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研究生:何育澤
研究生(外文):Yu-Tse Ho
論文名稱:豆類與穀類基質應用於樟芝與銀耳共培養液態發酵與其生物活性探討
論文名稱(外文):Submerged Fermentation and Biological Activity of Tremella fuciformis and Antrodia cinnamomea Co-culture in Different Substrates of Beansand Grains
指導教授:徐泰浩徐泰浩引用關係林芳儀林芳儀引用關係
指導教授(外文):Tai-Hao HsuFang-Yi Lin
口試委員:柳源德陳南吟
口試日期:2012-07-12
學位類別:碩士
校院名稱:大葉大學
系所名稱:生物產業科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:94
中文關鍵詞:Tremella fuciformisAntrodia cinnamomea共培養生物活性
外文關鍵詞:Tremella fuciformisAntrodia cinnamomeaco-culturebiological activity
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銀耳(Tremella fuciformis Berk),俗稱白木耳,銀耳多醣具多種生物活性如抗過敏及美白等。樟芝(Antrodia cinnamomea),俗稱牛樟菇,樟芝三萜類化合物亦具多種生物活性如抗腫瘤、抗氧化等。但依目前文獻未曾有樟芝與銀耳共培養之探討,因此本研究利用不同農產品探討銀耳與樟芝進行共培養後對生物活性成分之影響。本實驗先以搖瓶試驗探討紅豆、綠豆、麥片及薏仁萃取液為培養基比較單一培養與共培養對生物活性之影響,進一步以不同的溫度、震盪轉速及進氣量探討其生物活性產物之差異,最後以最適培養條件下所得之代謝產物行抗氧化試驗及對HeLa之抗腫瘤試驗。結果顯示搖瓶試驗中以綠豆萃取液能產出較高生質量及胞內多醣,分別為4.35mg/mL、1.38mg/mL且較單一培養時產量較為佳。在於不同溫度及轉速中分別以25℃、180rpm能產出較高生質量、胞內多醣及粗三萜。進氣量方面則以2.0vvm時能產出較高生質量及胞內多醣,分別為9.49mg/mL、1.8mg/mL但粗三萜產量無顯著差異。
抗氧化試驗中亞鐵螯合能力、DPPH清除能力、還原能力皆依濃度增加而有增加趨勢,菌絲體甲醇取物當10mg/mL時亞鐵螯合能力為66%,10mg/mL時DPPH清除能力為40%;10mg/mL時還原力吸光值達1.51。HeLa子宮頸癌細胞之MTT試驗中菌絲體熱水萃取物與胞外多醣在24小時對HeLa細胞無顯著影響,但經48小時及72小時後細胞相對存活率則有下降之趨勢。結果顯示菌絲體熱水萃取物當濃度為100μg/mL~500μg/mL依濃度增加對細胞相對存活率具有下降之趨勢,而高濃度時細胞相對存活率則與濃度不成正比關係。胞外多醣方面當濃度為100μg/mL時對細胞相對存活率具有下降之現象,而濃度為300μg/mL~700μg/mL依濃度增加而有增值之現象,而當濃度為900μg/mL、1000μg/mL細胞相對存活率則具下降之趨勢。研究結果顯示Tremella fuciformis Berk與Antrodia cinnamomea共培養後對於多醣、三萜具有增加且具抗氧化能力及對HeLa細胞具有抑制現象,因此在未來研究可特別針對免疫活性進行探討,以提升市面上保健食品之功能性。

