Process for the preparation of opl structured esters and use thereof

Abstract

The application provides a preparation method and application of OPL structure ester, and belongs to the technical field of oil processing. The application utilizes dodecyl glucoside-lipase catalysis to carry out acidolysis reaction on oil rich in palmitic acid and oleic acid and linoleic acid, and combines with ultrasonic treatment, so that the content of OPL in the product reaches 42.66-47.88%, the content of palmitic acid at sn-2 position reaches 73.26-78.13%, and the acyl migration rate is as low as 2.16-2.51%. The application effectively overcomes the technical problems of low mass transfer efficiency, long reaction time, significant acyl migration side reaction and insufficient palmitic acid retention rate at sn-2 position in the enzyme acidolysis reaction, has the advantages of high reaction efficiency, good product quality and controllable cost, and has a wide application prospect in the field of food, medicine or health care products for digestion or lubricating the intestines.

Description

Technical Field

[0001] This invention belongs to the field of oil and fat processing technology, specifically relating to a method for preparing an OPL-structured ester and its application. Background Technology

[0002] Human milk fat not only provides approximately 40-50% of the energy for infants' growth and development, but also provides essential fatty acids and vitamins, playing a vital role in the development of the infant's nervous system, retina, and the maturation of the immune system. 1-Oleic-2-palmitic-3-linoleic triglyceride (OPL) is one of the most abundant triglycerides in breast milk. Its unique fatty acid distribution can effectively promote the absorption of fatty acids and calcium in infants, relieve constipation, and reduce infant crying, showing broad application prospects in the infant formula industry.

[0003] Enzymatic acid hydrolysis is the main method for preparing OPL structured esters, offering advantages such as mild reaction conditions, high regioselectivity, and easy separation of byproducts. However, existing enzymatic acid hydrolysis processes have the following shortcomings:

[0004] First, the reaction system is a hydrophobic heterogeneous system, resulting in low mass transfer efficiency between the immobilized lipase and the liquid substrate, leading to a slow reaction rate and long reaction time (usually 4-6 hours).

[0005] Second, in order to achieve a high conversion rate, a large amount of lipase is added (generally 10-12%), which increases the cost of enzyme preparations.

[0006] Third, excessively long reaction times can easily trigger acyl migration side reactions, reduce the relative content of palmitic acid at the sn-2 position, and affect the nutritional value of the product.

[0007] To address the aforementioned limitations in mass transfer and excessively long reaction times, studies have introduced ultrasound technology into enzymatic reaction systems. The cavitation, mechanical, and thermal effects generated by ultrasound in liquid media can enhance substrate-enzyme mixing and mass transfer, thereby increasing the reaction rate. However, the inhibition of acyl migration is limited when ultrasound is used alone, and shortening the reaction time does not completely eliminate acyl migration. Furthermore, methods involving screening and modifying lipases are costly and time-consuming.

[0008] Therefore, without significantly increasing process complexity and cost, it is urgent to develop a method for preparing OPL-structured esters to further inhibit acyl migration and improve the retention rate of palmitic acid at the sn-2 position in OPL-structured esters. Summary of the Invention

[0009] The technical problem to be solved: In existing enzymatic acid hydrolysis methods for preparing OPL-structured esters, the low mass transfer efficiency, long reaction time leading to significant acyl migration side reactions, and insufficient retention of palmitic acid at the sn-2 position are problems encountered. The purpose of this invention is to provide a method for preparing OPL-structured esters and its applications. By utilizing the hydrogen bonds and electrostatic interactions between dodecyl glucoside and lipase molecules, dodecyl glucoside is bound to the lipase, increasing the α-helix and β-sheet content and reducing the random coil content, thereby enhancing the enzyme's conformational rigidity. Furthermore, the cavitation effect generated by ultrasonic treatment enhances the mass transfer between the substrate and enzyme, shortens the reaction time, and forms a synergistic effect. This improves reaction efficiency while increasing the relative content of OPL and palmitic acid at the sn-2 position in the product, and reduces acyl migration.

