Method for catalytic synthesis of OPDHA structural lipid by lipase

Abstract

The application relates to the technical field of functional lipid biological processing, and discloses a method for synthesizing OPDHA structural fat by catalysis of lipase, which comprises the following steps: S1: mixing fish oil, a phosphate buffer and Lipozyme RM IM lipase, and then stirring and reacting in a constant-temperature water bath to promote hydrolysis and release of DHA in the fish oil, so that a sample rich in free DHA is obtained; and S2: using specific immobilized lipase to catalyze an acidolysis reaction among glyceryl tripalmitate, oleic acid and the sample rich in free DHA, so that OPDHA structural fat is finally obtained. The product highly simulates the sn-2 palmitic acid positioning characteristics of breast milk fat in the glyceride stereoscopic configuration, and reasonably introduces oleic acid and DHA to sn-1,3 positions, so that the nutritional demands of infants and young children for high-efficiency absorption of palmitic acid and physiological efficiency of DHA are considered from the molecular structure level, and a more comprehensive breast milk fat solution is provided.

Description

Technical Field

[0001] This invention belongs to the technical field of functional lipid bioprocessing, and more specifically relates to a method for synthesizing OPDHA structured lipids catalyzed by lipase. Background Technology

[0002] Docosahexaenoic acid (DHA) plays a crucial role in brain and visual development, and fish oil is an important source of DHA. However, in natural fish oil, DHA usually exists in the form of triglycerides along with various other fatty acids. Its content is limited, and its position within the triglycerides is random, which may not achieve optimal bioavailability, thus restricting its application in high-purity health supplements or pharmaceuticals. Traditional chemical purification methods are often cumbersome and subject to harsh conditions, easily leading to DHA oxidation or isomerization, and lacking selectivity. Therefore, developing a purification method that is mild, highly selective, and efficient in enriching DHA is of great value.

[0003] Triglycerides account for over 98% of breast milk fat, with the sn-2 position mostly composed of saturated fatty acids, especially palmitic acid, while the sn-1 and sn-3 positions are composed of unsaturated fatty acids. This unique structure is beneficial for infants' absorption of lipids and calcium, promoting bone development and gut health, and is also crucial for the development of the infant's brain and retina. Traditional formula milk powder often uses a physical mixture of vegetable oils, fish oils, or algal oils to mimic the fatty acid composition of breast milk, but its triglyceride stereostructure (sn-position distribution) is significantly different from that of breast milk. Current technology lacks an efficient synthetic method that can simultaneously and effectively position palmitic acid at the sn-2 position and rationally introduce DHA and oleic acid into the sn-1 and sn-3 positions of structured lipids. Chemical synthesis suffers from poor position selectivity, harsh reaction conditions, and the easy generation of byproducts. Therefore, developing an enzymatic process with high position selectivity, mild conditions, and precise synthesis of OPDHA structured lipids is of great significance. Summary of the Invention

[0004] The main objective of this invention is to address the aforementioned problems by providing a method for synthesizing OPDHA structured lipids using lipase catalysis. This method offers mild, highly selective, and efficient synthesis of OPDHA structured lipids, closely mimicking the localization pattern of key functional fatty acids in breast milk fat. It solves the problems of fat absorption, calcium absorption, and stool softening in infant formula, while also introducing bioactive DHA to provide a more comprehensive solution for breast milk-like fat.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution: A method for synthesizing OPDHA structured lipids catalyzed by lipase includes the following steps: S1: Fish oil, phosphate buffer, and Lipozyme RM IM lipase were mixed and stirred in a constant temperature water bath. Ethanol was then added to terminate the enzyme reaction. Hexane and water were added for extraction, and the aqueous phase was separated and retained. The pH of the aqueous phase was adjusted to 1.0, and hexane was added for extraction. The organic phase was separated and retained. The solvent was removed by rotary evaporation of the organic phase to obtain a sample rich in free DHA. S2: Tripalmitoyl glycerol, oleic acid and a sample rich in free DHA are mixed, then immobilized lipase is added and the mixture is heated in a constant temperature water bath. After the reaction is completed, the mixture is centrifuged and the upper oil is collected and dissolved in hexane. KOH-ethanol solution is added to remove free fatty acids, thus obtaining OPDHA structured lipids.

