Method for synthesizing 1, 3-dilinoleic acid-2-palmitic acid triglyceride

Abstract

The invention belongs to the technical field of fat synthesis, and particularly discloses a method for synthesizing 1, 3-dilinoleic acid-2-palmitic acid triglyceride, which comprises the following steps: S1, dissolving palm stearate in a solvent, and purifying to obtain PPP; s2, mixing the PPP purified in the step S1 with LA, dissolving, and adding immobilized lipase ANL-MARE to carry out primary reaction; after the primary reaction is completed, carrying out acid removal; and then adding LA again to carry out secondary reaction, filtering to remove enzyme after the reaction is completed, and separating and purifying to obtain the LPL structure lipid. According to the method, PPP-rich palm stearin and linoleic acid are used as raw materials, and immobilized lipase is utilized to catalyze two-step acidolysis reaction in a solvent-free system to synthesize the structured triglyceride rich in LPL. Compared with a common one-step acidolysis method, the method has the advantages that the relative content of LPL is increased by 10.27%, the relative content of sn-2PA is kept unchanged, the usage amount of enzyme is effectively reduced, and the LPL efficiently synthesized through enzymatic catalysis of the method has the potential of being used for production of human milk fat substitutes.

Description

Technical Field

[0001] The present invention belongs to the technical field of fat synthesis, and particularly relates to a method for synthesizing 1,3-dilinoleoyl-2-palmitoyl glycerol triester. Background Art

[0002] Breast milk is an ideal nutrient for infant growth and development, containing fat, protein, carbohydrates, minerals, and other bioactive substances, and is the first choice food for newborn infants. Fat accounts for 3-5% of breast milk, but provides more than 50% of the energy required by infants and young children. Among them, 98% is composed of triglycerides (TAGs), and has a unique molecular structure. Most of the sn-2 position is occupied by saturated fatty acids, while most of the unsaturated fatty acids are distributed at the sn-1,3 positions. This triglyceride with a special structure can enhance the digestion and absorption of lipids and minerals by infants and young children, promote bone development and intestinal health, promote lipid metabolism and reduce lipid accumulation, and plays a key role in the early growth and development and even long-term health of infants. Breast milk lipids

[0003] The main TAGs in HMF-breast milk lipids are 1,3-dioleoyl-2-palmitoyl glycerol (OPO) and 1-oleoyl-2-palmitoyl-3-linoleoyl glycerol (OPL). 1,3-dilinoleoyl-2-palmitoyl glycerol (LPL) also accounts for a certain proportion. They can enhance TAG metabolism and promote the absorption of fat and calcium by infants. The highest content component in mature breast milk abroad is OPO, followed by OPL. In contrast, the highest content components in Chinese breast milk are OPL and OPO, followed by LPL, and there are also slight differences among different regions in China, which may be related to different eating habits and subsequent physiological and metabolic effects. In recent years, affected by work pressure, personal factors, etc., situations such as insufficient breast milk and nutritional deficiencies often occur, and formula milk has become an ideal supplementary food for the healthy growth and development of infants. However, the oils and fats in formula milk are mostly directly prepared from natural vegetable oils, and only simulate breast milk fat well in terms of macro fatty acids, while there are still significant differences from breast milk fat in terms of micro fatty acids, the content of palmitic acid at the Sn-2 position, and TAG composition. Human milk fat substitutes rich in OPO, OPL, and LPL can not only simulate the composition of human milk fat, but also provide health benefits for infants. Therefore, developing a structured lipid with a fatty acid composition and distribution similar to breast milk fat is an inevitable trend to improve infant nutrition and health.