[0010] Technical solution: This invention provides a method for preparing an OPL-structured ester, comprising the following steps:

[0011] S1. Dissolve alkyl glucoside and lipase in PBS buffer, stir well and incubate in the dark; after incubation, collect the retentate after ultrafiltration of the mixture, and dry it to obtain alkyl glucoside-lipase.

[0012] S2. Free fatty acids are obtained by mixing oleic acid and linoleic acid. Palmitic acid-rich oils and free fatty acids are then mixed evenly, and alkyl glucoside-lipase is added to prepare a reaction system. The system is stirred under ultrasonic assistance, and after the reaction is complete, it is separated and purified to obtain an OPL-structured ester. Ultrasonic assistance can be any of the following methods:

[0013] (1) After adding alkyl glucoside-lipase, the mixture is subjected to ultrasonic pretreatment and then stirred for reaction;

[0014] (2) Throughout the entire reaction process after the addition of alkyl glucoside-lipase, continuous ultrasonic treatment was performed.

[0015] Preferably, in step S1, the alkyl glucoside is dodecyl glucoside; the lipase is a 1,3-specific lipase or a non-specific lipase; the mass ratio of alkyl glucoside to lipase is 6~15:1; the incubation temperature is 23~26℃, the incubation time is 2~6 h; and the molecular weight cutoff of ultrafiltration is 8~20 kDa.

[0016] Preferably, the drying method in step S1 is freeze drying or vacuum drying.

[0017] Preferably, the palmitic acid-rich oil in step S2 is any one or more of palm oil, palm stearin, and palmitic acid glycerides.

[0018] Furthermore, the palm stearin contains ≥75% palmitic acid, ≥65% relative palmitic acid at the sn-2 position, and ≥80% tripalmitoylglycerol (PPP) content.

[0019] Preferably, the free fatty acids in step S2 are obtained by mixing oleic acid and linoleic acid or by mixing vegetable oils and then saponifying and acidifying them.

[0020] Furthermore, the mass ratio of oleic acid to linoleic acid is 1:1~2.

[0021] Furthermore, the mass ratio of oleic acid to linoleic acid is 1:1.2~1.3.

[0022] Preferably, in step S2, the molar ratio of palmitic acid-rich oil to free fatty acids is 1:6~16.

[0023] Preferably, the amount of dodecyl glucoside-lipase added in step S2 is 2-12% of the total substrate mass.

[0024] Preferably, the reaction system in step S2 is a solvent-free system, an organic solvent system, a eutectic solvent system, or an ionic liquid system.

[0025] Preferably, in step S2, the temperature of the stirring reaction is 45~65℃, the stirring reaction time is 1~8 h, and the stirring speed is 300~800 rpm.

[0026] Furthermore, the temperature of the stirring reaction is 50~60℃, the stirring reaction time is 2~4 h, and the stirring speed is 500~700 rpm.

[0027] Preferably, the ultrasound in step S2 is performed using an ultrasonic bath or a probe-type ultrasonic device.

[0028] Furthermore, the ultrasound is performed using a probe-type ultrasound device.

[0029] Preferably, when the ultrasound-assisted method is (1), the molar ratio of palmitic acid-rich oil and free fatty acid in step S2 is 1:10~14; the amount of alkyl glucoside-lipase added is 4~10% of the total substrate mass; the ultrasound power is 100~600 W, the ultrasound duty cycle is 10~80%, and the ultrasound time is 1~30 min.

[0030] Furthermore, the ultrasonic power is 200~400 W, the ultrasonic duty cycle is 50~70%, and the ultrasonic time is 3~18 min.

[0031] Preferably, when the ultrasound-assisted method is (2), the molar ratio of palmitic acid-rich oil and free fatty acid is 1:12~15; the amount of alkyl glucoside-lipase added is 8~12% of the total substrate mass; the ultrasound power is 60~180 W, the ultrasound duty cycle is 20~90%, and the ultrasound time is 1~180 min.

[0032] Furthermore, the ultrasonic power is 100~160 W, the ultrasonic duty cycle is 60~80%, and the ultrasonic time is 90~150 min.

[0033] The present invention also provides an OPL structured ester prepared by the above preparation method, wherein the content of palmitic acid at the sn-2 position of the OPL structured ester is 73.26~78.13% and the acyl mobility is 2.16~2.51%.