[0006] First, this invention promotes the hydrolysis of DHA in fish oil by selecting the lipase Lipozyme RM IM and adjusting the hydrolysis conditions, converting it into free fatty acids. In the subsequent separation step, the free fatty acids are enriched in the aqueous phase as carboxylate ions, while being separated from the large amount of unhydrolyzed or partially hydrolyzed neutral esters. The aqueous phase is then acidified to pH 1.0 to completely protonate the fatty acids, and they are recovered by back-extraction with n-hexane, ultimately yielding a mixture of free fatty acids with a significantly higher DHA content than the original fish oil.

[0007] Then, a specific immobilized lipase was used to catalyze the acidolysis reaction between trimalmitoyl glycerol, oleic acid, and the DHA-rich free fatty acid mixture obtained in the first step. Under the strictly sn-1,3 region-selective catalysis of the immobilized enzyme, the palmitic acid at the sn-1 and sn-3 positions of the trimalmitoyl glycerol molecule was directionally replaced by free oleic acid and DHA in the system, while the palmitic acid at the sn-2 position was completely retained due to steric hindrance recognition by the enzyme, ultimately yielding an OPDHA-structured lipid. This product highly mimics the sn-2 palmitic acid positioning characteristics of breast milk fat in its triglyceride stereoconfiguration, while rationally introducing oleic acid and DHA to the sn-1,3 positions, thus addressing the nutritional needs of infants for efficient palmitic acid absorption and DHA physiological efficacy at the molecular structure level.

[0008] More preferably, in S1, the amount of lipase Lipozyme RM IM added is 2-3% of the fish oil mass; the pH value of the reaction system obtained after mixing fish oil, phosphate buffer and lipase Lipozyme RM IM is 6.0-7.0, and the water-to-oil ratio is 1.5-2.5; the temperature of the constant temperature water bath is 30-40℃; and the stirring reaction time is 8-15h.

[0009] More preferably, in S2, the molar ratio of the tripalmitoyl glycerol to the total molar ratio of oleic acid and DHA in the sample rich in free DHA is 1:12-14; the molar ratio of oleic acid to DHA in the sample rich in free DHA is 1:0.8-1; the amount of immobilized lipase added is 7-10% of the total substrate mass; the temperature of the constant temperature water bath is 55-60℃; and the heating reaction time is 2-4 hours.

[0010] More preferably, in S2, the immobilized lipase is Lipozyme RM IM lipase.

[0011] More preferably, in step S2, the method for preparing the immobilized lipase includes the following steps: (1) Dissolve hexadecyltrimethylammonium bromide and urea in water to obtain a template agent solution; mix tetraethyl orthosilicate, octadecyltriethoxysilane and 3-aminopropyltriethoxysilane to obtain a mixed solution; add the mixed solution dropwise to the template agent solution and perform a hydrothermal reaction. After the reaction is completed, filter and collect the product, wash and calcine to obtain a mesoporous SiO2 support with a pore size of 5-10 nm. (2) After the mesoporous SiO2 support swells in sodium carbonate buffer, sodium chloroacetate is added and reacted; the support is collected by filtration, washed until neutral, and then zinc chloride solution is added and reacted; the support is collected by filtration, and washed until no free Zn is found. 2+ Cl - The zinc ion chelated mesoporous SiO2 support was detected, dried, and obtained. (3) The zinc ion chelate mesoporous SiO2 carrier was added to phosphate buffer to swell, the free enzyme was added, and the immobilized enzyme was adsorbed; the immobilized enzyme was collected by filtration and dried to obtain immobilized lipase.

[0012] Immobilized lipase was first synthesized using an organic-inorganic hybrid mesoporous SiO2 support, the pore walls of which were simultaneously modified with octadecyl long chains and active amino groups. The octadecyl groups provided strong hydrophobic microdomains, enabling the adsorption of lipase through hydrophobic interactions and facilitating the opening of its "cap" domain, resulting in its active conformation. The active amino group was subsequently converted into an iminodiacetic acid (IDA) chelating group via a mild carboxymethylation reaction, which was then coordinated with Zn. 2+ This forms immobilized metal ion affinity adsorption sites. When free lipase comes into contact with this carrier, the enzyme molecule binds to Zn via histidine residues on its surface. 2+ On the one hand, coordination occurs; on the other hand, the enzyme binds to the octadecyl chain through hydrophobic regions. These two forces work synergistically to firmly and orderly immobilize the enzyme within and on the surface of the mesoporous channels. This dual-mode immobilization strategy not only improves enzyme loading and operational stability, but the presence of the hydrophobic interface also pre-activates the lipase, enabling it to exhibit higher initial activity in subsequent catalytic reactions.

