TWI901501B - A process for extracting and separating high-concentration DHA and EPA from algae oil using a simulated moving bed (SMB) - Google Patents

Abstract

A process for extracting and separating high-concentration DHA and EPA from algae oil using a simulated moving bed (SMB) mainly comprises: subjecting glycerol-based algae oil to an ethyl esterification reaction to form ethyl ester-based algae oil; introducing the ethyl ester-based algae oil into a first simulated moving bed, wherein the first simulated moving bed adsorption device comprises a plurality of beds containing adsorbent materials; introducing a desorbent stream containing a desorbent into the simulated moving bed; The first simulated moving bed removes C16 and C18 ethyl ester fatty acids, retaining the raffinate of ethyl ester DHA and EPA. The raffinate of ethyl ester DHA and EPA is introduced into the second simulated moving bed to separate the ethyl ester DHA and EPA. After enzymatic translipidation and molecular distillation, high-purity, high-concentration DHA and EPA with a concentration greater than 80% are isolated.

Description

A process for extracting and separating high-concentration DHA and EPA from algae oil using a simulated moving bed (SMB)

The present invention relates to a process for extracting and separating high-concentration DHA and EPA from algae oil using a simulated moving bed (SMB), and more particularly, to a process for separating high-purity, high-concentration DHA and EPA with a concentration greater than 80% using multiple SMBs.

Note: DHA, short for Docosahexaenoic Acid, is an essential omega-3 fatty acid. It's crucial for the human body, but the body cannot produce it naturally and requires dietary supplementation. According to a medical study, DHA, like lutein, supports eye health, helps pregnant women supplement their babies with DHA, aids their growth, provides nutritional support through breastfeeding, enhances concentration and responsiveness in schoolchildren and office workers, and helps promote metabolism and regulate physiological functions. EPA (Eicosapentaenoic acid), like DHA, is an essential omega-3 fatty acid that cannot be synthesized by the body and must be supplemented through the diet. EPA has the following benefits: helping circulation in the body, promoting metabolism, regulating physiological functions and constitution to help maintain health, nourishing and strengthening the body to help maintain spirits, and providing nutrients needed to maintain health before and after childbirth or illness.

Microalgae oils are rich in omega-3 unsaturated fatty acids. Bioscientists are using various methods to produce large quantities of omega-3 fatty acids from microalgae. DHA and EPA, among them, have garnered significant attention as fish oil alternatives. In addition to DHA and EPA, algae oils also contain other unsaturated fatty acids (C16:0), monounsaturated fatty acids (C18:1), and various other fatty acids. Recent clinical studies have shown that DHA and EPA have different functions in the human body. Therefore, isolating high levels of DHA and EPA from algae oil is a crucial issue for the pharmaceutical raw material industry.

Simulated Moving Bed (SMB) is a continuous chromatographic separation technology that can separate two mixtures into pure substances. Compared with traditional batch chromatography, this method continuously separates and extracts the two parts, thereby obtaining a higher yield of pure substances. Simulated Moving Bed technology was developed by UOP (Universal Oil Products) in the 1960s and is widely used in the petrochemical industry. In 2005, the global pharmaceutical industry had approximately 1,500 tons of pharmaceutical intermediates using Simulated Moving Bed technology. Simulated Moving Bed is a continuous process with high product concentration, excellent separation effect, high yield, low solvent consumption, and easy scale-up. To understand the principles of a simulated moving bed (TMB), one must first understand the True Moving Bed (TMB), as shown in Figure 1. The TMB's design primarily allows countercurrent contact between the flushing liquid and the solids. The entire cascade is divided into multiple sections based on the inlet and outlet locations. For example, consider four sections (Sections 1 through 4). The functions of each section are as follows: Section 1: Clean mobile phase washes the solid adsorbent. Section 2: Concentrates adsorbents with strong surface retention on the solid, shown in the diagram as component A. Section 3: Concentrates adsorbents with weak surface retention on the solid, shown in the diagram as component B. Section 4: Clean mobile phase washes the stationary phase.