Tremella fuciformis Berk, more commonly known as white fungus contains poLysaccharides with a variety of biological activities such as: anti-allergy, anti-tumor, and skin whitening. Antrodia cinnamomea, which is more commonly known as niu-chang-chih or chang-chih, also possesses a variety of biological activities, such as anti-tumor and anti-oxidation properties. According to the Literature, however, there has not been a study with Antrodia cinnamomea and Tremella fuciformis co-cultured. In this study, different Beansand Grains were used to explore the effects of T. fuciformis and A. cinnamomea cultured on the bioactive component. In this study, the first shake flask test of red beans, green beans, oatmeal and job’s tears were employed as extract mediums both separately and co-cultured and biological activity was measured. Other variables that were explored were different temperatures, shock speed and feed rate difference. Finally, the optimal culture conditions were determined from the metabolites using an antioxidant test and from a HeLa anti-tumor test. The results show that to achieve higher biomass and intracellular polysaccharide production in the shake flask experiment the green bean extract provided the best yield among the single cultures (4.35mg/mL, 1.38mg/mL). The optimal temperatures and shock speed were 25℃ and 180rpm respectively; they were found to produce a high biomass and greater intracellular polysaccharide and crude triterpenoid production. In terms of feed rate difference a higher biomass and intracellular polysaccharide production was found at 2.0vvm (9.49mg/mL, 1.8mg/mL, respectively) but in crude triterpenoids production there was no significant difference.
Anti-oxidation was measured by determining the ferrous ion cheLating ability, the DPPH scavenging capacity and the reducing capacity concentration increase. The ferrous ion chelating ability of the methanol extract of mycelium when 10mg/mL was 66%. The DPPH scavenging ability was 40% of the concentration when 10mg/mL, and the reducing ability was 1.51 of the concentration when 10mg/mL. An MTT assay of HeLa cervical cancer cell in a hot water extract of mycelium and extracellular polysaccharides in a 24 hour period found no significant effects on HeLa cells. The reLative survival rates of cells after 48 hours and 72 hours decreased gradually, however. The results showed that when the concentration of hot water in the extract of mycelium was increased from 100μg/mL to 500μg/mL, a downward trend in cells’ relative survival was found. Exopolysaccharides were also examined, and it was found when the concentration was 100μg/mL the cells’ relative survival rate would decline, but among concentrations of 300μg/mL to 700μg/mL the cells’ survival rate would increase. Concentrations of 900μg/mL to 1000μg/mL aLso displayed a decreased cellular relative survival rate. The results show that in the co-cuLtured Antrodia cinnamomea, polysaccharides and triterpenoids increase, an antioxidant capacity was found as well as the phenomenon of inhibition of HeLa cells. In the future, further research is warranted to study its specific immune activity in order to enhance its use in health food products.

封面內頁
簽名頁
中文摘要 iii
英文摘要 v
誌謝 vii
目錄 viii
表目錄 xiv

1.前言 1
2.文獻回顧 2
2.1銀耳生物學特性 2
2.2銀耳生物活性成份 2
2.2.1銀耳多醣 2
2.3樟芝簡介 3
2.3.1樟芝之特徵與形態 4
2.3.2樟芝活性成份 5
2.4豆類與穀類簡介 6
2.4.1綠豆簡介 6
2.4.2紅豆之簡介 7
2.4.3麥片之簡介 7
2.4.4薏仁之簡介 8
2.5共培養 9
2.6影響真菌代謝之因素 10
2.6.1碳源 10
2.6.2氮源 11
2.6.3培養溫度 11
2.6.4攪拌與通氣 12
2.6.5無機鹽類 13
2.6.6接種量 13
3.1實驗架構流程圖 14
3.2實驗菌株 15
3.2.1真菌菌株 15
3.2.2細胞株 15
3.3實驗藥品 15
3.3.1農產品: 16
3.4儀器設備 17
3.5實驗材料 18
3.5.1銀耳母株培養 18
3.5.2樟芝母株培養 18
3.5.3菌種保存 19
3.5.4農產品培養基配製 19
3.6實驗方法 19
3.6.1液態共生培養接菌量試驗 19
3.6.2搖瓶試驗 20
3.6.3五公升發酵槽 20
3.7分析方法 21
3.7.1菌絲體生物質量測定 21
3.7.2胞內多醣配製與測定 21
3.7.3胞外多醣配製與測定 22
3.7.4總醣測定 22
3.7.5粗三萜測定 23
3.7.6抗氧化活性分析 24
3.7.7細胞實驗 26
4.1搖瓶培養試驗 29
4.1.1液態培養樟芝菌絲體之形態變化 29
4.2液態培養銀耳菌絲體之形態變化 35
4.2.1以豆類與穀類萃取液培養銀耳探討生物活性成份之影響 38
4.3液態共培養之菌絲體形態變化 41
4.4以最適豆類與穀類比較共培養與單一培養生物活性成份之響 44
4.5接菌量在共培養對生物質量及胞內外多醣之影響 48
4.6震盪培養轉速對生物活性成份之影響 50
4.7共培養在不同溫度下對生物活性成份之影響 55
4.8五公升發酵槽共培養試驗 60
4.8.1五公升發酵槽液態共培養 60
4.8.2以五公升發酵槽探討不同通氣量對生物性成份之影 響 62
4.9抗氧化試驗 64
4.9.1亞鐵螯合能力試驗 64
4.9.2清除DPPH能力試驗 67
4.9.3還原力試驗 70
4.10細胞毒性試驗 73
5.結論 80
參考文獻 82