[0034] This invention also provides the application of the above-mentioned OPL structure ester in the preparation of food, medicine or health products that aid digestion or promote bowel movement.

[0035] Beneficial effects:

[0036] 1. This invention utilizes dodecyl glucoside to increase the α-helix and β-sheet content and reduce the random coil content in the lipase protein structure, thereby enhancing the conformational rigidity and order of the lipase and improving its thermal stability. This makes the lipase less prone to conformational changes during the enzymatic acid hydrolysis to prepare OPL-structured esters. Furthermore, ultrasonic treatment is introduced to enhance the mass transfer efficiency between the substrate and the lipase through ultrasonic cavitation, ensuring sufficient contact between the free fatty acids and the substrate in the reaction system. This significantly shortens the reaction time, effectively inhibits acyl migration side reactions, and reduces the probability of palmitic acid at the sn-2 position being replaced during the reaction. The final product has high OPL content, high sn-2 palmitic acid content, and low acyl migration rate, more closely resembling the natural proportions of breast milk.

[0037] 2. The synergistic effect of ultrasonic treatment and dodecyl glucoside-lipase effectively overcomes the technical contradiction in traditional processes where it is difficult to simultaneously improve reaction conversion rate and inhibit acyl migration. Ultrasonic treatment improves the mass transfer efficiency of the reaction system, while dodecyl glucoside improves the properties of the oil-water interface, promoting sufficient contact between the substrate and the enzyme, allowing the reaction to reach equilibrium in a shorter time. This significantly improves production efficiency, shortens the process cycle, and effectively reduces energy consumption and production costs.

[0038] 3. Due to the effective suppression of acyl migration side reactions, the amount of non-target ester structures (such as OPO, OPP, LPL, LPP, etc.) generated in the reaction system is reduced, and the content and purity of OPL ester structures in the product are significantly improved. This not only simplifies the subsequent purification process, but also improves the consistency of product quality and batch stability.

[0039] 4. This invention provides a flexible process solution: the ultrasonic pretreatment method requires less lipase, and the lipase can be reused after ultrafiltration recovery, resulting in low production costs; the whole ultrasonic treatment method has a shorter reaction time, making it suitable for rapid large-scale production.

[0040] 5. Due to the shortened reaction time, the exposure time of lipase to environments such as high temperature and organic solvents is reduced, resulting in improved enzyme activity retention and increased reusability. Simultaneously, dodecyl glucoside protects the enzyme's active site through conformational stabilization and improves the oil-water interface properties, reducing the interference of interfacial adsorption on the conformation of the enzyme's active site. This lowers the cost of enzyme use and facilitates its widespread application in industrial production. Attached Figure Description

[0041] Figure 1 The effect of different surfactants on the relative enzyme activity of lipase;

[0042] Figure 2 The results are circular dichroism spectra of lipase and dodecyl glucoside-lipase. Detailed Implementation

[0043] The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the present invention, but the present invention is not limited to these embodiments:

[0044] The enzyme activity of the Aspergillus niger lipase involved in the embodiments and comparative examples of this invention is 20,000 U / g; the immobilized lipase Lipozyme TL IM was purchased from Novozymes Biotechnology Beijing Co., Ltd.

[0045] Example 1

[0046] This example illustrates the effect of dodecyl glucoside on the activity of Aspergillus niger lipase. The specific method includes the following steps:

[0047] S1. Prepare dodecyl glucoside solutions of 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, and 5 mg / mL using PBS buffer (50 mmol / L, pH 7.4). Dilute Aspergillus niger lipase with different concentrations of dodecyl glucoside solutions 15,000 times and then measure the enzyme activity. Calculate the relative enzyme activity of Aspergillus niger lipase in the PBS reaction system without added surfactant as 100% (blank control).

