臺灣博碩士論文加值系統

許多研究指出食藥用菇類之多醣體成分具有抗腫瘤、刺激免疫活性、抗氧化、降血糖等生物活性功能，本實驗選用深具療效之桑黃菌（Phellinus igniarius PI）為試驗菌種，探討深層培養條件及不同型式發酵槽對其胞外多醣體生成與發酵液之生物活性（抑菌力、分子量、抗氧化力及β-1,3-D-glucan相對含量）的影響。
研究結果顯示，最適P . igniarius PI菌絲體與胞外多醣生成之深層培養條件為：培養溫度為30℃，最適碳源為1%（w/v）glucose、最適氮源為1%（w/v）yeast extract、添加0.3%（w/v）corn steep liquor生長因子、培養液起始pH 5.4及1.0（glucose / yeast extract，w/w）之碳氮比，以上述最適條件之搖瓶培養可於第8天達到最高菌絲體乾重及（9.39 g/l）胞外多醣產量（1.93 g/l），分別為基礎培養基產量之2.4及10.1倍。
發酵參數顯示攪拌式發酵槽具有最高之比生長速率（μ=0.88 day-1），而氣泡式發酵槽則具有最高之菌絲體產率（Yx/s=122.29 mg/g）及胞外多醣生成速率（Qp=0.39 g/l/day）；比多醣產率（Yp/x）及多醣產率（Yp/s）則呈現氣泡式>攪拌式>氣舉式發酵槽之趨勢。
抑菌力測定結果顯示在所使用之六種病原菌測定菌株（ (Bacillus cereus BCRC 10250, Escherichia coli BCRC 10239, Micrococcus luteus BCRC 10449, Pseudomonas aeruginosa BCRC 10261, Staphylococcus aureus BCRC 10451 and Salmonella typhimurium BCRC 10241）上並沒有任何抑菌效果。

搖瓶試驗與三種發酵槽所得之胞外多醣體分子量皆相同（約4000 Da），而氣泡式發酵槽所得分子量4000 Da之部分其相對含量為三者最高。
搖瓶試驗及三種不同發酵槽之發酵液在相同濃度（10 g/l）時，以攪拌式與氣泡式發酵槽具分別具最佳之DPPH自由基清除能力（86.94%）與總抗氧化能力（93.26%），彼等抗氧化力之IC50分別為4.83與1.52 g/l。β-(1→3)-D-glucan相對含量之測定亦以氣泡式發酵槽最高（19.22μg/ml LE）。
綜上所述，以具較低剪切力及較佳混合攪拌效果之氣泡式發酵槽培養桑黃菌，最能有效地將所消耗之葡萄糖轉換成胞外多醣與菌絲體，亦較攪拌式或氣舉式發酵槽有更高產量及較佳生物活性之胞外多醣體。以較廉價之食品級培養基取代試藥級培養基模擬工業化量產，可降低業界生產桑黃菌胞外多醣體之成本，並可以提高菌絲體（5.76 g/l）及胞外多醣體（3.74 g/l）之產量，分別為試藥級培養基之1.54及1.67倍，且可提供良好之pH緩衝效果。

The exopolysaccharides (EPS) from many mushrooms have been reported to possess important biological functions including anti-tumor, immuno-stimulating, hypoglycemic and antioxidative capacities. Phellinus igniarius PI, a potently medicinal and EPS-producing mushroom, was selected in this research to study the effect of submerged culturing condition and different types of fermentors on the production and the bioactivity (molecular weight, relative β-(1→3)-D-glucan content, antimicrobial and antioxidative activities) of EPS by P. igniarius PI.
Results showed that factors for optimal submerged-culturing condition were : incubation temperature of 30℃, glucose at 1% (w/v), yeast extract at 1% (w/v), corn steep liquor at 0.3% (w/v), initial pH of 5.4, and a C/N ratio (glucose / yeast extract) of 1.0. Under this optimized culturing condition, maximal yields of mycelial biomass (9.39 g/l) and EPS (1.93 g/l) were reached on the 8th day of incubation from shake flask culture and increased 2.4 and 10.1 times, respectively, as compared with those of the basal medium.
Among the three types of fermentors, the stirred-tank fermentor showed the highest specific growth rate (μ=0.88 day-1). The air bubble fermentor showed the highest biomass yield (Yx/s=122.29 mg/g) and EPS formation rate (QP=0.39 g/l/day). As for specific EPS yield (Yp/x) and EPS yield (Yp/s), results showed an increasing tendency of air bubble fermentor > stirred-tank fermentor > air lift fermentor.
The EPS of P. igniarius PI demonstrated no antibacterial activies against the six bacterial strains (Bacillus cereus BCRC 10250, Escherichia coli BCRC 10239, Micrococcus luteus BCRC 10449, Pseudomonas aeruginosa BCRC 10261, Staphylococcus aureus BCRC 10451 and Salmonella typhimurium BCRC 10241) tested.
Same molecular weight (4000 Da) was found in EPS from the shake flask and three different types of fermentors. The EPS from culture of air bubble fermentor had higher content of molecular weight of 4000 Da.
Under same concentration (10g/l), the fermented cultures from stirred-tank and air bubble fermentors showed the highest DPPH scavenging effect (86.94%, IC50=4.83g/l) and total antioxidant capacity (93.26%, IC50=1.52g/l), respectively, among those from shake flask and three different types of fermentors.The EPS from air bubble fermentor also had the highest relative β-(1→3)-D-glucan contents.
As mentioned above, using air bubble fermentor, which has lower shearing force and better mixing effect as compared with stirred-tank and air bubble fermentors, could produce higher yield of EPS from Phellinus igniarius PI with better bioactivity. Replacing reagent-grade medium components with food-grade components not only reduced the production cost of EPS but also increased the yields of mycelial biomass (5.76 g/l) and exopolysaccharide (3.74 g/l) 2.4 and 10.1 times, respectively. In addition, the food-grade medium components offered a better buffering effect.

