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研究生: 何筱琪
Ho, Hsiao-Chi
論文名稱: 利用乳酸菌醱酵絲瓜水產胜肽及多醣類之研究
Study of Loofah water using Lactobacillus fermentation for producing peptides and polysaccharides
指導教授: 謝寶全
學位類別: 碩士
Master
系所名稱: 農學院 - 食品科學系所
Department of Food Science
論文出版年: 2022
畢業學年度: 110
語文別: 中文
論文頁數: 186
中文關鍵詞: 絲瓜水醱酵胜肽多醣
外文關鍵詞: loofah water, fermentation, peptide, polysaccharide
DOI URL: http://doi.org/10.6346/NPUST202200301
相關次數: 點閱:41下載:0
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  • 絲瓜水(Loofah water),別名為天蘿水,需經過靜置、醱酵、過濾等過程製得而成。由於採集過程常有異物、雜質且製作過程相當繁瑣,故本實驗為利用圓筒絲瓜(Luffa cylidrica)及稜角絲瓜(Luffa acutangular)果實採自然醱酵分離出乳酸菌,篩選其具有產胜肽(peptides)及多醣類(polysaccharides)能力之菌株,再以此菌株製備絲瓜水。由圓筒絲瓜及稜角絲瓜中於MRS broth培養基上純化出之乳酸菌株,以AL22之菌株於圓筒絲瓜水中醱酵後,其胜肽量392.82 ± 2.70 mg/L及多醣量3.69 ± 0.01 g/L為最高,而DPPH自由基清除能力為9.69 ± 0.18 %,經API 50 CHL及16S rRNA菌種鑑定結果為植物型乳酸桿菌(Lactobacillus plantarum)。L. plantarum AL22在耐胃酸、耐膽鹽與抑菌能力試驗中皆有一定的耐受性及抑菌能力,可推測出此菌株具有益生菌潛力。L. plantarum AL22的最適生長條件為接種4 % (v/v)種菌於含有添加0.5 % (w/v)葡萄糖、2 % (w/v)蛋白腖、pH為6的絲瓜汁(圓筒絲瓜:純水=1:8 (w/v))中,並於37 ℃培養20小時為最佳生長條件。此外,在安全性評估試驗中,安姆氏試驗結果(Ames test)顯示,絲瓜水醱酵液及其製品在生理代謝前後,對Salmonella typhimurium TA98, TA100均不具有突變性,且絲瓜水醱酵液及其製品可抑制LPS (1 μg/mL)誘導後RAW 264.7分泌NO,具有抗發炎之潛力。因此,絲瓜水醱酵製品不僅可縮短製程時間及護膚外亦具有商業潛力。

    Loofah water, also known as Tianluo water, is made by the process of standing, fermenting, filtering and others. As there are often foreign substances and impurities during the loofah collection, and the production process of loofah water is complicated. The lactic acid bacteria strain for this study was isolated from natural fermentation of the pulp of Luffa cylindrica and Luffa acutangular, and screened by the ability of producing peptides and polysaccharides. The selected strain was then used to prepare loofah water. Lactic acid bacteria were purified from Luffa cylindrica and Luffa acutangular on MRS broth medium. The fermentation of AL22 strain in Luffa cylindrica liquid had highest peptides (392.82 ± 2.70 mg/L), polysaccharides (3.69 ± 0.01 g/L), and 9.69 ± 0.18 % of DPPH free radical scavenging ability. The strain was identified as Lactobacillus plantarum by API 50 CHL and 16S rRNA. As L. plantarum AL22 showed resistance to gastric acid and bile salt, and displayed antimicrobial activity in the agar diffusion test, it can be inferred that this strain has probiotic potential. Inoculated 4 % (v/v) of L. plantarum AL22 in pH 6 loofah juice (Luffa cylindrica : distilled water = 1:8 (w/v)) with 0.5 % (w/v) glucose and 2 % (w/v) tryptone, and cultured at 37 ℃ for 20 hours was the optimum growth condition. Additionally, the result of Ames test of fermented loofah juice was non-mutagenicity to Salmonella typhimurium TA98, TA100.