[0048] S2. Assay for lipase activity: 45 μL of substrate (4 mmol / L) p-NPP solution and 405 μL of PBS buffer (50 mmol / L, pH 7.4) were mixed thoroughly and preheated in a 40℃ water bath for 5 min. 50 μL of Aspergillus niger lipase was added, and the mixture was vortexed to mix thoroughly. After reacting for 10 min, 500 μL of anhydrous ethanol was added and the reaction was stopped by placing the mixture on ice. The mixture was centrifuged at 4℃ and 10000 rpm for 5 min, and the supernatant was collected. The absorbance was measured at 410 nm to calculate the enzyme activity. A blank control was set up using PBS instead of the enzyme solution. Under certain conditions, the enzyme activity (U / mL) was calculated based on the amount of Aspergillus niger lipase required to release 1 μmol of p-NP per minute.

[0049] Comparative Example 1

[0050] The difference between this comparative example and Example 1 is that in this comparative example, dodecyl glucoside is replaced with polyethylene glycol 6000, while the other steps are the same as in Example 1.

[0051] Comparative Example 2

[0052] The difference between this comparative example and Example 1 is that in this comparative example, dodecyl glucoside is replaced with sodium dodecyl sulfate, while the other steps are the same as in Example 1.

[0053] like Figure 1 As shown, when the nonionic surfactants dodecyl glucoside (Example 1) and polyethylene glycol 6000 (Comparative Example 1) were at low concentrations (1 mg / mL), the relative enzyme activities of *Aspergillus niger* lipase were 146.7% and 92.8%, respectively. With increasing concentration, dodecyl glucoside promoted the activity of *Aspergillus niger* lipase, increasing the relative enzyme activity to 238.6%, while polyethylene glycol 6000 inhibited the activity, decreasing the relative enzyme activity from 92.8% to 53.4%. The anionic surfactant sodium dodecyl sulfate showed an increasing relative enzyme activity at concentrations of 1–2 mg / mL, reaching a peak of 132.2% at 2 mg / mL. With increasing sodium dodecyl sulfate concentration, the *Aspergillus niger* lipase activity decreased from 132.2% to 107.3%, indicating that high concentrations of sodium dodecyl sulfate inhibited *Aspergillus niger* lipase activity. At 5 mg / mL, the relative enzyme activity of Aspergillus niger lipase treated with dodecyl glucoside was much higher than that of polyethylene glycol 6000 and sodium dodecyl sulfate. This may be related to its unique glycosyl structure, which can form a protective layer on the enzyme molecule surface through hydrogen bonding, thereby stabilizing the enzyme conformation and promoting effective contact between the enzyme and the substrate.

[0054] Example 2

[0055] This embodiment describes a method for preparing dodecyl glucoside-Aspergillus niger lipase, including the following steps:

[0056] S1. Dissolve dodecyl glucoside and Aspergillus niger lipase in 0.01 mol / L PBS buffer (pH 7.4) at a mass ratio of 7.5:1, stir at 200 rpm for 10 min at 4℃, and incubate at 24℃ in the dark for 4 h.

[0057] S2. After incubation, the mixture was transferred to an ultrafiltration centrifuge tube (molecular weight cutoff 10 kDa) and centrifuged at 4°C and 8000 rpm for 20 min. The retentate was collected and freeze-dried to obtain dodecyl glucoside-Aspergillus niger lipase.

[0058] S3. Dilute dodecyl glucoside-Aspergillus niger lipase to 0.20 mg / mL with 0.01 mol / L phosphate buffer (pH 7.4), place in a quartz cuvette with an inner diameter of 1 mm, set a resolution of 0.2 nm, a band width of 1.0 nm, and a sensitivity of 20 mdeg; perform circular dichroism spectroscopy with a wavelength range of 190–260 nm, a scan rate of 50 nm / min, and a scan temperature of 25 °C, and record the CD spectra. Circular dichroism is expressed as the average residue ellipticity θ, in deg·cm. 2 / mol), and used CDN software to analyze CD spectral data to obtain the contents of α-helices, β-sheets, β-turns and random coil structures.

[0059] like Figure 2 As shown, Aspergillus niger lipase and dodecyl glucoside-Aspergillus niger lipase exhibit two negative peaks near 208–210 nm and 220–222 nm, displaying typical α-helix structural characteristics. After dodecyl glucoside binds to Aspergillus niger lipase, the intensity of the negative peaks at 210 nm and 220 nm increases, and the α-helix characteristic signal significantly increases. The intensity of the negative peak near 215 nm also increases, and the β-sheet characteristic signal is significantly enhanced.

