Method for preparing docosahexaenoic acid lysophosphatidylcholine through enzyme catalysis

Abstract

The invention belongs to the technical field of phospholipid preparation, and particularly relates to a method for preparing docosahexaenoic acid lysophosphatidylcholine through enzyme catalysis. The method comprises the following steps: (1) synthesizing DHA-PC: taking PC and DHA as substrates, and reacting under the action of CALB to generate DHA-PC; and (2) synthesizing DHA-LPC: taking the DHA-PC obtained in the step (1) as a raw material, and reacting under the action of Lipozyme TL IM to generate the DHA-LPC. The DHA-LPC can be directly prepared through a two-step enzyme method, algae oil rich in DHA is used as a DHA receptor, the problem that the method mentioned in the background technology is limited by the yield of marine organisms is solved, the average yield of the DHA-PC can reach 43.35%, and the yield of the DHA-LPC can reach 33.13%. The average free radical scavenging capacity of the 6% DHA-PC emulsion can reach 70%; and the average free radical scavenging rate of the 6% DHA-LPC emulsion is 83.93%.

Description

Technical Field

[0001] The invention belongs to the technical field of phospholipid preparation, and particularly relates to a method for preparing docosahexaenoic acid lysophosphatidylcholine by enzyme catalysis. Background Art

[0002] Phospholipids rich in DHA are mostly extracted from marine organisms and their eggs. The steps are as follows: take dried marine organism tissue, extract it with an organic solvent, and then evaporate it to obtain crude oil. Add n-hexane to dissolve it again, and then use cold acetone to precipitate phospholipids. Evaporate it to obtain a precipitate, and then blow dry it with nitrogen to obtain phospholipids rich in DHA. The above method can extract phospholipids rich in DHA in marine organisms, but the specific phospholipid molecules cannot be determined. In addition, this method is limited by the production of marine organisms, and the extraction rate is low and the production cost is high.

[0003] There are also documents that disclose a method for enzymatically synthesizing docosahexaenoic acid phosphatidylcholine (hereinafter referred to as "DHA-PC"), which is to synthesize DHA-PC by catalyzing the fatty acyl exchange between DHA algae oil and PC by Antarctic Candida lipase B in a n-hexane-water two-phase system, and optimizing factors such as enzyme amount, temperature, two-phase volume ratio, substrate mass ratio, pH, reaction time, etc. Under the optimal conditions, the maximum yield of DHA-PC is 21%, which is higher than the DHA content of most commercially available DHA-PLs supplements (about 12%).

[0004] In the above methods, the maximum yield of DHA-PC still needs to be improved.

[0005] There is little literature disclosure on the method of preparing docosahexaenoic acid-lysophosphatidylcholine (hereinafter referred to as "DHA-LPC").

[0006] Therefore, it is necessary to improve the above method and invent a preparation method of docosahexaenoic acid lysophosphatidylcholine with high extraction rate and relatively low cost. Summary of the invention

[0007] In order to solve the above technical problems, the present invention can directly prepare DHA-LPC through a two-step enzymatic method, and the yield of DHA-PC can reach 43.35%, and the yield of DHA-LPC can reach 33.13%.

[0008] The method for preparing docosahexaenoic acid lysophosphatidylcholine by enzyme catalysis, the optimized steps are as follows:

[0009] (1) Synthetic Docosahexaenoic Acid Phosphatidylcholine (DHA-PC)

[0010] Take 4 round-bottom flasks, add 100 mg of phosphatidylcholine (PC) and 1000 mg of DHA algae oil respectively, then add 10 mL of n-hexane, sonicate for 10 min to completely dissolve the substrate in n-hexane, add 2 ml of pH 7.0 phosphate buffer, continue sonication for 5 min to completely disperse the substrate, then add 80 μl of immobilized lipase solution (CALB), put the round-bottom flasks in a constant temperature shaking incubator, and react at 50 ° C in the dark for 24 h;

[0011] After the reaction is completed, the upper organic phase is taken, 5g of active Al2O3 is added, and then placed on a shaker to adsorb in the dark for 12 hours; after the adsorption is completed, the active Al2O3 is collected to a 50ml centrifuge tube, 10ml of 4°C pre-cooled acetone is added to shake and wash to wash away the unreacted algae oil, and the process is repeated 3 times, and then 10ml of pre-cooled methanol is added to elute the active Al2O3 to obtain the docosahexaenoic acid phosphatidylcholine adsorbed on the active Al2O3, and the eluate is collected, and the process is repeated 3 times; then the eluate is centrifuged at 7000r / min at 4°C for 10min, the upper clear liquid is taken, and the solvent is evaporated to obtain the docosahexaenoic acid phosphatidylcholine;

[0012] (2) Synthetic Docosahexaenoic Acid Lysophosphatidylcholine (DHA-LPC)

[0013] Take 4 round-bottom flasks, add 200 mg of DHA-PC prepared in (1) and 10 ml of n-hexane, and then add 40 mg of Lipozyme TL IM enzyme equilibrated with a saturated salt solution, the salt solution used is LiCl (water activity Aw = 0.13), MgCl2·6H2O (Aw = 0.33), NaCl (Aw = 0.75) and K2SO4 (Aw = 0.97); put them in a 40°C magnetic stirring water bath under nitrogen protection and react for 4 hours respectively. After the reaction is completed, the mixture is centrifuged at 6000 r / min for 10 minutes, the supernatant is taken, and then filtered with a 0.45 μm PTFE filter to ensure that Lipozyme TL IM is completely removed, thereby terminating the reaction;

[0014] The mixture obtained after the reaction is loaded into a silica gel column, and the free fatty acids are firstly removed by eluting with an eluent, and then the phospholipids are recovered with methanol, and the solvent is rotary evaporated to obtain docosahexaenoic acid lysophosphatidylcholine; in the eluent, the volume ratio of chloroform to methanol is 10:1.