[0004] In recent years, enzymatic synthesis of structured lipids has received extensive attention due to its high catalytic efficiency, mild reaction conditions, and environmental friendliness. The synthesis methods mainly include acidolysis, alcoholysis-esterification, ester-ester exchange, etc. Enzymatic acidolysis has the advantages of mild reaction and simple process. It is the most direct and effective way to incorporate the desired fatty acids into specific positions of TAG to obtain TAG rich in special fatty acids, and is currently widely used in industrial production. At present, several commercial lipid products rich in OPO have been incorporated into infant formula as essential nutritional supplements. The OPO of human milk fat substitute lipids from foreign enterprises almost occupies the entire market of infant products in China. At the same time, the research on OPL has gradually increased, but the research on LPL is still very limited. As a structured triglyceride with a corresponding proportion in Chinese breast milk, LPL contains the essential fatty acid linoleic acid (LA), which can be converted into biologically active ω-6 polyunsaturated fatty acids and plays an important role in the brain and retina. At the same time, linoleic acid is also an indispensable component of certain skin ceramides and is of great significance for maintaining the epidermal water barrier of the skin. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a method for synthesizing 1,3-dilinoleoyl-2-palmitoyl glycerol.

[0006] The present invention uses immobilized lipase ANL-MARE as a biocatalyst, and uses PPP and linoleic acid to efficiently synthesize LPL by two-step enzymatic catalysis.

[0007] In order to achieve the above invention object, the present invention provides the following technical solutions:

[0008] A method for synthesizing 1,3-dilinoleoyl-2-palmitoyl glycerol, comprising the following steps:

[0009] S1. Dissolve palm stearin in a solvent and purify to obtain tripalmitin.

[0010] S2. After mixing and dissolving the tripalmitin purified in step S1 with linoleic acid, add immobilized lipase ANL-MARE for a first reaction; after the first reaction is completed, remove the acid; then add linoleic acid again for a second reaction, and after the reaction is completed, filter to remove the enzyme, and obtain LPL structured lipid after separation and purification.

[0011] 1,3-Dilinoleoyl-2-palmitoyl glycerol, CAS registration number: 2190-16-1.

[0012] Preferably, in step S1, palm stearin is dissolved at 60 °C, mixed with acetone solvent in a ratio of 5 mL / g, then allowed to stand and crystallize in a 35 °C water bath for 3.5 h, and the solvent is removed by vacuum filtration to obtain PPP.

[0013] Preferably, based on the mass of the substrate for one reaction, the amount of immobilized lipase ANL-MARE added accounts for 3% to 5%.

[0014] Preferably, the total molar ratio of tripalmitin to linoleic acid as the substrate is 1:1 to 15.

[0015] Preferably, the reaction time for one reaction is 4 to 8 hours, and the reaction temperature is 40 to 70 °C.

[0016] Preferably, the reaction temperature and the amount of linoleic acid added in the second reaction are the same as those in the first reaction.

[0017] Preferably, the operation for removing acid is as follows: Add 10 mL / g of n-hexane, and then add 0.5 mol / L KOH and 30% aqueous ethanol solution to neutralize the free fatty acids.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] In this paper, palm stearin rich in PPP and linoleic acid are used as raw materials. In a solvent-free system, immobilized lipase is used to catalyze two-step acidolysis reactions to synthesize structured triglycerides rich in LPL. Compared with the ordinary one-step acidolysis method, the relative content of LPL in the LPL structured fat synthesized by the two-step enzymatic acidolysis method increases by 10.27%, while the relative content of sn-2PA remains unchanged, and the amount of enzyme used is effectively reduced. The LPL efficiently synthesized by this method has the potential for use in the production of human milk fat substitutes. Description of the Drawings

[0020] Figure 1 Content of PPP in palm stearin before and after fractionation (%).

[0021] Figure 2 Effect of substrate molar ratio on LPL content.

[0022] Figure 3 Effect of enzyme addition amount on LPL content.

[0023] Figure 4 Effect of reaction temperature on LPL content.

[0024] Figure 5 Effect of reaction time on LPL content.

