TW201912788A - A lipase packed bed bioreactor for continuous synthesis of docosahexaenoates and eicosapentaenoates - Google Patents

Abstract

The present invention relates to a process for continuous synthesis of docosahexaenoic acid esters and eicosapentaenoic acid esters by using a lipase-packed-bed bioreactor and using immobilized lipase as a catalyst. The fatty acid, contents more than 30% of total docosahexaenoic acid and eicosapentaenoic acid, was used as raw materials for esterification reaction. After pass through the lipase-packed-bed bioreactor with alcohols or esters, the fatty acid was esterified to docosahexaenoic acid esters and eicosapentaenoic acid esters. The present invention is a method for continuous synthesis of docosahexaenoic acid esters and eicosapentaenoic acid esters by esterification reaction using a lipase packed-bed bioreactor, and the continuous production can reduce the production cost and improve the efficiency and yield.

Description

   Lipase packed bed bioreactor for continuous synthesis of docosahexaenoate and eicosapentaenoate   

The invention relates to a method for manufacturing docosahexaenoic acid esters and eicosapentaenoic acid esters, and more particularly to a lipase packed bed bioreactor for continuous production of docosahexan Methods for enoates and eicosapentaenoates.

Fish oil is rich in long-chain n-3 polyunsaturated fatty acids (PUFA), such as docosahexaenoic acid (DHA; C22: 6) and eicosapentaenoic acid; EPA; C20: 5). The biological effects of DHA and EPA have been extensively studied and can lower blood pressure, blood lipids, cholesterol, treat cardiovascular disease, anti-inflammatory and inhibit allergic reactions, promote the formation of the human brain and retina and delay the aging of the brain.

In fish oil, 10-25% of the total fatty acids in its triglycerides are n-3 polyunsaturated fatty acids, and its medical and health effects are not ideal. As a health food-grade application, the total content of DHA and EPA is generally greater than 30%, the total content of DHA and EPA for pharmaceutical grade applications must be greater than 60%. If raw fish oil is to be produced as a health product or medicine, physical or chemical methods need to be used to increase the content of DHA and EPA, thereby enhancing its medical and health effects.

The production of the currently disclosed DHA esters and EPA esters, such as the method mentioned in the literature of Poisson et al. (Journal of Biotechnology 91 (2001) 75-81), uses fats and oils rich in DHA and EPA as raw materials, and ethanol It uses lipase for transesterification, but this method has the disadvantages of long time, batch production, low synthesis conversion rate and low DHA and EPA content of the product.

In order to overcome the shortcomings of long time, batch production, low synthesis conversion rate and low DHA and EPA content of the product, the present invention provides a fast, effective and continuous operation for a long time to produce high content of DHA esters and EPA Ester method.

Embodiments of the present invention provide a method for continuously producing esters containing DHA and EPA using a lipase packed bed bioreactor, which increases the chance of collision between enzymes and substrates, and improves the conversion rate of ester synthesis moles.

In order to achieve the above purpose and effect, the creative department includes the following steps, using fatty acids with high content of DHA and EPA (the total content of DHA and EPA is greater than 30%) as raw materials and alcohols or esters to pump to the lipase filling In the bed bioreactor, the reactor is filled with immobilized lipase to continuously synthesize DHA esters and EPA esters.

The total content of DHA and EPA of fatty acids used in this method is greater than 30%.

The general formula of alcohol used in this method is R1OH, and R1 may be C1-20 alkyl.

The general formula of esters used in this method is R2 COOR3, R2 and R3 may be the same or different C1-20 alkyl.

In this method, the temperature is 30-80 ° C.

The residence time of the reactants in this method is 0.1-60 minutes.

In this method, ultrasonic assistance can be used to increase the conversion rate of the synthetic mole, in which the ultrasonic frequency is 37 to 80 Hz.

The enzyme packed in the packed bed reactor in this method is immobilized lipase to catalyze the synthesis of DHA and EPA esters by fatty acids and alcohols or esters.

Therefore, this creation can be said to be a very practical and progressive creation, which is worthy of promotion by the industry and made public.

The first figure is a device diagram of a lipase packed bed bioreactor according to the first embodiment of the present invention.

The second diagram is a gas chromatography analysis diagram of the fatty acid raw material used in the present invention.

The third diagram is a liquid chromatography analysis diagram of the fatty acid raw material used in Example 1 of the present invention.

The fourth diagram is a liquid chromatography analysis diagram of the product produced after the fatty acid raw material flows through the lipase packed bed bioreactor according to Embodiment 1 of the present invention.

The present invention relates to a method for continuously synthesizing DHA esters and EPA esters using a lipase packed bed bioreactor, using fatty acids and alcohols or esters to pump to the lipase packed bed bioreactor, please Referring to the first figure, after catalyzed by lipase, DHA esters and EPA esters are synthesized. Among the fatty acids used, the total content of DHA and EPA is greater than 30%. The general formula of alcohol used is R1OH, and R1 may be C1-20 alkyl. The general formula of the esters used is R2 COOR3, R2 and R3 may be the same or different C1-20 alkyl.