[0013] Meanwhile Zn 2+ By regulating the spatial conformation of the enzyme's active site through electron transfer, the hydrophobic pocket structure of the active site becomes more compatible with the molecular structures of DHA and oleic acid, enhancing the enzyme's specific binding ability to DHA and oleic acid and promoting the acyl transfer reaction at the sn-1,3 positions. Furthermore, Zn... 2+ The coordination effect of the enzyme can stabilize the binding of palmitic acid at the sn-2 position to the enzyme active site, inhibit the migration of palmitic acid from the sn-2 position to the sn-1,3 position, reduce the generation of by-products, and further improve the structural selectivity.

[0014] Furthermore, the pore size limitation of the mesoporous SiO2 support allows enzyme molecules to enter the pores and be firmly physically confined and adsorbed. This "pore confinement effect" significantly enhances the multiple weak interactions (hydrophobic and coordination interactions) between the enzyme and the pore wall, effectively preventing enzyme leakage and thermal desorption during use, and improving operational stability. Simultaneously, substrate molecules can freely diffuse into the mesoporous pores, contacting the active sites of lipase molecules immobilized on the inner surface of the pores and undergoing catalytic reactions. It also provides ample channels for the diffusion of reactants to the enzyme active sites within the pores and the release of products outwards. If the pore size is too small, although the enzyme may still barely enter, severe internal diffusion restriction will occur during the catalytic reaction, leading to rapid depletion of substrate concentration and product accumulation within the pores, significantly reducing the apparent reaction rate and macroscopic enzyme activity. If the pore size is too large, the confinement effect weakens, and enzyme molecules easily escape from the pores. Therefore, the pore size limitation of the mesoporous SiO2 support can simultaneously accommodate enzyme molecules and ensure free mass transfer between substrate and product, balancing high enzyme loading and catalytic activity.

[0015] More preferably, in step (1), the concentration of hexadecyltrimethylammonium bromide in the template agent solution is 0.1-0.3 mol / L, and the concentration of urea is 0.5-2.0 mol / L; the molar ratio of tetraethyl orthosilicate, octadecyltriethoxysilane, and 3-aminopropyltriethoxysilane in the mixture is 1:0.02-0.2:0.05-0.2.

[0016] More preferably, in step (1), the amount of the mixture and template agent solution added is calculated based on the molar ratio of tetraethyl orthosilicate and hexadecyltrimethylammonium bromide of 1:0.1-0.3; the hydrothermal reaction is carried out at 80-120℃ for 24-72h; and the calcination is carried out by heating to 500-600℃ and holding for 4-8h.

[0017] More preferably, in step (1), the particle size of the mesoporous SiO2 support is 5-50 μm, more preferably 10-30 μm.

[0018] More preferably, in step (2), the pH of the sodium carbonate buffer solution is 10-11; the bath ratio of the mesoporous SiO2 support added to the sodium carbonate buffer solution is 1:15-30 g / mL; the swelling time is 1-4 h; the mass ratio of sodium chloroacetate to mesoporous SiO2 support is 3-5:1; the reaction temperature for adding sodium chloroacetate and reacting is 50-70℃, and the time is 8-12 h.

[0019] More preferably, in step (2), the bath ratio of the carrier added to the zinc chloride solution is 1:10-20 g / mL; the concentration of the zinc chloride solution is 0.1-0.5 mol / L; the reaction temperature for adding the zinc chloride solution and reacting is 25-35℃, and the reaction time is 4-8h.

[0020] Further preferably, in step (3), the pH of the phosphate buffer is 6.0-7.5; the bath ratio of the zinc ion chelated mesoporous SiO2 carrier added to the phosphate buffer is 1:10-30 g / mL; the swelling time is 0.5-2 h; and the free enzyme is derived from thermophilic fungi. Rhizomucor miehei The immobilization adsorption temperature is 20-40℃, and the time is 4-12h; the immobilized lipase has a free enzyme loading of 30-150 mg / g, more preferably 50-120 mg / g.