[0025] Figure 6 Effect of reaction method on LPL synthesis: (A) Relative content of LPL; (B) Relative content of sn-2PA. Detailed Embodiments

[0026] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments and comparative examples of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0027] Unless otherwise specified, the test methods used in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0028] Example 1:

[0029] 1. Materials and Methods

[0030] 1.1 Materials and Reagents

[0031] Palm stearin at 58°C, other melting points are also available from Yihai Kerry Guangzhou Grain and Oil Co., Ltd.;

[0032] Linoleic acid LA from Shanghai Ron Reagent Co., Ltd.;

[0033] A mixture of 37 fatty acid methyl ester standards from Shanghai SigmaAldrich Co., Ltd.;

[0034] Immobilized lipase ANL-MARE (derived from Aspergillus niger lipase, immobilized on macroporous acrylic resin)

[0035] 1.2 Study on the two-step enzymatic acidolysis synthesis process of LPL structured lipid

[0036] 1.2.1 Fractionation of PPP

[0037] The solvent fractionation method was used to prepare PPP. The palm stearin was fully dissolved at 60°C, mixed with acetone at a ratio of 5 mL / g, and allowed to crystallize in a 35°C water bath for 3.5 h. Then the mixture was quickly vacuum filtered, and the residual solvent was removed using a rotary evaporator to obtain high-content PPP.

[0038] 1.2.2 Synthesis of LPL by two-step enzymatic acidolysis method

[0039] Mix the fractionated PPP and LA, completely dissolve them at 60 °C, then place them in a thermostatic water bath oscillator, add immobilized lipase ANL-MARE (the mass of the reaction substrate in one reaction, w / w), and carry out the constant-temperature reaction with rotation at 200 rpm. The end point of the first round of reaction is half of the total reaction duration. At this time, carry out deacidification, re-add LA, and no longer add immobilized lipase ANL-MARE. Then carry out the second round of acidolysis reaction. The reaction temperature and reaction time conditions are the same as those in the first round. Finally, filter to remove the enzyme, separate and purify the product to obtain LPL structured lipid. During the synthesis of LPL structured lipid, use the relative content of LPL in total TAG and the mass fraction of palmitic acid at the sn-2 position in total palmitic acid (sn-2PA, relative content) as indicators to evaluate the content of LPL and the phenomenon of acyl transfer in the product. All reactions are repeated 3 times.

[0040] 1.2.3 Single-factor experiments

[0041] According to the method in the previous section, investigate the effects of factors such as the total molar ratio of PPP to LA (1:4, 1:6, 1:8, 1:10, 1:12), the addition amount of immobilized lipase ANL-MARE in one reaction (6%, 8%, 10%, 12%, 14%), the reaction time of the total reaction (1 h, 2 h, 4 h, 6 h, 8 h), and the reaction temperature of one reaction (45 °C, 50 °C, 55 °C, 60 °C, 65 °C) on the synthesis of LPL structured lipid by the two-step method.

[0042] Table 1 Factor levels of single-factor experiment design

[0043]

[0044]

[0045] 1.2.4 Response surface experiment optimization

[0046] In order to study the relationship between the synthesis conditions and the reaction degree, select 3 factors that have a significant impact on the relative content of LPL and the relative content of sn-2PA: substrate molar ratio (A), enzyme addition amount (B), and reaction time (C). Conduct a Box-Behnken design with three factors and three levels to optimize the two-step enzymatic acidolysis process for synthesizing LPL. Carry out regression analysis on the response variables, and fit the following second-order polynomial model equation to describe the relationship between the response variables and the independent variables. The levels of each factor are shown in Table 2.

[0047] Table 2 Factor levels of response surface experiment design

[0048]

[0049] Note: ① The molar ratio of substrate A is the total molar ratio of the substrates in the first and second reactions. If the reaction is carried out step by step, for example, the total molar ratio of the substrates of tripalmitin and linoleic acid is 1:10, then 5 equivalents of linoleic acid are added in the first reaction, and the remaining 5 equivalents of linoleic acid are added in the second reaction; ② The addition amount of enzyme B is the amount added in the first reaction, and no enzyme needs to be added in the second reaction; ③ The reaction time C is the time of the total reaction (the first and second reactions).

[0050] 1.2.5 Purification of LPL

[0051] To purify the structured lipid, 10 mL / g of n-hexane was added to the crude product, and then 0.5 mol / L KOH-30% ethanol aqueous solution was added to neutralize the free fatty acids. The organic phase was collected, washed with saturated sodium chloride solution, then the water was removed by anhydrous sodium sulfate, and finally the organic solvent was removed by rotary evaporation at 50 °C to obtain the structured lipid.