When a feed containing two adsorbent components, A and B, enters a device with a continuously upward-flowing solid adsorbent, the adsorbents are adsorbed by the solid and then washed away by the downward-flowing mobile phase. Component A, with its stronger retention, is adsorbed by the solid and moves upward, while component B, with its weaker retention, moves downward with the mobile phase. Therefore, the upper section of the feed, the second section in Figure 1, primarily concentrates the more strongly retentive component A, while the lower section of the feed, the third section in Figure 2, concentrates the less retentive component B by displacing the more strongly retentive component. The fourth section in Figure 1 primarily utilizes a solid adsorbent to remove the weakly retentive component B remaining in the mobile phase, preventing it from being carried out of the bed and continuing to circulate, potentially causing cumulative contamination. The first section, on the other hand, uses the mobile phase to flush the strongly retentive component A from the solid adsorbent, preventing it from being carried out of the bed and continuing to circulate, potentially causing contamination. Analogous to the structure of a distillation tower, the second section can be called the enriching section; the third section can be called the stripping section; and the first and fourth sections are analogous to the condenser and reboiler. The first and second sections perform desorption operations, while the third and fourth sections perform adsorption operations.

In light of this, the applicant, through continuous research and experimentation, came up with a process for extracting and separating high-concentration DHA and EPA from algae oil using a simulated moving bed (SMB). By using multiple SMBs, they were able to separate greater than 80% of high-purity, high-concentration DHA and EPA.

The primary objective of this invention is to provide a process for extracting and separating high-concentration DHA and EPA from algae oil using a simulated moving bed (SMB). By performing at least two SMB cycles, greater than 80% of high-purity, high-concentration DHA and EPA can be separated.

The aforementioned process method for extracting and separating high-concentration DHA and EPA from algae oil using a simulated moving bed (SMB) mainly comprises: subjecting glycerol-based algae oil to an ethyl esterification reaction to form ethyl ester-based algae oil; introducing the ethyl ester-based algae oil into a first simulated moving bed, wherein the first simulated moving bed adsorption device comprises a plurality of beds containing adsorbent materials; introducing a desorbent stream containing a desorbent into the simulated moving bed; The first simulated moving bed removes C16 and C18 ethyl ester fatty acids, retaining the raffinate of ethyl ester DHA and EPA. The raffinate of ethyl ester DHA and EPA is introduced into the second simulated moving bed to separate the ethyl ester DHA and EPA. After enzymatic translipidation and molecular distillation, high-purity, high-concentration DHA and EPA with a concentration greater than 80% are isolated.

The aforementioned process utilizes a simulated moving bed (SMB) to extract and separate high-concentration DHA and EPA from algae oil. The first SMB separation primarily removes fatty acids other than C20:n and C22:n, while the second SMB separation separates EPA and DHA, yielding high-purity, high-concentration EPA and DHA.

The aforementioned process for extracting and separating high-concentration DHA and EPA from algae oil using a simulated moving bed (SMB) reactor utilizes ethylated algae oil, ethyl palmitate, ethyl stearate, ethylated fatty acids (EPA ethyl ester (~90%), and DHA ethyl ester (~80%)) as catalysts for the transesterification reaction.

The aforementioned process for extracting and separating high-concentration DHA and EPA from algal oil using a simulated moving bed (SMB) involves a catalytic transesterification reaction, including the following steps: weighing 10 grams of ethylated fatty acid starting material and 1 gram of glycerol, adding them sequentially to a concentration bottle; adding 0.6-1.0 grams of IM-100 enzyme, and conducting a re-transesterification reaction at a vacuum of -400 mmHg, a temperature of 60-80°C, and a rotation speed of 220-260 rpm; terminating the reaction at irregular intervals during the reaction process, separating the reaction solution by centrifugation, sampling 20 μL, and then continuing the re-transesterification reaction with the enzyme and the remaining reaction solution; and diluting 5 μL of the sampled reaction solution with 995 μL of hexane for HPLC analysis.

Please refer to Figure 3, which is a preparation flow chart of the present invention. As shown in the figure, the preparation process of the present invention sets up three ethylated fatty acid components that need to be separated, namely EPA, DHA, and (C16:0+C18:1). The remaining small amount of ethylated fatty acids are temporarily ignored during the screening process under the simulated moving bed (SMB) operating conditions. The retention order of the three ethylated fatty acids in the simulated moving bed (SMB) is from weak to strong: EPA~DHA~(C16:0+C18:1). The present invention ethyl-esterifies glycerol-based algae oil and then separates EPA and DHA in the algae oil through two simulated moving bed (SMB) operations. The first SMB separation mainly removes fatty acids other than C20:n and C22:n, while the second simulated moving bed (SMB) separation separates EPA and DHA to obtain high-purity EPA and DHA.

