A method for simultaneously preparing monoglyceride rich in palmitoleic acid and diglyceride rich in oleic acid

Abstract

The present invention proposes a method for simultaneously preparing monoglyceride rich in palmitoleic acid and diglyceride rich in oleic acid, and belongs to the technical field of compound preparation and extraction. The method comprises the following steps: 1) pre-reacting anhydrous dimethyl sulfoxide, 4-dimethylaminopyridine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and Sn1,3-specific lipase, then adding low-ester pectin, reacting to obtain a first reaction solution; after dialysis, freeze-drying the solution obtained in the dialysis bag to obtain Sn1,3-specific lipase pectin immobilized enzyme; 2) weighing sea buckthorn fruit oil and anhydrous glycerol and adding them to a stoppered conical flask, after ultrasonic-assisted mixing, adding the above-mentioned Sn1,3-specific lipase pectin immobilized enzyme to the stoppered conical flask, filling with nitrogen, reacting to obtain a second reaction solution, suction filtering, and separating and purifying the liquid obtained by suction filtration to obtain a product. The obtained products include both Sn-1 monoglycerides rich in palmitoleic acid and Sn-1,3 diglycerides rich in oleic acid.

Description

Technical Field

[0001] The invention belongs to the technical field of compound extraction, and in particular relates to a method for simultaneously preparing monoglyceride rich in palmitoleic acid and diglyceride rich in oleic acid. Background Art

[0002] Palmitoleic acid (POA) is a 16-carbon omega-7 monounsaturated fatty acid, as shown in Formula I. Studies have shown that omega-7 fatty acids can prevent diabetes, reduce inflammation, regulate cholesterol levels in the body, help control weight, maintain skin health, etc. The content of palmitoleic acid in ordinary plants is low, usually not more than 5%. At present, palmitoleic acid is mainly derived from seafood such as whale oil and cod liver oil. The content of palmitoleic acid in fish oil is relatively high, at 6.0%-10%. However, with the international ban on whaling and the scarcity of fishery resources, the source of palmitoleic acid is also very limited. Plants with high content of palmitoleic acid include cat's feces seeds, cat's claws and sea buckthorn fruit oil. The content of palmitoleic acid in sea buckthorn fruit oil is as high as 26-35%. Common methods for enriching palmitoleic acid include hydrolyzing oils into free fatty acids, and then purifying them by urea inclusion, supercritical fluid extraction and other methods to obtain products rich in free palmitoleic acid;

[0003]

[0004] Sn1,3 diglyceride is a structural ester with functions such as weight control. Under the action of pancreatic lipase, triglycerides are hydrolyzed into free fatty acids and 2-monoglycerol in the intestine, and then resynthesized into triglycerides under the action of glyceryl transferase, entering the blood through the intestinal lymph. Sn1,3 diglyceride is hydrolyzed into 1-monoglycerol or 3-monoglycerol in the intestine. They have poor affinity for glyceryl transferase and are difficult to resynthesize triglycerides in small intestinal epithelial cells. Instead, they are directly hydrolyzed into glycerol and free fatty acids, and decomposed into water and carbon dioxide through the β-oxidation pathway, which enables diglycerides to reduce fat accumulation in the body and lower postprandial blood lipids.

[0005] It can be seen that palmitoleic acid and Sn1,3 diglyceride both have certain use value, but their extraction methods are relatively complicated and the extraction rate is low, which limits their large-scale use. Summary of the invention

[0006] The invention provides a method for simultaneously preparing monoglyceride rich in palmitoleic acid and diglyceride rich in oleic acid.