[0015] The beneficial effects of the present invention are:

[0016] (1) DHA-LPC can be directly prepared by a two-step enzymatic method, using DHA-rich algae oil as the DHA receptor, thus solving the problem of being restricted by the yield of marine organisms in the methods mentioned in the background art;

[0017] (2) Compared with the method described in the background art, the average yield of DHA-PC in the present invention can reach 43.35%, and the yield of DHA-LPC can reach 33.13%;

[0018] (3) DHA-PC retains the emulsifying properties of phospholipids and has improved antioxidant activity. The emulsifying properties and antioxidant activity of DHA-LPC are further improved compared with DHA-PC. The average free radical scavenging ability of 6% DHA-PC emulsion can reach 70%; the average free radical scavenging rate of 6% DHA-LPC emulsion is 83.93%. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The effect of pH on the enzyme-catalyzed synthesis of DHA-PC;

[0020] Figure 2 The effect of substrate mass ratio on enzyme-catalyzed synthesis of DHA-PC;

[0021] Figure 3 The effect of temperature on the enzyme-catalyzed synthesis of DHA-PC;

[0022] Figure 4 The effect of enzyme amount on the enzyme-catalyzed synthesis of DHA-PC;

[0023] Figure 5 The effect of hydrolysis time on the enzyme-catalyzed synthesis of DHA-LPC;

[0024] Figure 6 The effect of temperature on the enzyme-catalyzed synthesis of DHA-LPC;

[0025] Figure 7 The effect of enzyme amount on the enzyme-catalyzed synthesis of DHA-LPC;

[0026] Figure 8 Fourier transform infrared spectroscopy analysis of DHA-PC;

[0027] Fig. 9 This is the antioxidant activity analysis diagram of DHA-PC emulsion;

[0028] Fig.10 Fourier transform infrared spectroscopy analysis of DHA-LPC;

[0029] Fig.11 This is an analysis diagram of the antioxidant activity of DHA-LPC emulsion. DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0031] In this application, the experimental reagents and materials are as follows:

[0032] Table 1 Experimental reagents and materials

[0033]

[0034] The instruments and equipment are as follows:

[0035] Table 2 Main instruments and equipment

[0036]

[0037] The raw materials or equipment used in other testing processes not listed are all common commercially available raw materials or equipment and will not be described in detail here.

[0038] Example 1

[0039] Synthesis of DHA-PC and investigation of its influencing factors

[0040] In this example, the effects of pH, two-phase volume ratio, substrate mass ratio, temperature, and enzyme amount on the synthesis of DHA-PC were investigated as follows:

[0041] 1.1 Effect of pH on the synthesis of DHA-PC

[0042] (1) Take 4 round-bottom flasks, add 100 mg PC, 1000 mg DHA algae oil, and 10 mL n-hexane respectively, sonicate for 10 min to completely dissolve the substrate in n-hexane, add 2 ml phosphate buffer of different pH (pH = 6.5, 7.0, 7.5, 8.0), continue sonication for 5 min to completely disperse the substrate, then add 120 μl CALB enzyme solution, place the round-bottom flasks in a constant temperature shaking incubator, and react at 50°C in the dark for 24 h.

[0043] (2) After the reaction, take the upper organic phase, add 5g of activated alumina and place it on a shaker in the dark for adsorption for 12h. After the adsorption is completed, collect the active Al2O3 into a 50ml centrifuge tube, add 10ml of 4℃ pre-cooled acetone and shake to wash, the purpose is to wash away the unreacted algae oil, repeat 3 times, then add 10ml of pre-cooled methanol to elute the active Al2O3, the purpose is to obtain DHA-PC adsorbed on the active Al2O3, collect the eluate, repeat 3 times; then centrifuge the eluate at 4℃ 7000r / min for 10min, take the upper clear liquid, evaporate the solvent to obtain DHA-PC, and store it at -20℃ until analysis.

[0044] As attached Figure 1As shown in the figure, there is a certain correlation between the yield of DHA-PC and pH value. As the pH value gradually increases, the yield of DHA-PC shows an increasing trend. When the pH is 7, the yield of DHA-PC reaches the highest, with a yield of 31.9%, and then the yield decreases with the increase of pH.

[0045] 1.2 Effect of substrate mass ratio on the synthesis of DHA-PC

[0046] Take 4 round-bottom flasks, add 100 mg PC and different masses of algae oil (algae oil mass is 100 mg, 500 mg, 1000 mg, 1500 mg), add 10 mL of n-hexane, sonicate for 10 min to completely dissolve the substrate in n-hexane, add 2 ml of pH = 7 phosphate buffer, continue sonication for 5 min to completely disperse the substrate, then add 120 μl of CALB enzyme solution, put the round-bottom flask in a constant temperature shaking incubator, and react at 50 ° C in the dark for 24 h. After the reaction is completed, the operation steps are the same as (2) in 1.1.

[0047] As attached Figure 2 As shown in the figure, with the increase of substrate mass ratio, the yield continues to increase. When DHA:PC reaches 10:1, the yield reaches a maximum of 28.22%. After that, the substrate mass ratio continues to increase and the yield decreases. This may be because in a system with stable reaction conditions, the enzyme will combine with the reaction substrate to form an intermediate complex, and then decompose into products and free enzymes. When the substrate concentration is low, most enzymes are in a free state. As the substrate concentration increases, the probability of substrate binding to the enzyme increases, and the yield increases accordingly. When the enzyme reaches saturation, the reaction rate no longer increases, and too much substrate cannot bind to the enzyme and is free in the reaction system, resulting in a decrease in the fluidity of the system, which in turn reduces the probability of intermolecular collisions, resulting in a decrease in reaction rate and a decrease in yield.