[0060] Table 1. Relative contents of the secondary structures of Aspergillus niger lipase and dodecyl glucoside-Aspergillus niger lipase

[0061]

[0062] As shown in Table 1, the relative contents of α-helix, β-sheet, β-turn, and random coil in the non-Aspergillus niger lipase were 52.5%, 12.4%, 14.5%, and 17.2%, respectively. In the dodecyl glucoside-Aspergillus niger lipase, the relative contents of α-helix and β-sheet increased by 9.2% and 4.1%, respectively, while the relative contents of β-turn and random coil decreased by 5.7% and 4.9%, respectively. The increased contents of α-helix and β-sheet enhance the overall rigidity and order of the enzyme molecule structure, contributing to the stabilization of the conformation of the catalytic active site.

[0063] Table 2. Thermal stability of lipase and dodecyl glucoside-Aspergillus niger lipase

[0064]

[0065] As shown in Table 2, the enzyme activity retention rate of free lipase after 10 h in a 60℃ water bath was 17.4%, indicating that it had basically lost its activity; the enzyme activity retention rate of dodecyl glucoside-Aspergillus niger lipase was 79.2%, indicating that dodecyl glucoside can improve the thermal stability of Aspergillus niger lipase.

[0066] Example 3

[0067] This embodiment describes the preparation of 1-oleic-2-palmitic-3-linoleic acid triglyceride (OPL) via enzymatic acid hydrolysis assisted by ultrasonic pretreatment. The specific method includes the following steps:

[0068] S1. 300 g of vegetable oil (the mass ratio of camellia seed oil to sunflower seed oil was 1:2.4) was placed in a round-bottom flask, and 74 g of potassium hydroxide, 300 mL of distilled water and 200 mL of 95% ethanol were added. The mixture was refluxed at 80℃ and 200 rpm for 1 h for saponification. After saponification, 3 mol / L hydrochloric acid was added to adjust the pH to 1.0. After standing and separating the layers, 100 mL of n-hexane was added to the oil phase, and the residual hydrochloric acid was washed away with distilled water. The solvent was removed by rotary evaporation at 50℃ and 40 rpm, and the mixture was dehydrated with anhydrous sodium sulfate to obtain free fatty acids (FFAs). The ratio of oleic acid to linoleic acid in the obtained FFAs was 1:1.25.

[0069] S2. Take 5 g of palm stearin (PS), mix PS and FFAs at a molar ratio of 1:12, add 6% of total mass of dodecyl glucoside-Aspergillus niger lipase, pretreat for 6 min under ultrasonic power of 300 W and duty cycle of 60%, then react with magnetic stirring at 55℃ for 3 h. After the reaction is completed, filter with an ultrafiltration membrane with a molecular weight cutoff of 20 kDa to recover the dodecyl glucoside-Aspergillus niger lipase and obtain the acid hydrolysis product.

[0070] S3. After concentrating the acid hydrolysis product, spot it onto a thin-layer chromatography plate to separate triglycerides (TAG). The developing solvent is n-hexane-diethyl ether-acetic acid (volume ratio 80:20:1). After scraping off the TAG band, extract it twice with diethyl ether, combine the extracts, and blow the solvent dry with nitrogen to obtain the OPL structured ester.

[0071] Example 4

[0072] The difference between this embodiment and embodiment 3 is that the molar ratio of PS to FFAs in this embodiment is 1:8, and the remaining steps are the same as in embodiment 3.

[0073] Example 5

[0074] The difference between this embodiment and Embodiment 3 is that in this embodiment, the amount of dodecyl glucoside-lipase added is 4% of the total substrate mass, and the remaining steps are the same as in Embodiment 3.

[0075] Example 6

[0076] The difference between this embodiment and embodiment 3 is that the stirring reaction temperature in this embodiment is 50°C and the stirring reaction time is 4.5 h, while the remaining steps are the same as in embodiment 3.

