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Title: 黃耆混合寡醣及其乳酸發酵產物之生理活性探討
Authors: Huang, Li-Yeh
黃立曄
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: 黃耆;北 (白) 耆;晉 (紅) 耆;黃耆多醣;黃耆寡醣;黃耆寡醣乳酸發酵;抑制雙醣酶活性;抗凝血活性;抗氧化;非洲猿猴腎細胞 (Vero) 抗腸病毒 71 型;小鼠巨噬細胞 (Raw 264.7) 抗發炎;人類結腸癌細胞 (Caco-2) 吸收運輸
Astragalus;Hedysarum;Astragalus polysaccharide;Astragalus oligosaccharide;Astragalus oligosaccharide lactic fermented product;Anticoagulant activity;Disaccharidase activity;Antioxidant activity;Vero cell anti-enterovrius 71;RAW 264.7 anti-inflammatory;Caco-2 cell, Absorption and transportation
Date: 2016
Issue Date: 2018-08-22T06:41:20Z
Abstract: 研究目的為測試白耆 (北耆) 混合寡醣 (Astragalus mixing oligosaccharides,APS-1035K)、紅耆(晉耆) 混合寡醣 (Hedysarum mixing oligosaccharides,HPS-1035K) 及其乳酸發酵產物於抑制雙乳醣酶、抗凝血、抗氧化、抗病毒、抗發炎、Caco-2腸道吸收與運輸變化之生理活性影響。2 株海洋細菌MA103 和 MAEF108 以及 3 株枯草細菌分離株ST-BO-108、ST-BO-109、和 ST-BO-224 經分別使用 3 % (w/v) 白耆與紅耆粉末誘導,測定 a-澱粉 (a-amylase)、葡萄糖苷內切酶 (endoglycosidase)、外切型纖維素酶 (excellulase)、聚甘露糖酶 (mannanase)、與聚木糖酶 (xylanase) 之酵素活性,實驗結果顯示澱粉酶酵素活性 MA103 為 0.47 U與1.58 U,MA108 為 0.06 U 與 1.34 U,ST-BO-108 為 0.48 U 與 0.49 U,ST-BO-109 為 1.00 U與0.45 U,ST-BO-224得之澱粉酶活性為 0.45 U 與 0.50 U。將上述 5 種白耆粉末誘導酵素液水解白耆熱萃液與 5 種紅耆粉末誘導酵素液水解紅耆熱萃液,結果顯示皆以被誘導 MA103 之酵素效果最佳,白耆水解液之總糖量與還原糖量增加到 10.30 mg/mL與3.03 mg/mL,紅耆水解液之總糖量與還原糖量增加到 11.57 mg/mL與3.82 mg/mL。將上述 2 種熱萃液與 10 種水解寡醣液經薄層色層分析 (thin layer chromatography, TLC) 試驗結果顯示所得白耆及紅耆水解寡醣產物皆含有 1-6 醣,並以 2 醣含量較多。將上述 12 種樣品以 HPLC 分析,結果得到白耆、紅耆熱萃液皆在27.62 min (DP 1.5, M. W. 260.85 Da) 出現相同波峰時間,白耆熱萃液經白耆粉末誘導 MA103 酵素水解 24 hr 結果為11.41 min (DP 1.78, M. W. 291.77 Da); 白耆寡醣水解液分別為25.28 min (DP 3.82, M. W. 686.88 Da)、27.65 min (DP 1.43, M. W. 257.63 Da),皆顯示水解後波峰明顯上升。紅耆熱萃液經紅耆粉末誘導 MA103 酵素水解 24 hr結果紅耆水解產物的波峰時間為 11.29 min (DP 2.17, M. W. 356.54 Da) 顯示波峰明顯上升; 紅耆寡醣水解液為 25.27 min (DP 3.83, M. W. 689.73 Da)、27.63 min (DP 1.44, M. W. 259.77 Da) 顯示水解後波峰略為上升。以 5 株乳酸菌發酵白耆多醣液 (APS)、紅耆多醣液 (HPS)、白耆寡醣液 (APS-1035K)、及紅耆寡醣液 (HPS-1035K) 發酵 30 hr 之結果顯示 BCRC14023、BCRC14068、BCRC14940、BCRC10695、與BCRC12327 等5株乳酸菌於發酵 15 - 25 hr 之 pH 值皆能降至 4.6 以下,且乳酸菌量皆能達到 107 CFU/mL,因 BCRC12327能以短時間 5 hr 降至pH值 4.6 以下,乳酸分泌較高,相較其他乳酸菌,表現活性較佳,故選擇 BCRC12327 做為後續混合寡醣乳酸發酵之實驗。抗凝血試驗中以APS、 HPS、 APS-1035K、和 HPS-1035K 測試實驗,結果顯示多醣效果最佳為 APS 在濃度 5 mg/mL 對APTT 延遲時間為 266 sec 並有明顯抑制凝結反應,APS 與 HPS 在濃度 5 mg/mL 對PT 延遲時間為 66 sec 與 48 sec 皆有明顯抑制凝結反應,而 APS 與 HPS 在濃度 5 mg/mL 對 TT 雖無延遲時間但有些微抑制凝結效果。寡糖效果最佳為 APS-1035K 在濃度 20 mg/mL 對APTT 延遲時間為 42 sec 雖 HPS-1035K 與控制組相比沒有延遲時間,兩者皆有明顯抑制凝結效果,三種雙醣酶抑制試驗以APS、HPS、APS-1035K、和HPS-1035K 測試在濃度 2 mg皆達到較佳的抑制力,抑制乳糖酶活性以 HPS 為 99.54%; 抑制麥芽糖酶以 APS-1035K 為 99.34%; 抑制蔗糖酶以 APS-1035K 為 76.48%。抗氧化活性試驗以 APS、HPS、APS-1035K、HPS-1035K、白耆寡醣乳酸發酵產物(APS-1035K-LFS)、紅耆寡醣乳酸發酵產物 (HPS-1035K-LFS) 測試實驗,結果顯示在濃度 10 mg/mL 結果為最佳,分別對 DPPH 自由基清除效應以 HPS-1035K最佳為 83.40%,相當等於171.95 ug/mL Trolox,還原力以 HPS 最佳為吸光光值 0.68 相當等於34.19 ug/mL Trolox,對亞鐵離子螯合能力以 HPS-1035K 最佳為84.97%。在抗腸病毒 71 型的試驗中以 APS、HPS、APS-1035K、HPS-1035K、APS-1035K-LFS、HPS-1035K-LFS 測試實驗,結果顯示經預防處理組 HPS-1035K濃度 0.2 ug/mL較佳可提升細胞存活率至 98%,治療處理組 APS-1035k-LFS濃度 2 ug/mL較佳,可提升細胞存活率至 93%,共同處理組 APS-1035k 濃度 0.2 ug/mL較佳,可提升細胞存活率至 92%。抗發炎試驗以濃度 200 g/mL達到較好效果,分別以 APS-1035K、HPS-1035K、APS-1035k-LFS、與HPS-1035k-LFS處理後皆有降低 NO 生成量之效果,分別可降至 41.35 M,41.62 uM,41.65 uM,41.41 uM。 在Caco-2 細胞吸收運輸試驗以APS、HPS、APS-1035K、HPS-1035K、APS-1035K-LFS、HPS-1035K-LFS 測試實驗,結果顯示運輸率最佳以APS-1035K-LFS 為48.82% 與 HPS-1035K-LFS 為46.25%; 細胞吸收率最佳以 APS 為64.83% 與 HPS 為 74.55%。