[0021] Compared with the prior art, the present invention has the following advantages: (1) By utilizing the strict sn-1,3 region selectivity of enzymes, DHA and oleic acid are directionally introduced into the sn-1,3 position while retaining palmitic acid at the sn-2 position. This achieves a green process for efficiently preparing OPDHA structured lipids, a human milk fat analog, from natural fish oil. The resulting structured lipids simultaneously simulate the key configuration of palmitic acid at the sn-2 position of human milk fat and meet the nutritional fortification requirements of DHA, taking into account both calcium absorption and brain visual development function in infants and young children. (2) Modification of the carrier improves the enzyme's loading stability, active conformation retention and reusability, and replacing copper with zinc significantly improves food safety; at the same time, it can achieve effective pore confinement and structural protection of enzyme molecules, and fully ensure the free diffusion of substrate and product molecules, avoiding internal diffusion restrictions and improving catalytic efficiency. (3) The prepared OPDHA structured lipid has a palmitic acid content of 60% or more at the sn-2 position, and an oleic acid and DHA content of 40% or more and 50% or more at the sn-1 and 3 positions, respectively. Attached Figure Description

[0022] Figure 1 A comparison chart of DHA content under different lipase conditions; Figure 2A comparison chart of DHA content under different reaction pH conditions; Figure 3 A comparison chart of DHA content under different water-oil ratios; Figure 4 A comparison of the fatty acid composition and content of OPDHA synthesized under different immobilized lipase conditions. Figure 5 A comparison of the fatty acid composition and content of OPDHA synthesized under different substrate molar ratios; Figure 6 A comparison chart of fatty acid composition and content of OPDHA synthesized under different oleic acid to DHA molar ratios. Figure 7 A comparison chart of fatty acid composition and content of OPDHA synthesized under different reaction temperature conditions; Figure 8 A comparison chart showing the fatty acid composition and content of OPDHA synthesized under different reaction time conditions. Detailed Implementation

[0023] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0024] Example 1 (Step S1, selecting different lipases) (1) Weigh 3g of fish oil into different centrifuge tubes, add phosphate buffer with pH 6.5 (control the water-oil ratio to 1.5), and then add 2% of Lipozyme RM IM lipase, Novozym 435 lipase and Lipozyme TL IM lipase according to the mass of fish oil to conduct parallel experiments. Place the centrifuge tubes in a 35℃ water bath and stir at 500r / min for 10h. After the reaction is completed, add 10mL of 95wt% ethanol to terminate the reaction.

[0025] (2) Then add 20 mL of n-hexane and 10 mL of deionized water, shake to extract, transfer the mixture to a separatory funnel, separate and discard the n-hexane layer (mainly unhydrolyzed glycerides), retain the aqueous phase, add 3 M hydrochloric acid to the aqueous phase, adjust the pH to 1.0, at which point the free fatty acids are completely precipitated.

[0026] (3) Add 10 mL of n-hexane to extract the precipitated fatty acids, and remove the solvent by rotary evaporation at 40 °C to obtain a product rich in free DHA. After methyl esterification, the fatty acid composition of the product is analyzed by gas chromatography-mass spectrometry.

[0027] like Figure 1As shown, Novozym435 lipase and LipozymeRMIM lipase exhibit high affinity and selectivity for long-chain unsaturated fatty acids, retaining more DHA during enzymatic hydrolysis. The DHA contents obtained by these two lipases are 95.20% and 98.20%, respectively. LipozymeTLIM lipase has a relatively low affinity for long-chain unsaturated fatty acids, favoring short-chain or medium-chain fatty acids during hydrolysis, resulting in poor DHA hydrolysis and a final product DHA content decreasing to 91.73%. To retain more DHA, LipozymeRMIM lipase was chosen for the subsequent reactions.

[0028] Example 2 (Step S1, selecting different reaction pH) The same preparation method as in Example 1 and Lipozyme RM IM lipase were used, but in step (1), "adding phosphate buffer with pH 6.5" was changed to "adding phosphate buffer with pH values ​​of 4.5, 5.5, 6.5, 7.5 and 8.5 respectively for parallel experiments", and other conditions remained unchanged.

[0029] like Figure 2 As shown, when the external environment is above or below the enzyme's optimal pH, the enzyme activity weakens, leading to a decrease in the rate of enzymatic reaction. When the pH of the reaction system is in the range of 4.5-8.5, the DHA content initially increases with increasing pH, then decreases with further increases in pH. The highest DHA content is observed at a pH of 6.0-7.0.

[0030] Example 3 (Step S1, selecting different water-oil ratios) The same preparation method as in Example 1 and Lipozyme RM IM lipase were used, but in step (1), “controlling the water-oil ratio to 1.5” was changed to “controlling the water-oil ratio to 0.5, 1, 1.5, 2, 2.5 respectively for parallel experiments”, while other conditions remained unchanged.