[0077] Example 7

[0078] The difference between this embodiment and embodiment 3 is that the ultrasonic power in this embodiment is 400 W, the ultrasonic duty cycle is 55%, and the ultrasonic time is 10 min. The remaining steps are the same as in embodiment 3.

[0079] Example 8

[0080] This embodiment describes the preparation of OPL via ultrasound-assisted enzymatic acid hydrolysis. The specific method includes the following steps:

[0081] S1. Oleic acid and linoleic acid were mixed evenly at a mass ratio of 1:2 to obtain FFAs. 5 g of PS was taken, and PS and FFAs were mixed at a molar ratio of 1:13.2. 10% of the total mass of dodecyl glucoside-Aspergillus niger lipase was added. The reaction was carried out under ultrasonic power of 140 W, duty cycle of 70%, and 55 °C for 120 min. After the reaction was completed, the mixture was filtered through an ultrafiltration membrane with a molecular weight cutoff of 20 kDa to recover the dodecyl glucoside-Aspergillus niger lipase and obtain the acid hydrolysis product.

[0082] S2. After concentrating the acid hydrolysis product, spot it onto a thin-layer chromatography plate to separate TAG. The developing solvent is n-hexane-diethyl ether-acetic acid (volume ratio 80:20:1). After scraping off the TAG band, extract it twice with diethyl ether, combine the extracts, and blow the solvent dry with nitrogen to obtain the OPL structured ester.

[0083] Example 9

[0084] The difference between this embodiment and embodiment 8 is that the mass ratio of oleic acid to linoleic acid in this embodiment is 1:1, and the remaining steps are the same as in embodiment 8.

[0085] Example 10

[0086] The difference between this embodiment and embodiment 8 is that in this embodiment, the amount of dodecyl glucoside-Aspergillus niger lipase added is 8% of the total substrate mass, and the remaining steps are the same as in embodiment 8.

[0087] Example 11

[0088] The difference between this embodiment and embodiment 8 is that the ultrasonic power in this embodiment is 120 W, the ultrasonic duty cycle is 75%, and the ultrasonic time is 135 min. The remaining steps are the same as in embodiment 8.

[0089] Comparative Example 3

[0090] The difference between this comparative example and Example 8 is that ultrasonic treatment is not used in this comparative example, while the other steps are the same as in Example 8.

[0091] Comparative Example 4

[0092] The difference between this comparative example and Example 8 is that in this comparative example, dodecyl glucoside-Aspergillus niger lipase is replaced with free Aspergillus niger lipase, while the other steps are the same as in Example 8.

[0093] Comparative Example 5

[0094] The difference between this comparative example and Example 8 is that in this comparative example, the dodecyl glucoside-Aspergillus niger lipase is replaced with the immobilized enzyme Lipozyme TL IM. After the reaction, the immobilized enzyme is removed by centrifugation. The remaining steps are the same as in Example 8.

[0095] Comparative Example 6

[0096] The difference between this comparative example and Example 8 is that, in this comparative example, dodecyl glucoside-Aspergillus niger lipase is replaced with Tween20 modified porcine pancreatic lipase. The preparation method of Tween20 modified porcine pancreatic lipase includes the following steps:

[0097] Tween20 was dissolved in anhydrous ethanol at 40°C, and 2 times the volume of water was added and mixed well. The amount of Tween 20 was 5%. Porcine pancreatic lipase was slowly added with stirring, and stirring was continued. The mixture was centrifuged at 3500 rpm for 5 min. The precipitate was freeze-dried to obtain Tween20-modified porcine pancreatic lipase. The remaining steps were the same as in Example 8.

[0098] Table 3 Product Indicators of OPL

[0099]

