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研究生:蕭美玲
研究生(外文):Meiling Hsiao
論文名稱:深層培養條件與不同型式發酵槽對巴西蘑菇菌AgaricusblazeiMurrill胞外多醣體生成之影響
論文名稱(外文):Effect of submerged-culturing conditions and different types of fermentors on exopolysaccharide production by Agaricus blazei Murrill
指導教授:閻立平閻立平引用關係
指導教授(外文):Lipyng Yan
學位類別:碩士
校院名稱:東海大學
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:2007
畢業學年度:95
語文別:中文
論文頁數:127
中文關鍵詞:巴西蘑菇深層培養發酵槽菌絲體胞外多醣
外文關鍵詞:Agaricus blazei Murrillsubmerged culturefermentormycelial biomassexopolysaccharide
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摘要
巴西蘑菇(Agaricus blazei Murrill)為食藥用真菌,原產於巴西,近年來多被開發為機能性食品。在菌絲體深層培養中所生成的代謝產物被認定具有抗腫瘤、提升免疫力及抗氧化能力等生理活性。本研究探討深層培養條件及不同型式發酵槽對巴西蘑菇胞外多醣體生成與發酵液機能性功能(抑菌力、分子量及抗氧化能力)的影響。
研究結果顯示,最適Agaricus blazei Murrill菌絲體與胞外多醣生成之深層培養條件: 培養溫度為25℃、添加0.3%(w/v)之生長因子corn steep liquor、培養液起始pH=5.4、載液量為50ml、最適碳源為4%(w/v)之fructose、最適氮源為0.4%(w/v)之yeast extract及10(fructose / yeast extract,w/w)之碳氮比,以上述最適條件之搖瓶培養,可於第 14天時達到最高菌絲體乾重及胞外多醣產量,分別為25.2 g/l與0.302 g/l。
發酵槽培養可較搖瓶培養縮短最大胞外多醣與菌絲體生成時間(7~10 vs. 14天)及增加胞外多醣生成量(3.20~6.96 vs. 0.302 g/l)與菌絲體乾重量(42.01~46.70 vs. 25.22 g/l)。
攪拌式發酵槽雖具最高之比生長速率(μ=0.476 day-1),但其胞外多醣生成速率(Qp=0.46 g/l/day)為最低;氣舉式發酵槽其比生長速率(μ=0.365 day-1)雖為三種發酵槽中之最低,然具最高之胞外多醣生成速率(Qp=0.77 g/l/day)。比多醣產率(Yp/x)、多醣產率(Yp/s)及菌絲體產率(Yx/s)皆呈現氣舉式>氣泡式>攪拌式發酵槽之趨勢。
三種發酵槽發酵液之抑菌力及抑菌種類均較搖瓶培養液為高及增加,抑菌力大小則有氣舉式>氣泡式>攪拌式之趨勢。會受發酵液抑制之菌株有Bacillus cereus、Salmonella typhimurium、Staphylococcus aureus、Enterococcus faecalis、Enterobacter aerogenes、Escherichia coli、Proteus vulgaris、Listeria monocytogenes、Shigella dysenteriae及Pseudomonas aeruginosa等十株菌,其中以Bacillus cereus受到之抑菌作用最大,此發現可作為將來發酵液製成生物抗菌製劑及防腐劑之參考。
三種發酵槽所得之胞外多醣其主要分子量(2.5×105及3.0×103Da)及分子量分佈範圍(7.4×104~2.1×106 Da)皆相同。氣舉式發酵槽所
三種發酵槽發酵液之抗氧化力(自由基清除能力)與正對照組抗氧化劑相等甚或超過。相同發酵液濃度下,以氣舉式者可得最高之抗氧化力。
綜合上述試驗結果,以胞外多醣生成量、菌絲體產量、多醣及菌絲體產率、抑菌力及抗氧化力而言,皆呈現氣舉式>氣泡式>攪拌式發酵槽之結果;生成胞外多醣之分子量,則三者發酵槽皆相同。因此,以具較低剪切力及較佳對流循環之氣舉式發酵槽培養,會較氣泡式或攪拌式發酵槽能有更高產量及較佳機能性之巴西蘑菇胞外多醣體。
Abstract
Agaricus blazei Murrill, an edible and medicinal mushroom originally grown in Brazil, was recently developed as a functional food. The metabolites of this mushroom from submerged culture were proved to have unique biological activity such as antitumor, antioxidative and immunity-stimulating capabilities. This research was aimed to study the effects of submerged-culturing conditions and different types of fermentors on the production and functional properties (molecular weight, antibacterial and antioxidative activities) of exopolysaccharide (EPS) by A. blazei.
Results showed that the optimal submerged-culturing conditions were : incubation temperature of 25℃, initial pH of 5.4, culture volumes of 50ml, corn steep liquor at 0.3% (w/v), fructose at 4% (w/v), yeast extract at 0.4% (w/v), and a C/N ratio (fructose / yeast extract) of 10. The maximal yields of EPS and mycelial biomass, 0.302 and 25.2 g/l, respectively, were reached on the 14th day of incubation from shake flask culture under the optimized culturing conditions.
A shorter incubation time for maximal EPS and mycelial production (7~10 vs. 14 days), greater EPS yield (3.20~6.96 vs. 0.302 g/l) and higher mycelial biomass (42.01~46.70 vs. 25.22 g/l) were obtained from cultures of fermentors as compared with those from shake flask culture.