[0048] 1.3 Effect of temperature on the synthesis of DHA-PC

[0049] Take 4 round-bottom flasks, add 100 mg PC, 1000 mg DHA algae oil, 10 mL n-hexane, ultrasonicate for 10 min to completely dissolve the substrate in n-hexane, add 2 ml pH = 7 phosphate buffer, continue ultrasonicate for 5 min to completely disperse the substrate, then add 120 μl CALB enzyme solution, put the round-bottom flasks in a constant temperature shaking incubator at different temperatures (temperatures of 40°C, 45°C, 50°C, 55°C), and react for 24 h in the dark. After the reaction is completed, the operation steps are the same as (2) in 1.1.

[0050] As attached Figure 3As shown in the figure, as the temperature continues to rise, the DHA-PC yield increases accordingly. When the temperature rises to 50°C, the DHA-PC yield reaches a maximum of 35.80%. On the one hand, this may be because the enzyme activity increases with the increase in temperature, thereby increasing the enzymatic rate; on the other hand, the increase in temperature accelerates the molecular thermal motion speed, increases the probability of collision between molecules, and accelerates the reaction rate. When the temperature continues to rise, the yield decreases, which may be because the high temperature denatures the enzyme structure and reduces the reaction rate.

[0051] 1.4 Effect of enzyme amount on DHA-PC synthesis

[0052] Take 4 round-bottom flasks, add 100 mg PC, 1000 mg DHA algae oil, 10 mL n-hexane, sonicate for 10 min to completely dissolve the substrate in n-hexane, add 2 ml pH = 7 phosphate buffer, continue sonication for 5 min to completely disperse the substrate, then add different amounts of CALB enzyme solution (enzyme amount is 80 μl, 120 μl, 160 μl, 200 μl), react at 50 ° C in the dark for 24 h. After the reaction is completed, the operation steps are the same as (2) in 1.1.

[0053] As attached Figure 4 As shown in the figure, with the increase of enzyme addition, the yield shows a trend of obvious increase and then gradually stabilized. When the enzyme addition amount is 120μl, the yield is the highest at 41.09%. This may be because the more enzymes are added, the more intermediate complexes can be formed by combining with the substrate, thereby increasing the rate of enzymatic reaction and the yield. When the enzyme in the reaction system is completely combined with the substrate, the enzyme will be free in the system and the reaction rate will tend to be stable. Excessive enzyme will increase the viscosity of the reaction system, causing the reaction rate to decrease and the yield to decrease. This may be the reason for the decrease in yield when the enzyme addition amount is 200μl.

[0054] 1.5 Conduct orthogonal experiment based on the single factor in 1.1-1.4

[0055] Based on the results of the single factor experiment, pH value, substrate mass ratio, reaction temperature and enzyme addition were selected as the key factors affecting the conversion rate of DHA-PC synthesis by fatty acyl exchange reaction. The yield of DHA-PC was used as the main measurement index, and the experiment was carried out according to the orthogonal experimental scheme designed in Table 3. The results of the orthogonal experiment are summarized in Table 4, and the experimental results are analyzed.

[0056] Table 3 Fatty acyl exchange reaction orthogonal level table L9 (3 4 )

[0057] level Temperature(A) / ℃ pH(B) Substrate mass ratio (C) Enzyme amount (D) / μl 1 45 7 1:5 80 2 50 7.5 1:10 120 3 55 8 1:15 160

[0058] Table 4 Range analysis results of orthogonal experiment

[0059]

[0060]

[0061] According to the results of the range analysis in Table 4, the influence of each factor on the transesterification reaction is C>A>D>B, that is, substrate mass ratio>temperature>enzyme amount>pH, and the best combination can be obtained as A2B1C2D1, that is, temperature 50°C, pH 7, substrate mass ratio (PC:DHA algae oil) 1:10, enzyme amount 80μl. Under this reaction condition, the reaction of synthesizing DHA-PC was carried out many times, and the average yield of DHA-PC was 43.35%.

[0062] Example 2

[0063] Synthesize DHA-LPC and investigate its influencing factors:

[0064] The effects of hydrolysis time, temperature and enzyme amount on the synthesis of DHA-LPC are as follows:

[0065] 2.1 Effect of hydrolysis time on the synthesis of DHA-LPC

[0066] (1) Take 4 round-bottom flasks, add 200 mg DHA-PC and 10 ml n-hexane, and then add 40 mg (20% substrate mass) of Lipozyme TL IM enzyme balanced with saturated salt solution. The salt solutions used are LiCl (water activity Aw = 0.13), MgCl2·6H2O (Aw = 0.33), NaCl (Aw = 0.75) and K2SO4 (Aw = 0.97); under nitrogen protection, put them in a 40°C magnetic stirring water bath and react for 2h, 4h, 6h, and 8h respectively.

[0067] (2) After the reaction, the mixture was centrifuged at 6000 r / min for 10 min, the supernatant was taken, and then filtered with a 0.45 μm PTEE filter to ensure that Lipozyme TL IM was completely removed to terminate the reaction. The mixture obtained after the reaction was loaded into a silica gel column, first eluted with chloroform: methanol (10:1, v / v) to remove free fatty acids, and then phospholipids were recovered with methanol, and the solvent was rotary evaporated to obtain DHA-LPC, which was stored at -20°C until analysis.