[0007] The present invention provides a method for simultaneously preparing monoglyceride rich in palmitoleic acid and diglyceride rich in oleic acid, comprising the following steps:

[0008] 1) pre-reacting anhydrous dimethyl sulfoxide, 4-dimethylaminopyridine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and Sn1,3-specific lipase, then adding low-ester pectin to carry out esterification reaction to obtain a first reaction solution; placing the first reaction solution in a dialysis bag, and after dialysis, freeze-drying the solution obtained in the dialysis bag to obtain Sn1,3-specific lipase pectin-immobilized enzyme;

[0009] 2) Weighing seabuckthorn fruit oil and anhydrous glycerol and adding them into a stoppered conical flask, after ultrasonic-assisted mixing, adding the above-mentioned Sn1,3-specific lipase pectin immobilized enzyme into the stoppered conical flask, filling with nitrogen, sealing, heating, stirring, reacting, and obtaining a second reaction liquid; filtering the second reaction liquid, separating and purifying the filtered liquid to obtain a product, wherein the product is a mixture of monoglycerides rich in palmitoleic acid and diglycerides rich in oleic acid.

[0010] Furthermore, in step 1), the immobilized Sn1,3-specific lipase is Rhizomucor miehei lipase, and the enzyme activity is 10075.68 U / mL.

[0011] Further, in step 1), the molar ratio of anhydrous dimethyl sulfoxide, 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1000-3000:1:5-20;

[0012] In step 1), the volume ratio of Sn1,3-specific lipase to anhydrous dimethyl sulfoxide is 1:10-100;

[0013] In step 1), the mass ratio of Sn1,3-specific lipase to low-ester pectin is 1:1-10.

[0014] Furthermore, in step 1), the pre-reaction time is 2-8 hours; and the esterification reaction time is 12-36 hours.

[0015] Furthermore, in step 2), the molar ratio of the seabuckthorn fruit oil to glycerol is (1-2):(1-4).

[0016] Furthermore, in step 2), the added amount of the Sn1,3-specific lipase pectin-immobilized enzyme is 5wt.%-20wt.% of the mass of seabuckthorn fruit oil.

[0017] Further, in step 2), the conditions for the ultrasonic-assisted mixing are that the ultrasonic frequency is 40 KHz and the ultrasonic time is 1-5 min;

[0018] In step 2), the temperature is raised to 30°C to 70°C;

[0019] In step 2), the reaction time is 4 h to 16 h.

[0020] Furthermore, the method further comprises step 3), wherein the product is molecularly distilled, and the obtained light phase is monoglyceride, and the obtained heavy phase is diglyceride.

[0021] Furthermore, in step 3), the pressure of the molecular distillation is 5.0 Pa, the heating surface temperature of the molecular distillation is 170° C., and the condensing surface temperature of the molecular distillation is 5° C.

[0022] The present invention also provides a product prepared by any of the above methods, which contains both monoglyceride rich in palmitoleic acid and diglyceride rich in oleic acid.

[0023] The present invention has the following advantages:

[0024] The present invention provides a method for simultaneously preparing monoglycerides rich in palmitoleic acid and diglycerides rich in oleic acid. The method comprises the following steps: microbial Sn1,3-specific lipase is grafted onto pectin through an esterification reaction to immobilize the lipase, wherein the esterification reaction is generated by the carboxyl group of the enzyme protein and the hydroxyl group of the pectin, and 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are used as initiators. Then, sea buckthorn fruit oil is used as a raw material, and the Sn1,3-specific lipase pectin-immobilized enzyme is used to perform an enzyme catalytic reaction. The obtained product includes both Sn-1 monoglycerides rich in palmitoleic acid, wherein the content of monoglycerides in the obtained product can reach 72.05%, wherein the content of palmitoleic acid is as high as 57.01%, and Sn-1,3 diglycerides rich in oleic acid, wherein the content of oleic acid can reach 61.23%. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0026] Figure 1 The filtrate of Example 1 of the present invention 13 CNMR spectrum, among which, Figure 1 (a) is the filtrate 3 The characteristic peak of carbonyl in CNMR spectrum, Figure 1 (b) is the filtrate 13 CNMR spectrum glycerol skeleton carbon characteristic peaks;

[0027] Figure 2 This is a full-wavelength ultraviolet scanning diagram of the monoglyceride obtained after molecular distillation of the filtrate in Example 1 of the present invention;

[0028] Figure 3 This is the GC-MS total ion current diagram of the monoglyceride obtained after molecular distillation of the filtrate in Example 1 of the present invention;