    It was also found that fermented loofah juice and product have anti-inflammatory potential, which could inhibit the secretion of NO from RAW264.7 cells treated with LPS (1 μg/mL). Therefore, fermented loofah juice not only has short production time and skin care but also has commercial potential.

    摘要 II
    Abstract III
    謝誌 IV
    目錄 VI
    圖目錄 XII
    表目錄 XVI
    附錄 XVIII
    壹、前言 1
    貳、文獻回顧 2
    一、絲瓜 2
    (一)絲瓜簡介 2
    (二) 圓筒絲瓜(Luffa cylindrica) 2
    (三) 稜角絲瓜(Luffa acutangula) 3
    (四) 圓筒絲瓜及稜角絲瓜之成分分析 3
    二、皮膚與天然保濕因子 8
    (一) 皮膚之基本構造 8
    (二) 天然保濕因子(Natural Moisturizing Factor, NMF) 10
    三、自由基 12
    (一) 自由基定義 12
    (二) 活性氧化物 (Reactive oxygen species, ROS) 概述 12
    四、自由基對皮膚老化造成之影響 13
    (一) 內源性老化(endogenous aging) 14
    (二) 外源性老化(exogenous aging) 15
    五、抗氧化機制與預防皮膚之老化 18
    (一) 抗氧化機制 18
    (二) 預防皮膚之老化 19
    六、乳酸菌 21
    (一) 乳酸菌簡介 21
    (二) 乳酸菌分類 21
    (三) 乳酸菌代謝途徑 22
    (四) 乳酸菌於化妝品上之應用 25
    七、生物活性肽 27
    (一) 生物活性肽簡介 27
    (二) 生物活性肽功能 27
    (三) 生物活性肽之應用 28
    八、多醣 32
    (一) 多醣簡介 32
    (二) 植物多醣 32
    (三) 胞外多醣 33
    九、化妝品衛生安全管理法之規範 35
    參、材料與方法 36
    一、實驗架構 36
    二、實驗材料 38
    (一) 原料 38
    (二) 實驗細胞株 38
    (三) 實驗菌株 38
    (四) 試藥 38
    (五) 培養基 40
    (六) 儀器設備 45
    三、實驗方法 47
    (一) 乳酸菌篩選 47
    (二) 乳酸菌之菌種鑑定 49
    (三) 益生菌特性試驗 50
    (四) 乳酸菌最適培養條件 51
    (五) 乳酸菌於絲瓜水最適培養條件探討 53
    (六) 絲瓜水醱酵液製品之製備 54
    (七) 安姆氏試驗(Ames test) 54
    (八) 細胞培養 59
    (九) 細胞一氧化氮生成量之檢測 (Nitric oxide assay) 60
    四、分析方法 61
    (一) 菌數分析(Total count) 61
    (二) 可滴定酸測定 (Titratable acidity) 61
    (三) pH測定 (pH value) 61
    (四) 耐胃酸試驗 (Acid tolerance) 62
    (五) 耐膽鹽試驗 (Bile salt tolerance) 62
    (六) 還原糖含量測定 (Reducing sugar) 62
    (七) 胜肽含量測定 (Peptides assay) 63
    (八) 多醣含量測定 (Polysaccharides assay) 63
    (九) DPPH清除自由基能力測定(DPPH free radical
    scavenging activity assay) 64
    (十) ABTS清除自由基能力測定(ABTS free radical
    scavenging activity assay) 65
    五、儲藏性試驗 66
    六、感官特性(Organoleptic characteristics) 66
    七、統計分析 66
    肆、結果討論 67
    一、絲瓜水中醱酵菌株之篩選分離 67
    二、乳酸菌菌株篩選 67
    (一) 由分離菌株中篩選出提升絲瓜水中胜肽及
    多醣量之菌株 73
    (二) 分離於A絲瓜水中提升胜肽及多醣產量