[0031] like Figure 3 As shown, when the water-to-oil ratio is 0.5-1.5, the oil-water interface area increases with increasing water content, leading to a continuous increase in hydrolysis efficiency and promoting the forward hydrolysis reaction. When the water-to-oil ratio exceeds 1.5, the fish oil hydrolysis efficiency decreases slightly. This is because, on the one hand, the increased water content dilutes the enzyme concentration in the reaction system, thus weakening the hydrolysis efficiency, indicating the existence of an optimal oil-water interface reaction area; on the other hand, the increased water content dilutes the fish oil, inhibiting the forward reaction. In summary, a water-to-oil ratio of 1.5-2.5 represents the optimal conditions for fish oil hydrolysis in the system of this invention, achieving superior hydrolysis efficiency and high DHA content.

[0032] Example 4 (Preparation of immobilized lipase I) (1) Dissolve hexadecyltrimethylammonium bromide and urea in water to obtain a template solution. The concentration of hexadecyltrimethylammonium bromide in the template solution is 0.2 mol / L and the concentration of urea is 1.0 mol / L. Tetraethyl orthosilicate, octadecyltriethoxysilane and 3-aminopropyltriethoxysilane are mixed in a molar ratio of 1:0.1:0.15 to obtain a mixed solution. The mixed solution is added dropwise to the template solution so that the molar ratio of tetraethyl orthosilicate in the mixed solution and hexadecyltrimethylammonium bromide in the template solution is 1:0.2. The dropwise addition is stopped, and a hydrothermal reaction is carried out in a reactor at 100°C for 48 h. After the reaction is completed, the product is collected by filtration, washed, heated to 600°C at 5°C / min, and calcined for 6 h to obtain a mesoporous SiO2 support with a pore size of 5-10 nm. (2) The mesoporous SiO2 support was added to a sodium carbonate buffer solution with a pH of 10.5 and swollen for 2 h at a bath ratio of 1:20 g / mL; sodium chloroacetate was added and reacted at 60 °C for 8 h at a mass ratio of 3:1 to sodium chloroacetate and mesoporous SiO2 support; the support was collected by filtration and washed until neutral, and then added to a 0.3 mol / L zinc chloride solution at a bath ratio of 1:20 g / mL and reacted at 30 °C for 6 h; the support was collected by filtration and washed until no free Zn was found. 2+ Cl - The zinc ion chelated mesoporous SiO2 support was detected, dried, and obtained. (3) The zinc ion chelated mesoporous SiO2 carrier was added to phosphate buffer at pH 6.5 and swollen for 1 h at a bath ratio of 1:20 g / mL; free enzyme (from thermophilic fungi) was added. Rhizomucor miehei The 1,3-specific lipase was immobilized and adsorbed at 25°C for 6 h; the immobilized enzyme was collected by filtration, dried, and immobilized lipase I was obtained, with a free enzyme loading of 88 mg / g.

[0033] Example 5 (Preparation of immobilized lipase II, without zinc ion chelation on the carrier) (1) Dissolve hexadecyltrimethylammonium bromide and urea in water to obtain a template solution. The concentration of hexadecyltrimethylammonium bromide in the template solution is 0.2 mol / L and the concentration of urea is 1.0 mol / L. Tetraethyl orthosilicate, octadecyltriethoxysilane and 3-aminopropyltriethoxysilane are mixed in a molar ratio of 1:0.1:0.15 to obtain a mixed solution. The mixed solution is added dropwise to the template solution so that the molar ratio of tetraethyl orthosilicate in the mixed solution and hexadecyltrimethylammonium bromide in the template solution is 1:0.2. The dropwise addition is stopped, and a hydrothermal reaction is carried out in a reactor at 100°C for 48 h. After the reaction is completed, the product is collected by filtration, washed, heated to 600°C at 5°C / min, and calcined for 6 h to obtain a mesoporous SiO2 support with a pore size of 5-10 nm. (2) The mesoporous SiO2 support was added to phosphate buffer at pH 6.5 and swollen for 1 h at a bath ratio of 1:20 g / mL; free enzyme (from thermophilic fungi) was added. Rhizomucor miehei The 1,3-specific lipase was immobilized and adsorbed at 25°C for 6 h; the immobilized enzyme was collected by filtration, dried, and immobilized lipase I was obtained, with a free enzyme loading of 79 mg / g.

[0034] Example 6 (Step S2, selecting different immobilized lipases) S1: Using the same preparation method as in Example 1 and employing Lipozyme RM IM lipase, a sample rich in free DHA was obtained.