[0029] Figure 4 This is the GC-MS total ion current diagram of diglyceride obtained after molecular distillation of the filtrate in Example 1 of the present invention;

[0030] Figure 5 Schematic diagram of the glycerol hydrolysis reaction principle of the present invention. DETAILED DESCRIPTION

[0031] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0032] An embodiment of the present invention provides a method for simultaneously preparing monoglycerides rich in palmitoleic acid and diglycerides rich in oleic acid, comprising the following steps:

[0033] 1) pre-reacting anhydrous dimethyl sulfoxide, 4-dimethylaminopyridine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and Sn1,3-specific lipase, then adding low-ester pectin to carry out esterification reaction to obtain a first reaction solution; placing the first reaction solution in a dialysis bag, and after dialysis, freeze-drying the solution obtained in the dialysis bag to obtain Sn1,3-specific lipase pectin-immobilized enzyme;

[0034] 2) Weighing seabuckthorn fruit oil and anhydrous glycerol and adding them into a stoppered conical flask, after ultrasonic-assisted mixing, adding the above-mentioned Sn1,3-specific lipase pectin immobilized enzyme into the stoppered conical flask, filling with nitrogen, sealing, heating, stirring, reacting, and obtaining a second reaction liquid; filtering the second reaction liquid, separating and purifying the filtered liquid to obtain a product, wherein the product is a mixture of monoglycerides rich in palmitoleic acid and diglycerides rich in oleic acid.

[0035] In the embodiment of the present invention, sea buckthorn fruit oil and glycerol are mixed in a certain ratio, lipase pectin immobilized enzyme is added, and the temperature and time are controlled to carry out enzyme catalysis reaction, so as to prepare a mixed product containing monoglyceride rich in palmitoleic acid and diglyceride rich in oleic acid. Figure 5 shown.

[0036] In step 1) of the embodiment of the present invention, since free lipase has defects such as poor solubility and cannot be reused, immobilizing the enzyme is an important method to improve the enzyme potency. In the present invention, lipase is covalently cross-linked to low-ester pectin through an esterification reaction to prepare a lipase pectin-immobilized enzyme.

[0037] In terms of the enzyme-carrier grafting method, the common enzyme-carrier grafting reaction is mediated by glutaraldehyde and covalently linked through the reaction of amino and carbonyl groups. The grafting reaction mechanism of the Sn1,3-specific lipase and the carrier of the present invention is the esterification reaction produced by the carboxyl group of the enzyme protein and the hydroxyl group of pectin. 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are initiators, and anhydrous dimethyl sulfoxide is used as a solvent to dissolve the reactants to participate in the reaction.

[0038] Furthermore, in step 1), the immobilized Sn1,3-specific lipase is Rhizomucor miehei lipase, and the enzyme activity is 10075.68 U / mL. Rhizomucor miehei lipase, model number is Palatase 2000L, purchased from Novozymes (Beijing) Biotechnology Co., Ltd. The Mw of low-ester pectin is 1.5×10 5 About, galacturonic acid content ≥74%.

[0039] Furthermore, in step 1), the molar ratio of anhydrous dimethyl sulfoxide (DMSO), 4-dimethylaminopyridine (DMAP) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) is 1000-3000:1:5-20.

[0040] Furthermore, in step 1), the volume ratio of Sn1,3-specific lipase to anhydrous dimethyl sulfoxide is 1:10-100.

[0041] Further, in step 1), the mass ratio of Sn1,3-specific lipase to low-ester pectin is 1:1-10. In the embodiment of the present invention, pectin is of biological origin and has biocompatibility. Compared with other non-biological immobilized enzyme carriers, such as inorganic carriers silicon dioxide, ferrous oxide, etc., it highlights the characteristics of organic and green. In addition, pectin has porosity and can adsorb substrates to increase the substrate concentration around the enzyme in the catalytic reaction, thereby improving the reaction efficiency.

[0042] Furthermore, in step 1), the pre-reaction is carried out at room temperature; the pre-reaction time is 2-8h.