Among the three types of fermentors, the agitated fermentor showed the highest specific growth rate (μ=0.476 day-1) and the lowest EPS formation rate (Qp=0.46 g/l/day). On the contrary, the air lift fermentor demonstrated the lowest specific growth rate (μ=0.365 day-1) and the highest EPS formation rate (Qp=0.77 g/l/day). As for specific EPS yield (Yp/x), EPS yield (Yp/s) and biomass yield (Yx/s), results showed an increasing tendency of air lift fermentor > air bubble fermentor > agitated fermentor.
Cultures of fermentors had higher and broader spectrum of antibacterial activies than shake flask culture. The inhibitory capability also demonstrated the same order as : air lift fermentor > air bubble fermentor > agitated fermentor. The bacterial strains susceptible to fermented cultures were : Bacillus cereus, Salmonella typhimurium, Staphylococcus aureus, Enterococcus faecalis, Enterobacter aerogenes, Escherichia coli, Proteus vulgaris, Listeria monocytogenes, Shigella dysenteriae and Pseudomonas aeruginosa. Among the ten strains, B. cereus was the most susceptible one. This finding could be applied to use the culture broth of A. blazei as a potential biopreservative.
Same major molecular weights (2.5×105 and 3.0×103 Da) and molecular weight distribution (7.4×104~2.1×106 Da) were observed for EPS from the three batch cultures of fermentors. The EPS from culture of air lift fermentor had higher content of molecular weight of 2.5×105 Da.
The antioxidative activity (DPPH scavenging effect) of the three fermented cultures was equal or higher than that of control antioxidants. Under the same concentration, the fermented culture of air lift fermentor presented the highest antioxidative activity.
As far as the EPS yield, the mycelial biomass production, the formation rates of EPS and mycelial biomass, and the antibacterial and antioxidative activities of fermented cultures were concerned, all above results showed a trend of air lift fermentor > air bubble fermentor > agitated fermentor. In conclusion, using the air lift fermentor, which has lower shearing force and better convective circulation as compared with agitated and air bubble fermentors, could produce higher yield of EPS from A. blazei with better functional properties.
目錄
中文摘要.........................Ι
英文摘要.........................IV
壹、前言.........................1
貳、文獻整理.......................3
一、真菌菇類之簡介....................3
(一)菇菌之種類.....................3
(二)菇菌之生態特性...................4
(三)菇菌之機能性....................5
二、真菌菇類多醣之簡介..................7
(一)多醣體之結構....................9
(二)β-D-glucan之免疫功能............... 9
(三)β-D- glucan多醣體結構與活性之關係........ 12
(四)(1→3)鍵結的β-D- glucan主鏈與活性關係..... 13
三、巴西蘑菇之簡介....................14
(一)巴西蘑菇之發現...................14
(二)巴西蘑菇的分類地位與形態特徵............15
(三)巴西蘑菇的化學成分.................15
(四)巴西蘑菇之藥理研究.................17
四、食藥用真菌深層培養條件之探討.............22
(一)深層發酵培養之定義.................22
(二)深層發酵培養與傳統固態培養之比較..........22
(三)深層發酵培養之物理及化學因子............23
五、發酵槽(Fermentor)之簡介..............29
(一)攪拌式發酵槽(Agitated fermentor)........29
(二)氣泡式發酵槽(Air bubble fermentor).......30
(三)氣舉式發酵槽(Air lift fermentor)........31
參、材料與方法......................33
一、試驗材料.......................33
(一)試驗菌種......................33
(二)培養基.......................33
(三)重要藥品......................35
二、試驗儀器.......................36
三、試驗方法.......................38
(一)試驗流程......................38
(二)菌種貯存......................39
(三)菌種活化......................39
(四)種菌培養......................39
(五)平板培養基試驗...................39
(六)種菌懸浮液之製作..................39
(七)搖瓶試驗......................40
(八)發酵槽試驗.....................41
四、分析方法.......................43
(一)菌絲體乾重測定...................43
(二)pH值之測定.....................43
(三)胞外多醣之測定...................43
(四)殘糖分析......................44
(五)發酵參數之分析...................44
(六)分子量之測定....................46
(七)抑菌能力之測定...................47
(八)抗氧化力之測定...................47
(九)統計分析......................48
肆、結果與討論......................49
一、固體培養基試驗....................49
二、搖瓶試驗.......................52
(一)培養溫度對巴西蘑菇胞外多醣體生成之影響.......52
(二)生長因子對巴西蘑菇胞外多醣體生成之影響.......56
(三)載液量對巴西蘑菇胞外多醣體生成之影響........59
(四)起始pH值對巴西蘑菇胞外多醣體生成之影響.......61
(五)碳源對巴西蘑菇胞外多醣體生成之影響.........65
(六)氮源對巴西蘑菇胞外多醣體生成之影響.........67
(七)碳氮比對巴西蘑菇胞外多醣體生成之影響........69
(八)搖瓶培養時間對巴西蘑菇胞外多醣體生成之影響.....72
(九)巴西蘑菇搖瓶培養液抑菌力之測定...........75
三、不同型式發酵槽培養試驗................79
(一)胞外多醣體生成之影響................80
(二)發酵液抑菌力之測定.................89
(三)多醣體分子量之測定.................95
(四)發酵液抗氧化力之測定................102
伍、結論.........................105
陸、參考文獻.......................108
柒、附錄.........................120
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