[0035] S2: 0.1 mol of tripalmitoyl glycerol (PPP), 0.6 mol of oleic acid, and a sample rich in free DHA (containing 0.6 mol of DHA) were added to a 50 mL spiral centrifuge tube. The molar ratio of tripalmitoyl glycerol to the total molar ratio of oleic acid and DHA was 1:12, and the molar ratio of oleic acid to DHA was 1:1. Then, 10% of the total mass of the substrate in the centrifuge tube was added (Lipozyme RM IM lipase, immobilized lipase I, and immobilized lipase II were added in parallel experiments). The centrifuge tube was placed in a water bath at 60℃ and stirred at 500 rpm for 6 h. After the reaction, the centrifuge tube was centrifuged at 8000 rpm for 5 min, and the upper oil layer was collected. The collected oil was dissolved in 50 mL of n-hexane, and 40 mL of 0.8 mol / L KOH (30% ethanol) solution was added. The mixture was magnetically stirred for 5 min and then allowed to stand for separation. The upper n-hexane phase was collected, and the solvent was removed by rotary evaporation. The lower aqueous phase was extracted once more with 15 mL of n-hexane. The organic phases were combined and evaporated to dryness to obtain the crude product. The crude product was subjected to fatty acid composition analysis at the sn-2 position and fatty acid composition analysis.

[0036] Depend on Figure 4 As shown in A, B, and C, immobilized lipase I exhibits a stronger ability to bind oleic acid and DHA, facilitating the insertion of DHA and oleic acid. The content of oleic acid and DHA at the sn-1 and 3 positions of the product is significantly higher than that of the other two enzymes, and the content and relative content of palmitic acid at the sn-2 position are also significantly higher. This indicates that immobilized lipase I has a lower migration rate for palmitic acid at the sn-2 position and is more stable, while the other two enzymes caused acyl group migration. In immobilized lipase II, the carrier lacks zinc ion chelation, which not only reduces the enzyme loading capacity but also significantly affects the enzyme's specific binding ability to DHA and oleic acid, leading to a decrease in the content of DHA and oleic acid at the sn-1 and 3 positions. Furthermore, the specific binding ability of immobilized lipase II to DHA and oleic acid is even lower than that of Lipozyme RM IM lipase, possibly due to the lack of Zn. 2+Specific optimization of the active site cannot achieve enhanced selectivity for DHA and oleic acid. In fact, simple hydrophobic adsorption may lead to conformational distortion of some enzyme molecules and non-specific adsorption inactivation, which may weaken the background selectivity.

[0037] Example 7 (Step S2, selecting different substrate molar ratios) The same preparation method as in Example 6 and immobilized lipase I were used, but the phrase "the molar ratio of trimalmitoyl glycerol to the total molar ratio of oleic acid and DHA is 1:12" was changed to "parallel experiments were conducted at molar ratios of trimalmitoyl glycerol to the total molar ratio of oleic acid and DHA of 1:8, 1:10, 1:12, 1:14, and 1:16 respectively". The total molar amount of trimalmitoyl glycerol, oleic acid, and DHA was 2.2 mol, and the molar ratio of oleic acid to DHA was 1:1. Other conditions remained unchanged.

[0038] Depend on Figure 5 As shown in section A, the contents of both oleic acid and DHA reach their maximum values ​​at a molar ratio of 1:12. Figure 5 As shown in equation B, the content of palmitic acid at position 2 first increases and then decreases as the molar ratio decreases. Since the enzyme used is a hydrolase at positions 1 and 3, the content of palmitic acid at position 2 should remain constant, indicating that acyl migration occurred during the reaction. The relative content of palmitic acid at position 2 first increases and then decreases as the substrate molar ratio decreases. Figure 5 As shown in Figure C, the contents of oleic acid and DHA at positions 1 and 3 show a trend of first increasing and then decreasing as the molar ratio decreases. When the molar ratio is 1:12-14, the incorporation rates of DHA and oleic acid are relatively high, and the content of palmitic acid at position 2 remains relatively high.

[0039] Example 8 (Step S2, selecting different molar ratios of oleic acid to DHA) The same preparation method as in Example 6 and immobilized lipase I were used, but the phrase "the molar ratio of oleic acid to DHA is 1:1" was changed to "parallel experiments were conducted at molar ratios of oleic acid to DHA of 1:0.6, 1:0.8, 1:1, 1:1.2, and 1:1.4 respectively". The total molar amount of oleic acid and DHA was 1.2 mol, and other conditions remained unchanged.

