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研究生:龍明有
研究生(外文):ming-yeou Lung
論文名稱:樟芝發酵生產多醣體及其抗氧化特性之研究
論文名稱(外文):Exopolysaccharide Production and Antioxidant Property of Antrodia camphorata in Batch Fermentation
指導教授:徐敬衡徐敬衡引用關係
指導教授(外文):Chin-Hang Shu
學位類別:博士
校院名稱:國立中央大學
系所名稱:化學工程與材料工程研究所
學門:工程學門
學類:化學工程學類
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:163
中文關鍵詞:樟芝抗氧化多醣
外文關鍵詞:polysacchrideantioxidantAntrodia camphorata
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樟芝(Antrodia camphorata)又稱牛樟芝(niu-chang-chih),為一種特有之真菌,只寄生於腐朽中空的牛樟樹(Cinnamomum kanehirai Hayata)上,樟芝是非常重要的中國藥用真菌擔子菌(Basidiomycetes),可用於治療食物及藥物之中毒、腹痛、高血壓、肝癌等疾病。目前有潛力之化合物如固醇類、三怗類已被分離及鑑定出其成分,而且從樟芝子實體及液態培養之菌絲體萃取出之多醣體已證明具有抗B型肝炎病毒(anti-hepatitis B virus)之活性。雖樟芝具有相當潛力之藥學應用,但是很少資訊報導有闗於利用製程之觀點來生產具生物活性之化合物。這也許是因為樟芝寄生專一性(只寄生在牛樟樹)、自然產量很少及很少用人工栽培成功。因此為了解決此問題,樟芝液態培養生產生物活性物質如多醣體為很好之途徑。本研究是利用樟芝之液態發酵藉有機酸及pH之調控來提昇多醣之產量,同時檢測其生物活性。為了程序之設計與最適化,此研究也提出數學模式來模擬pH對樟芝多醣生產之效應。
結果顯示,在搖瓶培養,添加六種有機酸對細胞成長及多醣產量之影響,除丙酮酸(pyruvic acid)外,其餘酸都對細胞成長有抑制作用。在添加六種有機酸之條件下所生成之多醣具有較低之算術平均分子量(Mn),添加琥珀酸(succinic acid)能增加30% 之多醣產量,且在添加3.0g/L琥珀酸時可得最佳之產率。隨著琥珀酸添加量從0到5g/L則EPS之產率隨之上升。在氣舉式生物反應器(air-lift bioreactor)培養,在添加3.0g/L琥珀酸時可提升28%的EPS產率及可得較高之EPS分子量(Mn)大約為310kDa,此外,添加3.0g/L琥珀酸能抑制EPS之降解(degradation)而可得較高之分子量(Mn)。
在攪拌式發酵槽培養,培養條件pH對樟芝發酵液(filtrates)及菌絲體(mycelia)甲醇萃取液之抗氧化及清除自由基活性影響很大。發酵液及菌絲體甲醇萃取液的抗氧化活性是明顯和總多酚含量、多醣含量及蛋白質和多醣比值有關。樟芝發酵生產抗氧化成分之最佳條件是在pH 5.0,在此條件下,菌絲體甲醇萃取液有最大的總多酚含量及多醣含量各為152.2 mg/g 及 33.5%。和樟芝子實體比較,在培養條件pH 5.0之下,可得較高之抗氧化劑產量。從最初培養液萃取而得之甲醇萃取液含非常低的抗氧化活性可證明發酵液及菌絲體甲醇萃取液的抗氧化活性主要是來自發酵程序。因此,從研究成果證明利用液態發酵生產抗氧化劑可替代樟芝子實體。
在搖瓶及攪拌式發酵槽培養,不同pH培養條件對樟芝的成長、 EPS生合成及EPS之分子量影響很大。在控制pH的攪拌槽培養,最佳的細胞成長條件是在pH 4.0,細胞產率(cell yield) 是0.3g/g,但最適EPS生合成的pH為5.0,其產率為5.05mg/g.在較低的pH培養條件,可得相當高的分子量(Mn)及較低產率之EPS,然而,相當低的分子量(Mn)及較高產率之EPS是在較高pH培養條件得到。在搖瓶培養得到的EPS其分子量(Mn)是比在攪拌式發酵槽培養的還高。兩階 段pH操作證實可提升EPS產率為148mg/L及有高的分子量(Mn) 2.18 x 105 Da.