[0100] As shown in Table 3, the products prepared in Examples 3-11 of this invention have an OPL content of 42.66-47.88%, a relative content of palmitic acid at the sn-2 position of 73.26-78.13%, and an acyl migration rate of 2.16-2.51%. Compared with the comparative examples, this invention has significant advantages in product purity, position specificity, and reaction selectivity. In Comparative Example 3 (without ultrasonic treatment), the OPL content and relative content of palmitic acid at the sn-2 position are reduced, while the acyl migration rate is increased. This indicates that ultrasonic treatment can effectively promote substrate mass transfer and improve reaction efficiency, while also helping to maintain the original positional distribution of palmitic acid at the sn-2 position and reducing the occurrence of acyl migration side reactions. In Comparative Example 4 (free Aspergillus niger lipase), the OPL content and relative content of palmitic acid at the sn-2 position are relatively low, while the acyl migration rate is as high as 3.72%, indicating that the free enzyme has poor stability in the reaction system, low catalytic efficiency, and poor position selectivity. Although the contents of each index of Comparative Example 5 (immobilized lipase Lipozyme TL IM C) and Comparative Example 6 (Tween20 modified porcine pancreatic lipase) are better than those of other comparative examples, they are still not as good as those of the embodiments. This indicates that the dodecyl glucoside-lipase used in this invention is superior to commercial immobilized enzymes and Tween20 modified porcine pancreatic lipases in terms of catalytic efficiency and sn-2 position specificity, which is more conducive to improving the catalytic activity and position selectivity of the enzyme.

[0101] 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 person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A method for preparing an OPL-structured ester, characterized in that, Includes the following steps: S1. Dissolve alkyl glucoside and lipase in PBS buffer, stir well and incubate in the dark; after incubation, collect the retentate after ultrafiltration of the mixture, and dry it to obtain alkyl glucoside-lipase. S2. Free fatty acids are obtained by mixing oleic acid and linoleic acid. Palmitic acid-rich oils and free fatty acids are then mixed evenly, and alkyl glucoside-lipase is added to prepare a reaction system. The system is stirred under ultrasonic assistance, and after the reaction is complete, it is separated and purified to obtain an OPL-structured ester. Ultrasonic assistance can be any of the following methods: (1) After adding alkyl glucoside-lipase, the mixture is subjected to ultrasonic pretreatment and then stirred for reaction; (2) Throughout the entire reaction process after the addition of alkyl glucoside-lipase, continuous ultrasonic treatment was performed.

2. The method for preparing the OPL-structured ester according to claim 1, characterized in that: In step S1, the alkyl glucoside is dodecyl glucoside; the lipase is 1,3-specific lipase or non-specific lipase; the mass ratio of alkyl glucoside to lipase is 6~15:1; the incubation temperature is 23~26℃, the incubation time is 2~6 h; and the molecular weight cutoff of ultrafiltration is 8~20 kDa.

3. The method for preparing the OPL-structured ester according to claim 1, characterized in that: In step S2, the palmitic acid-rich oil is any one or more of palm oil, palm stearin, and palmitic acid glycerides; the mass ratio of oleic acid to linoleic acid in the free fatty acids is 1:1~2; and the molar ratio of the palmitic acid-rich oil to the free fatty acids is 1:6~16.

4. The method for preparing the OPL-structured ester according to claim 1, characterized in that: In step S2, the amount of alkyl glucoside-lipase added is 2-12% of the total substrate mass.

5. The method for preparing the OPL-structured ester according to claim 1, characterized in that: The reaction system in step S2 can be a solvent-free system, an organic solvent system, a eutectic solvent system, or an ionic liquid system.

6. The method for preparing the OPL-structured ester according to claim 1, characterized in that: In step S2, the temperature of the stirring reaction is 45~65℃, the stirring reaction time is 1~8 h, and the stirring speed is 300~800 rpm.

7. The method for preparing the OPL-structured ester according to claim 1, characterized in that: When the ultrasound-assisted mode is (1), the ultrasound power is 100~600 W, the ultrasound duty cycle is 10~80%, and the ultrasound time is 1~30 min.

8. The method for preparing the OPL-structured ester according to claim 1, characterized in that: When the ultrasound-assisted mode is (2), the ultrasound power is 60~180 W, the ultrasound duty cycle is 20~90%, and the ultrasound time is 1~180 min.

9. The OPL-structured ester prepared by the preparation method according to any one of claims 1 to 8, characterized in that, The content of palmitic acid at the sn-2 position in the OPL structure ester is 73.26~78.13%, and the acyl migration rate is 2.16~2.51%.

10. The use of the OPL structured ester according to claim 9 in the preparation of food, medicine or health products that aid digestion or promote bowel movement.

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