目 錄
摘 要.............................................................I
Abstract..........................................................III
壹、前 言............................................................1
貳、文獻回顧..........................................................3
一、食藥用菇類（medicinal mushrooms）簡介..............................3
(一)食藥用菇類的種類...................................................3
(二)食藥用菇類的生態特性...............................................4
(三)食藥用菇類的活性物質...............................................5
(四)食藥用菇類的利用...................................................7
二、食藥用菇類多醣簡介.................................................8
(一)多醣體之結構......................................................9
(二)β-D-glucan之免疫活性..............................................9
三、桑黃菌種介紹.....................................................14
(一)桑黃菌的簡介.....................................................14
(二)桑黃菌多醣體的組成份與相關研究.....................................16
四、食藥用菇類之深層培養..............................................22
(一)深層培養之定義...................................................22
(二)深層培養與傳統固態培養之差異.......................................23
(三)影響深層培養的因素................................................23
(四)深層發酵之應用...................................................27
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.........................................................36
二、試驗儀器.........................................................37
三、試驗方法.........................................................39
(一)試驗流程.........................................................39
(二)菌種貯存.........................................................40
(三)菌種活化.........................................................40
(四)種菌培養.........................................................40
(五)種菌懸浮液之製作..................................................40
(六)平板培養基試驗...................................................41
(七)搖瓶試驗.........................................................41
(八)發酵槽試驗.......................................................43
四、分析方法.........................................................44
(一)菌絲體乾重測定...................................................44
(二)pH值之測定.......................................................44
(三)胞外多醣之測定...................................................44
(四)殘糖分析.........................................................45
(五)發酵參數之分析...................................................46
(六)分子量之測定.....................................................47
(七)抑菌能力之測定...................................................48
(八)抗氧化力之測定...................................................49
(九)β-(1→3)-D-glucan相對含量測定....................................51
(十)統計分析.........................................................52
肆、結果與討論.......................................................53
一、平板培養基試驗 ...................................................53
(一)培養溫度及不同培養基對桑黃菌菌絲體生成之影響.........................53
(一)碳源對桑黃菌PI胞外多醣生成之影響...................................66
(二)氮源對桑黃菌PI胞外多醣體生成之影響..................................68
(三)生長因子對桑黃菌PI胞外多醣體生成之影響..............................70
(四)培養基起始pH值對桑黃菌PI胞外多醣體生成之影響.........................72
(五)碳氮比對桑黃菌PI胞外多醣體生成之影響................................76
(六)搖瓶培養時間對桑黃菌胞外多醣體生成之影響.............................78
三、不同型式發酵槽培養試驗.............................................81
(一)胞外多醣體生成之影響..............................................81
四、發酵液抑菌力之測定................................................90
五、多醣體分子量之測定................................................90
六、發酵液抗氧化力之測定..............................................93
(一)DPPH自由基清除能力測定............................................95
(二)總抗氧化能力測定..................................................96
七、β-(1→3)-D-glucan相對含量測定.....................................96
八、模擬工業化量產之發酵槽試驗........................................102
伍、結論...........................................................106
陸、參考文獻........................................................109
柒、附錄...........................................................119

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