[0052] To completely remove the residual impurities, the solvent crystallization method was used to purify LPL. Absolute ethanol (3 ml / g) was added to the product, dissolved at 60 °C, then crystallized at 25 °C for 7 min and filtered by suction. Absolute ethanol and water (3 / 1, v / v) were added again, dissolved at 60 °C, crystallized at 30 °C for 7 min, filtered by suction and dried under vacuum to obtain LPL.

[0053] 1.2.6 Analysis of triglyceride composition

[0054] The composition of triglycerides in the product was analyzed by a reversed-phase high-performance liquid chromatograph equipped with an evaporative light scattering detector (RP-HPLC-ELSD). The method is as follows: Air was used as the nebulizing gas; the temperature of the ELSD detector was 40 °C; the carrier gas pressure was 3.5 bar; the gain was 6; chromatographic column: Dionex C30 (250 mm × 4.6 mm, 5 μm); mobile phase: phase A was acetonitrile, phase B was isopropanol; gradient elution program: 0 - 10.0 min, 95% - 60% A, 5% - 40% B; 10.0 - 60.0 min, 60% A, 40% B; 60.0 - 61.0 min, 60% - 25% A, 40% - 75% B; 61.0 min - 66.0 min, 25% A, 75% B; 66.0 min - 67.0 min, 25% - 95% A, 75% - 5% B; flow rate: 1.0 mL / min; column temperature: 30 °C; injection volume: 20 μL.

[0055] 1.2.7 Analysis of fatty acid composition

[0056] 1.2.7.1 Fatty acid methylation

[0057] Dissolve 20 mg of the sample in 1.0 mL of chromatographic grade n-hexane, add 0.5 mL of 2 mol / L potassium hydroxide-methanol solution to react, vortex and oscillate, draw the upper layer, filter through a membrane after removing water, and detect and analyze by gas chromatography.

[0058] 1.2.7.2 Gas chromatography analysis conditions

[0059] Chromatographic column: DB-WAX capillary column (60 m × 0.25 mm × 0.25 μm); detector temperature 250 °C; inlet temperature 250 °C; temperature programming: hold at 50 °C for 1 min, rise to 175 °C at 22.5 °C / min, and finally rise to 230 °C at 4 °C / min, hold for 20 min; helium flow rate 1.0 mL / min; inject 1 μL; split ratio 1:20.

[0060] 1.2.7.3 Analysis of sn-2 fatty acid composition

[0061] Add 2 mL of Tris-HCl buffer (pH = 8.0), 0.5 mL of 0.05% cholate solution, and 0.2 mL of 2.2% calcium chloride solution to the product, vortex and oscillate for 2 min, add 50 mg of pancreatic lipase, react at 40 °C for 3 min. After the reaction, add 1 mL of 6 mol / L HCl and 2 mL of ether to the sample, centrifuge at 4000 r / min, draw the upper organic layer, dry with anhydrous sodium sulfate, and concentrate to 200 μL under nitrogen. Spot the sample on thin-layer chromatography, the developing agent is n-hexane: anhydrous ether: glacial acetic acid (50:50:1, v / v / v), develop with 2,7-dichlorofluorescein, identify under ultraviolet light, scrape off 2-MAG, extract twice with anhydrous ether, dry the anhydrous ether by blowing with nitrogen, and then methylate the fatty acids of the sample. After detection and analysis by gas chromatography, the calculation formula for the relative content of palmitic acid at the sn-2 position of the glycerol backbone is as follows:

[0062]

[0063] 1.2.8 Data analysis

[0064] All reactions and tests were repeated 3 times, all data were expressed as mean ± standard error, one-way ANOVA was performed using SPSS 26, with P < 0.05 considered statistically significant, and the difference analysis was performed using Duncan's multiple range test. Response surface test analysis was performed using software Design-Expert V 8.0.6. Graphs were drawn using software Origin 2021.