The aim of this study is to investigate biological activities of Astragalus mixing oligosaccharides (APS-1035K), Hedysarum mixing oligosaccharides (HPS-1035K), and that the lactic acid bacteria fermented oligosaccharides products as anticoagulant, disaccharidase, antioxidant, antivirus, and anti-inflammatory, as well as to on intestinal absorption and transport by intestinal model established with the human colon cancer cell line Caco-2. 2 strain Marine bacteria MA103 and MAEF108, and 3 strain Bacillus subtilis bacterial isolates ST-BO-108, ST-BO-109, ST-BO-224 and through the use individual 3 % (w/v) of Astragalus and Hedysarum powder induce , measured -amylase, within endoglycosidase, excellulase, mannanase, xylanase of enzyme activity, the amylase enzyme activity of MA103 could reach 0.47 U and 1.58 U, the amylase activity of MA108 were 0.06 U and 1.34 U, the amylase activity of ST-BO-108 were 0.48 U and 0.49 U, the activity of ST-BO-109 were 1.00 U and 0.45 U, the amylase activity of ST-BO-224 were 0.45 U and 0.50 U. Above 5 Astragalus powder were induced enzyme hydrolysis Astragalus extract heat solution and 5 Hedysarum powder was induced enzyme hydrolysis Hedysarum extract heat liquid. The results showed that the enzymes begin to be induced MA103 best, so Astragalus hydrolyzate raise total sugar and reducing sugar 10.30 mg/mL and 3.03 mg/mL, Hedysarum hydrolyzate raise total sugar and reducing sugar 11.57 mg/mL and 3.82 mg/mL. Above Astragalus and Hedysarum extract heat solutions and 10 hydrolyzed oligosaccharide solution by thin layer chromatography (TLC) of the results obtained show the Astragalus and Hedysarum oligosaccharide hydrolysates were havding 1-6 sugar, and high content of 2 sugar concentration. The aforementioned 12 samples were analyzed by HPLC, the result of Astragalus and Hedysarum heat extracts are at 27.62 min (DP 1.5, MW 260.85 Da) exhibit the same peak time, Astragalus heat extract slution by Astragalus powder induced MA103 enzyme hydrolysis 24 hr result of 11.41 min (DP 1.78, MW 291.77 Da); Astragalus oligosaccharide hydrolyzate were 25.28 min (DP 3.82, MW 686.88 Da), 27.65 min (DP 1.43, MW 257.63 Da), were displayed wave front increased significantly after hydrolysis. Hedysarum heat extract sloution by Hedysarum powder induced MA103 enzyme hydrolysis 24 hr results Hedysarum hydrolyzate wave front time of 11.29 min (DP 2.17, MW 356.54 Da) show the wave front increased significantly; Hedysarum oligosaccharide hydrolyzate were 25.27 min (DP 3.83, MW 689.73 Da), 27.63 min (DP 1.44, MW 259.77 Da) show a slight rise in the wave front after hydrolysis. The 5 lactic acid bacteria ferment of Astragalus polysaccharide (APS), Hedysarum polysaccharide (HPS), Astragalus was mixed oligosaccharides (APS-1035K), and Hedysarum mixed oligosaccharides (HPS-1035K) results 30 hours of fermentation liquid display BCRC14023, BCRC14068, BCRC14940, BCRC10695, and BCRC12327 and other five lactic acid bacteria in the fermentation 15 - pH value of