[0040] Depend on Figure 6 As shown in section A, when the molar ratio of oleic acid to DHA is 1:1, the contents of both palmitic acid and oleic acid reach their maximum values. Figure 6 As shown in equation B, as the ratio of oleic acid to DHA decreases from 1:0.6 to 1:1.4, the relative values ​​of oleic acid and palmitic acid at positions 2 and 2 show a trend of first increasing and then decreasing. For example... Figure 6As shown in Figure C, when the molar ratio of oleic acid to DHA is 1:0.6-1, the distribution of oleic acid and DHA shows a slight upward trend, while palmitic acid is relatively low. OPDHA content increases with increasing oleic acid and DHA content. When the total fatty acid content is constant, a higher oleic acid to DHA ratio increases the oleic acid insertion rate, easily leading to an increase in OPO content in the product. Conversely, a lower oleic acid to DHA ratio easily generates a large amount of DHAPDHA lipids. Therefore, choosing a molar ratio of oleic acid to DHA of 1:1 results in higher oleic acid and DHA content at positions 1 and 3, and the palmitic acid content at position sn-2 also remains high, leading to a higher content of the resulting OPDHA product.

[0041] Example 9 (Step S2, selecting different reaction temperatures) The same preparation method as in Example 6 and immobilized lipase I were used, but the "temperature of 60°C" was changed to "parallel experiments were conducted at temperatures of 50°C, 55°C, 60°C, 65°C, and 70°C respectively", while other conditions remained unchanged.

[0042] like Figure 7 As shown in Figure A, the content of oleic acid and DHA in the product decreases at 60-70℃, while palmitic acid increases. For reaction systems containing polyunsaturated fatty acids, high temperatures lead to severe oxidation, thereby reducing their nutritional value. Figure 7 As shown in Figure B, at temperatures above 60℃, the relative content of palmitic acid at position 2 decreases, indicating a higher content of palmitic acid at positions 1 and 3. This suggests that while higher temperatures improve the synthesis efficiency of structured lipids, they may also accelerate acyl migration, affecting the stability and activity of lipases. Therefore, to achieve high efficiency and minimize side effects, the reaction temperature should be maintained between 55-60℃.

[0043] Example 10 (Step S2, selecting different reaction times) The same preparation method as in Example 6 and immobilized lipase I were used, but the "stirring reaction for 6 hours" was changed to "parallel experiments were conducted at reaction times of 2 hours, 4 hours, 6 hours, 8 hours, and 10 hours respectively", while other conditions remained unchanged.

[0044] like Figure 8 As shown in Figure A, the contents of oleic acid and DHA reached their maximum after 4 hours of reaction, while the content of palmitic acid increased with the extension of reaction time. Figure 8 As shown in Figure B, due to the effect of acyl migration, the palmitic acid content at position 2 shows an increasing trend, and the relative palmitic acid content at position 2 first increases and then decreases with increasing reaction time. Figure 8As shown in Figure C, when the reaction time is less than 4 hours, the contents of oleic acid at the sn-1 and 3 positions and DHA in the reaction products increase with increasing reaction time. In the initial stage of the reaction, fatty acid acyl donors gradually replace palmitic acid, and the reaction reaches equilibrium at 4 hours. When the reaction time exceeds 4 hours, the contents of palmitic acid at the sn-1 and 3 positions generally show an increasing trend, and the longer the reaction time, the more vigorous the reverse reaction. Therefore, overall, a reaction time of 2-4 hours yields better results.

[0045] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0046] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for synthesizing OPDHA structured lipids catalyzed by lipase, characterized in that, Includes the following steps: S1: Fish oil, phosphate buffer, and Lipozyme RM IM lipase were mixed and stirred in a constant temperature water bath. Ethanol was then added to terminate the enzyme reaction. Hexane and water were added for extraction, and the aqueous phase was separated and retained. The pH of the aqueous phase was adjusted to 1.0, and hexane was added for extraction. The organic phase was separated and retained. The solvent was removed by rotary evaporation of the organic phase to obtain a sample rich in free DHA. S2: Tripalmitoyl glycerol, oleic acid and a sample rich in free DHA are mixed, then immobilized lipase is added and the mixture is heated in a constant temperature water bath. After the reaction is completed, the mixture is centrifuged and the upper oil is collected and dissolved in hexane. KOH-ethanol solution is added to remove free fatty acids, thus obtaining OPDHA structured lipids.

2. The method for synthesizing OPDHA structured lipids catalyzed by lipase as described in claim 1, characterized in that, In S1, the amount of lipase Lipozyme RM IM added is 2-3% of the fish oil mass; the pH value of the reaction system obtained after mixing fish oil, phosphate buffer and lipase Lipozyme RM IM is 6.0-7.0, and the water-to-oil ratio is 1.5-2.5; the temperature of the constant temperature water bath is 30-40℃; and the stirring reaction time is 8-15h.