    之菌株 76
    (三) 分離於B絲瓜水中提升胜肽及多醣產量
    之菌株 76
    (四) 由分離菌株中篩選出具有高抗氧化能力之菌株 76
    三、篩選菌株之基本特性試驗與鑑定 80
    (一) 顯微鏡觀察 80
    (二) API 50 CHL初步鑑定 80
    (三) 16S rRNA序列鑑定 80
    四、乳酸菌菌株之益生菌特性探討 84
    (一) 耐胃酸試驗 84
    (二) 耐膽鹽試驗 86
    (三) 抑菌能力試驗 86
    五、乳酸菌之最適培養條件探討 90
    (一) L. plantarum-NPUST-AL22(Ho)之生長曲線探討 90
    (二) L. plantarum-NPUST-AL22(Ho)生長之最適碳源
    種類與濃度之探討 92
    (三) L. plantarum -NPUST-AL22(Ho)生長之最適氮源
    種類與濃度之探討 95
    (四) 不同pH值之培養基對L. plantarum-NPUST
    -AL22(Ho)生長之影響 98
    (五) 不同接種量對L. plantarum-NPUST-AL22(Ho)
    在培養基中生長之影響 98
    (六) 不同培養溫度對L. plantarum-NPUST-AL22(Ho)
    在培養基中生長之影響 101
    六、乳酸菌於不同比例絲瓜水中生長與代謝之探討 103
    (一) L. plantarum-NPUST-AL22(Ho)於不同比例
    絲瓜水中生長之影響 103
    (二) L. plantarum-NPUST-AL22(Ho)於不同比例
    絲瓜水中胜肽量、多醣量及還原糖量之探討 105
    (三) L. plantarum-NPUST-AL22(Ho)於不同比例
    絲瓜水中清除DPPH自由基能力之探討 109
    (四) L. plantarum-NPUST-AL22(Ho)於不同比例
    絲瓜水中清除ABTS自由基能力之探討 111
    七、乳酸菌於最適比例絲瓜水中生長與代謝之探討 113
    (一) L. plantarum-NPUST-AL22(Ho)於最適比例
    絲瓜水中之生長曲線 113
    (二) L. plantarum-NPUST-AL22(Ho)於最適比例
    絲瓜水中代謝產物之變化 115
    (三) 絲瓜水中添加碳氮源對L. plantarum-NPUST
    -AL22(Ho)於生長之影響 117
    (四) 絲瓜水中添加碳氮源對L. plantarum-NPUST
    -AL22(Ho)生長代謝之影響 119
    (五) L. plantarum-NPUST-AL22(Ho)於最適碳氮源
    絲瓜水培養基中之生長曲線 126
    (六) L. plantarum-NPUST-AL22(Ho)於添加絲瓜水
    培養基中最適碳氮源之代謝情形 128

    八、分析絲瓜水醱酵液製品總胜肽量、總多醣量
    、清除DPPH能力與清除ABTS能力 130
    九、絲瓜水醱酵液之安姆氏試驗 132
    (一) 試驗菌株基因型之確認 132
    (二) 絲瓜水醱酵液及其製品之毒性試驗 135
    (三) 絲瓜水醱酵液及其製品之致突變性試驗 138
    (四) 絲瓜水醱酵液之抗致突變能力 141
    十、細胞存活率試驗(MTT assay) 145
    (一) 絲瓜水醱酵液及絲瓜水醱酵液製品對小鼠
    巨噬細胞(RAW 264.7)生長之影響 145
    (二) 絲瓜水醱酵液及絲瓜水醱酵液製品對經LPS
    誘導之小鼠巨噬細胞(RAW 264.7)生長之影響 148
    十一、細胞型態變化 150
    (一) 絲瓜水醱酵液(fermented A-loofah juice, FAL)
    對RAW264.7及經LPS誘導後之細胞型態變化 150
    (二) 絲瓜水醱酵液製品(fermented A-loofah juice product
    , FALP)對RAW264.7之細胞型態變化 150
    十二、絲瓜水醱酵液及絲瓜水醱酵液製品對經LPS誘導
    小鼠巨噬細胞(RAW 264.7)一氧化氮生成量之測定
    (Nitiric oxide assay) 154
    十三、儲藏性試驗 156
    十四、感官特性探討 163
    伍、結論 165
    陸、未來展望 167
    柒、附錄 168
    捌、參考文獻 171
    玖、作者簡介 186

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