臺灣博碩士論文加值系統

在人類母乳的油脂中含有20-25%的棕櫚酸(C16:0)以及30-35%的油酸(C18:1)，其中超過60%的棕櫚酸在甘油中的第二個位置上。嬰兒食品中主要的結構性脂質為1,3-Oleoyl-2-palmitoyl glycerol(OPO)，所以本研究希望能從便宜的油脂中得到此結構脂質。由於棕櫚油本身就富含棕櫚酸及油酸，於是本研究利用棕櫚油得到高純度的棕櫚酸和油酸再利用棕櫚酸合成棕櫚酸的三酸甘油酯，製備結構脂質OPO所需的基質。首先將棕櫚油皂化然後利用低溫結晶法純化棕櫚酸及油酸，棕櫚酸純度可達到87.8%回收率85.4%；油酸純度可達到94.5%回收率77.5%。若將低溫結晶法濃縮得到的棕櫚酸再經選擇性酯化反應後，則可得到含94.2%棕櫚酸乙基酯回收率91.4%。以此含94.2%棕櫚酸乙基酯與甘油進行酯化反應，可得到酯化程度88.2%，產物甘油酯中TG含量為79.7%，如果先將乙基酯水解為FFA，再與甘油反應可得到酯化程度97.7%，產物甘油酯中TG含量為98.5%，經矽膠管柱純化後，TG含量達100%。

Human milk fat contains 20-25% palmitic acid (C16:0), 30-35% of oleic acid (C18:1) and more than 60% of the palmitic acid occurs in the sn-2 position of the glycerol backbone. Structured lipid 1,3 — Oleoyl-2- palmitoylglycerol (OPO) is an important ingredient in infant formula. In order to obtain an economically feasible process for the synthesis of OPO structured lipid it is utmost important that starting substrates must be isolated from inexpensive sources by commercially viable methods. Palm oil is a chief and rich source of these fatty acids. Thus, in the present investigation the purification of palmitic acid and oleic acid from the saponified palm oil and lipase assisted enzymatic synthesis of triplamitin were carried out. The method consists of several steps. Firstly, low temperature solvent fractionation of fatty acids from saponified palm oil which gives 87.5% pure palmitic acid and 94.5% pure oleic acid with corresponding recovery of 85.4% and 77.5%, respectively. Secondly, the enrichment of palmitic acid via the lipase catalyzed selective esterification. This step gives 94.2% pure ethyl palmitate with recovery of 91.4%. Finally, the synthesis of tripalmitin from ethyl palmitate and palmitic acid with glycerol by lipase catalysis. Esterification of glycerol with ethyl palmitate gives 79.7% triglycerides with 88.2% degree of esterification while, direct esterification of glycerol with palmitic acid after hydrolysis of ethyl palmitate gives 98.5% triglycerides with 97.7% degree of esterification. Separation of the reaction mixture by silica gel column chromatography resulted in triglyceride with 100% purity.

目錄
中文摘要.................................................Ⅰ
英文摘要.................................................Ⅱ
目錄.....................................................Ⅲ
圖索引...................................................Ⅸ
表索引.................................................ⅩⅡ
符號索引...............................................ⅩⅢ
第一章 緒論..............................................1
1.1酵素催化作用的優點.....................................1
1.2酵素於有機溶劑中的催化反應.............................2
1.3酵素固定化的優點.......................................4
1.4脂解酵素之專一性.......................................6
1.5酵素催化油脂之反應.....................................7
1.6脂肪酸的分類及構造.....................................8
1.7長鏈三酸甘油酯的代謝...................................9
1.8結構脂質..............................................11
1.9內容簡介..............................................13
第二章 文獻回顧.........................................15
2.1棕櫚油及棕櫚酸........................................15
2.1.1棕櫚油的來源........................................15
2.1.2棕櫚油之特性........................................16
2.1.3棕櫚油的發展趨勢....................................20
2.1.4母乳中的脂肪組成....................................21
2.1.5結構性脂質(OPO).....................................23
2.1.6油脂的選擇..........................................23
2.2脂肪酸的濃縮..........................................26
