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研究生:鄭萬禎
研究生(外文):Cheng Wan Jen
論文名稱:利用批式發酵生產巴西洋菇菌絲體及胞外多醣之研究
論文名稱(外文):Study of mycelium and exopolysaccharide formation by Agaricus blazei Murill in batch fermentation
指導教授:方繼方繼引用關係
指導教授(外文):Tony J. Fang Ph. D.
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:103
中文關鍵詞:Agaricus blazei Murrillfunctional foodsumberged fermentationexopolysaccharideebiomass
外文關鍵詞:巴西洋菇機能性食品深層發酵胞外多醣生質
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巴西洋菇(Agaricus blazei Murrill),原產於巴西,即目前在日本等地為人所熟知的 ”姬松茸”,目前廣泛的栽培於日本及中國大陸等地。早在過去,巴西洋菇就因其藥用特性在世界各地被當成食品或機能性食品來使用。
在菇類的深層培養中,有許多種類的胞外多醣被認為具有生理活性。利用深層發酵技術培養巴西洋菇菌絲體可以比栽培其子實體更快速獲得其菌體,還能額外獲得其胞外產物。根據前人的研究,食藥用真菌在深層發酵時,其菌體生長及胞外多醣生成會受到發酵環境因子的影響。因此本研究的目的在於評估發酵條件對巴西洋菇於深層培養時菌體生長及胞外多醣生成造成的影響。
研究結果顯示,利於巴西洋菇菌體量之生成並具顯著差異(p<0.05)的發酵條件因子有:起始pH值在5~6.5之間、載液量為70 ml或90 ml(於250 ml有溝錐形瓶中)、較佳之碳源種類為果糖優於葡萄糖及蔗糖、較佳之氮源種類為peptone、yeast extract及casein。另外將碳氮比調整為15時,可以得到最高之菌量。
而可以獲得高胞外多醣量並具顯著差異(p<0.05)的發酵條件有:起始pH值6、載液量為50 ml(於250 ml有溝錐形瓶中)、最佳的碳源、氮源分別為麥芽糖及酵母萃取物,而碳氮比40最適合胞外多醣生成。
在巴西洋菇的搖瓶培養,最初培養15天僅能獲得1g/L的菌體量,當進行到碳氮源之探討時,發現在8天的培養後可以在碳氮比為15時得到15.2 g/L之最高菌量,相當於最初菌量之15倍。
當將搖瓶試驗探討時對菌體生長的有利因子結合,利用5公升桌上型攪拌式發酵槽進行驗證的小規模放大培養時,發現只要4天即可得到10 g/L的菌體量。
The mushroom Basidiomyceta Agaricus blazei Murrill, a native mushroom in Brazil, is popularly known in Japan as “Himematsutake” or “Kawariharatake”. It has been widely cultivated and studied in Japan. At present, A. blazei is often consumed as food or functional food in different parts of the world, especially because of its imputed medicinal properties.
Several kinds of exo-polysaccharides, produced from submerged culture of mushroom, have been recognized as active ingredients. Submerged fermentation technique was useful to obtain the biomass and products fast than cultivated the fruit body of the A. blazei. According to the present paper, the growth and production of the biomass and exopolysaccharide by edible and medicinal fungi are influenced by the fermentation environmental parameters. The objective of this work was to evaluate the effects of fermentation factors on the submerged fermentation of A. blazei.
Our study showed the factors that can yielded significant higher biomass formation(ρ<0.05) were as follows:initial pH 5~6.5, cultivation volume of 70 ml or 90 ml(in 250 ml flask with baffle). Carbon sources that can supported high biomass were in the following order: fructose > glucose > sucrose. Peptone was found to support the highest biomass formation, followed by casein and yeast extract. When the C/N ratio of the medium was adjusted to 15, the highest biomass was obtained.
The highest exopolysaccharide production was obtained in the following cultivation conditions: initial pH 6.0, cultivation volume of 50 ml (in 250 ml flask with baffle), with maltose and yeast extract as the carbon and nitrogen source, respectively. And the best C/N ratio of medium for condition of exopolysaccharide production ratio of medium was 40.