For the device, please refer to the first figure. This example is the first example. The fatty acid raw materials used are analyzed by gas chromatography as shown in the second figure. The DHA and EPA contents are 53% and 17%, respectively. Ethyl acetate is used as a solvent to configure the fatty acid to a concentration of 100 mM, and it is evenly mixed with a magnetic stirrer (as shown in (a) of the first figure), and the immobilized lipase is packed in a packed bed bioreactor (as shown in the first figure (c )), Use the pump (as shown in the first figure (b)) to introduce the reaction solution with a volume flow of 1 mL / min into the packed bed bioreactor, which is a metal column with a length of 25 cm and an inner diameter 0.46cm, the temperature is set to the reaction temperature of 60 ℃, the product continuously flows out of the lipase packed bed bioreactor and is collected (as shown in the first figure (e)), the collected product is detected by liquid chromatography and the molar conversion is calculated The liquid chromatograms of the product before the reaction and after passing through the column are shown in the third and fourth figures, respectively, and the molar conversion of the DHA ethyl ester and EPA ethyl ester products is 98%.

Example 2 The fatty acid raw materials used have DHA and EPA contents of 53% and 17%, respectively, and the fatty acids are prepared to a concentration of 300 mM with ethyl acetate as the solvent, and mixed uniformly with a magnet stirrer, and the immobilized lipase is filled into the filling In the bed bioreactor (as shown in the first picture (c)), the pump (as shown in the first picture (b)) is used to introduce the reaction solution with a volume flow rate of 3 mL / min into the packed bed bioreactor. The vessel is a metal column with a length of 25 cm, an inner diameter of 0.46 cm, and the temperature is set at a reaction temperature of 60 ° C. The molar conversion of the DHA ethyl ester and EPA ethyl ester product is 95%.

Embodiment 3: The fatty acid raw materials used have DHA and EPA contents of 53% and 17%, respectively. The ethyl acetate is used as the solvent to configure the fatty acid to a concentration of 500 mM, and the magnetic stirrer is used to mix evenly, and the immobilized lipase is filled into the filling. In the bed bioreactor (as shown in the first picture (c)), the pump (as shown in the first picture (b)) is used to introduce the reaction solution with a volume flow rate of 5 mL / min into the packed bed bioreactor. The vessel is a metal column with a length of 25 cm, an inner diameter of 0.46 cm, and the temperature is set to a reaction temperature of 60 ° C. The molar conversion of the DHA ethyl ester and EPA ethyl ester product is 86%. Under the same conditions, after the lipase packed bed bioreactor is assisted by 37 Hz ultrasound, the molar conversion of DHA ethyl ester and EPA ethyl ester product can be 93%.

In Example 4, under the conditions of Example 1, the operation was continued for 5 days for testing, and the molar conversion rate of the DHA ethyl ester and EPA ethyl ester products was still 98%.

In the fifth embodiment, under the conditions of the first embodiment, the lipase packed bed bioreactor was assisted by 37 Hz ultrasound, and the operation was continued for 5 days for testing. The molar conversion rate of the DHA ethyl ester and EPA ethyl ester product was still 98%.

In the sixth embodiment, the ultrasound-assisted product in the third embodiment and the product in the fifth embodiment are each taken 100ml of the product in a vacuum concentrator at 80 ° C to remove ethyl acetate and acetic acid, respectively, to obtain 15.9g and 3.15g of product The molar conversion of the esterification reaction is 98% and 93%, respectively.

Example 7: The fatty acid raw materials used have DHA and EPA contents of 53% and 17%, respectively, and the fatty acid is formulated with ethyl acetate as the solvent to a concentration of 400mM, and immobilized lipase 25mg is added for batch reaction at a temperature of 50 ° C. , Shaking and stirring at 150rpm / min. After eight hours of reaction, the molar conversion of the esterification reaction was 97%.

Although the present invention has been disclosed as several embodiments above, it is not intended to limit the present invention. Any person with ordinary knowledge in this technical field can do any modification and retouching without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope defined in the appended patent application.

Claims (9)

    A method for catalyzing the synthesis of DHA esters and EPA esters using a lipase packed bed bioreactor, which includes the following steps: using fatty acids with high content of DHA and EPA (the total content of DHA and EPA is greater than 30%) as raw materials and alcohol The esters or esters are pumped into the lipase packed bed reactor. The reactor is filled with immobilized lipase. Through the lipase catalytic reaction, the products of DHA esters and EPA esters are continuously synthesized.         According to the method described in item 1 of the patent application range, the total content of DHA and EPA of the fatty acids used is greater than 30%. .         The method of synthesizing DHA esters and EPA esters according to the lipase packed bed reactor described in item 1 of the patent scope, wherein the general formula of alcohols used is R1OH, and R1 may be C1-20 alkyl.         The method of synthesizing DHA esters and EPA esters according to the lipase packed bed reactor described in item 1 of the patent scope, wherein the general formula of the esters used is R2 COOR3, R2 and R3 may be the same or different C1-20 alkyl.         According to the method of synthesizing DHA esters and EPA esters in the lipase packed bed reactor as described in item 1 of the patent application scope, ultrasonic waves can be used to increase the conversion rate. The frequency of ultrasonic waves is 37 to 80 Hz.         The method for synthesizing DHA esters and EPA esters according to the lipase packed bed reactor described in item 1 of the patent application scope, wherein the temperature is 30-80 ° C.         The method for synthesizing DHA esters and EPA esters according to the lipase packed bed reactor described in item 1 of the patent application scope, wherein the residence time of the reactants is 0.1-60 minutes.         The method for synthesizing DHA esters and EPA esters according to the lipase packed bed reactor described in item 1 of the patent scope, wherein the enzyme packed in the packed bed reactor is immobilized lipase.         According to the method for synthesizing DHA esters and EPA esters described in item 1 of the patent application scope, immobilized lipase can be used under batches, the amount of immobilized lipase added is 0.01-5%, and the reaction temperature 30-80 ℃, reaction time 1-8 hours.