Kazuaki Maruyama

Enrichment of polyunsaturated fatty acids withGeotrichum candidum lipase

Three lipases, isolated previously in our laboratory, each with different fatty acid and positional specificities, and a known lipase fromCandida cylindracea were screened for concentrating docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids in glycerides.Geotrichum candidum lipase was found to be suitable for their concentration in glycerides. Tuna oil was treated at 30°C with this lipase for 16 h, and 33.5% hydrolysis resulted in the production of glycerides containing 48.7% of DHA and EPA. The hydrolysis was not increased despite adding further lipase, so the glycerides were extracted, and the reaction was repeated. The second hydrolysis produced glycerides containing 57.5% of DHA and EPA in a 54.5% yield, with recovery of 81.5% of initial DHA and EPA. Of the total glycerides, 85.5% were triglycerides. These results showed thatG. candidum lipase was effective in producing glycerides that contained a high concentration of polyunsaturated fatty acids in good yield.

Production of structured lipid containing docosahexaenoic and caprylic acids using immobilized Rhizopus delemar lipase

Triglycerides with medium-chain fatty acids at the 1- and 3-positions and functional fatty acids at the 2-position are referred to as structured lipids. An attempt was made to produce a structured lipid containing docosahexaenoic and caprylic acids using 1,3-positional specific Rhizopus delemar lipase, which was immobilized on a ceramic carrier by coprecipitation with acetone. When a reaction mixture containing 5 g tuna oil and 10 g caprylic acid, 0.6 g immobilized lipase, and 0.3 g water was incubated at 30°C for 2 d with shaking, acidolysis proceeded effectively but hydrolysis occurred simultaneously. This hydrolysis could be repressed by omitting water from the subsequent reaction mixture. The immobilized lipase could be reused 14 times without any significant loss of activity, and approximately 65% of the fatty acids at the 1- and 3-positions in the tuna oil were exchanged for caprylic acid. All the triglyceride products contained one or two caprylic acid molecule(s). Docosahexaenoic acid bound at the 1(3)-position in tuna oil was not exchanged because of the low activity of the R. delemar lipase on it.

Enrichment of arachidonic acid: Selective hydrolysis of a single-cell oil fromMortierella withCandida cylindracea lipase

Three lipases, isolated previously in our laboratory, and a known lipase fromCandida cylindracea were screened for the enrichment of arachidonic acid (AA). The enzyme fromC. cylindracea was the most effective for the production of oil with high concentration of AA. When a single-cell oil fromMortierella alpina, containing 25% AA, was hydrolyzed with this lipase for 16 h at 35°C, the resulting glycerides contained 50% AA at 52% hydrolysis. After this, no further hydrolysis occurred, even with additional lipase. However, when the glycerides were extracted from the hydrolyzate and were hydrolyzed again with new lipase, the resulting oil contained 60% AA, with a recovery of 75% of its initial AA content. Triglycerides were the main components of the resulting oil. The release of each fatty acid from the oil depended on the hydrolysis rate of its ester. The fatty acid, whose ester is the poorest substrate for the enzyme, is concentrated in the glycerides.

Selective hydrolysis of polyunsaturated fatty acid-containing oil withGeotrichum candidum lipase

Polyunsaturated fatty acids (PUFA), especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), can be concentrated in glycerides by hydrolyzing tuna oil withGeotrichum candidum lipase, the main components in the resulting oil being triglycerides. The reaction mechanism of this selective hydrolysis was investigated. Although the lipase acted well on the esters of oleic, linoleic, and α-linolenic acids, it did not affect the esters of γ-linolenic acid, arachidonic acid, EPA, and DHA as much. The action of PUFA-glycerides was mono-> di- > triglycerides. Furthermore, the condensation of PUFA-partial glycerides and PUFA occurred even in the presence of a large amount of water, and the partial glycerides converted to the triglycerides by transacylation. These results suggested that the PUFA-rich triglycerides were accumulated in the glyceride fraction by the following mechanism: The PUFA-partial glycerides generated by the hydrolysis were converted to PUFA-triglycerides by condensation and transacylation reactions. As the PUFA-triglycerides formed were the poor substrates of lipase, they were accumulated in the reaction mixture.