數學模式來模擬pH對樟芝生物質量、EPS生產及EPS分子量之效應。一個簡單數學模式被提出就是利用logistic、Luedeking-Piret及modified Luedeking-Piret 方程式分別來模擬細胞的成長、EPS生產及glucose的消耗。在這數學模式,和pH有關的參數是被求出。成長速率常數和EPS分子量是用exponential equation來模擬。最大生物質量(maximum biomass)、對EPS生成與成長無關參數(non-growth-associated constants for product formation)及EPS產率可用 Gaussian equation 來描述。modified Luedeking-Piret 的參數則用quadratic expression來模擬。本研究所提出的數學模式能夠精確地描述實驗數據。
Antrodia camphorata (Chinese name, niu-chang-chih or chang-chih) is an exclusive fungus parasitic on the inner cavity of the endemic species Cinnamomum kanehirai Hayata and an important traditional Chinese medicinal fungus (Basidiomycetes) for the treatment of human diseases such as food and drug intoxication, diarrhea, abdominal pain, hypertension, itchy skin and liver cancer. Some bioactive compounds of A. camphorata including sesquiterpene lactone, steroids and triterpenoids have been isolated and characterized. Recently, polysaccharides extracted from fruiting bodies and mycelial cultures of A. camphorata have been shown to have anti-hepatitis B virus activities. In spite of these potential pharmaceutical applications, relatively rare information regarding the process aspects of producing these bioactive compounds has been published. This might be partially due to host specificity, rarity in nature and little success in artificial cultivation. Therefore, the submerged culture might be the major route of production of valuable metabolites including exopolysaccharide. Our research is attempting to promote the production of exopolysaccharides by A. camphorata in a batch culture through organic acids and pH regulation. Also, the biological activities of exopolysaccharides were examined. In order to design and optimize both laboratory scale and industrial scale processes, mathematical models were also developed to describe the pH effect.
Our results could be concluded that five out of six organic acid supplemented cultures showed negative effects on cell growth except the pyruvic acid supplemented culture, and lower number average molecular weight (Mn) of EPS were obtained in all organic acid supplemented cultures in shaker flasks. EPS production was enhanced by 31% due to the supplement of succinic acid. An optimum product yield was achieved at 3.0 g dm-3 succinic acid; however, the specific production of EPS increased monotonically as succinic acid was supplemented from 0 to 5 g/L. Enhancement of EPS yield by 28 % and a higher Mn of EPS around 310 kDa due to the supplement of succinic acid were also demonstrated in an air-lift bioreactor. Besides, a novel fermentation process resistant to EPS degradation was proposed by the organic acid supplementation.
Culture pH significantly affected antioxidant and scavenging free radical activities of methanolic extracts from mycelia and filtrates. Antioxidant activities of methanolic extracts from mycelia (MEM) and filtrates (MEF) have been successfully correlated with total polyphenol content, polysaccharide content and protein/ polysaccharide ratios. The optimal culture pH for antioxidants production by Antrodia camphorata was 5.0, and the maximum total polyphenol and polysaccharide/protein ratio in MEM were 152.2 mg/g and 33.5%, respectively. Higher amounts of antioxidants were obtained in the submerged culture at pH 5.0 as compared with that of fruiting body. Besides, the maximum polysaccharide in MEF was 55.3 mg/g. The relatively low antioxidant ability of methanolic extracts from culture medium indicated that the antioxidant abilities of MEM and MEF were mainly derived from the fermentation process. Besides, an alternative approach to produce the antioxidants of A. camphorata by submerged culture was proposed.
