sn-2 位置組成分析

甘油酯中 TG 的 FFA 組成及 sn-2 位置 FFA 之組成如表 4 所示。由表中可知若以 GLA 含量為 91.83 mol%之 TG 進行酸解反應，辛酸在 sn-2 位置佔 22.6 mol%，表示有醯基轉 移的現象發生。所欲合成 sn-1,3 位置為辛酸，sn-2 位置為 GLA 的 SL，經計算佔 TG 中 的 65.22 mol%。

圖 18 是 TG 之 GLA 含量(GLA=91.83 mol%及 97.94 mol%)對酸解反應之影響。由圖 中可知在反應之初，TG 中若有較高的 GLA (97.94 mol%)，辛酸混入率會較低，這可能 是 Lipozyme IM 對 GLA 的反應性較低所致，但反應 60 h 後則差異很小。以 GLA 含量 為 97.94 mol%之 TG 進行酸解反應，在反應 60 h 後，甘油酯產物中 TG 佔有 99.27%，

由表一可知此時 TG 中含有 65.65 mol%的辛酸及 33.37 mol%的 GLA，sn-2 位置的組成 分析顯示辛酸在 sn-2 位置有 17.31 mol%，表示有醯基轉移發生。此時在所欲合成 sn-1,3 位置為辛酸，sn-2 位置為 GLA 的 SL (1,3-dicapryloyl-2-γ-linolenoyl-glycerol)，經計算佔 TG 中的 72.84 mol%。

結論

本研究以由天然油脂所得的游離脂肪酸(FFA)為原料，以低溫溶劑結晶法及脂解酵 素催化的選擇性酯化法，以提高 FFA 中 PUFA 之含量。濃縮後之 FFA 以脂解酵素 Novozym 435 催化的酯化反應合成三酸甘油酯，再以脂解酵素 Lipozyme IM 催化此三酸 甘油酯及辛酸之酸解反應以合成 sn-2 位置為 GLA，sn-1,3 位置為辛酸之結構脂質 (1,3-dicapryloyl-2-γ-linolenoyl-glycerol)。各步驟之最佳操作條件及結果如下：

1. 兩階段低溫溶劑結晶法濃縮 GLA：第一階段濃縮：溶劑 = 氰化甲烷，溶劑 = 250 mL，FFA = 4 g，貯存時間 = 48 h，操作溫度 = -40℃。第二階段濃縮：溶劑 = 30%

氰化甲烷+ 70%丙酮，溶劑 = 110 mL，FFA = 1 g，貯存時間 = 24 h，操作溫度 = -80

℃。可將 GLA 由皂化後的 23.4%提升至 92.1%，回收率為 89.3%。

2. 以經兩階段低溫溶劑結晶法濃縮之 FFA(含 92.1% GLA)為選擇性酯化反應之基質。

反應條件：Lipozyme IM = 20 mg，溶劑 = 己烷(以分子篩除水)，丁醇 = 100 mM (以 分子篩除水)，FFA = 100 mM，溫度 = 37 ℃，反應體積 = 5 mL，攪拌轉速 = 600 rpm，反應時間 = 40 min。能進一步提升 GLA 含量至 99.1%，此選擇性酯化反應之 回收率為 81.6%，是以 GLA 純化之後總回收率高達 72.8%。

3. 酯化反應合成 triGLA 之基質為含 98.38% GLA 的 FFA。反應條件：溫度= 40℃，21 mg

分子篩，己烷體積 = 5 mL，FFA = 1.5 mmol，Glycerol/FFA = 6/1 mol/mol，20 mg Novozym 435，反應時間 = 9 h。產物甘油酯中 TG 佔 94.43%，此 TG 之脂肪酸組成 中，GLA 佔 97.94 mol%。

4. 以上述之 triGLA 為酸解反應之基質。反應條件：溫度= 50℃，Caprylic acid/TG = 12/1

10

mol/mol，150 mg Lipozyme，TG = 30 mM，溶劑 = 己烷，反應時間 = 60 h。產物甘 油酯中 TG 佔 99.27%，辛酸在甘油酯中的含量可達 65.65 mol%，分析三酸甘油酯 sn-2 位置之成份結果顯示，結構脂質(1,3-dicapryloyl-2-γ-linolenoyl-glycerol)在 TG 中佔有 72.84 mol%。