[0043] Furthermore, in step 1), the esterification reaction is carried out at room temperature; the esterification reaction time is 12-36h.

[0044] Furthermore, in step 1), the molecular weight cutoff of the dialysis bag is 1×10 4 ~2×10 4 g / cm 3 Preferably, the molecular weight cutoff of the dialysis bag is 1.4×10 4 g / cm 3 .

[0045] Furthermore, in step 1), during dialysis, the dialysis time is 2 to 4 days, during which the dialysis fluid is replaced 1 to 4 times.

[0046] In the embodiment of the present invention, in step 2), sea buckthorn fruit oil is used as raw material, and Sn1,3-specific lipase pectin immobilized enzyme is used to carry out enzyme catalysis reaction. Due to the use of such specific immobilized enzyme, the catalytic product includes both monoglycerides rich in palmitoleic acid and diglycerides rich in oleic acid.

[0047] Furthermore, in step 2), the molar ratio of the seabuckthorn fruit oil to glycerol (TAG / Gly) can be (1-2):(1-4). Preferably, the molar ratio of the seabuckthorn fruit oil to the glycerol substrate can be 2:1, 1:1, 1:2, 1:3, 1:4, etc. The seabuckthorn fruit oil is obtained by separating, extracting and refining the seabuckthorn fruit juice after the seabuckthorn fruit is squeezed and the seeds are removed.

[0048] Furthermore, in step 2), the added amount of the Sn1,3-specific lipase pectin-immobilized enzyme is 5wt.%-20wt.% of the mass of seabuckthorn fruit oil.

[0049] Furthermore, in step 2), the conditions for the ultrasonic-assisted mixing are that the ultrasonic frequency is 40 KHz and the ultrasonic time is 1-5 min.

[0050] Furthermore, in step 2), the temperature is raised to 30°C to 70°C.

[0051] Furthermore, in step 2), the reaction time is 4 h to 16 h.

[0052] Specifically, in step 2), the stirring is achieved by placing a magnetic rotor, and the stirring rate is 300 r / min.

[0053] In one embodiment of the present invention, the process further comprises step 3), wherein the product is molecularly distilled, and the obtained light phase is monoglyceride, and the obtained heavy phase is diglyceride.

[0054] Furthermore, in step 3), the pressure of the molecular distillation is 5.0 Pa, the heating surface temperature of the molecular distillation is 170° C., and the condensing surface temperature of the molecular distillation is 5° C.

[0055] An embodiment of the present invention further provides a product prepared by any of the above methods containing both monoglyceride rich in palmitoleic acid and diglyceride rich in oleic acid.

[0056] The product obtained in the embodiment of the present invention is used to determine the monoglyceride content in the filtrate by periodic acid oxidation method. 13 CNMR was used to detect the diglyceride composition in the filtrate, and gas phase mass spectrometry was used to detect the fatty acid composition of monoglyceride and diglyceride after molecular distillation.

[0057] The results showed that the monoglyceride content in the filtrate could reach 72.05%, of which the palmitic acid content was as high as 57.01%. In addition, the monoglyceride produced by lipase glycerolysis was initially a monoglyceride at the Sn-2 position. However, Sn-2 monoglyceride (2-MAGs) was unstable, and then acyl migration occurred to form a relatively stable monoglyceride at the Sn-1 position. Compared with Sn-2 monoglyceride, Sn-1 monoglyceride is not easily converted into triglyceride and absorbed by the body again, and is a more popular structure for preparing fats and oils with weight loss effects. Figure 5 shown.

[0058] The diglyceride in the filtrate is mainly Sn-1,3 diglyceride (Sn-1,3DAG), and the oleic acid content in the diglyceride can reach 61.23%.

[0059] The present invention will be described in detail below with reference to the accompanying drawings and embodiments.