Figure 4 shows the simulated moving bed (SMB) configuration of the present invention. As shown, the present invention utilizes a simulated moving bed (SMB) apparatus with six columns connected in series and a chromatographic apparatus using 95% ethanol as the mobile phase (or eluent). This invention connects six chromatographic columns in series, with columns C1 and C2 forming the first section; columns C3-C4 forming the second section; and columns C5-C6 forming the third section. The simulated moving bed (SMB) device of this invention has two inlets: the feed (sample feed, i.e., the C5 inlet) and the desorbent (eluent feed, i.e., the C1 inlet); and two outlets: the extract (extract, i.e., the C2 outlet) and the raffinate (extract raffinate, i.e., the C6 outlet). If all feed and outlet positions are simultaneously switched to the next column after a period of time, fixed reverse movement can be simulated. For example, the feed port is switched from C5 to C6, and the remaining ports are also switched to the next column. At the same time, the flushing agent and feed continue to flow continuously to the raffinate end. If the positions of the feed ports are continuously switched, the solid will flow in the opposite direction and circulate repeatedly, thus achieving a process of continuous countercurrent flow contact between the stationary phase and the mobile phase.

The preparation process of the present invention primarily comprises the following steps: 1. Ethylation Reaction A1: Prepare glycerol-based algae oil 11, ethylate the glycerol-based algae oil 11 to form ethyl ester-based algae oil 12, and recover solvent 2 by vacuum distillation. In this embodiment, the ethyl esterification method comprises: a. Weigh 350 g of algae oil and add it to a reactor; b. Weigh 3.5 g of sodium hydroxide and dissolve it in 2625 mL of anhydrous ethanol; c. Purge the reactor with nitrogen, then add anhydrous ethanol containing sodium hydroxide, heat to 60°C, and stir to react; d. Start timing when the reaction solution temperature reaches 60°C, and react for 2 hours; e. After the reaction is completed, vacuum concentrate the solution (50°C) and centrifuge at 10,000 rpm for 10 minutes to separate the glycerol; f. Wash the ethyl esterified algae oil with warm water (40°C) (add an equal volume of DDW) to remove any residual alkaline catalyst, centrifuging for 10 minutes each time. min, washed to neutrality, then dehydrated with anhydrous magnesium sulfate and filtered. 2. First Simulated Moving Bed (SMB) A2: The ethyl ester algal oil 12 from Step 1 enters the first simulated moving bed (SMB) where molecular distillation removes the C16 and C18 low-molecular-weight fatty acids (low-molecular-weight fatty acids C16:0-EE + C18:1-EE) 3, retaining the C20:n and C22:n fatty acids to form ethyl ester EPA + DHA (EPA-EE + DHA-EE) 4. The solvent 2 is then recovered by vacuum distillation. The C16 and C18 low-molecular-weight fatty acids 3 can be used in soaps, detergents, and other daily necessities, as well as in feed. After the first simulated moving bed (SMB) separation, ethyl ester EPA+DHA4 should elute from the R-terminal (Raffinate), while C16 and C18 low-molecular-weight fatty acids 3 should elute from the E-terminal (Extract). Solvent 2 is recovered by vacuum distillation. The resulting ethyl ester EPA+DHA4 product has a purity of 1.00 and a recovery rate of 95.7%.

The experiment was conducted at the flow rate of the first simulated moving bed (SMB) separation with a switching time of 4'28". The resulting area fractions, purities, and recoveries are shown in Tables 1 and 2. The results show that both C16 and C18 low-molecular-weight fatty acids 3 appeared at the Extract outlet, while ethyl ester-type EPA+DHA 4 appeared at the Raffinate outlet. The purities at the Raffinate and Extract outlets were 1.000 and 0.918, respectively, indicating excellent separation. The majority of impurities remained at the Extract outlet. The recovery at the Raffinate outlet was 0.957, indicating high production efficiency. The primary objective of the experiment was to separate EPA and DHA. Since EPA and DHA were not yet separated at the Raffinate outlet, the resulting solution was concentrated, and the semi-finished product was subjected to a second separation. Table 1 Table 2 3. Second simulated moving bed (SMB) A3: The ethyl ester EPA+DHA 4 from Step 2 above enters the second simulated moving bed (SMB) to separate the ethyl ester EPA (EPA-EE) 5 and the ethyl ester DHA (DHA-EE) 6. After this second SMB separation, the ethyl ester EPA (EPA-EE) 5 should elute from the R-terminal (Raffinate) and the ethyl ester DHA (DHA-EE) 6 should elute from the E-terminal (Extract). Solvent 2 is recovered by vacuum distillation.