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13

圖 1 在以氰化甲烷為溶劑之一階段低溫溶劑結晶法中，溶劑量對濃縮 Borage Oil-FFA 中 GLA 的影響。操作條件：FFA = 3 g，貯存時間 = 24 h。(□,■) = -20℃，(○,●) = -30℃，

(△,▲) = -40℃。空心符號：GLA content，實心符號：GLA yield。

圖 2 在以氰化甲烷及丙酮混合物為溶劑之一階段低溫溶劑結晶法中，操作溫度及溶劑中 的氰化甲烷含量對濃縮 Borage Oil-FFA 中 GLA 的影響。操作條件： FFA = 3 g，溶劑 = 90 mL，貯存時間=24 h。(□,■) = -50℃，(○,●) = -60℃，(△,▲) = -70℃，(▽,▼) = -80

℃。空心符號：GLA content，實心符號：GLA yield。

20 40 60 80 100 120 140 160 180 200

GLA content (area%)

Solvent (mL)

GLA content (area%)

Acetonitrile content in solvent (vol%)

50

14 h。○：ALA content，●：ALA yield；□：EPA + DHA content，■：EPA + DHA yield。

40 60 80 100 120 140 160 180 200

GLA content (area%)

Solvent (mL)

PUFA content (area%)

Solvent (mL)

15

圖 5 FFA 與醇之選擇性酯化反應時，醇鏈長對 GLA 含量及回收率之影響。反應條件：

Lipozyme IM = 20 mg，溫度 = 37℃，FFA conc. = Alcohol conc. = 100 mM，溶劑 = 己烷，

反應體積 = 5 mL，轉速 = 600 rpm，己烷及醇均先以分子篩除水。(□,■) = Ethanol，(○,

●) = 1-Butanol，(△,▲) = 1-Hexanol，(▽,▼) = 1-Octanol，(◇,◆) = 1-Decanol，(☆,★) = 1-Dodecanol。空心符號：GLA content，實心符號：GLA yield。

圖 6 FFA 與丁醇之選擇性酯化反應時，溫度對 GLA 含量及回收率之影響。反應條件除 溫度及醇為丁醇外，其餘條件與圖 5 相同。(□,■) = 25℃，(○,●) = 37℃，(△,▲) = 50

℃。空心符號：GLA content，實心符號：GLA yield。

0 2 0 40 60 8 0 1 0 0 1 2 0

GLA content (area%)

R eactio n tim e (m in)

GLA content (area%)

Reaction time (min)

30

16 content，■：GLA yield。

0 2 0 4 0 6 0 8 0 1 0 0 1 2 0

GLA content (area%)

R e a ctio n tim e (m in )

GLA content (area%)

Butanol concentration (mM)

75

17

圖 9 FFA 與丁醇之選澤性酯化反應時，起始水含量對 GLA 含量及回收率之影響。除水 含量及醇為丁醇外，其餘反應條件與圖 5 相同。 (□,■)=分子篩除水，(○,●) = 1% H2O w/w (水重/反應物重)，(△,▲) = 2% H₂O w/w，(▽,▼) = 3% H₂O w/w。空心符號：GLA content，實心符號：GLA yield。

圖 10 FFA 與甘油之酯化反應時，基質莫耳比對酯化程度的影響。反應條件：溫度 = 50

GLA content (area%)

Reaction time (min)

30

Degree of Esterification

Reaction time (h)

18

圖 11 FFA 與甘油之酯化反應時，基質莫耳比對產物甘油酯中 TG 含量的影響。反應條件 與圖 10 相同。

圖 12 FFA 與甘油之酯化反應時，分子篩對酯化程度的影響。反應條件：除己烷 = 5 mL，

分子篩加入量，Glycerol/FFA = 6/1 mol/mol，FFA = 1.5 mmol 外，溫度 = 40℃外，其餘 條件與圖 10 相同。

Reaction time (h)

0 10 20 30 40 50

mg of molecular sieve added 0

7 21

Degree of esterification (%)

Reaction time (h)

19

圖 13 FFA 與甘油之酯化反應時，分子篩對產物甘油酯中 TG 含量的影響。反應條件與 圖 12 相同。

圖 14 FFA 與甘油之酯化反應時，GLA 含量對酯化程度的影響。反應條件：除己烷 = 5 mL，分子篩加入量，Glycerol/FFA = 6/1 mol/mol ，FFA = 1.5 mmol 外，溫度 = 40℃外，

其餘條件與圖 10 相同。

0 10 20 30 40 50

0 10 20 30 40 50 60 70 80 90 100

mg of molecular sieve added 0

7 21

TG content in glycerides (%)

