變物造成DNA傷害之阻斷劑 16
V-1-2-1. 致原致突變物之不活化作用 17
V-1-2-2. 對代謝活化酵素系統之抑制作用 17
V-1-2-3. 對原致突變物代謝活化物之不活化作用 17
V-1-2-4. 促進解毒代謝系統 17
V-1-3. 抗氧化劑 17
V-2. 生物抗致突變物 18
V-2-1. 作用於DNA修補之化合物 18
V-2-2. 抑制腫瘤促進與增殖作用之抑制劑 18
VI. 抗致突變性之檢測方法 18
VI-1. Histidine requirement 19
VI-2. rfa mutation 19
VI-3. uvrB mutation 20
VI-4. R-factor 20
VI-5. pAQ1 plasmid 20
三、材料與方法 21
I. 實驗材料 21
I-1. 山藥 21
I-2. 試驗菌株 21
I-3. 培養基 21
I-4. 材料與儀器設備 24
I-5. 藥品 25
II. 乳酸菌菌株之保存與活化 26
II-1. 菌株的保存 26
II-2. 菌株的活化 27
III. 基隆山藥及山藥酸乳酪凍乾粉末之一般成分分析 27
III-1. 水分含量 27
III-2. 灰分含量 27
III-3. 粗脂肪含量 27
III-4. 粗蛋白質含量 27
III-5. 總醣量的測定 27
IV. 降低山藥酸乳酪離水現象之生產條件探討 28
IV-1. 山藥酸乳酪的製備 28
IV-2. 降低山藥酸乳酪離水現象之測試 28
IV-2-1. 山藥酸乳酪酸鹼值之測定 28
IV-2-2. 山藥酸乳酪可滴定酸度之測定 28
IV-2-3. 山藥酸乳酪乳清離水現象之測定 29
V. 減少山藥酸乳酪中澱粉沉積現象之探討 29
V-1. 山藥水煮液之製備 29
V-2. 以多醣分解酵素降解山藥水煮液 29
V-3. 還原醣之測定 29
V-4. 山藥水解液之膠體過濾層析分析 30
V-5. 山藥酸乳酪發酵試驗 30
V-5-1. 微生物含量之測定 30
V-6. 山藥酸乳酪之貯藏試驗 31
V-6-1. 乳酸菌菌數之測定 31
V-6-2. 山藥酸乳酪澱粉沉積現象之觀察 31
VI. 山藥及山藥酸乳酪之抗氧化性之探討 31
VI-1. 山藥及山藥酸乳酪冷、熱水萃取物之製備 31
VI-2. 清除 DPPH 自由基能力之測定 31
VI-3. 硫氫酸鐵法抗氧化活性之測定 32
VI-4. 螯合亞鐵離子能力之測定 32
VII. 安氏試驗法 32
VII-1. Salmonella typhimurium TA100 之保存與活化 32
VII-1-1. 試驗菌株之保存 33
VII-1-2. 試驗菌株之活化 33
VII-2. S. typhimurium TA100 基因型態之確認 33
VII-2-1. 組胺酸需求性之確認 33
VII-2-2. rfa 突變測試 33
VII-2-3. uvrB突變測試 34
VII-2-4. R-factor 之測試 34
VII-3. 毒性試驗 34
VII-4. 致突變實驗 34
VII-5. 抗致突變性試驗 35
VII-6. 統計分析 35
四、結果與討論 36
I. 基隆山藥及山藥酸乳酪凍乾粉末之一般成分 36
II. 以產胞外黏性物質乳酸菌發酵山藥脫脂牛乳 36
II-1. 產胞外黏性物質 (EAS) 乳酸菌菌種組合之篩選 36
II-2. 降低山藥酸乳酪離水現象生產條件之探討 37
II-2-1. pH 值 37
II-2-2. 可滴定酸度 38
II-2-3. 離水現象 38
III. 減少山藥酸乳酪澱粉沉積現象之探討 40
III-1. 利用多醣分解酵素降解山藥水煮液 40
III-1-1. 還原醣含量 40
III-1-2. 膠體過濾層析分析 41
III-2. 發酵與貯藏試驗 42
III-2-1. 發酵試驗 42
III-2-2. 貯藏試驗 43
III-2-3. 澱粉沉積現象之觀察 44
IV. 山藥及山藥酸乳酪抗氧化活性之探討 45
IV-1. 清除 DPPH 自由基之能力 45
IV-2. 硫氰酸鐵法抗氧化活性測定 45
IV-3. 亞鐵離子螯合效果 46
V. 山藥及山藥酸乳酪抗致突變性之探討 47
V-1. S. typhimurium TA100 試驗菌株基因型態之確認 47
V-2. 毒性試驗 48
V-3. 致突變性試驗 48
V-4. 抗致突變性試驗 48
五、結論 52
六、參考文獻 54
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