臺灣博碩士論文加值系統

黏紅酵母菌Rhodotorula glutinis (BCRC 22360)是一株生產油脂外也富含高價值的β-胡蘿蔔素，β-胡蘿蔔素是天然的抗氧化劑，利用分子中含有多個雙鍵能與具有不成對電子的自由基結合來中斷脂質過氧化連鎖反應，進而保護細胞不被破壞。R. glutinis細胞生長迅速、可達高密度細胞培養，因此具有相當高發展潛力。在綠色能源意識高漲下，使得生質柴油產量大幅提升，同時累積大量的副產物粗甘油，而粗甘油中含有未反應的脂肪酸、醇類及催化劑等，在純化的製程上較為複雜且高成本，因此使用粗甘油作為培養基的碳源可作為解決粗甘油的方法。本實驗分別探討固態及液態發酵對於 R. glutinis菌體生長以及其代謝產物β-胡蘿蔔素的影響。固態方面，基質選用同為蛋雞飼料的玉米，期望可直接應用於增色飼料中，減少萃取、純化等成本；液態方面，利用生質柴油副產物作為培養基碳源。藉由改變環境因子來提高兩種發酵型態的β-胡蘿蔔素產量。
在固態搖瓶實驗中，分別做了基質選擇、環境因子的影響，藉此增加β-胡蘿蔔素的提升，其中使用玉米作為基質表現較佳，在環境因子最適化的實驗中，以含水量65 %、粒徑小於0.85 mm培養96 hr效果較好，β-胡蘿蔔素可達到2.45 mg/kg發酵物；在將滅菌後基質進行切塊的實驗中，有進行切塊的β-胡蘿蔔素從原本2.45 mg/kg發酵物提升至2.96 mg/kg發酵物；在基質中添加碳源(蔗糖)以及氮源(硫酸銨)的實驗中，相較於未添加者，β-胡蘿蔔素由2.96 mg/kg發酵物顯著提升至3.67 mg/kg發酵物。
由固態搖瓶放大至2 L固態旋轉瓶，分別進行了單瓶基質量、改變培養方式的探討，在單瓶基質量的實驗中，以200 g的基質量最符合成本以及效益，在有2 L/min的通氣下，β-胡蘿蔔素可達到4.14 mg/kg發酵物；在改變培養方式的實驗中，間歇性通入飽和氣體的方式對β-胡蘿蔔素的產量有明顯助益，β-胡蘿蔔素可從4.14 mg/kg發酵物提升至6.39 mg/kg發酵物。
在液態搖瓶實驗中，進行了添加誘導物(柑橘類果皮萃取液、植物油)以及改變C/N ratio的實驗，在添加柑橘類果皮萃取液的實驗中，於各時段添加萃取液對於β-胡蘿蔔素並無明顯助益，菌量受到萃取溶劑(乙醇)以及萃取成分(檸檬烯)的抑制而降低；在添加植物油的實驗中，以軟棕櫚油2.5 %添加效果最好，菌量及β-胡蘿蔔素分別得到11.5 g/L、0.33 mg/g；在改變C/N ratio的實驗中，隨著C/N ratio提高，菌量以及β-胡蘿蔔素皆有所提升，實驗結果以碳源(粗甘油60 g/L)氮源(硫酸銨1 g/L)較好，可得到菌量8.2 g/L、 β-胡蘿蔔素0.26 mg/g 。
由搖瓶油脂添加實驗放大至5 L攪拌式發酵槽，可以發現軟棕櫚油2.5 %的添加對於菌體生長有很顯著的效益，其菌量可達到29.8 g/L，β-胡蘿蔔素則與控制組差異不大，皆可得到約0.35 mg/g的濃度；放大至5 L氣舉式發酵槽，其菌量可得24.5 g/L，β-胡蘿蔔素可得0.29 mg/g。兩發酵槽對比，5 L攪拌式發酵槽對於菌量、β-胡蘿蔔素以及脂質方面效果皆較為出色。
由搖瓶改變C/N ratio實驗放大至50 L氣舉式發酵槽，藉由饋料方式Fed batch來提高培養期間培養基中的C/N ratio，β-胡蘿蔔素並無太大差異約在0.2 mg/g，而lipid content有了顯著提升，由40 %提升至接近60 %，應是菌體將多餘碳源轉而累積油脂。
蛋雞餵養實驗中，將固態發酵及液態發酵物添加至飼料中，添加比例為10 %，連續餵養144 hr，固態部分，蛋黃所含β-胡蘿蔔素為2.18 mg/kg egg yolk，液態部分為3.73 mg/kg egg yolk，對比於控制組的1.53 mg/kg egg yolk分別提高2.5倍及1.4倍。

Rhodotorula glutinis (BCRC 22360) is an oleaginous yeast that can accumulate high content of total lipid. In addition for total lipids production, R. glutinis was well known as having high content of β-carotene, which is a natural antioxidants to protect cells from being damaged by free radical through the interruption of lipid peroxidation chain reaction. Even though, the high cost of commercializing biosiesel production has been a thorny issue by using the microbial oils as the feedstock. In this study, the effects of liquid submerged fermentation on the cell growth and β-carotene production was investigated by using crude glycerol as carbon source. The corn is used as the substrate to reduce the cost for the extraction and the purification process.