Reaction time (h)

0 10 20 30 40 50

0 10 20 30 40 50 60 70 80 90 100

92.1% GLA 98.4% GLA

Degree of esterification (%)

Reaction time (h)

20

圖 15 FFA 與甘油之酯化反應時，GLA 含量對酯化程度的影響。對產物甘油酯中 TG 含 量的影響。反應條件與圖 14 相同。

圖 16 基質莫耳比對酸解反應的影響。反應條件：反應溫度 = 50℃，TG = 30 mM，

溶劑 = 己烷，酵素量 = 60 mg Lipozyme IM，反應體積 = 5 mL，磁石攪拌速度 = 600 rpm， TG 及己烷均事先以分子篩除水。Caprylic acid/TG (mol/mol)：(□,■) = 4/1，(○,

●) = 8/1，(△,▲) = 12/1，(▽,▼) = 16/1。空心符號：C8:0 content，實心符號：GLA

TG content in glycerides (%)

Reaction time (h)

0 10 20 30 40 50 60 70 80

C8:0 content in glycerides (mol%)

Reaction time (h)

30

21

圖 17 溫度對酸解反應的影響。反應條件除溫度、酵素量 = 150 mg 與基質比 = 12/1 mol/mol 外，其餘條件與圖 16 相同。(□,■) = 30℃，(○,●) = 40℃，(△,▲) = 50℃，(▽,

▼) = 60℃。空心符號：C8:0 content，實心符號：GLA content。

圖 18 GLA 含量對酸解反應的影響。反應條件除溫度、酵素量 = 150 mg、TG 之 GLA 含量與基質比 = 12/1 mol/mol 外，其餘條件與圖 16 相同。GLA 含量：(□,■) = 91.83 mol%，(○,●) = 97.94 mol%。空心符號：C8:0 content，實心符號：GLA content。

0 1 0 2 0 3 0 4 0 5 0 6 0

C8:0 content in glycerides (mol%)

R ea ctio n tim e (h )

C8:0 content in glycerides (mol%)

Reaction time (h)

30

22

表 1 Borage oil-FFA 濃縮前及濃縮後的脂肪酸組成

Content (area%) Fatty Acid

FFAI^a FFAII^b FFAIII^c 16:0 10.34±0.14 0.07±0.01 0.04±0.01 18:0 3.61±0.03 0.02±0.01 0.02±0.01 18:1 15.89±0.06 1.05±0.04 0.09±0.02 18:2 37.28±0.08 6.71±0.13 0.65±0.12 GLA 23.35±0.07 92.09±0.13 99.13±0.15

20:1 4.25

±

0.05 0.06

±

0.13 0.06

±

0.01

22:1 3.16±0.16 ND^d ND

24:1 2.12

±

0.09 ND ND

GLA

recovery (%) - 89.28±1.20 81.58±2.20 Overall GLA

recovery (%) 89.28±1.20 72.83±1.97

a: 琉璃苣油皂化所得之 FFA。

b: FFAI 經兩階段濃縮後之 FFA。第一階段濃縮：溶劑 = 氰化甲烷，溶劑 = 250 mL，FFA = 4 g，貯存時 間 = 48 h，操作溫度 = -40℃。第二階段濃縮：溶劑 = 30%氰化甲烷 + 70%丙酮，溶劑 = 110 mL，FFA

= 1 g，貯存時間 = 24 h，操作溫度 = -80℃。

c: FFAII 經選擇性酯化反應後所得之 FFA。反應條件：Lipozyme IM = 20 mg，溶劑 = 己烷(以分子篩除水)，

丁醇 = 100 mM (以分子篩除水)，FFA = 100 mM，溫度 = 37 ℃，反應體積 = 5 mL，轉速 = 600 rpm，

反應時間 = 40 min。

d: not detected.