The effects of culture pH ranging from pH 3.0 to 6.0 on cell growth, exopolysaccharide biosynthesis and molecular weight distribution of exopolysaccharides of A. camphorata were examined both in shake flask culture and in a stirred tank fermenter. In a controlled pH stirred tank fermentation, the optimum pH for cell growth was 4.0 with a cell yield at 0.3 g/g while that for exopolysaccharide formation was 5.0 with a product yield at 5.05 mg/g. A relatively high molecular weight exopolysaccharide with a lower yield was obtained at low pH values while a relatively low molecular weight exopolysaccharide with a high yield was obtained at higher pH values. The average molecular weight of the exopolysaccharide in the flask culture was higher than that in the stirred tank fermenter. A two stage pH process that maximized product formation was demonstrated with a high product yield of 148 mg/liter with the relatively high average molecular weight of 2.18 x 105.
Fermentation kinetics of pH effects on growth and polysaccharide production of Antrodia camphorata was studied in a pH-controlled batch system. A simple model was proposed by using the logistic equation for cell growth, the Luedeking-Piret equation for polysaccharide production and a modified Luedeking-Piret equation for glucose consumption. The pH dependence of the parameters in this model was evaluated. The growth rate constants and the average molecular weight of polysaccharides were modeled with an exponential equation. The maximum biomass concentration, non-growth-associated constants for product formation and product yields were modeled with a Gaussian equation. The parameters of the modified Luedeking-Piret equation were modeled with a quadratic expression. The model developed in this study accurately described the experimental data.
Abstract
(Chinese).......................................................................................................I
Abstract (English) ………………………………………………………… …IV
Contents……………………………………………………………..……….VII
List of tables……………………………………………………………………XII
List of figures………………………………………………………………….XIV
Nomenclature………………………………………………………………XVIII

Chapter 1 Introduction
1.1 Research motives………………………………………………………..1
1.2 Research aims……………………………………………………………3
Chapter 2 Literature review

2.1 Nomenclature of Antrodia camphorata ………………………………….5
2.2 Classification of Antrodia camphorata…………………………………..5
2.3 Growth environment and characteristic of Antrodia camphorata…………6
2.3.1 Growth environment…………………………………………….6
2.3.2 Morphology and characteristic……………….…………………..6
2.4 Chemical compounds in A. camphorata………………………………….10
2.4.1 Non-volatile taste components………………………………….10
2.4.2 Sesquiterpene lactone, steroids, and triterpenoids…………….11
2.4.3 Maleic and succinic acid derivatives……………………………14
2.4.4 Polysaccharide………………………………………………….17
2.5 Biological activities of A. camphorata………………………………….19
2.6 Antitumor and immunomodulating polysaccharide in medicinal
mushrooms………………………………………………………….23
2.6.1 The numbers of mushrooms with antitumor polysaccharides…...23
2.6.2 Biosynthesis of polysaccharide in mushrooms…………………23
2.6.3 Purification procedures for polysaccharides in medicinal
mushrooms……………………………………………..…25
2.6.3 Structural composition of antitumor polysaccharides
in mushrooms…………………..……………….…………….29
2.6.4 Correlation of structure and antitumor activities of
mushroom polysaccharides…………………………………….30
2.6.5 Mechanisms of antitumor and immunomodulating action by
polysaccharides in medicinal mushrooms…………………….32
2.7 Antioxidant properties of medicinal mushrooms……………………….36
2.8 Exopolysaccharide production of fungi in submerged culture………….41
2.8.1 Effects of culture medium compositions………………………41
2.8.1.1 Effect of carbon source on exopolysaccharide formation..41
2.8.1.2 Effect of nitrogen on exopolysaccharide production…..42
2.8.1.3 Organic acids effects……………………………………..44
2.8.2 Effects of environmental conditions…………………………….44
2.8.2.1 Effect of pH …………………………………………….44