2.2.1脂肪酸的濃縮方法....................................26
2.3富含棕櫚酸之三酸甘油酯的合成..........................32
2.3.1化學法合成TG........................................32
2.3.2脂解酵素催化合成TG的反應............................33
2.3.3脂解酵素催化的甘油酯化反應..........................34
2.3.4有機溶劑對甘油酯化反應的影響........................35
第三章 實驗藥品及儀器..................................37
3.1實驗藥品..............................................37
3.2儀器及設備............................................39
3.3 實驗方法.............................................40
3.3.1酵素固定化..........................................40
3.3.2脂肪酸的製備........................................41
3.3.2.1油脂的皂化........................................41
3.3.2.2皂化值(S.V.)......................................42
3.3.2.3酸值(A.V.)........................................43
3.3.2.4酵素水解棕櫚油....................................43
3.3.2.5 TMSH甲酯化試劑的製備.............................45
3.3.2.6脂肪酸甲基酯化步驟................................45
3.3.2.7氣相層析儀的分析條件..............................46
3.3.3棕櫚酸及油酸的濃縮..................................47
3.3.3.1低溫溶劑結晶法分離飽和及不飽和脂肪酸步驟..........47
3.3.3.2 兩階段低溫溶劑結晶法濃縮油酸之步驟...............47
3.3.3.3 三階段低溫溶劑結晶法濃縮油酸之步驟...............48
3.3.3.4 選擇性酯化反應...................................48
3.3.3.5選擇性酯化反應產物中脂肪酸及酯的組成分析..........48
3.3.4 棕櫚酸三酸甘油酯的合成.............................49
3.3.4.1 脂肪酸乙基酯之水解反應...........................49
3.3.4.2 FFA或FFA乙基酯與甘油之酯化反應...................50
3.3.4.3 以薄層火燄離子分析儀(TLC-FID)分析產物中甘油酯
組成.............................................50
3.3.5 以矽膠充填管柱分離甘油酯混合物.....................51
3.3.6 酵素活性的測定.....................................52
3.3.6.1 脂解酵素之酯化活性測定...........................52
3.3.6.2 Candida rugosa脂解酵素之水解活性測定.............53
第四章 結果與討論.......................................54
4.1酵素之活性............................................54
4.2棕櫚油脂肪酸之組成....................................54
4.2棕櫚油之水解..........................................54
4.3.1酵素篩選............................................55
4.3.2反應溫度............................................55
4.3.3酵素量的影響........................................58
4.4低溫結晶法分離飽和及不飽和脂肪酸......................58
4.5二階段低溫結晶法去除液相中的飽和脂肪酸................62
4.6三階段低溫結晶法......................................64
4.7選擇性酯化反應........................................64
4.7.1酵素篩選............................................65
4.7.2醇鏈長的影響........................................68
4.7.3酵素固定化..........................................68
4.7.4溫度之影響..........................................68
4.7.5轉速的影響..........................................72
4.7.6水含量的影響........................................72
4.7.7基質莫耳比的影響....................................72
4.7.8產物脂肪酸及酯的分離................................73
4.8三棕櫚酸甘油酯的合成..................................76
4.8.1脂肪酸乙基酯與甘油的酯化反應........................77
4.8.1.1是否使用溶劑之影響................................77
4.8.1.2酵素篩選..........................................77
4.8.1.3溫度的影響........................................77
4.8.1.4水含量的影響......................................82
4.8.1.5 結論.............................................82
4.8.2棕櫚酸乙基酯之水解反應..............................84
4.8.2.1酵素篩選..........................................84
4.8.2.2溫度的影響........................................84
4.8.2.3水含量的影響......................................84
4.8.3富含棕櫚酸之FFA與甘油反應...........................87
第五章 結論..............................................91
參考文獻.................................................93