[0060] Embodiment 1 A method for simultaneously preparing monoglyceride rich in palmitoleic acid and diglyceride rich in oleic acid comprises the following steps:

[0061] In a 500 mL conical flask, 150 mL (2.11 mol) of anhydrous dimethyl sulfoxide (DMSO), 10 mL of Rhizomucor miehei lipase (Palatase 2000L, enzyme activity 10075.68 U / mL, Novozymes), 4-dimethylaminopyridine (DMAP, 0.8 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, 12 mmol) were added and pre-reacted at room temperature (25±2°C) for 5 h; then 12 g of low-ester pectin (galacturonic acid content ≥ 74%, Shanghai Yien Chemical Technology Co., Ltd.) was added and reacted at room temperature for 28 h for esterification immobilization reaction. After the reaction was completed, the reaction solution was placed in a dialysis bag (retention molecular weight of 1.4×10 4 g / cm 3 ), dialyzed for 3 days, during which the dialysate was replaced twice. The solution in the dialysis bag was then freeze-dried to obtain Sn1,3-specific lipase pectin-immobilized enzyme.

[0062] The refined seabuckthorn fruit oil and anhydrous glycerol were mixed at a molar ratio of 1:3, and ultrasonic assisted mixing was performed at 40KHz for 5 minutes. Then, 8wt.% (based on seabuckthorn fruit oil) of lipase pectin immobilized enzyme was added, a magnetic stirrer was added, nitrogen was filled, the mixture was sealed, the temperature was raised to 52°C, magnetic stirring was performed, the stirring rate was 300r / min, the reaction was performed for 14 hours, and after the reaction was completed, the solid lipase was recovered by suction filtration, and the obtained filtrate was a mixture of monoglyceride rich in palmitoleic acid and diglyceride rich in oleic acid.

[0063] In addition, the filtrate was molecularly distilled at a pressure of 5.0 Pa, a heating surface temperature of 170° C., a condensing surface temperature of 5° C., and the obtained light phase was monoglyceride, and the heavy phase was diglyceride.

[0064] Embodiment 2 A method for simultaneously preparing monoglyceride rich in palmitoleic acid and diglyceride rich in oleic acid comprises the following steps:

[0065] In a 500 mL conical flask, 120 mL (1.69 mol) of anhydrous dimethyl sulfoxide (DMSO), 8 mL of Rhizomucor miehei lipase (Palatase 2000L, enzyme activity 10075.68 U / mL, Novozymes), 4-dimethylaminopyridine (DMAP, 1 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, 10 mmol) were added and pre-reacted at room temperature (25±2°C) for 6 h; then 10 g of low-ester pectin (galacturonic acid content ≥ 74%, Shanghai Yien Chemical Technology Co., Ltd.) was added and reacted at room temperature for 26 h for esterification immobilization reaction. After the reaction was completed, the reaction solution was placed in a dialysis bag (retention molecular weight of 1.4×10 4 g / cm 3 ) for 2 days, during which the dialysate was changed 3 times. The solution in the dialysis bag was then freeze-dried to obtain Sn1,3-specific lipase pectin-immobilized enzyme.

[0066] The refined seabuckthorn fruit oil and anhydrous glycerol were mixed at a molar ratio of 1:2, and ultrasonic assisted mixing was performed at 40KHz for 4 minutes. Then, 10wt.% (based on seabuckthorn fruit oil) of lipase pectin immobilized enzyme was added, a magnetic stirrer was added, nitrogen was filled, the mixture was sealed, the temperature was raised to 50°C, magnetic stirring was performed, the stirring rate was 300r / min, the reaction was performed for 12 hours, and after the reaction was completed, the solid lipase was recovered by suction filtration, and the obtained filtrate was a mixture of monoglyceride rich in palmitoleic acid and diglyceride rich in oleic acid.

[0067] In addition, the filtrate was molecularly distilled at a pressure of 5.0 Pa, a heating surface temperature of 170° C., a condensing surface temperature of 5° C., and the obtained light phase was monoglyceride, and the heavy phase was diglyceride.