[0065] 2 Results and analysis

[0066] 2.1. Fractionation of PPP

[0067] Palm stearin is rich in PPP. As a food-grade raw material, PPP is a good substrate source for the synthesis of sn-2 palmitate. Therefore, obtaining a large amount of PPP as the reaction substrate is an important step in the enzymatic synthesis of LPL. As Figure 1 shown, PPP was enriched by solvent fractionation, and the content of PPP in the fractionation product increased from 73.53% to 92.96%. The above results indicate that this method can effectively enrich PPP in palm stearin and can be used as a good raw material source for the subsequent synthesis of LPL.

[0068] 2.2 Single-factor experiments

[0069] 2.2.1 Effect of substrate molar ratio on the synthesis of LPL

[0070] First, the effect of the molar ratio of the substrate PPP to the fatty acid acyl donor on the enzymatic synthesis of LPL was investigated. As Figure 2 shown, as the substrate molar ratio increased from 1:4 to 1:8, the relative content of LPL in the product increased significantly from 43.50% to 66.20% (P < 0.05), and the relative content of palmitic acid (PA) at the sn-2 position increased from 61.00% to 71.00%. The higher concentration of the fatty acid acyl donor effectively promoted the acyl exchange process under enzymatic catalysis by increasing the molecular contact probability in the reaction system, replaced the palmitic acid on PPP, increased the effective collision between the lipase and the substrate, and thus improved the synthesis efficiency of LPL. When the substrate molar ratio continued to increase, the relative contents of LPL and sn-2PA in the acidolysis product changed little (P > 0.05), indicating that the reaction had reached a dynamic equilibrium state at this time. Further analysis showed that the excessive fatty acyl donor not only changed the system pH value and then inhibited the catalytic efficiency of the lipase, but also significantly increased the difficulty and economic cost of the subsequent deacidification process. In summary, 1:8 was selected as the subsequent substrate molar ratio.

[0071] 2.2.2 Effect of enzyme dosage on the synthesis of LPL

[0072] As Figure 3As shown, the effect of lipase addition amount on the biosynthesis efficiency of LPL was evaluated. When the dosage of ANL-MARE lipase increased from 6% to 12%, the relative content of LPL showed a significant upward trend (P < 0.05), and the relative content of sn-2PA increased first and then stabilized. When the enzyme addition amount increased from 12% to 14%, the relative contents of LPL and sn-2PA in the target product did not show significant changes (P > 0.05), indicating that the binding sites between enzyme molecules and substrates had reached a saturated state at this time. Further analysis showed that under the condition of fixed substrate concentration, appropriately increasing the enzyme loading amount could effectively increase the binding frequency between substrate molecules and the enzyme active center, thus significantly increasing the synthesis efficiency of LPL. However, when the reaction tended to equilibrium, the addition of excessive lipase not only could not improve the catalytic efficiency, but might instead trigger acyl migration, leading to increased hydrolysis of triacylglycerol (TAG). To avoid the waste caused by excessive enzyme preparations and the accumulation of reaction by-products, 12% was selected as the optimal enzyme addition amount.

[0073] 2.2.3 Effect of reaction temperature on the synthesis of LPL

[0074] As Figure 4 shown, from 45°C to 50°C, the relative contents of LPL and sn-2PA increased significantly (P < 0.05). At 60°C, the relative content of LPL showed a downward trend (P < 0.05). At 65°C, the relative contents of LPL and sn-2PA both decreased significantly (P < 0.05). This change characteristic was mainly closely related to the optimal catalytic temperature range (40 - 60°C) of the ANL-MARE immobilized enzyme. When the reaction temperature was too low, the PPP present in the substrate was not fully dissolved, resulting in an increase in the viscosity of the system, which significantly hindered the effective formation of the enzyme-substrate complex and led to an incomplete reaction. Moderate heating could improve the rheological properties of the system, increase the effective collision frequency by enhancing molecular thermal motion, and thus accelerate the reaction rate. However, too high a temperature not only exacerbated the non-specific migration of acyl groups, but was more likely to cause a change in the protein spatial conformation of the enzyme, resulting in a decrease in catalytic activity. Therefore, on the premise of ensuring reaction efficiency and product quality, the reaction temperature should be as low as possible. 50°C was selected as the reaction condition.

