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研究生: 邱靖瑜
Chiu, Ching-Yu
論文名稱: 兔腸道益生菌的篩選與活性分析
Screening and Activity Analysis of Probiotics in Rabbit Intestine
指導教授: 徐志宏
Shyu, Douglas J. H.
學位類別: 碩士
Master
系所名稱: 農學院 - 生物科技系
Department of Biological Science and Technology
畢業學年度: 109
語文別: 中文
論文頁數: 72
中文關鍵詞: 益生菌乳酸菌活性分析
外文關鍵詞: Probiotics, Lactic acid bacteria, Activity analysis
DOI URL: http://doi.org/10.6346/NPUST202100447
相關次數: 點閱:46下載:0
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  • 市售發酵製品等相關的益生菌產品常見添加菌種有嗜酸乳桿菌 (Lactobacillus acidophilus)、乾酪乳桿菌 (Lactobacillus casei)、副乾酪乳桿菌 (Lactobacillus paracasei)、保加利亞乳酸桿菌 (Lactobacillus bulgaricus)、比菲德氏菌 (Bifidobacterium spp.)、嗜熱鏈球菌(Streptococcus thermophilus)、植物乳桿菌 (Lactiplantibacillus plantarum) 等。益生菌對宿主來體內的免疫系統來說是外來物質,所以能否適應速主體內腸胃道環境對益生菌來說是一大考驗,若對胃酸及膽鹽的耐受性極低那益生菌也沒辦法順利到達後腸被吸收,也達不到促進及改善健康的效果,因此益生菌篩選條件主要挑選能適應腸胃道環境、存活率佳的菌株,再針對其他益生菌篩選標準去做試驗分析。本實驗選出7株已鑑定完畢的菌株,其中兩株作為對照組比對用,模擬腸胃道環境做了耐酸性、耐膽鹽的存活率試驗,還有測定生長曲線、溶血性試驗、真空冷凍乾燥存活率、活性測試定性及定量 (CMC分解酶、Xylan分解酶、Pectin分解酶、Starch分解酶、Protein分解酶)、吸附性試驗 (疏水性、自動聚合) 等,來綜合評估各個菌株的可開發性、應用方面及後續開發潛能。結果為,LP5、LP19、PTA22、LP1、LP2有些微α-溶血,PAL44、SL45並無觀察到溶血;冷凍乾燥存活率為PAL44最佳;耐酸性試驗LP5、LP19、PTA22均表現出良好耐酸性;耐膽鹽試驗PAL44、SL45表現良好耐膽鹽性;活性分析試驗在CMCase、Xylanase、Pectinase、Protease中LP5、LP19、PTA22、LP1、LP2均有表現出活性,在Amylase中PAL44、SL45有表現出活性;在吸附性試驗的疏水性試驗中,PAL44表現出最高疏水性;在自動聚合試驗中PAL44、SL45表現較佳,且7株菌的自動聚合率都有隨著時間增加。實驗過後決定將PTA22列入開發菌株;PAL44、SL45活性分析不佳,但有文獻指出可朝海洋生物益生菌及合成金銀奈米粒子的方面去做研究分析。

    Commercially available fermented products and other related probiotic products commonly added bacteria include Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus bulgaricus, Bifidobacterium spp., Streptococcus thermophilus, Lactiplantibacillus plantarum, etc. Probiotics are foreign substances to the host’s immune system, so whether they can adapt to the gastrointestinal environment of the main body is a big test for probiotics. If the tolerance to gastric acid and bile salts is extremely low, then probiotics There is no way to smoothly reach the hindgut to be absorbed, and the effect of promoting and improving health cannot be achieved. Therefore, the probiotic screening conditions are mainly to select strains that can adapt to the gastrointestinal environment and have good survival rates, and then test against other probiotic screening standards. analyze. This experiment selected 7 strains that have been identified, two of which were used as a control group to simulate the gastrointestinal tract environment for acid resistance and Bile salt survival test, as well as growth curve determination, hemolysis test, and vacuum freezing. Dry survival rate, activity test qualitative and quantitative (CMCase activity test, xylanase activity test, pectinase activity test, amylase activity test, protease activity test), adsorption test (hydrophobicity test、auto-aggregation), etc., to comprehensively evaluate the viability of each strain development, application and subsequent development potential. The results are that LP5, LP19, PTA22, LP1, LP2 have some slight α-hemolysis, and PAL44 and SL45 have no hemolysis; freeze-drying survival rate is the best for PAL44; acid resistance test LP5, LP19, PTA22 all show good acid resistance ; bile salt tolerance test PAL44, SL45 showed good bile salt tolerance; activity analysis test showed activity in CMCase, xylanase, pectinase, protease, LP5, LP19, PTA22, LP1, LP2, and PAL44, SL45 in amylase In the hydrophobicity test of the adsorption test, PAL44 showed the highest hydrophobicity; in the automatic polymerization test, PAL44 and SL45 performed better, and the automatic polymerization rate of the 7 strains increased with time. After the
    experiment, it was decided to include PTA22 as the developed strain; PAL44 and SL45 activity analysis was not good, but someliterature pointed out that research and analysis could be done in terms of marine probiotics and synthetic gold and silver nanoparticles.