[0068] Embodiment 3 A method for simultaneously preparing monoglyceride rich in palmitoleic acid and diglyceride rich in oleic acid comprises the following steps:

[0069] In a 500 mL conical flask, 150 mL (2.11 mol) of anhydrous dimethyl sulfoxide (DMSO), 10 mL of Rhizomucor miehei lipase (Palatase 2000L, enzyme activity 10075.68 U / mL, Novozymes), 4-dimethylaminopyridine (DMAP, 0.8 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, 12 mmol) were added and pre-reacted at room temperature (25±2°C) for 5 h; then 12 g of low-ester pectin (galacturonic acid content ≥ 74%, Shanghai Yien Chemical Technology Co., Ltd.) was added and reacted at room temperature for 28 h for esterification immobilization reaction. After the reaction was completed, the reaction solution was placed in a dialysis bag (retention molecular weight of 1.4×10 4 g / cm 3 ), dialyzed for 3 days, during which the dialysate was replaced twice. The solution in the dialysis bag was then freeze-dried to obtain Sn1,3-specific lipase pectin-immobilized enzyme.

[0070] The refined seabuckthorn fruit oil and anhydrous glycerol were mixed at a molar ratio of 1:4, and ultrasonic assisted mixing was performed at 40KHz for 5 minutes. Then, 9wt.% (based on seabuckthorn fruit oil) of lipase pectin immobilized enzyme was added, a magnetic stirrer was added, nitrogen was filled, the mixture was sealed, the temperature was raised to 50°C, magnetic stirring was performed, the stirring rate was 300r / min, the reaction was performed for 10 hours, and after the reaction was completed, the solid lipase was recovered by suction filtration, and the obtained filtrate was a mixture of monoglyceride rich in palmitoleic acid and diglyceride rich in oleic acid.

[0071] In addition, the filtrate was molecularly distilled at a pressure of 5.0 Pa, a heating surface temperature of 170° C., a condensing surface temperature of 5° C., and the obtained light phase was monoglyceride, and the heavy phase was diglyceride.

[0072] Comparative Example 1

[0073] Same as Example 1, except that porcine pancreatic lipase (15-35 U / mg) was used to replace the Rhizomucor miehei lipase.

[0074] The same method is used to immobilize porcine pancreatic lipase, and then the same conditions are used to catalyze the reaction of seabuckthorn fruit oil and anhydrous glycerol. After the reaction is completed, the filtrate is filtered and the obtained filtrate is a mixture of monoglyceride and diglyceride.

[0075] Test Example 1 Analysis of Monoglyceride Content in Products by Periodic Acid Oxidation Method

[0076] Accurately weigh the filtrate obtained in Example 1 (including a mixture of monoglycerides rich in palmitoleic acid and diglycerides rich in oleic acid) into a 100 mL volumetric flask, add dichloromethane, shake well, and then make up to volume. After dissolving the sample, transfer it to a separatory funnel, wash the volumetric flask with 50 mL of 5% glacial acetic acid solution, transfer the washing liquid to the separatory funnel, shake vigorously for 1 min, let it stand for 1 h, and separate the layers.

[0077] Accurately pipette 25mL of the lower layer sample solution into a 500mL iodine volumetric flask, add 25mL of 0.01mol / L periodic acid solution, shake vigorously, and react in the dark for 30min. After taking it out, use a pipette to transfer 10mL of 15% KI solution. Shake vigorously, place in a dark place away from light for 3min, then add 100mL of distilled water, titrate with 0.01mol / L Na2S2O3 solution until light yellow, then add 1% starch solution that has been boiled and cooled, and continue to titrate until the blue fades. Record the consumption volume V1 (mL) of the Na2S2O3 solution. Perform a blank experiment without adding a sample and record the consumption V0 of the Na2S2O3 solution.

[0078] The monoglyceride content in the sample is calculated according to formula 1:

[0079]

[0080] Where:

[0081] X-----monoglyceride content, %;

[0082] c-----Concentration of Na2S2O3 standard solution, (mol / L);

[0083] M r -----Specific relative molecular mass of monoglyceride, selected according to fatty acid composition;

[0084] m-----mass of the sample, g;

[0085] r-----The ratio of the volume of the dichloromethane phase (100 mL) to the volume used for determination (50 mL or less), in milliliters per milliliter (mL / mL).