In shake flask culture of A. blazei Murrill, 1 g/L of biomass was observed after 15 days of incubation. However, 15.2 g/L of biomass, a 15-fold increase, was obtained in C/N ratio of medium was 15 after 8 days submerged culture.
When A. blazei was cultivated in a bench top fermentor with 200 r.p.m. agitation and 2 v.v.m. aeration rate, 10 g/L of biomass formation was obtained after 4 days of grow
壹、前言…………………………………………………………….…………1
貳、文獻整理……………………………………………………….…………2
一、巴西洋菇的簡介……………………………………………………..2
(一)、巴西洋菇的發現與由來………………………………………2
(二)、巴西洋菇的分類與型態特徵…………………………………2
(三)、巴西洋菇的機能性……………………………………………3
(四)、巴西洋菇多醣的介紹………………………………………....6
(五)、巴西洋菇多醣體之相關研究………………………………..12
二、深層發酵食藥用真菌菌絲體及多醣體生產之探討………………14
(一)、接種量與種菌型態………………………………….………17
(二)、碳源種類及濃度…………………………………….………18
(三)、氮源種類及濃度…………………………………….……...20
(四)、碳氮比……………………………………………………….22
(五)、溫度………………………………………………………….22
(六)、起始pH值及pH值控制……………………………………...24
(七)、攪拌與通氣…………………………………………………25
(八)、無機鹽類…………………………………………….……...32
參、材料與方法……………………………………………………………...34
一、實驗材料……………………………………………………………34
(一)、實驗菌株……………………………………………………..34
(二)、基礎培養基…………………………………………………..34
(三)、修飾培養基…………………………………………………..34
(四)、藥品清單……………………………………………………..35
(五)、儀器清單……………………………………………………..36
(六)、電腦套裝軟體………………………………………………..37
二、實驗設計大綱………………………………………………………38
三、實驗方法……………………………………………………………39
(一)、菌種保存……………………………………………………..39
(二)、菌種活化……………………………………………………..39
(三)、繼代培養……………………………………………………..39
(四)、種菌培養……………………………………………………..40
(五)、種菌懸浮液的製作…………………………………………..40
(六)、實驗室小量栽培試驗………………………………………..40
(1).母種培養基………………………………………………40
(2).原種培養基……………………………………….……...42
(3).栽培種培養基……………………………………………42
(4).接菌與栽培………………………………………………42
(七)、批式培養方法與條件………………………………………..43
(1).種菌試驗培養條件……………………………….……...43
(2).搖瓶試驗基礎培養條件…………………………………43
(3).5L桌上型攪拌式發酵槽試驗培養條件………………...44
四、分析方法……………………………………………………………45
(一)、分析方法流程圖…………………………………….………45
(二)、菌體乾重(Dry cell weight)分析…………………….………46
(三)、pH值的測定…………………………………………………46
(四)、胞外多糖(Exopolysaccharide)的萃取與分析………………46
(五)、殘糖分析…………………………………………….………48
(1).DNS法…………………………………………………...48
(2).HPLC殘糖分析法……………………………….………49
(六)、菌體收率(Y x/s)之計算…………………………………….50
肆、結果與討論…………………………………………………….………..51
一、實驗室小量栽培試驗………………………………………………51
二、種菌條件之探討……………………………………………………53
(一)、植物油之添加………………………………………………..53
(二)、生長促進物質的添加………………………………………..56
(三)、種菌之生長曲線……………………………………………..58
三、搖瓶試驗……………………………………………………………61
(一)、起始pH值之探討……………………………………………61
(二)、載液量之探討………………………………………………..65
(三)、碳源種類之探討…………………………………….………68
(四)、最適葡萄糖濃度之探討…………………………….………71
(五)、最適果糖濃度之探討……………………………….………74
(六)、雙碳源培養基之探討………....…………………….………76
(七)、不同雙碳源濃度組合培養基之探討……………………….77
(八)、氮源種類之探討…………………………………….………81
(九)、碳氮比(C/N ratio)的探討…………………………………...84
四、5L桌上型攪拌式發酵槽試驗………………………………87
伍、結論……………………………………………………………………...90
陸、未來展望………………………………………………………………...92
柒、參考文獻………………………………………………………………...94
捌、附錄一………………………………………………………………….103
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