摘要
本研究旨以酵母菌與乳酸菌組合菌發酵南瓜子仁懸浮液，並分析發酵產品抗氧化活性。5株酵母菌株於25℃及5株乳酸菌株於37℃培養48小時，所得酵母菌菌數與乳酸菌菌數均分別達8 log cfu/mL與9 log cfu/mL以上；酵母菌株發酵液pH值5.04~6.1、乳酸菌株則為pH值3.75~4.0，皆低於控制組的 6.57。酵母菌株及乳酸菌株發酵液可滴定酸度分別為0.19%~0.32%與0.365%~0.396%，皆高於控制組的 0.09%，顯示酵母菌株與乳酸菌株可於南瓜子液中生長良好。擇優選出酵母菌及乳酸菌各2株，於30℃進行酵母菌與乳酸菌混株發酵南瓜子液48小時，各組發酵液總酚含量(約290 g/100 mL)皆顯著高於各單株菌發酵液所得(約190 g/100 mL)。南瓜子液添加黃糖、白糖、紅糖之培養液培養4組混合菌株，顯示添加黃糖所得總酚含量高於紅糖所得(約10 g/100 mL差異)，後者又高於白糖所得。由酵母菌+乳酸菌的4種組合測試不同黃糖濃度及發酵溫度的影響，建立發酵南瓜子液的發酵條件如下：10% (w/v)南瓜子液添加5%(v/v)黃糖，以酵母菌SC21686與乳酸菌FKR2526混株於30℃發酵72 hr，所得總酚含量為298.24 g/100 mL，高於單株酵母菌(266.43 g/100 mL)或乳酸菌(285.23 g/100 mL)發酵液所得。酵母菌SC21686與乳酸菌FKR2526混株發酵南瓜子液乾燥粉末之乙醇萃取物對DPPH清除率的IC50 為1.95 mg/mL，於 5 mg/mL濃度時所得清除率為94.4%。



關鍵詞：南瓜子仁；酵母菌； 乳酸菌 ；混株發酵； 抗氧化活性

Abstract
The purpose of this study was to ferment the pumpkin seed kernel (PSK) suspension with yeast and lactic acid bacteria (LAB) combination, and to analyze the antioxidant activity of the fermented product. After incubation of 5 yeast strains 25℃ and 5 LAB strains at 37℃ in PSK suspension for 48 hours, and counts for yeast and LAB were above 8 log cfu/mL and 9 log cfu/mL, respectively. The pH value of the fermentation broth of yeast strains was 5.04~6.1, and the pH value of LAB strains was 3.75~4.0, which were both lower than 6.57 of the control group. The titratable acidity of the fermentation broth of yeast strains and LAB strains was 0.19%~0.32% and 0.365%~0.396% respectively, which were higher than 0.09% of the control group. These data indicated that yeast strains and LAB strains could grow well in PSK suspension. Two strains of yeast and LAB were selected, and the mixed strains of yeast plus LAB were fermented at 30°C for 48 hours. The total phenolic content (about 290 g/100 mL) of each combination group of fermented PSK broth was significantly higher than that of each single strain fermented PSK broth (about 190 g/100 mL). Four groups of mixed strains were cultured in the PSK suspension added with 3 type sugars of light brown sugar, white sugar and brown sugar. It was shown that the total phenolic content obtained by adding light brown sugar was higher than that obtained by brown sugar (about 10 g/100 mL difference), and the latter was higher than that obtained by white sugar. The effects of different light brown sugar concentrations and fermentation temperatures were tested by four combinations of yeast + LAB, and the fermentation conditions for PSK suspension were established as follows: 10% (w/v) PSK suspension was added with 5% light brown sugar, and the mixed strain of yeast SC21686 and lactic acid bacteria FKR2526 was fermented at 30°C for 72 hr, and the total phenolic content obtained was 298.24 g/100mL, which was higher than that (266.43 g/100mL) from single yeast strain SC21686 and that (285.23 g/100mL)from single LAB strain FKR2526. The IC50 value of the ethanol extract of the fermented PSK powders from the mixed strains of yeast SC21686 and LAB FKR2526 for DPPH radical scavenging activity was 1.95 mg/mL. The scavenging activity at 5 mg/mL of the ethanol extract of fermented PSK was 94.4%.