[0068] As attached Figure 5As shown in the figure, with the extension of reaction time, the hydrolysis rate of DHA-PC gradually increased. When the hydrolysis was performed for 4 hours, the highest hydrolysis rate was about 24%. With the increase of time, the hydrolysis rate decreased. It may be due to the occurrence of side reactions. Lipozyme IM acts on the sn-1 position of PC. With the increase of hydrolysis time, a large amount of sn-2 LPC is produced in the system. However, acetyl exchange occurs during the reaction, so that the group at the sn-2 position is transferred to the sn-1 position, and then it can be hydrolyzed by Lipozyme TL IM to produce GPC.

[0069] 2.2 Effect of temperature on the synthesis of DHA-LPC

[0070] Take 4 round-bottom flasks, add 200 mg DHA-PC and 10 ml n-hexane respectively, and then add 40 mg (20% substrate mass) of Lipozyme TL IM equilibrated with saturated salt solution for 24 hours. Under nitrogen protection, place in magnetic stirring water baths at 20℃, 30℃, 40℃, and 50℃ to react for 4 hours. After the reaction, operate the same as (2) in 2.1.

[0071] As attached Figure 6 As shown in the figure, as the temperature increases, the hydrolysis rate continues to increase. At 40°C, the hydrolysis rate is about 19.49%. As the temperature continues to rise, the hydrolysis rate shows a downward trend. This may be because Lipozyme TL IM is most active at 40°C. As the temperature increases, the molecular movement rate accelerates, and the increased probability of enzyme-substrate binding may also be one of the reasons for the high hydrolysis rate. As the temperature continues to rise, the enzyme activity decreases, resulting in a downward trend in the hydrolysis rate.

[0072] 2.3 Effect of enzyme amount on DHA-LPC synthesis

[0073] Take 4 round-bottom flasks, add 200 mg DHA-PC and 10 ml n-hexane, and add 20 mg, 30 mg, 40 mg, 50 mg (10%, 15%, 20%, 25% of the substrate mass) of Lipozyme TLIM after equilibration with saturated salt solution for 24 hours. Under nitrogen protection, place them in a 40°C magnetic stirring water bath for 4 hours. After the reaction, operate the same as (2) in 2.1.

[0074] As attached Figure 7 As shown, with the increase of enzyme amount, the hydrolysis rate increased. When the added amount was 30 mg, the hydrolysis rate reached 26.63%. Thereafter, with the continued increase of enzyme amount, there was no significant difference in the hydrolysis rate.

[0075] Example 3 DHA-LPC 31 P analysis

[0076] (1) Design of orthogonal experiment: In order to optimize the reaction conditions and improve the hydrolysis rate, the hydrolysis time, enzyme amount, and temperature were explored. Based on the results of the single factor experiment, orthogonal experiments were designed at different levels to perform P analysis on the hydrolysis products. 31 According to the results of the single factor experiment, the hydrolysis time (2h / 4h / 6h), temperature (30℃ / 40℃ / 50℃), enzyme amount (10%, 15%, 20%) were used as influencing factors, and the hydrolysis rate of DHA-PC was used as the measurement index to investigate the influence of the above three factors on the hydrolysis synthesis of DHA-LPC. The orthogonal experiment was designed according to Table 5. The hydrolysis products were analyzed by P 31 NMR analysis was used to analyze the composition of the products after DHA-PC hydrolysis. The results are shown in Table 6.

[0077] The method is as follows: First, 90 mg of the hydrolyzate was dissolved in 1 ml of deuterated chloroform (with 25 mg of triphenyl phosphate as an internal standard), followed by the addition of 1 ml of methanol and 1 ml of EDTA solution with a pH of 8.5. After vigorous shaking and mixing, the mixture was centrifuged at 3000 rpm for 5 minutes. Finally, the lower chloroform layer was removed and transferred to an NMR tube for subsequent analysis. The NMR spectroscopy scanning conditions were as follows: probe temperature of 29 ° C, pulse width of 22 μs, scan width of 9718 Hz, acquisition time of 1.2 s, relaxation delay of 10 s, and scan number of 256 times. The relative distribution of PC and LPC was expressed as a molar percentage relative to the sum of all phospholipids and lysophospholipids using the internal standard method.

[0078] (2) Data Statistical Analysis

[0079] Data are presented as mean ± standard deviation, and all experiments were repeated three times. GraphPadPrism 9, Orign 2018 and IBM SPSS Statistics 26 software were used for data analysis, and one-way analysis of variance and Waller-Dunca's multiple comparisons were used for statistical analysis. P < 0.05 and differences marked with different letters were statistically significant.

[0080] Table 5 Orthogonal levels of lipase hydrolysis reaction Table L9 (3 3 )

[0081] level Temperature(A) / ℃ Enzyme amount (B) (w / w) Time (C) / h 1 30 10％ 2 2 40 15％ 4 3 50 20％ 6

[0082] Table 6 Relative peak area of ​​hydrolysis products

[0083]

[0084] Table 7 Range analysis results of orthogonal experiment

[0085]

[0086]

[0087] According to the extreme results of the NMR P spectrum analysis, it can be seen from Tables 5 and 6 that the influence of each factor on the hydrolysis reaction is A>B>C, that is, temperature>enzyme amount>time, and the best combination can be obtained as A2B3C2, that is, temperature 40°C, enzyme amount 20% of the substrate mass, hydrolysis 4h, then DHA-PC is hydrolyzed to prepare DHA-LPC under this reaction condition.