At the flow rate of the first separation experiment, when the switching time was 4'15", the area fraction, purity and recovery rate were shown in Tables 3 and 4. The results showed that the purity of DHA at the extract end was 76.1%, and the purity of EPA at the raffinate end was 97.3%. The purity of ethyl ester EPA (EPA-EE) 5 and ethyl ester DHA (DHA-EE) 6 was greater than 80%. %, so the overall experimental flow rate was reduced. When the switching time was increased to 6'55" at the reduced flow rate, the area fraction, purity, and recovery rates were reported in Tables 3 and 4. The results show that the final purity of DHA on the extract side was 80.7%, and the purity of EPA on the raffinate side was 95.7%. The recoveries were 98.1% and 95.5%, respectively. These results met the restriction requirements, with both EPA and DHA purities exceeding 80%. Table 3 Table 4 4. Enzyme transesterification reaction A4. Ethyl ester EPA (EPA-EE) 5 and ethyl ester DHA (DHA-EE) 6, separated in step 3, were catalyzed using an immobilized enzyme. First, the effects of the ratio of glycerol to ethyl ester algal oil, enzyme addition, and reaction temperature on the re-transesterification reaction were investigated using ethyl esterified algal oil. Finally, operating conditions were selected that ensured a triglyceride content >50% in the reaction product. Under these selected operating conditions, a kinetic model for the transesterification reaction was established. By fitting the established kinetic model with experimental results, the reaction rate constant for the re-transesterification reaction was estimated, and it was inferred that the immobilized enzyme and reaction conditions selected in this study exhibited high catalytic activity for EPA ethyl ester and stearic acid mono- and diglycerides.

The enzyme transesterification reaction uses five ethylated fatty acid materials: ethylated algae oil, ethyl palmitate, ethyl stearate, EPA ethyl ester (~90%), and DHA ethyl ester (~80%). 10 grams of the ethyl ester fatty acid raw materials and 1 gram of glycerol were weighed and added to a concentration bottle in that order. 0.6-1.0 grams of IM-100 enzyme was then added. The transesterification reaction was performed at a vacuum of -400 mmHg, a temperature of 60-80°C, and a rotation speed of 220-260 rpm. The reaction was terminated at irregular intervals during the reaction process. After centrifugation, a 20-μL sample was taken. The enzyme and the remaining reaction solution were then subjected to further transesterification. 5 μL of the sampled reaction solution was diluted with 995 μL of hexane and analyzed by HPLC. Under the specified reaction conditions and procedures, ethylated algal oil, ethyl palmitate, and ethyl stearate all yielded products with triglyceride contents exceeding 50% after two days of reaction. However, ethyl ester EPA (EPA-EE)5 and ethyl ester DHA (DHA-EE)6 failed to yield products with triglyceride contents exceeding 50%. Furthermore, the conversion rate of ethyl ester DHA (DHA-EE) was found to be low, and the corresponding triglyceride ratio was the lowest. Clearly, the reaction conditions were not suitable for the re-transesterification of ethyl ester EPA (EPA-EE)5 and ethyl ester DHA (DHA-EE)6. In the future, further screening of reaction conditions for the re-transesterification reaction of ethyl ester EPA (EPA-EE) 5 and ethyl ester DHA (DHA-EE) 6 should be conducted to ensure that the triglyceride content of the reaction product is >50%. If further adjustment of the reaction conditions still fails to achieve a triglyceride content >50%, molecular distillation can be used to increase the triglyceride content to above 50%. 5. Molecular Distillation A5: After the aforementioned enzymatic transesterification reaction, ethyl ester EPA (EPA-EE) 5 and ethyl ester DHA (DHA-EE) 6 can be molecularly distilled to obtain high-purity triglyceride EPA (EPA-TG) 7 and high-purity triglyceride DHA (DHA-TG) 8.

This invention used glycerol-based algae oil 11 as the raw material and conducted two simulated moving bed (SMB) separation experiments. The results showed that the first separation experiment produced an EPA+DHA product with a purity of 1.00 and a recovery rate of 95.7%. The second separation experiment produced products with a purity of 95.7% EPA and 80.7% DHA, respectively, with recoveries of 98.1% and 95.5%. These validation results demonstrate that the simulated moving bed (SMB) process can produce algae oil products with EPA and DHA purities exceeding 80%.