圖4.1a-d複式顯微鏡觀察(1000X)Antrodia cinnmoamea菌絲生長之形態變化 30
圖4.2 a-d MIC-D觀察(22X)Antrodia cinnamomea菌絲球生長之形態變化 31
圖4.3以豆類與穀類培養Antrodia cinnamomea對生產生質量及生物活性成份之影響 34
圖4.4 a-d為複式顯微鏡觀察(1000X) Tremella fuciformis菌絲生長之形態變化 36
圖4.5 a-d以複式顯微鏡(1000X)及MIC-D(22X)觀察Tremella fuciformis菌絲生長之形態變化 37
圖4.6以豆類與穀類培養Tremella fuciformis生產生質量、生物活性成份之影響 40
圖4.7以複式顯微鏡(1000X)共培養第7天菌絲形態,白色鍵號為樟芝、黑色鍵號為銀耳 42
圖4.8 a-b為MIC-D(22X)觀察共培養菌絲球生長之形態變化 43
圖4.9以綠豆萃取液為培養基探討共培養與單一菌株培養對生物活性成份之影響 46
圖4.10麥片萃取液為培養基探討共培養與單一菌株培養對生物活性成份之影響 47
圖4.11接菌量對共培養後對生物活性成份之影響 49
圖4.12以麥片萃取液為培養基經共培養後比較不同溫度對生物活性成份之影響 57
圖4.13以綠豆萃取液為培養基經共培養後比較不同溫度對生物活性成份之影響 58
圖4.14以麥片加綠豆萃取液為培養基經共培養後比較不同溫度對生物活性成份之影響 59
圖4.15 a-d Tremella fuciformis、Antrodia cinnamomea共培養之五公升發酵槽生長形態變化 61
圖4.16五公升攪拌式發酵槽經共培養後不同進氣量對生物活性成份之影響 63
圖4.17單一培養及共培養菌絲體甲醇萃取物比較其亞鐵螯合力 66
圖4.18單一培養及共培養菌絲體甲醇萃取物比較其DPPH清除能力 69
圖4.19單一培養及共培養菌絲體甲醇萃取物比較其還原能力 72
圖4.20比較單一與共培養菌絲體熱水萃取液於1mg/mL與HeLa細胞共培養24、48、72小時對細胞存活率之影 響 76
圖4.21比較單一與共培養胞外多醣於1mg/mL與HeLa細胞共培養24、48、72小時對細胞存活率之影響 77
圖4.22比較單一與共培菌絲體熱水萃取液不同濃度下與HeLa細胞共培養72小時對細胞存活率之影響 78
圖4.23比較單一與共培胞外多醣不同濃度下與HeLa細胞共培養72小時對細胞存活率之影響 79

表4.1以綠豆萃取液為培養基經共培養後比較不同震盪轉速對生物活性成份之影響 52
表4.2以麥片萃取液為培養基經共培養後比較不同震盪轉速對生物活性成份之影響 53
表4.3以麥片加綠豆混合萃取液為培養基經共培養後比較不同震盪轉速生物活性成份之 54

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