[0088] From the above experimental data, we can conclude that:

[0089] (1) Synthetic DHA-PC

[0090] Take 4 round-bottom flasks, add 100 mg PC, 1000 mg DHA algae oil, and 10 mL n-hexane respectively, sonicate for 10 min to completely dissolve the substrate in n-hexane, add 2 ml of pH 7.0 phosphate buffer, continue sonication for 5 min to completely disperse the substrate, then add 80 μl CALB enzyme solution, place the round-bottom flasks in a constant temperature shaking incubator, and react at 50°C in the dark for 24 h;

[0091] After the reaction, take the upper organic phase, add 5g of activated alumina and place on a shaker to adsorb in the dark for 12h; after the adsorption is completed, collect the active Al2O3 to a 50ml centrifuge tube, add 10ml of 4℃ pre-cooled acetone to shake and wash, the purpose is to wash away the unreacted algae oil, repeat 3 times, then add 10ml of pre-cooled methanol to elute the active Al2O3, the purpose is to obtain DHA-PC adsorbed on the active Al2O3, collect the eluate, repeat 3 times; then centrifuge the eluate at 4℃ 7000r / min for 10min, take the upper clear liquid, and evaporate the solvent to obtain DHA-PC;

[0092] Under the conditions of temperature 50°C, pH 7, substrate mass ratio (PC:DHA algae oil) 1:10, and enzyme amount 80 μl, the DHA-PC synthesis yield was 43.35%;

[0093] (2) Synthetic DHA-LPC

[0094] Take 4 round-bottom flasks, add 200 mg of DHA-PC prepared in (1) and 10 ml of n-hexane, and then add 40 mg (20% substrate mass) of Lipozyme TL IM equilibrated with a saturated salt solution. The salt solutions used are LiCl (water activity Aw = 0.13), MgCl2·6H2O (Aw = 0.33), NaCl (Aw = 0.75) and K2SO4 (Aw = 0.97). Under nitrogen protection, place in a 40°C magnetic stirring water bath to react for 4 hours. After the reaction is completed, the mixture is centrifuged at 6000 r / min for 10 minutes, the supernatant is taken, and then filtered with a 0.45 μm PTFE filter to ensure that the Lipozyme TL IM is completely removed, thereby terminating the reaction;

[0095] The mixture obtained after the reaction was loaded into a silica gel column, and chloroform:methanol (10:1, v / v) was first used to elute to remove free fatty acids, and then methanol was used to recover phospholipids, and the solvent was rotary evaporated to obtain DHA-LPC;

[0096] It was detected that under the conditions of temperature of 40°C, hydrolysis for 4 hours, and enzyme amount of 20wt% of substrate concentration, the yield of DHA-LPC obtained was 33.13%.

[0097] Example 4

[0098] Testing of DHA-PC for antioxidant activity

[0099] The DHA-PC prepared by the method of Example 3 was prepared into an emulsion in the present invention, and its stability and antioxidant activity were tested, and Fourier transform infrared spectroscopy analysis was also performed, as shown below:

[0100] 4.1 Preparation of DHA-PC emulsion

[0101] PC, DHA-PC, and DHA-LPC were dispersed in deionized water and stirred magnetically for 3 hours at room temperature in the dark to obtain 2% PC, 2% DHA-PC, 4% DHA-PC, 6% DHA-PC (w / w), 2% DHA-LPC, 4% DHA-LPC, and 6% DHA-LPC (w / w) aqueous phases. MCT was used as the oil phase, and the oil phase and the aqueous phase (1:9, v / v) were ultrasonically prepared in an ice bath using an ultrasonic cell disruptor to obtain an emulsion, which was stored at room temperature until analysis. Ultrasonic conditions: 100w, ultrasonic 2s, interval 2s, total ultrasonic time 5min.

[0102] 4.2 Fourier transform infrared spectroscopy analysis

[0103] Its function is to explore whether DHA is successfully inserted into the PC structure. This paper analyzes the FITR infrared spectra of PC, DHA algae oil and DHA-PC. Figure 8 As shown, the characteristic absorption peak of PC is mainly located at 2949 cm -1 , 2846cm -1 , 1737cm -1 , 1252cm -1 and 975cm -1 ; The characteristic absorption peak of DHA algae oil is mainly located at 3020cm -1 , 2929cm -1 , 2857cm -1 , 1748cm -1 , 1153cm -1 and 719cm -1 ; The characteristic absorption peak of DHA-PC is mainly located at 3021cm -1 , 2930cm -1 , 2860cm -1 , 1739cm -1 , 1250cm -1 and 978cm -1 According to the infrared absorption peak characteristics of functional groups

[151] :In the CH stretching zone (3000-2700cm -1 )DHA-PC shows the characteristic absorption peak of DHA in the carbonyl stretching region (1900-1650cm -1 )、XH in-plane stretching zone (1475-1000cm -1 ) and CH in-plane stretching zone (1000-650cm -1 ) The characteristic absorption peak of PC appeared. The experimental results show that DHA-PC was synthesized by fatty acyl exchange reaction.

[0104] 4.3 Determination of DPPH free radical scavenging activity

[0105] Prepare 0.1mM DPPH solution, dilute the emulsion 100 times before the test, take 200μl of the diluted emulsion and put it into a centrifuge tube, then add an equal volume of DPPH solution, incubate at 20℃ in the dark for 30min, and then measure the absorbance As at 517nm; take 200μl of the diluted emulsion and put it into a centrifuge tube, then add an equal volume of ethanol solution, and measure the absorbance Ab in the same way; take 200μl of ethanol solution in a test tube, add an equal volume of DPPH solution, and measure the absorbance A0 in the same way. Set up 3 parallels for each group, take the average absorbance, and repeat the experiment 3 times. The antioxidant capacity of the emulsion against DPPH free radicals is calculated as follows:

[0106]

[0107] Fig. 9(a) The ability of different emulsions to scavenge DPPH free radicals was compared. The results showed that compared with the blank group, the addition of both PC and DHA-PC significantly increased the DPPH free radical scavenging rate of the emulsion, which indicates that the antioxidant activity of the emulsion is improved, which may be due to the addition of phospholipids. At the same concentration, the DPPH free radical scavenging rate of 2% DHA-PC emulsion was significantly higher than that of 2% PC emulsion, which may be due to the insertion of DHA in DHA-PC. Both PC and DHA have certain antioxidant activity, and the combination of the two exerts a synergistic effect to enhance the antioxidant function. The continuous improvement of the DPPH free radical scavenging ability of 2% DHA-PC, 4% DHA-PC, and 6% DHA-PC emulsions shows that the increase in the amount of DHA-PC can not only increase the storage stability of the emulsion, but also improve the antioxidant capacity of the emulsion.