The effects of medium ingredients and environmental cultivation conditions on the growth of cell and the content of β-carotene in the shaker flask experiments.It was found that under the condition of 65 % content of water, the particle sizes which less than 0.85 mm, and cultivated for 96 hours can obtain the best consequence that the amount of fermentates of β-carotene reach 2.45 mg/kg. By dicing the substrate after sterilizing can enhance β-carotene from 2.45 to 2.96 mg/kg fermentates significantly. By adding 20 g/L of surcrose and 5 g/L of ammonium sulfate can enhance β-carotene from 2.96 to 3.67 mg/kg fermentates significantly.
The effects on the growth of cell and β-carotene were evaluated by adding different volume of subsrtate in a 2 L rotary fermenter, powdering during the period of cultivating and methods of ventilation. While adding 200 g substrates in the 2 L rotary fermenter, the fermentates of β-carotene we obtained was 4.14 mg/kg and it fitted the economic benefits. The content of β-carotene was increased significantly from 4.14 to 6.389 mg/kg with the saturated gas pass into periodicity.
The effects of C/N ratio and adding inducer in the medium on the growth of cell and β-carotene in the shaker flask by cultivating in the liquid fermentation were evaluated. The result demonstrated that a monoterpene ethanol solution did not increase the content of β-carotene significantly. Additionly, the growth of the cell was suppressed. However, it was found that adding 2.5 % palm oil can promote the growth of cell and β-carotene, which could obtain 11.5 g/L of biomass and 0.33 mg/g of β-carotene. In C/N ratio experiment, it seems that the increase of C/N ratio can increase the cells growth and β-carotene content. It can get 8.2 g/L of biomass and 0.26 mg/g of β-carotene by using 60 g/L crude glycerol and 1 g/L ammonium sulfate.
The experiment of adding 2.5 % palm oil was discussed that scaled up by using the 5 L stirred tank reactor and 5 L airlift. It was found that adding palm oil can promote the growth of cell significantly, which could obtaine 29.8 g/L of biomass and 0.35 mg/g of β-carotene by using 5 L stirred tank reactor. While getting 24.5 g/L of biomass and 0.29 mg/g of β-carotene by using 5 L airlift. Comparing the two reactors, 5 L stirred tank reactor was better than another one.
The experiment of changing C/N ratio was discussed that scaled up by using the 50 L airlift. The fed batch operation with the disposable feeding could enhance the lipid content from 41 to 60 %, but not promote for synthesizing the β-carotene, which could obtain 0.2 mg/g of β-carotene.