Jieneng 25 hr to 4.6 or less, and the amount of lactic acid bacteria able to meet the 107 CFU/mL, can in a short time because BCRC12327 5 hrs lowered pH value below 4.6 and higher lactic acid secretion, compared to other lactic acid, better performance of the activity, so choose BCRC12327 as follow-up experiment mixing lactic acid fermentation of oligosaccharides. Anticoagulation tests to APS, HPS, APS-1035K, and HPS-1035K test experiments, the results showed the best results for the APS polysaccharide concentration 5 mg/mL of APTT delay time of 266 sec and a inhibit condensation reaction, APS and HPS concentration 5 mg/mL of PT delay time of 66 sec and 48 sec inhibit condensation reaction, and HPS and APS in concentration 5 mg/mL of TT Although there is no delay, but slightly the effect of inhibiting coagulation. Oligosaccharides best for the APS-1035K at a concentration 20 mg/mL of APTT delay time of 42 sec, although HPS-1035K compared to a control group with no delay time, a combination of both inhibit coagulation effect three disaccharide enzyme inhibition test polysaccharide APS, HPS, APS-1035K, and HPS-1035K test concentration of 2 mg in both achieve better inhibition, inhibition of lactase activity in HPS were 99.54%; maltose enzyme inhibition were to APS-1035K 99.34%; sucrose enzyme inhibition with APS-1035K were 76.48%. Antioxidant activity test with APS, HPS, APS-1035K, HPS-1035K, Astragalus oligosaccharides lactic acid fermentation product (APS-1035K-LFS),and Hedysarum oligosaccharides lactic acid fermentation product (HPS-1035K-LFS) experimental test results are shown in concentration of 10 mg/mL for the best results, respectively DPPH radical scavenging effect in HPS-1035K best of 83.40 percent, quite equal to 171.95 ug/mL Trolox, reducing power to the best of absorptiometry HPS quite equal to the value 34.19 0.68 ug/mL trolox, for ferrous ion chelating ability to HPS-1035K best of 84.97 percent. Anti-EV71 trial APS, HPS, APS-1035K, HPS-1035K, APS-1035K-LFS,and HPS-1035K-LFS test experimental results show that after prophylactic treatment group HPS-1035K concentration of 0.2 ug/mL preferred can improve cell viability to 98%, the treatment group treated APS-1035k-LFS concentration of 2 ug/mL preferred, can improve cell viability to 93%, common treatments APS-1035k concentration of 0.2 ug/mL preferred, can improve cell viability to 92%. Anti-inflammatory tests at a concentration of 200 ug/mL to achieve better results, respectively, APS-1035K, HPS-1035K, APS-1035k-LFS, after HPS-1035k-LFS Jie dealing with the effect of reducing the amount of generation of NO, respectively, can be fell 41.35 uM, 41.62 uM, 41.65 uM, 41.41 uM. In Caco-2 cells to absorb transportation test with APS, HPS, APS-1035K, HPS-1035K, APS-1035K-LFS,and HPS-1035K-LFS testing laboratory the results showed that the best transport rates with APS-1035K-LFS were 48.82% and HPS-1035K-LFS were 46.25%; the rate of cellular uptake of the best in APS were 64.83% and HPS were 74.55%.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0040342009.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/49034
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