[0108] 4.4 ABTS free radical scavenging activity

[0109] Mix ABTS solution (7.4 mM) and potassium persulfate solution (2.6 mM) at a ratio of 1:1 (v / v) and allow to react for 12-16 hours in the dark to generate ABTS· + . Dilute ABTS with deionized water. + The ABTS working solution was obtained when the absorbance was 0.7±0.02 (734nm). Before the test, the emulsion was diluted 50 times, 10μl of the diluted emulsion was added to 190μl of ABTS working solution in a 96-well plate and mixed, incubated in a 37°C water bath for 1h, and the absorbance A1 was measured at 415nm; 10μl of the diluted emulsion was added to 190μl of deionized water in a 96-well plate and mixed, and A2 was measured in the same way; 200μl of ABTS working solution was added to a 96-well plate and A0 was measured in the same way. Three parallels were set up for each group, the absorbance was averaged, and the experiment was repeated 3 times. The antioxidant capacity of the emulsion against ABTS free radicals is calculated as follows:

[0110]

[0111] Fig. 9 (b) The ability of different emulsions to scavenge ABTS free radicals was compared. The changing trends of the ability of different groups of emulsions to scavenge ABTS free radicals were similar to those of the ability to scavenge DPPH free radicals. The results showed that the ABTS scavenging rate of emulsions stabilized with phospholipids as emulsifiers increased, and with the increase in the amount of phospholipids added, the ABTS free radical scavenging rate also increased. The ABTS scavenging capacity of 6% DHA-PC emulsion reached the highest 86.57%.

[0112] 4.5 Determination of Fe 2+ Chelating activity

[0113] According to Hermund's method

[150] Determination of sample chelated Fe2+ The activity of the emulsion was diluted 50 times before the test. After taking 100 μL of the emulsion to a 96-well plate, 135 μL of distilled water was added, and then 5 μL of FeCl2 solution (2 mM) was added. The mixture was mixed and kept for 3 minutes. Then 10 μL of phenanthroline solution was added, and the absorbance As was measured at 562 nm. After taking 100 μL of the emulsion to a 96-well plate, 135 μL of distilled water was added, and the absorbance Ab was measured at 562 nm. After taking 235 μL of distilled water to a 96-well plate, 5 μL of FeCl2 solution was added, the mixture was mixed and kept for 3 minutes. Then 10 μL of phenanthroline solution was added, and the absorbance A0 was measured at 562 nm. Three parallels were set up for each group, and the absorbance was averaged. The experiment was repeated 3 times. Fe of the emulsion 2+ The chelating capacity is calculated as follows:

[0114]

[0115] This application compares the Fe 2+ Chelating ability, results such as Fig. 9 (c) As shown in the previous results, the addition of phospholipids significantly increased the Fe 2+ The chelating capacity of 2% PC emulsion is higher than that of 2% PC emulsion. 2+ The chelating capacity is similar to that of 6% DHA-PC emulsion, which indicates that PC is effective in chelating Fe 2+ More advantages in terms of.

[0116] 4.6 Determination of ferric reducing ability (FRAP)

[0117] Prepare HAc-NaAc buffer solution (300mM pH=3.6), TPTZ solution (10mM), and FeCl3 solution (20mM), mix the above three reagents at a ratio of 10:1:1 (v / v / v) to prepare FRAP working solution, and store in the dark. Prepare and use immediately. Plot the standard curve: Take 100μL of each of 0.2μmol / mL, 0.4μmol / mL, 0.8μmol / mL, 1.6μmol / mL, and 3.2μmol / mL FeSO4 solution, mix them with 100μL TPTZ solution and 1mL HAC-NaAC buffer solution, take 200μL of the mixture and add it to a 96-well plate, incubate in a 37℃ water bath in the dark for 30min, measure the absorbance at 593nm, and plot Fe 2+ Concentration-absorbance standard curve.

[0118] Sample determination: before the test, the emulsion was diluted 100 times, 100 μL of the diluted emulsion was taken into a centrifuge tube, and then 600 μL of FRAP working solution was added, and the mixture was shaken, and then 200 μL was taken into a 96-well plate, and incubated in a 37°C water bath in the dark for 30 min, and the absorbance As was measured at 593 nm; 100 μL of the diluted emulsion was taken into a centrifuge tube, and then 600 μL of deionized water was added, and the absorbance Ab was measured in the same way; the absorbance Ar of the emulsion was calculated (Ar=As-Ab), and Ar was substituted into the standard curve equation to deduce the ferrous sulfate equivalent, which is the FRAP value of the emulsion.

[0119] The FRAP values ​​of the five emulsions are as follows Fig. 9 As shown in (d), the FRAP value of the emulsion varies with the type and amount of emulsifier added. Compared with the blank group, the FRAP value of the emulsion in the phospholipid group increased significantly. At the same mass concentration, the FRAP value of the emulsion in the DHA-PC group was higher (P<0.05); as the mass of DHA-PC increased, the FRAP value of the emulsion increased further. The results showed that the 6% DHA-PC emulsion had the best antioxidant activity. The standard curve is shown in the attached figure. Fig. 9 (e) as shown.