The above embodiments are merely for illustrating preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Slight modifications and changes may be made without losing the essence of the present invention or departing from the spirit and scope of the present invention.

In summary, this invention uses glycerol-based algae oil as the raw material, separating DHA and EPA via a first simulated moving bed process. Low-molecular-weight fatty acids (C16 and C18) are removed by molecular distillation, leaving the ethyl ester DHA and EPA residue. This ethyl ester DHA and EPA residue is introduced into a second simulated moving bed process to separate the ethyl ester DHA and EPA. Subsequently, through enzymatic translipidation and molecular distillation, high-purity DHA and EPA with a purity exceeding 80% are isolated. This practical design is truly innovative and creative. We have filed a patent application in accordance with the law and hope that the FDA will review and approve the patent as soon as possible. We are deeply grateful.

11: Glycerol-based algae oil 12: Ethyl ester glycerol 2: Vacuum distillation to recover solvent 3: C16 and C18 low molecular weight fatty acids 4: Ethyl ester EPA + DHA (EPA-EE + DHA-EE) 5: Ethyl ester EPA (EPA-EE) 6: Ethyl ester DHA (DHA-EE) 7: Triglyceride EPA (EPA-TG) 8: Triglyceride DHA (DHA-TG) A1: Ethyl esterification reaction A2: First simulated moving bed (SMB) reaction A3: Second simulated moving bed (SMB) reaction A4: Enzyme transesterification reaction A5: Molecular distillation

Figure 1 is a schematic diagram of a conventional TMB. Figure 2 is a diagram of a conventional simulated moving bed (SMB) configuration. Figure 3 is a flow chart of the preparation process of the present invention. Figure 4 is a diagram of the simulated moving bed (SMB) configuration of the present invention.

11: Glycerol algae oil

12: Ethyl ester algae oil

2: Vacuum distillation to recover solvent

3: C16 and C18 low molecular weight fatty acids

4: Ethyl ester type EPA+DHA (EPA-EE+DHA-EE)

5: Ethyl ester EPA (EPA-EE)

6: Ethyl ester DHA (DHA-EE)

7: Triglyceride-type EPA (EPA-TG)

8: Triglyceride DHA (DHA-TG)

A1: Ethyl esterification reaction

A2: First Simulation of a Mobile Bed (SMB)

A3: Second Simulated Moving Bed (SMB)

A4: Enzyme transesterification reaction

A5: 4th-grade molecular distillation

Claims (3)

A process for extracting and separating high-concentration DHA and EPA from algae oil using a simulated moving bed (SMB) reactor comprises the following steps: Ethyl esterifying glycerol-based algae oil to form ethyl ester-based algae oil; Removing C16 and C18 low-molecular-weight ethyl ester fatty acids from the ethyl ester-based algae oil using a first simulated moving bed (SMB) cycle to retain ethyl ester-based EPA + DHA (EPA-EE + DHA-EE); Separating ethyl ester-based EPA (EPA-EE) and ethyl ester-based DHA (DHA-EE) from the ethyl ester-based EPA + DHA (EPA-EE + DHA-EE) using a second simulated moving bed (SMB) cycle; The separated ethyl ester EPA and ethyl ester DHA are subjected to a transesterification reaction catalyzed by an immobilized enzyme; high-purity triglyceride EPA (EPA-TG) and high-purity triglyceride DHA (DHA-TG) are then obtained by distillation. A process for extracting and separating high-concentration DHA and EPA from algae oil using a simulated moving bed (SMB) as described in claim 1, wherein the transesterification reaction uses ethylated algae oil, ethyl palmitate, ethyl stearate, ethylated fatty acids of EPA ethyl ester (~90%) and DHA ethyl ester (~80%) as catalysts. The process for extracting and separating high-concentration DHA and EPA from algal oil using a simulated moving bed (SMB) as described in claim 1 or 2, wherein the transesterification reaction comprises the following steps: Weighing 10 grams of the ethylated fatty acid starting material and 1 gram of glycerol, and adding them sequentially into a concentration bottle; Adding 0.6-1.0 grams of IM-100 enzyme, and conducting a transesterification reaction at a vacuum of -400 mmHg, a temperature of 60-80°C, and a rotation speed of 220-260 rpm.