23

表 2 以不同溶劑系統利用一階段低溫溶劑結晶法濃縮皂化琉璃苣油所得 FFA 之結果比 較

Content (area%) Fatty Acid

Acetonitrile^a 30% Acetonitrile^b Acetone^c 16:0 0.18±0.04 0.08±0.02 0.10±0.01

18:0 ND^d ND ND

18:1 1.60±0.24 1.82±0.07 2.14±0.11 18:2 10.37±1.03 8.29±0.07 10.51±0.49 18:3 n6 (GLA) 87.43±1.56 88.94±0.24 86.49±0.55 20:1 0.39±0.27 0.80±0.08 0.60±0.03 22:1 ND 0.08±0.00 0.13±0.01

24:1 ND ND 0.02±0.01

GLA recovery (%) 28.62±0.41 61.95±2.16 38.03±3.95

a:溶劑 = 30 mL 氰化甲烷，FFA = 3 g，操作溫度 = -40℃

b:溶劑 = 30%氰化甲烷 + 70%丙酮，溶劑 = 90 mL，FFA = 3 g，操作溫度 = -80℃

c:溶劑 = 60 mL 丙酮，FFA = 3 g，操作溫度 = -80℃

d:ND：not detected

24

表 3 Borage oil-FFA 經兩階段低溫溶劑結晶法濃縮後之 FFA 組成 Content (area%) Fatty Acid

1^st stage^a 2^nd stage^b 1^st stage^c 2^nd stage^d 16:0 0.12±0.00 0.05±0.01 0.43±0.04 0.07±0.01 18:0 ND^e ND 0.08±0.03 ND 18:1 4.34±0.07 0.69±0.05 12.54±0.05 0.66±0.01 18:2 28.63±0.10 5.31±0.13 50.98±0.04 5.53±0.05 18:3 n6(GLA) 66.09±0.07 93.91±0.18 32.73±0.10 93.7±0.04 20:1 0.70

±

0.14 0.04

±

0.00 2.61

±

0.08 0.05

±

0.00 22:1 0.12

±

0.02 ND 0.55

±

0.02 ND 24:1 ND ND 0.10±0.02 ND Recovery (%) of

GLA 93.04±0.10 92.41±0.41 93.44±0.28 87.79±0.41 Overall recovery

(%) of GLA 93.04 85.98 93.44 82.03

a: 溶劑 = 250 mL 氰化甲烷，FFA = 4 g，操作溫度 = -40℃，貯存時間 = 48 h。

b: 溶劑 = 30%氰化甲烷 + 70%丙酮，溶劑 = 90 mL，FFA = 1 g，操作溫度 = -80℃。

c: 溶劑 = 200 mL 氰化甲烷，FFA = 4 g，操作溫度 = -20℃。

b: 溶劑 = 30%氰化甲烷 + 70%丙酮，溶劑 = 50 mL，FFA = 1 g，操作溫度 = -80℃。

e:ND：not detected.

表 4 酸解反應後三酸甘油酯之 FFA 組成

FFA^b (mol%) FFA^c (mol%) Fatty

Acid

Total sn-1,3 sn-2 Total sn-1,3 sn-2 C8:0 66.36±0.54 88.27 22.55±0.69 65.65±0.40 89.82 17.31±2.27 C16:0 0.13±0.02 0.08 0.24±0.05 0.16±0.20 0.15 0.18±0.01 C18:1 0.33±0.04 0.19 0.61±0.03 0.10±0.02 0.08 0.14±0.01 C18:2 1.67±0.04 1.15 2.72±0.31 0.72±0.06 0.45 1.28±0.17 GLA 31.51±0.46 10.32 73.89±0.40 33.37±0.45 9.51 81.10±2.10

a: 反應條件：除反應時間 = 60 h，酵素量 = 150 mg Lipozyme IM， Caprylic Acid/TG = 12/1 mol/mol 外，

其餘條件與圖 16 相同。

b: 酸解反應前 TG 含 91.83 mol% GLA。

c: 酸解反應前 TG 含 97.94 mol% GLA。

25

計劃成果自評

本計畫為三年期計畫，第一及第二年計畫主要目的是以琉璃苣油皂化所得游離脂肪 酸為原料，先以低溫溶劑結晶法，再利用脂解酵素催化之酯化反應將游離脂肪酸中之 GLA 含量從 23.4%提升至 99.1%，回收率達 72.8%。將此方法應用於其他多元不飽和脂 肪酸之濃縮純化時，除了從亞麻油中濃縮 ALA 尚未達成預定目標(>90%)之外，從琉璃 苣油中濃縮 GLA 及從單細胞油中濃縮 AA（或 DHA）效果都很好。GLA 及 AA（或 DHA）

之純度分別可達 99%及 95%，且回收率都超過 70%。第一年及第二年計畫之成果已發表

之純度分別可達 99%及 95%，且回收率都超過 70%。第一年及第二年計畫之成果已發表

在文檔中 多不飽和脂肪酸之純化與結構脂質之合成(III) (頁 18-34)