[0120] Comparison of free radical scavenging ability of emulsions, Fe 2+ Chelating ability and iron reducing ability, both PC and DHA-PC significantly improved the antioxidant activity of the emulsion. In addition, DHA-PC was more advantageous in scavenging DPPH and ABTS free radicals and had better iron reducing ability.

[0121] Example 5

[0122] Testing of DHA-LPC for antioxidant activity

[0123] The DHA-LPC prepared by the method in Example 3 was prepared into an emulsion, and Fourier transform infrared spectroscopy, emulsion stability, DPPH free radical scavenging activity, ABTS free radical scavenging activity, Fe 2+ Determination of chelating activity and iron reducing ability (the methods are the same as in Example 4).

[0124] 5.1 Fourier transform infrared spectroscopy analysis of DHA-LPC

[0125] As attached Fig.10 As shown, the characteristic peak of LPC is mainly located at 2919 cm -1 , 2849cm -1 , 1735cm -1 , 1471cm -1 , 1227cm -1 , 1052cm -1 , 971cm-1 , and 829cm -1 ; The characteristic absorption peak of DHA-PC is mainly located at 3016cm -1 , 2930cm -1 , 2856cm-1, 2118cm -1 , 1740cm -1 , 1454cm -1 , 1162cm -1 and 718cm -1 ; The characteristic absorption peak of DHA-LPC is mainly located at 3016cm -1 , 2928cm -1 , 2856cm -1 , 1735cm -1 , 1471cm -1 , 1240cm -1 , 1051cm -1 , 954cm -1 and 829cm -1 According to the infrared absorption peak characteristics of functional groups

[151] :In the CH stretching zone (3000-2700cm -1 )DHA-LPC retains the characteristic absorption peak of DHA in DHA-PC, in the carbonyl stretching region (1900-1650cm -1 )、XH in-plane stretching zone (1475-1000cm -1 ) and CH in-plane stretching zone (1000-650cm -1 ) The characteristic absorption peak of LPC appears. The experimental results show that DHA-PC is hydrolyzed into DHA-LPC by lipase.

[0126] 5.2 Analysis of Antioxidant Activity of DHA-LPC Emulsion

[0127] As can be seen from the figure, the free radical scavenging ability of the two emulsions showed a dose-dependent relationship, and the change range of the DPPH free radical scavenging ability of the DHA-LPC emulsion was significantly higher than that of the DHA-PC emulsion. At a mass concentration of 2%, the DPPH scavenging rates of the DHA-PC emulsion and the DHA-LPC emulsion were 45.12% and 57.70%, respectively. At a mass concentration of 6%, the DPPH scavenging rates of the two emulsions increased to 52.14% and 79.56%, respectively. The ABTS free radical scavenging ability also had a similar trend. With the increase of concentration, the ABTS free radical scavenging ability of the DHA-PC emulsion increased from 54.67% to 64.73%, and that of the DHA-LPC emulsion increased from 68.11% to 88.31%. In addition, at the same mass concentration, the free radical scavenging ability of the DHA-LPC emulsion was significantly higher than that of the DHA-PC emulsion, and the ability to scavenge ABTS free radicals was better than the ability to scavenge DPPH free radicals. The above results show that compared with DHA-PC, DHA-LPC has an enhanced ability to scavenge free radicals. This may be due to the different fatty acid composition. From the fatty acid composition results, it can be seen that DHA-LPC contains 17.33% C18:2 and 46.82% C22:6 (46.82%), and its unsaturated fatty acid content is significantly higher than that of DHA-PC.

[0128] Fe 2+ Chelating ability refers to the ability of a compound or molecule to bind to iron ions to form a stable complex or chelate. 2+ It will promote the occurrence of lipid peroxidation, and chelates can increase the stability of metal ions, thereby slowing down the occurrence of oxidation reactions. Fig.11 (c) It can be seen that the increase in mass concentration also improves the iron ion chelating ability of the emulsion. At the same mass concentration, the iron ion chelating ability of the DHA-LPC emulsion is stronger. This paper speculates that this may be because the insertion of DHA enhances the chelating ability of DHA-PC and DHA-LPC. PC and LPC themselves are more likely to bind to Fe 2+ The chelating ability is weaker. Fatty acid molecules contain carboxyl groups and can bind to Fe under certain conditions. 2+ Chelation. As the concentration increases, the number of fatty acid molecules in the system increases, and the chelating ability is improved. The fatty acid composition results show that the DHA content of DHA-LPC is higher than that of DHA-PC. Therefore, at the same mass concentration, there are more DHA molecules in the DHA-LPC emulsion, which may be the reason why the DHA-LPC emulsion has a strong ability to chelate iron ions. As mentioned earlier, the iron reducing ability method is a method to reflect the total antioxidant capacity of antioxidants. The higher the FRAP value, the stronger the reducing property. From Fig.11It can be seen that the changing trend of the FRAP value is similar to the changing trend of the free radical scavenging ability. The FRAP value of the emulsion with a mass concentration of 6% is the highest, and at the same mass concentration, the FRAP value of the DHA-LPC emulsion is significantly higher than that of the DHA-PC emulsion, indicating that the DHA-LPC emulsion has a stronger reducing ability.

[0129] The DPPH free radical scavenging ability, ABTS free radical scavenging ability, Fe 2+ The chelating ability and ferric iron reducing ability showed that DHA-PC also had certain antioxidant ability, but DHA-LPC emulsion had better oxidative stability, and the antioxidant ability of DHA-LPC was better than that of DHA-PC.