Enrichment of ethyl docosahexaenoate by selective alcoholysis with immobilized Rhizopus delemar lipase

Abstract We attempted to purify ethyl docosahexaenoate (E-DHA) by the alcoholysis of fatty acid ethyl esters with lipase. Fatty acid ethyl esters originating from tuna oil (E-DHA content, 23 mol%; E-tuna-23) were used as starting materials, and Rhizopus delemar lipase immobilized on a ceramic carrier was used as a catalyst which acted only very weakly on E-DHA. Because the immobilized lipase did not exhibit the alcoholysis activity, it was activated by shaking at 30°C for 24 h in the E-tuna-23/lauryl alcohol mixture to which water (2%) was added. The alcoholysis activity of the lipase increased markedly as a result of this pretreatment, but the hydrolysis activity also increased. The hydrolysis activity was completely repressed by repeating the reaction after transferring the immobilized enzyme into a fresh E-tuna-23/lauryl alcohol mixture without adding additional water. Several factors affecting the alcoholysis of E-tuna-23 were investigated to determine the optimum reaction conditions. When alcoholysis was conducted at 30°C with shaking in a reaction mixture containing E-tuna-23/lauryl alcohol (1 : 3, mol/mol) and the activated lipase (4% of the mixture volume), E-DHA was efficiently enriched in the ethyl ester fraction. By alcoholyzing E-tuna-23 with lauryl alcohol for 50 h under these reaction conditions, the E-DHA content was increased from 23 mol% to 52 mol% in a 90% yield. In addition, when the fatty acid ethyl esters, of which the E-DHA contents were 45 mol% and 60 mol%, were alcoholyzed for 50 h, the contents of E-DHA were increased to 72 mol% and 83 mol%, respectively. In these reactions, the recovery of E-DHA in the ethyl ester fraction was greater than 90%. We termed this new reaction system selective alcoholysis because advantage of the fatty acid specificity of the lipase was taken in this reaction system. To investigate the stability of the immobilized lipase, continual batch reactions were carried out by replacing the reaction mixture with a fresh E-tuna-23/lauryl alcohol mixture every 24 h. The decrease in the extent of alcoholysis was only 15% even after the 47th batch reaction.

Autoxidation Kinetic Analysis of Docosahexaenoic Acid Ethyl Ester and Docosahexaenoic Triglyceride with Oxygen Sensor

The application of ω-3 polyunsaturated fatty acids (PUFAs) as food additives is restricted by their chemically quite reactive properties. However, quantitative analyses of the oxidative kinetics of PUFAs are very few compared to other studies on food chemistry. In this study, the autoxidation kinetics of ethyl docosahexaenoate (DHAEE), docosahexaenoic triglyceride (DHA oil), and emulsified DHA oil were investigated with an oxygen sensor. The autocatalytic reaction rate constants for DHAEE, DHA oil, and the emulsified DHA oil with 20%(w/v) GA, 20% SSPS, or 20% SSPS containing 5% soy protein were obtained at 35, 50, and 70°C. A plot of the natural logarithm of the frequency factor, ln ka0, vs. the activation energy, Ea, demonstrated that ln ka0 against Ea fitted well with a single straight line both for the data from this study and for other reported results. This implies that the chemical compensation relationship holds between k a0 and E a for PUFA and emulsified DHA oil.

One-pot enzymatic synthesis of docosahexaenoic acid-rich triacylglycerols at the sn-1(3) position using by-product from selective hydrolysis of tuna oil

Docosahexaenoic acid (DHA)-rich oil has been industrially produced by selective hydrolysis of tuna oil with a lipase that acts weakly on DHA. The free fatty acids (FFAs) generated in this process as by-products contain a high DHA concentration (46wt%) but are treated as industrial waste. This study attempted to reuse these by-product FFAs using a one-pot process, and succeeded in producing triacylglycerols (TAGs) through the esterification of the by-product FFAs with glycerol using immobilized Rhizomucor miehei lipase. Regiospecific analysis of the resulting TAGs showed that the content of DHA at the sn-1(3) position (51.7mol%) was higher than the content of DHA at the sn-2 position (17.3mol%). The DHA distribution in TAGs synthesized in this study was similar to the DHA distribution in TAGs from seal oil.