目錄
中文摘要 I
Abstract III
目錄 V
圖目錄 X
表目錄 XII
第一章 緒論 1
第二章 文獻回顧 2
2.1 黏紅酵母菌-Rhodotorula glutinis 2
2.2 生質柴油副產物-粗甘油(Crude glycerol) 3
2.3 類胡蘿蔔素簡介 5
2.3.2 β-胡蘿蔔素的生理活性 8
2.3.3影響微生物生成β-胡蘿蔔素因子 10
2.3.4.1 油脂 12
2.3.4.2軟棕櫚油 13
2.3.5 固態發酵 14
2.3.5.1 固態發酵與液態發酵之差異 14
2.3.5.2 固態發酵之優缺點 15
2.3.5.3 固態發酵之應用 16
2.3.5.4 固態發酵培養重要參數 17
2.3.6 玉米 19
第三章 實驗材料與方法 20
3.1 實驗材料 20
3.1.1實驗酵母菌種及菌株 20
3.1.2實驗藥品 21
3.3 分析方法 25
3.3.1 菌體濃度分析方法 25
3.3.2甘油濃度分析方法 25
3.3.3總脂質濃度分析方法 25
3.3.4 β-胡蘿蔔素濃度分析方法 26
3.4 實驗方法 27
3.4.1 原始菌種保存 27
3.4.2培養基組成 27
3.4.2.1 種子培養基 (Seed culture medium, SM) 27
3.4.2.2 發酵培養基 (Fermentation medium, FM) 28
3.4.3 接菌 28
3.5 實驗架構 29
3.6 實驗培養條件 30
3.6.1 固態搖瓶批次發酵程序 30
3.6.1.1 不同基質之影響 30
3.6.1.2 菌體培養時間之影響 30
3.6.1.3 不同含水量之影響 31
3.6.1.4 不同粒徑大小之影響 31
3.6.1.5 滅菌過之基質進行切塊之影響 32
3.6.1.6添加碳氮源(蔗糖、硫酸銨)於基質中之影響 32
3.6.1.7 不同光照強度之影響 34
3.6.2 2 L旋轉瓶固態批次發酵程序 35
3.6.2.1 單瓶2L固態旋轉瓶填裝基質量之影響 35
3.6.2.2 改變培養方式 35
3.6.2.3 間歇性通入飽和氣體-進氣量之影響 37
3.6.3 深層液態搖瓶批次發酵程序 38
3.6.3.1 添加柑橘類果皮萃取液之影響 38
3.6.3.2添加植物油之影響 38
3.6.3.3 不同C/N ratio之影響 39
3.6.4 5 L攪拌式發酵槽批次發酵程序 40
3.6.4.1添加2.5 % 軟棕櫚油、1 g/L Tween 80之影響 40
3.6.5 5 L氣舉式發酵槽批次發酵程序 40
3.6.5.1添加2.5 % 軟棕櫚油、1 g/L Tween 80之影響 40
3.6.6 50 L氣舉式發酵槽發酵程序 41
3.6.6.1 Fed-batch- C/N ratio之影響 41
3.7.1搖瓶發酵培養裝置圖 (250 ml) 42
3.7.2 5 L氣舉式發酵槽批次發酵培養裝置圖 42
3.7.3 5 L攪拌式發酵槽批次發酵培養裝置圖 43
3.7.4 50 L氣舉式發酵槽饋料批次培養裝置圖 43
第四章 結果與討論 45
4.1 固態搖瓶批次發酵程序 45
4.1.1 不同基質之影響 45
4.1.2 菌體培養時間之影響 48
4.1.3 不同含水量之影響 49
4.1.4 不同粒徑大小之影響 50
4.1.5 滅菌過之基質進行切塊之影響 52
4.1.6添加碳氮源(蔗糖、硫酸銨)於基質中之影響 53
4.1.7 不同光照強度之影響 55
4.2 2 L旋轉瓶固態批次發酵程序 56
4.2.1 單瓶2 L固態旋轉瓶填裝基質量之影響 57
4.2.2 改變培養方式 58
4.2.3 間歇性通入飽和氣體-通氣量之影響 60
4.3 深層液態搖瓶批次發酵程序 61
4.3.1 添加柑橘類果皮萃取液之影響 61
4.3.2添加植物油之影響 63
4.3.3 不同C/N ratio之影響 68
4.4 5 L攪拌式發酵槽批次發酵程序 69
4.4.1添加2.5 % 軟棕櫚油、1 g/L Tween 80之影響 69
4.5 5 L 氣舉式發酵槽批次發酵程序 72
4.5.1添加2.5 % 軟棕櫚油、1 g/L Tween 80之影響 72
4.6 50 L氣舉式發酵槽發酵程序 74
4.6.1 Fed-batch-低C/N ratio之影響 74
4.7 蛋雞餵養實驗 76
第五章 結論與未來展望 82
5.1 結論 82
5.2 未來展望 85
參考文獻 86
附錄 91