[0075] 2.2.4 Effect of reaction time on the synthesis of LPL

[0076] As Figure 5As shown in the figure, in the initial stage of the reaction (0 - 4 h), the relative contents of LPL and sn - 2PA in the product showed a synchronous and significant increase (P < 0.05). At 4 h, the content of LPL reached 73.75%. From 4 to 6 h of the reaction, the relative contents of LPL and sn - 2PA increased slightly, but the difference was not significant (P > 0.05), indicating that the enzyme - catalyzed system had approached an equilibrium state. When the reaction reached 8 h, the relative content of sn - 2PA decreased slightly (P < 0.05). These observation results indicate that the reaction time is the main factor affecting acyl transfer. Further analysis shows that prolonging the reaction time will cause non - specific migration of palmitic acid groups at the sn - 2 position, which are gradually replaced by other fatty acid acyl donors, thereby reducing the purity of the reaction product and increasing the proportion of non - target products. Therefore, considering the balance between reaction efficiency and product quality, 4 h is preferably selected as the optimal reaction time, which can not only ensure the efficient synthesis of key products but also effectively reduce the side - reaction process.

[0077] 2.3 Response surface optimization experiment

[0078] 2.3.1 Establishment of regression model and variance analysis

[0079] On the basis of single - factor experiments, according to the Box - Behnken experimental design principle, three influencing factors, namely, the substrate molar ratio (A), enzyme dosage (B), and reaction time (C), were selected as independent variables, and the relative content of LPL (Y1) and the relative content of sn - 2PA (Y2) were used as response values. A three - factor and three - level response surface optimization experiment was designed and carried out to optimize the two - step enzymatic acidolysis synthesis reaction. The experimental results are shown in Tables 3 - 5. The change ranges of the relative content of LPL and the relative content of sn - 2PA are 41% - 78.82% and 61.76% - 73.49% respectively. In the present invention, the Design expert 8.0.6 software was used to perform multiple fitting regression analyses on the experimental data, and multiple regression equations (1) and (2) for the relative content of LPL and the relative content of sn - 2PA were obtained, which describe the relationship between the substrate molar ratio (A), enzyme loading (B), and reaction time (C).

[0080] Y1 = 68.36 + 7.27A + 2.96B + 12.71C + 0.3440AB + 1.19AC + 0.9640BC - 4.76A 2 - 0.3971B 2 - 7.02C 2

[0081] Y2 = 68.26 + 2.89A + 0.5266B + 3.33C + 0.7905AB + 0.3315AC + 0.3442BC - 0.4664A 2 + 0.1074B 2-0.49

[0082] 95C 2

[0083] Table 3 Response surface design and results

[0084]

[0085]

[0086] Table 4 ANOVA results of the regression model for the relative content of LPL

[0087]

[0088]

[0089] Note: A, B, and C represent the substrate molar ratio, enzyme loading, and reaction time, respectively; "**" represents extremely significant difference (P≤0.01), and "*" represents significant difference (P≤0.05).

[0090] Table 5 ANOVA results of the regression model for the relative content of sn-2PA

[0091]

[0092]

[0093] Note: A, B, and C represent the substrate molar ratio, enzyme loading, and reaction time, respectively; "**" represents extremely significant difference (P≤0.01), and "*" represents significant difference (P≤0.05).

[0094] 2.3.2 Response surface cross-interaction analysis

[0095] According to the F values and P values of each factor in Tables 4 and 5, for the LPL content, among the factors selected in the experiment, the first-order terms A and C have extremely significant effects on the results (P < 0.01), while B has a significant effect on the results (P < 0.05), and all interaction terms have no significant effect on the results (P > 0.05). The second-order term C 2 has an extremely significant effect (P < 0.01), A 2 has a significant effect on the results (P < 0.05), while B 2 has no significant effect on the results (P > 0.05). For the relative content of sn-2Pa, the first-order terms A and C both have extremely significant effects on the results (P<0.01), B has a significant effect on the results (P<0.05), the interaction term AB has a significant effect on the experiment (P<0.05), while the remaining interaction terms have no significant effect on the results (P > 0.05). The second-order terms A 2 、B 2 、C2 Its effect has no significant impact on the results (P > 0.05). In summary, the synthesis conditions, namely the substrate molar ratio, enzyme dosage, and reaction time, are the main factors affecting the synthesis of LPL, that is, C > A > B.