3. The method for synthesizing OPDHA structured lipids catalyzed by lipase as described in claim 1 or 2, characterized in that, In S2, the molar ratio of the tripalmitoyl glycerol to the total molar ratio of oleic acid and DHA in the sample rich in free DHA is 1:12-14; the molar ratio of oleic acid to DHA in the sample rich in free DHA is 1:0.8-1; the amount of immobilized lipase added is 7-10% of the total substrate mass; the temperature of the constant temperature water bath is 55-60℃; and the heating reaction time is 2-4 hours.

4. The method for synthesizing OPDHA structured lipids catalyzed by lipase as described in claim 1, characterized in that, In S2, the immobilized lipase is Lipozyme RM IM lipase.

5. The method for synthesizing OPDHA structured lipids catalyzed by lipase as described in claim 1, characterized in that, In S2, the method for preparing the immobilized lipase includes the following steps: (1) Dissolve hexadecyltrimethylammonium bromide and urea in water to obtain a template agent solution; mix tetraethyl orthosilicate, octadecyltriethoxysilane and 3-aminopropyltriethoxysilane to obtain a mixed solution; add the mixed solution dropwise to the template agent solution and perform a hydrothermal reaction. After the reaction is completed, filter and collect the product, wash and calcine to obtain a mesoporous SiO2 support with a pore size of 5-10 nm. (2) After the mesoporous SiO2 support swells in sodium carbonate buffer, sodium chloroacetate is added and reacted; the support is collected by filtration, washed until neutral, and then zinc chloride solution is added and reacted; the support is collected by filtration, and washed until no free Zn is found. 2+ Cl - The zinc ion chelated mesoporous SiO2 support was detected, dried, and obtained. (3) The zinc ion chelate mesoporous SiO2 carrier was added to phosphate buffer to swell, the free enzyme was added, and the immobilized enzyme was adsorbed; the immobilized enzyme was collected by filtration and dried to obtain immobilized lipase.

6. The method for synthesizing OPDHA structured lipids catalyzed by lipase as described in claim 5, characterized in that, In step (1), the concentration of hexadecyltrimethylammonium bromide in the template agent solution is 0.1-0.3 mol / L, and the concentration of urea is 0.5-2.0 mol / L; the molar ratio of tetraethyl orthosilicate, octadecyltriethoxysilane, and 3-aminopropyltriethoxysilane in the mixture is 1:0.02-0.2:0.05-0.

2.

7. The method for synthesizing OPDHA structured lipids catalyzed by lipase as described in claim 5 or 6, characterized in that, In step (1), the amount of the mixture and template agent solution added is calculated based on the molar ratio of tetraethyl orthosilicate and hexadecyltrimethylammonium bromide of 1:0.1-0.3; the hydrothermal reaction is carried out at 80-120℃ for 24-72h; the calcination is carried out by heating to 500-600℃ and holding for 4-8h.

8. The method for synthesizing OPDHA structured lipids catalyzed by lipase as described in claim 5, characterized in that, In step (2), the pH of the sodium carbonate buffer solution is 10-11; the bath ratio of the mesoporous SiO2 support added to the sodium carbonate buffer solution is 1:15-30 g / mL; the mass ratio of sodium chloroacetate to mesoporous SiO2 support is 3-5:1; the reaction temperature for adding sodium chloroacetate and reacting is 50-70℃, and the reaction time is 8-12 h.

9. The method for synthesizing OPDHA structured lipids catalyzed by lipase as described in claim 5 or 8, characterized in that, In step (2), the bath ratio of the carrier added to the zinc chloride solution is 1:10-20 g / mL; the concentration of the zinc chloride solution is 0.1-0.5 mol / L; the reaction temperature for adding the zinc chloride solution and reacting is 25-35℃, and the reaction time is 4-8 h.

10. The method for synthesizing OPDHA structured lipids catalyzed by lipase as described in claim 5, characterized in that, In step (3), the pH of the phosphate buffer is 6.0-7.5; the bath ratio of the zinc ion chelated mesoporous SiO2 carrier added to the phosphate buffer is 1:10-30 g / mL; the free enzyme is a 1,3-specific lipase from thermophilic fungi; the immobilization and adsorption temperature is 20-40℃, and the time is 4-12 h; the immobilized lipase has a free enzyme loading of 30-150 mg / g.

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