[0086] The result is rounded to two decimal places.

[0087] According to the above calculation method, the monoglyceride content in the filtrate obtained in Example 1 is 72.05%.

[0088] The testing method is the same as above. The monoglyceride content in the filtrate of Example 2 is 69.51%.

[0089] The testing method is the same as above. The monoglyceride content in the filtrate of Example 3 is 66.95%.

[0090] The testing method is the same as above. The monoglyceride content in the filtrate of Comparative Example 1 is 53.05%.

[0091] Ultraviolet characterization (UV) of monoglyceride obtained after molecular distillation of the product in Experimental Example 2

[0092] The monoglyceride obtained by molecular distillation of the filtrate product in Example 1 was characterized by UV. Figure 2 .

[0093] Depend on Figure 2 It can be seen that the maximum absorption peak is at 246nm, which is consistent with the monoglyceride standard. The sample after glycerolization is consistent with the standard control.

[0094] Analysis of fatty acid composition of monoglyceride of test example 3 product after molecular distillation

[0095] The fatty acid types and contents of the monoglycerides obtained after molecular distillation of the filtrate product in Example 1 were analyzed. Figure 3 And as shown in Table 1.

[0096] As can be seen from Table 1, the monoglyceride obtained in Example 1 has a palmitic acid content of up to 57.01%, which is 34.52% higher than the palmitic acid content in the raw material, achieving a large degree of enrichment, and is green and safe, and does not require large equipment, proving that the enzymatic glycerolysis reaction can be applied to industrial production.

[0097] In addition, the content of two functional fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids, in the monoglyceride after glycerol hydrolysis accounted for 65.78%, among which the content of monounsaturated fatty acids accounted for 60.52% and the content of palmitic acid accounted for 56.58%.

[0098] Table 1 Fatty acid types and contents in monoglycerides obtained in Experimental Example 1

[0099]

[0100]

[0101] Test Example 4 13 Structural analysis of diglycerides in CNMR analysis products

[0102] The filtrate obtained in Example 1 was 13 The CNMR spectrum carbonyl characteristic peak area analysis results in an enlarged image. Figure 1 (a) is the filtrate 3 The characteristic peak of carbonyl in CNMR spectrum, Figure 1 (b) is the filtrate 13 Characteristic peaks of glycerol skeleton carbon in CNMR spectrum.

[0103] Depend on Figure 1 (a) It can be obtained:3 The chemical shift of the carbonyl characteristic peak of the CNMR spectrum is in the range of 172-177. The characteristic peak chemical shift δ of FFA is 177.18, the characteristic peak chemical shift δ of 1-MAG is 174.23, the characteristic peak chemical shift δ of 2-MAG is 174.03, the characteristic peak chemical shift δ of 1,3-DAG is 173.88, the characteristic peak chemical shift δ of 1,2-DAG is 173.73 and 173.46, and the characteristic peak chemical shift δ of TAG is 173.29 and 172.84, which correspond to the carbonyl carbons at the Sn-1,3 positions and the Sn-2 positions of triglycerides, respectively.

[0104] Depend on Figure 1 (b) It is possible to obtain: 13 The chemical shift of the characteristic peak of the glycerol skeleton carbon in the CNMR spectrum is in the range of 61-75. The chemical shift δ of the characteristic peak of 2-MAG is 74.72; the chemical shift of the Sn-2 position of glycerol (G) is 72.28, and the chemical shift of the Sn-1,3 position is 63.08; the chemical shift δ of the characteristic peak of 1,2-DAG is 72.06, 62.29, and 61.17; there are three characteristic peaks of 1-MAG, and the chemical shift δ are 70.19, 64.97, and 63.37, respectively, of which the characteristic peak at 64.97 overlaps with the peak of 1,3-DAG at 64.92; the chemical shift δ of the characteristic peak of TAG is 68.90 and 62.08; the chemical shift δ of the characteristic peak of 1,3-DAG is 68.00 and 64.92, and the peak at 64.92 overlaps with the peak of 1-MAG.