[0096] 2.3.3 Determination and verification of the optimal synthesis conditions

[0097] The optimal conditions for the two-step acidolysis synthesis of LPL structured lipids were predicted using Design-expert 8.0.6 software. The obtained optimal process conditions were: reaction time 5 h, substrate molar ratio 1:9.76, enzyme dosage 13.71%, and reaction temperature 50 °C. Under these conditions, the theoretically predicted relative contents of LPL and sn-2PA were 79.14% and 73.41% respectively. To verify the reliability of the model and facilitate the synthesis experiment, the predicted optimal conditions were modified: reaction time 5 h, substrate molar ratio 1:10, enzyme dosage 13.70%, and reaction temperature 50 °C. Three parallel experiments were conducted under these conditions to verify the model. The average relative content of LPL was 78.05%, and the average relative content of sn-2PA was 71.66%. The strong correlation between the actual values and the predicted values confirmed the accuracy of the response model, which was sufficient to reflect the expected optimization effect of LPL synthesis.

[0098] 2.3.4 Compositions of FAs and TAGs in the product under the optimal synthesis conditions

[0099] Under the optimal conditions, LPL was synthesized by two-step enzymatic acidolysis using the specific sn-1,3-lipase ANL-MARE and then purified. The final relative content of LPL reached 78.05%. As shown in Table 6, the compositions of triglycerides in the product are listed. LPL is the component with the highest content, followed by PPL, accounting for 10.52%. PPP almost completely reacted, and the final ratio was only 0.10%. Table 7 lists the composition and distribution of fatty acids in the experimental product LPL. Among them, the most important unsaturated fatty acid, linoleic acid, accounts for 56.35%, the saturated fatty acid, palmitic acid, accounts for 38.62%, and the relative content of palmitic acid at the sn-2 position is 71.55%. Combining the high relative content of palmitic acid at the sn-2 position of the glycerol backbone, it can be seen that most of the LPL triglycerides exist in the form of sn-LPL. This is consistent with the expected results, indicating that this two-step enzymatic acidolysis reaction can prepare LPL structured lipids with a relatively high relative content of palmitic acid at the sn-2 position.

[0100] Table 6 Compositions of triglyceride components in the synthetic product (%)

[0101]

[0102] Table 7 Compositions of fatty acids in the synthetic product (%)

[0103]

[0104]

[0105] 2.3.5 Comparison between One-step and Two-step Syntheses of LPL

[0106] In the one-step acidolysis method, all raw material substrates and lipase are added at once for the reaction. Therefore, the amount of lipase added is twice that of the two-step acidolysis method. The total reaction duration, temperature, and other conditions are the same as those of the two-step acidolysis method in the examples until the reaction ends, and no deacidification treatment is involved in the middle.

[0107] The reaction route of the two-step acidolysis method is to make the reaction proceed in the forward direction by removing the palmitic acid formed in the first step and the excessive linoleic acid, so as to improve the reaction efficiency. Taking the total substrate molar ratio of 1:10, reaction temperature of 50 °C, reaction time of 5 h, and the enzyme addition amount in one reaction of 13.70% as the reaction conditions, the effects of the one-step and two-step methods on the LPL content in the product were compared, and the results are as Figure 6 shown. Under the optimal conditions, the relative content of LPL prepared by the one-step acidolysis method is 67.78%, and the relative content of sn-2PA is 71.50%. While the relative content of LPL prepared by the two-step enzymatic acidolysis method is 78.05%, and the relative content of sn-2PA is 71.66%. The relative content of LPL increased by 10.27% compared with the one-step method, and at the same time, there is no significant change in the relative content of sn-2PA. Since the amount of enzyme is added based on the total mass percentage of the reaction substrate (w / w), all fatty acid acyl donors are added at the beginning of the reaction in the one-step acidolysis method, and the total substrate mass is larger, resulting in an increase in the amount of enzyme added; the fatty acid donors in the two-step method are added step by step. In the first stage of the reaction, half of the total acyl donors are added, and the remaining acyl donors are added again in the subsequent stage of the reaction. This means that the amount of enzyme used is less than that of the one-step method. Without changing the amount of reaction substrate used, the two-step acidolysis method can reduce the amount of enzyme used while effectively increasing the content of LPL in the product, showing certain advantages compared with the traditional one-step acidolysis method.