作者簡介................................................103
圖索引
圖1-1 常見PUFA之結構式...................................10
圖1-2 論文研究流程圖.....................................14
圖2-1 生育醇與生育三烯醇結構式之比較.....................18
圖2-2 胡蘿蔔素異構物之結構式.............................19
圖3-1 酚酞在鹼性及酸性下的結構變化.......................44
圖3-2 TMSH製備以及甲酯化反應式...........................46
圖4-1 棕櫚油水解時，探討不同酵素對水解程度隨反應時間之
變化。.............................................56
圖4-2 以Candoda rugosa脂解酵素催化棕櫚油之水解時，反應
溫度對水解程度隨反應時間變化之影響。...............57
圖4-3 以Candoda rugosa脂解酵素水解棕櫚油時，酵素量對水
解程度隨反應時間變化之影響。.......................59
圖4-4 FFA與乙醇之選擇性酯化反應時PA含量及酯化程度隨時間
之變化。...........................................67
圖4-5 FFA與醇之選擇性酯化反應時，醇鏈長對Palmitate含量
及酯化程度隨時間之變化。...........................69
圖4-6 酵素固定化與否對FFA與乙醇之選擇性酯化反應影響。....70
圖4-7 FFA與乙醇之選擇性酯化反應時，溫度對Ethyl palmitate
含量及酯化程度隨時間之變化。.......................71
圖4-8 FFA與乙醇之選擇性酯化反應時，FFA對乙醇莫耳比對棕
櫚酸乙基酯含量及酯化程度隨時間之變化。.............74
圖4-9 在正己烷中，以SP 435催化甘油與乙基酯之酯化反應時
，產物中TG含量隨反應時間之變化。...................78
圖4-10 在正己烷中，以SP 435催化甘油與乙基酯之酯化反應
時，酯化程度隨反應時間之變化。....................79
圖4-11 在無溶劑系統中以不同酵素催化甘油與乙基酯之酯化
反應時，酯化程度及TG含量隨反應時間之變化。........80
圖4-12 以IM 60催化甘油與脂肪酸乙基酯之酯化反應時，探討
溫度對產物中TG含量及酯化程度隨反應時間之變化。....81
圖4-13 以IM 60催化甘油與脂肪酸乙基酯之酯化反應時，探討
水含量對TG含量及酯化程度隨反應時間之變化。........83
圖4-14 脂肪酸乙基酯水解時酵素種類對水解程度隨反應時間之
變化。............................................85
圖4-15 以SP 435水解脂肪酸乙基酯時，溫度對水解程度隨反應
時間之變化。......................................86
圖4-16 以SP 435水解脂肪酸乙基酯時，水含量對水解程度隨反
應時間之變化。....................................88
圖4-17 以IM 60催化甘油與乙基酯之酯化反應時，系統壓力對
產物中TG含量及酯化程度隨反應時間之變化。..........89
圖4-18 以IM 60及SP 435脂解酵素催化甘油與脂肪酸之酯化反
應時，酯化程度及TG含量隨反應時間之變化。..........90
表索引
表2-1 世界各種主要植物油生產量...........................20
表2-2 母乳中的脂肪酸組成.................................22
表2-3 各種油脂酯防酸組成與性質...........................24
表4-1 棕櫚油皂化所得FFA中各個脂肪酸之含量................55
表4-2 使用不同溶劑於低溫溶劑結晶法時，固、液相之組成.....60
表4-3 基質與溶劑比對低溫溶劑結晶法效果之影響.............61
表4-4 以丙酮為溶劑時溶劑量及溫度對低溫結晶法效果之影響...61
表4-5 以丙酮為溶劑經低溫溶劑結晶法後，固液相之脂肪酸組成.62
表4-6 以丙酮為溶劑進行二階段低溫結晶法時，溶劑量及溫度...63
表4-7 以丙酮為溶劑經二階段低溫結晶法處理後產物之組成.....63
表4-8 不同溶劑對第三階段低溫結晶法濃縮油酸效果之影響.....65
表4-9 以氰化甲烷為溶劑行第三階段低溫結晶法時溶劑量及溫度
對產物固相中油酸含量及回收率之影響.................66
表4-10 以氰化甲烷為溶劑經三階段低溫結晶法處理後產物組成..66
表4-11 棕櫚酸與乙醇之選擇性酯化，當基質莫耳比為2：1時，
酯化程度與回收率隨時間變化的關係..................75
表4-12 選擇性酯化後，利用低溫結晶法分離脂肪酸及酯........75
表4-13 選擇性酯化後組成..................................76
符號索引
A.V.：Acid value，酸值
C8:0：Caprylic acid，辛酸
C13:0：Tridecanoic acid，十三酸
C14:0：Myristic acid，肉豆蔻酸
C16:0：Palmitic acid (PA)，棕櫚酸
C18:0：Stearic acid，硬酯酸
C18:1：Oleic acid (OA)，油酸
C18:2：Linoleic acid, C18:2 n6，亞麻油酸
C18:3 n3：a-Linolenic acid (ALA)，a-亞麻酯酸
C18:3 n6：g-Linolenic acid (GLA)，g-亞麻酯酸
C20:0：cis-11-Eicosenoic acid，花生酸
C22:0：Docosanoic acid ，山酸
C22:4 n6：Arachidonic acid (AA) 花生四烯酸
C22:5 n3：Docosapentaenoic acid (DPA)，二十二碳五烯酸
DG：Diglyceride，二酸甘油酯
FFA：Free Fatty Acid，自由態脂肪酸
GC：Gas chromatography，氣相層析儀
HCl：Hydrochloric acid鹽酸
HMF：Human milk fat，母乳
KHP：Potassium hydrogen phthalate，酞酸氫鉀
KOH：Patassium Hydroxide，氫氧化鉀
LCFA：Long chain fatty acids，長鏈脂肪酸
MCFA：Medium chain fatty acids，中鏈脂肪酸
MG：Monoglyceride，單酸甘油酯
MTBE：Methyl tert-butyl ether，甲基三級丁基酯
NaOH：Sodium hydroxide，氫氧化鈉
N.D.：Not Detected
OPO：1,3-Oleyl-2-palmitoyl-glycerol
PUFA：Polyunsaturated Fatty Acid，多元不飽和脂肪酸
SCFA：Short chain fatty acids，短鏈脂肪酸
S.V.：Saponifacation value，皂化值
rpm：Round per minute，每分鐘轉數
TMSH：Trimethylsulfonium Hydroxide
TG：Triglyceride，三酸甘油酯
TP：Tripalmitin，三棕櫚酸甘油酯

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