[0105] The above NMR carbon spectrum results show that the obtained diglycerides are mainly diglycerides at the Sn-1,3 positions (1,3-DAG). Monoglycerides are mainly monoglycerides at the Sn-1 position, and the monoglycerides produced by lipase glycerolysis are initially monoglycerides at the Sn-2 position. However, Sn-2 monoglycerides (2-MAGs) are unstable, and then acyl migration occurs to form relatively stable monoglycerides at the Sn-1 position.

[0106] Analysis of fatty acid composition of diglyceride obtained after molecular distillation of test example 5

[0107] The fatty acid types and contents of diglycerides obtained after molecular distillation of the filtrate product in Experimental Example 1 are as follows: Figure 4 And as shown in Table 2.

[0108] As can be seen from Table 2, the oleic acid content of the diglyceride after enzymatic glycerolysis is as high as 61.23%, which is 9.64% higher than the oleic acid content in the raw material.

[0109] Table 2 Types and contents of fatty acids in seabuckthorn oil diglycerides

[0110]

[0111] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for simultaneously preparing monoglycerides rich in palmitoleic acid and diglycerides rich in oleic acid, characterized in that: The steps include: 1) pre-reacting anhydrous dimethyl sulfoxide, 4-dimethylaminopyridine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and Sn1,3-specific lipase, then adding low-ester pectin to carry out esterification reaction to obtain a first reaction solution; placing the first reaction solution in a dialysis bag, and after dialysis, freeze-drying the solution obtained in the dialysis bag to obtain Sn1,3-specific lipase pectin-immobilized enzyme, wherein the Sn1,3-specific lipase immobilized enzyme is Rhizomucor miehei lipase; 2) Weighing seabuckthorn fruit oil and anhydrous glycerol and adding them into a stoppered conical flask, after ultrasonic-assisted mixing, adding the above-mentioned Sn1,3-specific lipase pectin immobilized enzyme into the stoppered conical flask, filling with nitrogen, sealing, heating, stirring, reacting, and obtaining a second reaction liquid; filtering the second reaction liquid, separating and purifying the filtered liquid to obtain a product, wherein the product is a mixture of monoglycerides rich in palmitoleic acid and diglycerides rich in oleic acid, In step 1), the molar ratio of anhydrous dimethyl sulfoxide, 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1000-3000:1:5-20; the volume ratio of Sn1,3-specific lipase to anhydrous dimethyl sulfoxide is 1:10-100; the mass ratio of Sn1,3-specific lipase to low-ester pectin is 1:1-10; In step 2), the molar ratio of seabuckthorn fruit oil to glycerol is (1-2):(1-4); the added amount of the Sn1,3-specific lipase pectin-immobilized enzyme is 5wt.%-20wt.% of the mass of the seabuckthorn fruit oil.

2. The method according to claim 1, characterized in that In step 1), the enzyme activity of the immobilized Sn1,3-specific lipase is 10075.68 U / mL.

3. The method according to claim 1, characterized in that In step 1), the pre-reaction time is 2-8 hours; and the esterification reaction time is 12-36 hours.

4. The method according to claim 1, characterized in that: In step 2), the conditions for the ultrasonic-assisted mixing are that the ultrasonic frequency is 40 KHz and the ultrasonic time is 1-5 min; In step 2), the temperature is raised to 30°C to 70°C; In step 2), the reaction time is 4 h to 16 h.

5. The method according to claim 1, characterized in that The method further comprises step 3), wherein the product is molecularly distilled, and the obtained light phase is monoglyceride, and the obtained heavy phase is diglyceride.

6. The method according to claim 5, characterized in that In step 3), the pressure of the molecular distillation is 5.0 Pa, the heating surface temperature of the molecular distillation is 170° C., and the condensing surface temperature of the molecular distillation is 5° C.

7. A mixture of monoglycerides rich in palmitoleic acid and diglycerides rich in oleic acid prepared by the method according to any one of claims 1 to 6.

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