附錄 A 91
附錄 B 92
附錄C 93
作者簡歷 96

圖目錄
圖2-1 β-胡蘿蔔素結構 7
圖2-2 β-胡蘿蔔素代謝路徑 12
圖3-1 黏紅酵母菌Rhodotorula glutinis BCRC 22360生長在agar plate上之外觀 20
圖3-2 搖瓶實驗裝置圖 42
圖3-3 5 L氣舉式發酵槽裝置實圖(左)、示意圖(右) 42
圖3-4 5 L攪拌式發酵槽裝置圖 43
圖3-5 50 L氣舉式發酵槽裝置圖 43
圖3-6 固態旋轉發酵槽裝置圖 44
圖4-1 不同基質對於R. glutinis生產β-胡蘿蔔素之影響 46
圖4-2 黃豆(左)、糙米(中)、苦蕎麥(右)培養情形 46
圖4-3 紅薏仁(左)、紅豆(中)、綠豆(右)培養情形 46
圖4-4 玉米培養情形 46
圖4-5 不同基質本身v.s 發酵培養物之β-胡蘿蔔素濃度 47
圖4-6 R. glutinis生成β-胡蘿蔔素之時間變化量 48
圖4-7 不同含水量對於R. glutinis生產β-胡蘿蔔素之影響 49
圖4-8 不同粒徑大小對R. glutinis 生成β-胡蘿蔔素之影響 50
圖4-9 粒徑小的(less than 0.85 mm)生長情形 51
圖4-10 粒徑中的(between 0.85 mm and 2 mm)生長情形 51
圖4-11 粒徑大的(lager than 2 mm)生長情形 51
圖4-12 基質滅菌後切塊示意圖 52
圖4-13 滅菌過基質切塊之影響 52
圖4-14 固定硫酸銨添加量0.09 g改變蔗糖添加量(0.09、0.18、0.36 g)之影響 54
圖4-15 固定蔗糖添加量0.36 g改變硫酸銨添加量(0.09、0.18、0.36 g)之影響 54
圖4-16 不同光照度對R. glutinis生產β-胡蘿蔔素之影響 55
圖4-17 改變單瓶固態旋轉瓶之基質量對R. glutinis生產β-胡蘿蔔素之影響 57
圖4-18 不同培養方式(1) 於培養期間對基質進行磨碎；(2) 間歇性通入飽 59
圖4-19 間歇性通氣-進氣量之影響 60
圖4-20 柑橘類果皮萃取液添加時間點對R. glutinis之影響 62
圖4-21 不同植物油10 %以及1 g/L Tween 80對R. glutinis之影響 65
圖4-22 不同濃度之軟棕櫚油添加對R. glutinis之影響 65
圖4-23 不同程度之C/N ratio[(Low, 30:1)、(Medium, 60:1)、(High, 120:1)]對 68
圖4-24 5 L攪拌式發酵槽-添加軟棕櫚油2.5 %之影響 70
圖4-25 軟棕櫚油2.5 %添加-5 L氣舉式v.s 5 L攪拌式發酵槽 73
圖4-26 Fed batch-control 75
圖4-27 Fed batch-高C/N ratio之影響 75
圖4-28 蛋雞飼養實圖 77
圖4-29 蛋雞進食(混合菌粉)實圖 77
圖4-30 控制組蛋黃最終顏色變化 78
圖4-31 液態10 %蛋黃最終顏色變化 78
圖4-32 固態10 %蛋黃最終顏色變化 78

表目錄
表2-1 粗甘油微量元素分析表(Thompson et al., 2006) 3
表2-3 各種類胡蘿蔔素的功能 (孫證雄, 2011) 6
表2-4 常見類胡蘿蔔素的微生物來源(El-Banna 2012) 6
表3-1 黏紅酵母菌所使用之藥品清單 21
表3-2 實驗儀器清單 23
表3-3 種子培養基 27
表3-4 發酵培養基 28
表4-1 各基質培養前後，提升之β-胡蘿蔔素總量 47
表4-2 各油品脂肪酸組成 66
表4-3 各油品價格 67
表4-4 5 L攪拌式發酵槽添加2.5 %軟棕櫚油之動力學參數變化 70
表4-5 添加2.5 %軟棕櫚油脂肪酸組成變化 71
表4-6 5 L氣舉式發酵槽添加2.5 %軟棕櫚油之動力學參數變化 73
表4-7 50 L氣舉式發酵槽Fed batch之動力學參數變化 74
表4-8 固態及液態培養條件及所得菌體之β-胡蘿蔔素含量 76
表4-9 不同飼料連續餵養144 hr，雞蛋所含之β-胡蘿蔔素 79
表4-10 國際標準成人一天所需維生素A攝取量 (行政院衛生署) 79
表4-11 β-胡蘿蔔素部分，發酵槽實驗與文獻之比較 80

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