Claims

1. A method for preparing docosahexaenoic acid lysophosphatidylcholine by enzyme catalysis, characterized in that: The following steps are involved: (1) Synthesis of Docosahexaenoic Acid Phosphatidylcholine Phosphatidylcholine and docosahexaenoic acid algae oil were used as substrates, the substrates were completely dissolved in n-hexane, phosphate buffer was added, and the substrates were completely dispersed, and then immobilized lipase solution CALB was added, and the mixture was placed in a constant temperature shaking incubator to react in the dark; After the reaction is completed, the upper organic phase is taken, active Al2O3 is added, and then placed on a shaker to avoid light for adsorption; then the active Al2O3 is collected into a centrifuge tube, and pre-cooled acetone is added to shake and wash to wash away the unreacted algae oil, and then pre-cooled methanol is added to elute the active Al2O3, the eluate is collected, centrifuged, the upper clear liquid is taken, and the solvent is evaporated to obtain docosahexaenoic acid phosphatidylcholine; (2) Synthesis of Docosahexaenoic Acid Lysophosphatidylcholine Take the docosahexaenoic acid phosphatidylcholine prepared in (1), add n-hexane, then add immobilized lipase Lipozyme TL IM, react in a water bath under nitrogen protection, centrifuge, take the supernatant, filter to ensure that Lipozyme TL IM is completely removed, terminate the reaction, and load the mixture obtained after the reaction into a silica gel column, first elute with an eluent composed of chloroform and methanol to remove free fatty acids, then recover phospholipids with methanol, and rotary evaporate the solvent to obtain docosahexaenoic acid lysophosphatidylcholine.

2. The method for preparing docosahexaenoic acid lysophosphatidylcholine by enzyme catalysis according to claim 1, characterized in that: (1) The reaction conditions are as follows: temperature 45-55 °C, pH 6.5-7.5, the mass ratio of phosphatidylcholine and docosahexaenoic acid algae oil in the substrate is 1:5-15, and the enzyme amount is 80-200 μl.

3. The method for preparing docosahexaenoic acid lysophosphatidylcholine by enzyme catalysis according to claim 1, characterized in that: (1) The reaction conditions were as follows: temperature 50 °C, pH 7, the mass ratio of phosphatidylcholine and docosahexaenoic acid algae oil in the substrate was 1:10, and the enzyme amount was 80 μl.

4. The method for preparing docosahexaenoic acid lysophosphatidylcholine by enzyme catalysis according to claim 1, characterized in that: (2) The reaction conditions are as follows: temperature 30-50 °C, hydrolysis 2-6 h, and enzyme amount 10-25% of substrate concentration.

5. The method for preparing docosahexaenoic acid lysophosphatidylcholine by enzyme catalysis according to claim 1, characterized in that: (2) The reaction conditions were as follows: temperature 40 °C, hydrolysis 4 h, and enzyme amount 20% of the substrate concentration.

6. The method for preparing docosahexaenoic acid lysophosphatidylcholine by enzyme catalysis according to claim 1, characterized in that: In (2), the immobilized lipase Lipozyme TL IM is first equilibrated with a salt solution containing: LiCl with a water activity Aw of 0.13, MgCl2•6H2O with an Aw of 0.33, NaCl with an Aw of 0.75, and K2SO4 with an Aw of 0.

97.

7. The method for preparing docosahexaenoic acid lysophosphatidylcholine by enzyme catalysis as claimed in claim 1, comprising the following steps: (1) Synthesis of Docosahexaenoic Acid Phosphatidylcholine Take 4 round-bottom flasks, add 100 mg of phosphatidylcholine and 1000 mg of DHA algae oil respectively, then add 10 mL of n-hexane, sonicate for 10 min to completely dissolve the substrate in n-hexane, add 2 ml of pH 7.0 phosphate buffer, continue sonication for 5 min to completely disperse the substrate, then add 80 μl of immobilized lipase solution, place the round-bottom flask in a constant temperature shaking incubator, and react at 50°C in the dark for 24 h; After the reaction, the upper organic phase was taken, 5 g of active Al2O3 was added, and then placed on a shaker to avoid light for adsorption for 12 h; after the adsorption was completed, the active Al2O3 was collected into a 50 ml centrifuge tube, and 10 ml of 4 ℃ pre-cooled acetone was added for shaking and washing to wash away the unreacted algae oil, and the process was repeated 3 times, and then 10 ml of pre-cooled methanol was added to elute the active Al2O3 to obtain the docosahexaenoic acid phosphatidylcholine adsorbed on the active Al2O3, and the eluate was collected, and the process was repeated 3 times; then the eluate was centrifuged at 7000 r / min at 4 ℃ for 10 min, the upper clear liquid was taken, and the solvent was evaporated to obtain the docosahexaenoic acid phosphatidylcholine; (2) Synthesis of Docosahexaenoic Acid Lysophosphatidylcholine Take 4 round-bottom flasks, add 200 mg of DHA-PC prepared in (1) and 10 ml of n-hexane, then add 40 mg of Lipozyme TL IM equilibrated with saturated salt solution, and place in a 40°C magnetic stirring water bath under nitrogen protection for 4 hours. After the reaction, centrifuge the mixture at 6000 r / min for 10 min, take the supernatant, and filter it with a 0.45 μm polytetrafluoroethylene filter to ensure that Lipozyme TL IM is completely removed, thereby terminating the reaction. The salt solution contains: LiCl with a water activity Aw of 0.13, MgCl2•6H2O with an Aw of 0.33, NaCl with an Aw of 0.75, and K2SO4 with an Aw of 0.97; The mixture obtained after the reaction is loaded into a silica gel column, and the free fatty acids are firstly removed by eluting with an eluent, and then the phospholipids are recovered with methanol, and the solvent is rotary evaporated to obtain docosahexaenoic acid lysophosphatidylcholine; in the eluent, the volume ratio of chloroform to methanol is 10:1.

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