2.8.2.2 Effects of dissolved oxygen (DO)………………………45
2.8.2.3 Effects of temperature…………………………………..46
2.8.2.4 Effect of aeration and agitation…………………………47
2.8.3 Effect of other factors………………………………………………….49
2.9 Kinetic model……………………………………………………………50
Chapter 3 Materials and methods
3.1 Microorganism……………………………………………………………54
3.2 Effects of organic acids supplement……………………………………..54
3.2.1 Culture conditions………………………………………………54
3.2.2 Analysis methods……………………………………………….56
3.3 Antioxidant properties…………………………………………………..56
3.3.1 Culture conditions of Antrodia camphorata…………………..57
3.3.2 Preparation of methanol extraction from filtrate (MEF)…………57
3.3.3 Preparation of methanol extraction from mycelia (MEM)…….59
3.3.4 Preparation of methanol extraction from cultural medium
(MECM)……………………………………………….……….59
3.3.5 Preparation of methanol extraction from fruiting body of
Antrodia camphorata (MEFA)………….………………………59
3.3.6 Antioxidant activity……………………..………………………59
3.3.7 Scavenging effect on 1, 1-diphenyl-picrylhydrazyl (DPPH)
radical……………………………………………………………60
3.3.8 Reducing power…………………………...…………………….61
3.3.9 Chelating effects on ferrous ions………………………………61
3.3.10 Scavenging effect on superoxide anion………………………61
3.3.11 Scavenging effect on hydroxyl radical ………..……………….62
3.3.12 Determination of antioxidant components……………………62
3.4 pH effects ……………………………………………………………….64
3.4.1 pH control and culture conditions …………………………….64
3.4.2 Analysis methods………………………………………………64
3.5 Kinetics and modeling………………………………….………………66

3.5.1 Synopsis and aim…………………………………….……………66

3.5.2 Mathematical model…………………………………………….66
Chapter 4 Results and discussion
4.1 Effects of organic acids supplement……………………………………..68
4.1.1 Effects of organic acid supplementation on cell growth and EPS
production in shaker flask cultures…………………………….68
4.1.2 Effects of different succinic acid concentrations on cell growth
and EPS production in shaker flask cultures…………………….69
4.1.3 Effects of succinic acid supplementation on cell growth and
EPS production in an air-lift bioreactor………………………….70
4.1.4 Effects of different organic acids on the molecular weight of
EPS in shaker flask cultures……………………………………72
4.1.5 Effect of succinic acid supplementation on molecular weight of
EPS in an air-lift bioreactor………………………………………75
4.1.6 Effect of reaction vessel type on molecular weight of EPS………77
4.2 Effect of culture pH on the antioxidant properties
4.2.1 Effect of culture pH on cell growth and methanol extraction yield………………………………………………………………78
4.2.2 Antioxidant activity……………………………………………..79
4.2.3 Scavenging effect on 1, 1-diphenyl-picrylhydrazyl (DPPH) radical…………………………………………………………...80
4.2.4 Reducing power…………………………………………………81
4.2.5 Chelating effect on ferrous ions…………………………………84
4.2.6 Scavenging effect on hydroxyl free radicals……………………..84
4.2.7 Scavenging effect on superoxide anion……………………………87
4.2.8 Antioxidant components………………………………………….89
4.3 Effect of pH on the production and molecular weight distribution
of exopolysaccharide………………………………………………………92
4.3.1 Fermentation kinetics ……………….……………………………..92
4.3.2 Effect of pH control on fermentation kinetics ……………… ……92
4.3.3 Effect of initial pH on exopolysaccharide formation by
flask experiments ………………………………………………….95
4.3.4 Effect of pH on the cell growth of pH controlled fermentation……95
4.3.5 Effect of pH on exopolysaccharide formation of pH controlled
fermentation ………………………………………………………..97
4.3.6 Effect of pH on the molecular weight of exopolysaccharide……….97
4.3.7 Two-stage batch fermentation process for optimal exopolysaccharide
production …………………………………………….………….102
4.4 Kinetics and modeling of pH effects on polysaccharide production……105
4.4.1 Parameter estimation…………………………………………….105
4.4.2 Effects of pH………………………………………………………110
4.4.3 Modeling pH effect………………………………………………..112
4.4.4 Model Simulation and Validation………………………………….116
Chapter 5 Conclusion……………………………………..............................122
References………………………………………………………………………126
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