[0108] The present invention uses palm stearin rich in PPP and linoleic acid as raw materials, and under a solvent-free system, uses immobilized lipase ANL-MARE to catalyze two-step acidolysis reactions to synthesize structured triglycerides rich in LPL. The optimal process parameters are obtained through single-factor experiments and response surface optimization design: the molar ratio of PPP to fatty acid is 1:10, the addition amount of lipase ANL-MARE is 13.70%, react at 50 °C for 5 h. Under the optimal conditions, the content of LPL is 78.05%, and the relative content of sn-2PA is 71.66%. Compared with the ordinary one-step acidolysis method, the relative content of LPL synthesized by the two-step enzymatic acidolysis method increases by 10.27%, while the relative content of sn-2PA remains unchanged, and the amount of enzyme used is effectively reduced. Therefore, the LPL efficiently synthesized by enzymatic catalysis through this method has the potential for use in the production of human milk fat substitutes.

[0109] Obviously, the above-mentioned embodiments of the present invention are merely examples for clearly explaining the technical solutions of the present invention, rather than limitations on the specific implementation manners of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the claims of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. A method for synthesizing 1,3-dilinoleoyl-2-palmitoyl glycerol triester, characterized in that, It includes the following steps: S1. Add palm stearin to a solvent for dissolution, and purify to obtain tripalmitin. S2. After mixing and dissolving the tripalmitin obtained by purification in step S1 with linoleic acid, add immobilized lipase ANL-MARE for a primary reaction; after the primary reaction is completed, perform acid removal; then add linoleic acid again for a secondary reaction, and after the reaction is completed, filter to remove the enzyme, and obtain LPL structured lipid after separation and purification.

2. The method for synthesizing 1,3-dilinoleoyl-2-palmitoyl glycerol triester according to claim 1, wherein In step S1, dissolve palm stearin at 60 °C, mix it with acetone solvent at a ratio of 5 mL / g, then let it stand for crystallization in a 35 °C water bath for 3.5 h, remove the solvent by vacuum filtration to obtain tripalmitin.

3. The method for synthesizing 1,3-dilinoleoyl-2-palmitoyl glycerol triester according to claim 1, characterized in that, Based on the mass of the substrate in the primary reaction, the amount of immobilized lipase ANL-MARE added accounts for 3% - 5%.

4. The method for synthesizing 1,3-dilinoleoyl-2-palmitoyl glycerol triester according to claim 1, characterized in that, The total molar ratio of tripalmitin to linoleic acid as the substrate is 1:1 - 15.

5. The method for synthesizing 1,3-dilinoleoyl-2-palmitoyl glycerol triester according to claim 1, wherein The reaction time of the primary reaction is 4 - 8 hours, and the reaction temperature is 40 - 70 °C.

6. The method for synthesizing 1,3-dilinoleoyl-2-palmitoyl glycerol triester according to claim 5, characterized in that, The reaction temperature of the secondary reaction and the addition amount of linoleic acid are the same as those of the primary reaction.

7. The method for synthesizing 1,3-dilinoleoyl-2-palmitoyl glycerol triester according to claim 1, characterized in that, The operation of acid removal is: add 10 mL / g of n-hexane, and then add 0.5 mol / L KOH and 30% ethanol aqueous solution to neutralize free fatty acids.

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