    目錄

    摘要 I
    Abstract II
    謝誌 IV
    目錄 V
    圖目錄 VIII
    表目錄 IX
    第一章 緒論 10
    1.1 前言 10
    1.2 研究動機 12
    第二章 文獻回顧 14
    2.1 血培養基 (Blood Agar Plate) 14
    2.2 溶血性 (Hemolysis) 14
    2.2.1 α-溶血 (α-Hemolysis) 15
    2.2.2 β-溶血 (β-Hemolysis) 15
    2.2.3 γ-溶血 (γ-Hemolysis) 15
    2.3 纖維素 (Cellulose) 15
    2.4 羧甲基纖維素 (Carboxymethylcellulose) 16
    2.5 纖維素分解酶 (Cellulase) 16
    2.6 木聚醣 (Xylan) 17
    2.7 木聚醣酶 (Xylanase) 17
    2.8 果膠 (Pectin) 17
    2.9 果膠分解酶 (Pectinase) 18
    2.10 澱粉 (Starch) 18
    2.10.1直鏈澱粉 (Amylose) 19
    2.10.2 支鏈澱粉 (Amylopectin) 19
    2.11 澱粉酶 (Amylase) 20
    2.11.1 α-澱粉酶 (α-Amylase) 20
    2.11.2 β-澱粉酶 (β-Amylase) 20
    2.12 植物乳桿菌Lactiplantibacillus plantarum 20
    2.13 副球菌屬Paracoccus sp. 21
    2.14 緩慢葡萄球菌Staphylococcus lentus 21
    2.15 吸附性 (Adsorption) 21
    2.16 哺乳動物 (Mammal) 22
    2.17 前腸發酵動物 (Foregut fermenting Herbivore) 22
    2.18 後腸發酵動物 (Hindgut fermenting Herbivore) 23
    2.19 真空冷凍乾燥技術 (Vacuum freeze-drying) 23
    2.20 保護劑 (Cryoprotectants) 24
    第三章 材料與方法 25
    3.1實驗架構 25
    3.2材料來源 26
    3.2.1實驗菌株 26
    3.2.2儀器設備 26
    3.2.3實驗材料 26
    3.3實驗方法 29
    3.3.1 樣品處理 29
    3.3.2益生菌之固態及液態培養 29
    3.3.3生長曲線 (Bacterial growth curve) 29
    3.3.4溶血性測試 (Hemolytic test) 29
    3.3.5真空冷凍乾燥試驗 (Vacuum freeze-drying) 29
    3.3.6耐酸性試驗 (Acid tolerance) 30
    3.3.7耐膽鹽試驗 (Bile salt tolerance) 30
    3.3.8活性分析 (Activity analysis test) 30
    3.3.9吸附性試驗 (Adsorption) 33
    3.3.9.1疏水性試驗 (Hydrophobicity test) 33
    3.3.9.2自動聚合試驗 (Auto-aggregation) 34
    第四章 結果與討論 35
    4.1生長曲線 (Bacterial growth curve) 35
    4.2溶血性測試 (Hemolytic test) 39
    4.3真空冷凍乾燥試驗 (Vacuum freeze-drying) 39
    4.4耐酸性試驗 (Acid tolerance) 39
    4.5耐膽鹽試驗 (Bile salt tolerance) 45
    4.6活性分析 (Activity analysis test) 47
    4.6.1纖維素分解酶活性試驗 (CMCase activity test) 47
    4.6.2木聚糖分解酶活性試驗 (Xylanase activity test) 49
    4.6.3果膠分解酶活性試驗 (Pectinase activity test) 51
    4.6.4澱粉分解酶活性試驗 (Amylase activity test) 53
    4.6.5蛋白質分解酶活性試驗 (Protease activity test) 55
    4.7吸附性試驗 (Adsorption test) 57
    4.7.1疏水性試驗 (Hydrophobicity test) 57
    4.7.2自動聚合試驗 (Auto-aggregation) 59
    4.8總表 61
    第五章 結論 62
    未來展望 63
    第六章 參考文獻 65

    圖 1、實驗架構圖 25
    圖 2、37℃震盪培養27小時之生長曲線 37
    圖 3、LP1、LP5、LP19、PTA22、PAL44、SL45、LP1、LP2溶血性觀察結果 41
    圖 4、7株菌在pH 1.0、1.5、2.0、2.5、3.0下37℃震盪培養0、1、2、3、4、5、6小時之結果圖 43
    圖 5、在0.5%、1.0% Bile Salt下37℃震盪培養1、2、3、4、5、6、7、8小時之生長曲線 46
    圖 6、CMCase定性及定量結果 48
    圖 7、Xylanase定性及定量結果 50
    圖 8、Pectinase定性及定量結果 52
    圖 9、Amylase定性結果 54
    圖 10、Protease定性及定量結果 56
    圖 11、在室溫靜置1小時之疏水性結果 58
    圖 12、在37℃下靜置1、3、6、24小時之自動聚合結果 60

    表目錄
    表 1、MRS培養基之組成成分 27
    表 2、CMC培養基之組成成分 27
    表 3、Xylan培養基之組成成分 28
    表 4、Pectin培養基之組成成分 28
    表 5、Starch培養基之組成成分 28
    表 6、Skim milk培養基之組成成分 28
    表 7、真空冷凍乾燥存活率之結果 42

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