Purification of docosahexaenoic acid from tuna oil by a two-step enzymatic method: Hydrolysis and selective esterification |
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Authors: | Yuji Shimada Kazuaki Maruyama Akio Sugihara Shigeru Moriyama Yoshio Tominaga |
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Affiliation: | (1) Maruha Corp., 300-42 Ibaraki, Japan;(2) Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Joto-ku, 536 Osaka, Japan |
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Abstract: | Purification of docosahexaenoic acid (DHA) was attempted by a two-step enzymatic method that consisted of hydrolysis of tuna
oil and selective esterification of the resulting free fatty acids (FFA). When more than 60% of tuna oil was hydrolyzed with
Pseudomonas sp. lipase (Lipase-AK), the DHA content in the FFA fraction coincided with its content in the original tuna oil. This lipase
showed stronger activity on the DHA ester than on the eicosapentaenoic acid ester and was suitable for preparation of FFA
rich in DHA. When a mixture of 2.5 g tuna oil, 2.5 g water, and 500 units (U) of Lipase-AK per 1 g of the reaction mixture
was stirred at 40°C for 48 h, 83% of DHA in tuna oil was recovered in the FFA fraction at 79% hydrolysis. These fatty acids
were named tuna-FFA-Ps. Selective esterification was then conducted at 30°C for 20 h by stirring a mixture of 4.0 g of tuna-FFA-Ps/lauryl alcohol (1:2, mol/mol), 1.0 g water, and 1,000 U of Rhizopus delemar lipase. As a result, the DHA content in the unesterified FFA fraction could be raised from 24 to 72 wt% in an 83% yield.
To elevate the DHA content further, the FFA were extracted from the reaction mixture with n-hexane and esterified again under the same conditions. The DHA content was raised to 91 wt% in 88% yield by the repeated
esterification. Because selective esterification of fatty acids with lauryl alcohol proceeded most efficiently in a mixture
that contained 20% water, simultaneous reactions during the esterification were analyzed qualitatively. The fatty acid lauryl
esters (L-FA) generated by the esterification were not hydrolyzed. In addition, L-FA were acidolyzed with linoleic acid, but
not with DHA. These results suggest that lauryl DHA was generated only by esterification. |
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Keywords: | Docosahexaenoic acid hydrolysis lipase Pseudomonas sp Rhizopus delemar selective esterification tuna oil |
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