Key words: Pumpkin seed kernel, Yeast, Lactic acid bacteria, Mixed strain fermentation, Antioxidant activity

目錄
壹、前言 1
貳、文獻整理 2
一、南瓜介紹 2
1.南瓜的起源 2
2.南瓜的品種 2
3.南瓜的組成分 2
3.1南瓜皮 3
3.2南瓜果肉 3
3.3南瓜子 3
3.3.1南瓜子油 4
3.3.2南瓜子蛋白質 4
二、自由基 6
1.自由基的產生 6
2.自由基的種類 6
3.自由基對人體的影響 7
4.自由基的預防 8
三、抗氧化劑 9
四、活性胜肽 10
1.活性胜肽的產生 10
2.活性胜肽的功能 11
五、乳酸菌 12
1.乳酸菌的種類 12
2.乳酸菌對碳水化合物的代謝 12
3.乳酸菌發酵期間影響蛋白酶活性之因子 13
3.1發酵菌種 13
3.2發酵溫度 13
3.3發酵時間 14
4.乳酸菌之生理功能 14
4.1抑制致病菌、維持腸道菌相平衡 14
4.2改善乳糖不耐症 14
4.3降低血中膽固醇 15
4.4抗癌作用 15
4.5提升食物之營養價值 15
4.6抗氧化活性 15
4.7強化免疫系統 16
六、酵母菌 16
1.酵母菌營養源 17
1.1碳源 17
1.2氮源 17
1.3礦物質 18
1.4維生素 18
2.酵母菌發酵 19
參、實驗設計及材料與方法 20
一、實驗流程 20
二、材料與方法 21
(一)、材料 21
1.原料 21
2.菌株 21
3.化學藥品 21
3.1可滴定酸測定 21
3.2游離胺基酸測定 21
4.南瓜子發酵液微生物檢測 21
5.抗氧化測試 22
5.1總酚測定 22
5.2清除DPPH試驗 22
(二)、儀器設備 23
(三)、實驗方法 24
1.南瓜子粉 24
2.南瓜子懸浮液 24
3.菌株保存 24
3.1酵母菌株保存 24
3.2乳酸菌保存 24
4.菌株活化 24
4.1酵母菌株活化 24
4.2乳酸菌株活化 24
5.菌株篩選 24
5.1酵母菌 24
5.2乳酸菌 25
5.3乳酸菌與酵母菌組合菌株 25
6.活化菌液探討 25
6.1酵母菌 25
6.2乳酸菌 25
7.組合菌株發酵條件探討 25
7.1外加蔗糖的影響 25
7.1.1蔗糖種類 26
7.1.2蔗糖濃度 26
7.2發酵溫度 26
8.分析 26
8.1成份分析 26
8.1.1酸鹼值 26
8.1.2可滴定酸分析 26
8.1.3游離胺基態氮分析 26
8.2微生物分析 27
8.2.1好氣性總生菌數 27
8.2.2乳酸菌檢測 27
8.2.3酵母菌檢測 28
8.3抗氧化活性分析 28
8.3.1樣品萃取 28
8.3.2總酚之定量 28
8.3.3清除DPPH試驗 28
(四)、統計 29
肆、結果與討論 30
一、 單一菌株篩選 30
1. 酵母菌株篩選 30
2. 乳酸菌株篩選 32
二、 混合菌株發酵條件探討 33
1. 不同糖類對混株發酵南瓜子仁懸浮液的影響 33
2. 不同蔗糖濃度對混株發酵南瓜子仁懸浮液的影響 35
3. 不同培養溫度對混株發酵南瓜子仁懸浮液的影響 36
三、不同體積10%(w/v)南瓜子仁懸浮液混株發酵理化分析 38
四、單株酵母菌及乳酸菌及混合菌株發酵南瓜子仁懸浮液理化分析 39
五、混株發酵南瓜子仁懸浮液之抗氧化活性分析 40
伍、結論 42
陸、參考文獻 43

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