Preparation method and application of flaxseed diglyceride oil

Abstract

The invention discloses a preparation method and application of flaxseed diglyceride oil. The method comprises the following steps: mixing linseed oil with glycerol, and carrying out catalytic glycerolysis reaction by adopting a compound enzyme system consisting of lipase TL 100L and CALB according to a weight ratio of 9: 8 to generate an intermediate product mixture with high monoglyceride content, high diglyceride content and low triglyceride residual quantity, thereby laying a foundation for subsequent efficient purification; and centrifuging the reaction product to remove impurities, and sequentially carrying out two-stage molecular distillation purification. The content of diglyceride in the finally prepared flaxseed diglyceride oil product is higher than 80%. The process conditions are mild, high-purity products can be efficiently prepared, active ingredients of the linseed oil are reserved to the maximum extent, and the prepared diglyceride oil has a good application prospect in preparation of products with the function of lowering lipid and / or reducing blood sugar.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of oil processing, in particular to a preparation method of a flaxseed glycerol diester oil and application thereof. BACKGROUND

[0002] Flaxseed oil is rich in α-linolenic acid and other polyunsaturated fatty acids, and has many physiological functions such as regulating blood lipids and reducing blood sugar. However, the main component of traditional flaxseed oil is triglyceride, which is easy to recombine into fat in the body metabolism process, which is not conducive to the control of blood lipids and blood sugar. Glycerol diester, especially 1,3-glycerol diester, is considered as an oil component with health management potential due to its unique metabolic pathway of not being easy to accumulate body fat. Studies have shown that glycerol diester oil prepared from soybean oil, rapeseed oil and other raw materials shows certain effect in controlling weight and blood lipids.

[0003] The prior art mainly focuses on preparing DAG from common vegetable oils such as soybean oil and rapeseed oil. These raw material oils are mainly ω-6 fatty acids, and the DAG oil prepared therefrom has a “ceiling effect” in terms of metabolic benefits such as regulating blood lipids and anti-inflammatory. Although flaxseed oil is rich in ω-3 α-linolenic acid which is more metabolically active, it is more difficult to prepare high-purity DAG from flaxseed oil (because polyunsaturated fatty acids are easy to oxidize and more sensitive to process). SUMMARY

[0004] In view of the deficiencies of the prior art, the present application provides a preparation method of flaxseed glycerol diester oil. The method uses a specific compound lipase catalytic system in combination with a two-stage molecular distillation purification process, aiming to efficiently and mildly prepare flaxseed glycerol diester oil with high glycerol diester content and good quality. The present application also provides the application of the glycerol diester oil in preparing lipid-lowering and / or blood sugar-lowering products.

[0005] To achieve the above-mentioned purposes, the present application adopts the following technical solutions: A preparation method of flaxseed glycerol diester oil, comprising the following steps: Step S1, mixing flaxseed oil, glycerol and a compound lipase to occur an enzyme-catalyzed glycerolysis reaction to obtain a mixture rich in partial glycerides (mainly glycerol diesters and glycerol monoesters); Step S2, centrifuging the glycerol diester and glycerol monoester-containing mixture prepared in step S1 to collect an upper oil phase to remove most of the free glycerol, residual enzyme and water; Step S3, performing first-stage molecular distillation on the upper oil in step S2 to collect a heavy phase component to preliminarily enrich glycerol diesters and glycerol monoesters and remove light components such as free fatty acids, part of monoglycerides and volatile substances; Step S4, the heavy phase component in step S3 is subjected to a second molecular distillation, and the heavy phase component is collected, i.e. a high-purity linseed glyceride oil; the second distillation aims to further separate glycerol monesters and glycerol diesters to obtain a high-purity glyceride product.

[0006] As preferred, in step S1, the complex lipase is a mixture of lipase TL 100L and lipase CALB, and the weight ratio is 9:8. The specific ratio of the complex enzyme system takes advantage of the strong activity of TL 100L on glycerolysis reaction and the good selectivity of CALB, and synergistically acts to significantly improve the conversion efficiency of glyceride and the proportion of DAG in the product.

[0007] As preferred, in step S1, the weight ratio of linseed oil to glycerol is 4-6:1. This ratio helps the reaction to proceed in the direction of generating glyceride, while avoiding the separation difficulty caused by excessive glycerol.

[0008] As preferred, in step S2, the centrifugal rotation speed is 10000-15000 rpm / min to ensure effective separation of solid-liquid and light-heavy liquid phases.

[0009] As preferred, in step S3, the first molecular distillation is carried out under the following conditions: temperature 160-190℃, pressure 8-9 Pa, and time 150-180 minutes.

[0010] As preferred, in step S4, the second molecular distillation is carried out under the following conditions: temperature 210-230℃, pressure 8-9 Pa, and time 40-60 minutes. The design of two-stage distillation conditions aims to realize gradient separation and finally efficiently obtain high-purity glyceride.

[0011] A linseed glyceride oil prepared by any of the above methods, characterized in that the content of glyceride is higher than 80%.

[0012] The above linseed glyceride oil is used in the preparation of products with lipid-lowering and / or glucose-lowering functions.

[0013] Compared with the prior art, the beneficial effects of the present application are: (1) A complex catalytic system composed of lipase TL 100L and CALB with a specific weight ratio (9:8) is adopted to fully exert the synergistic effect of the two enzymes and generate an intermediate product mixture with high glycerol monoester content, high glyceride content, and low residual triglyceride, laying a foundation for subsequent efficient purification.

[0014] (2) Through the combined purification process of "centrifugal impurity removal → primary molecular distillation preliminary enrichment → secondary molecular distillation deep purification", especially the optimization of the temperature, pressure and time parameters of the two-stage molecular distillation, the content of glycerol diglyceride in the product can be efficiently increased to more than 80%, and the active nutritional ingredients in flaxseed oil are better retained.

[0015] (3) The whole preparation process is mild, avoiding the destruction of polyunsaturated fatty acids in flaxseed oil under high temperature and high pressure, and the product has good quality and high nutritional value.

[0016] (4) Animal experiments have confirmed that the flaxseed glycerol diglyceride oil prepared by the above specific method shows unexpected synergistic hypoglycemic and hypolipidemic activities, and has potential application value in regulating blood lipids and blood sugar. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 To compare the tail vascular staining intensity of each group of zebrafish. DETAILED DESCRIPTION

[0018] The embodiments of the present application are not limited thereto.

[0019] Example 1 Step S1: Take 83 g of flaxseed oil, 17 g of glycerol, and 20 g of complex lipase (a mixture of lipase TL100L and lipase CALB with a weight ratio of 9:8) in a flask, and place it in a gas bath shaker. The reaction is carried out at a temperature of 40℃ and a rotation speed of 150 rpm / min for 8 hours. Stop the reaction. Step S2: After the reaction is completed, centrifuge the mixture at a rotation speed of 10000 rpm / min for 5 minutes, and carefully collect the upper oil phase.

[0020] Step S3: The collected upper oil phase is subjected to first molecular distillation. The distillation temperature is set to 180℃, the system pressure is 8.5 Pa, and the distillation time is 180 minutes. The heavy phase component (mainly monoglyceride and diglyceride) is collected.

[0021] Step S4: The heavy phase component obtained in step S3 is subjected to second molecular distillation, and the distillation temperature is set to 220℃, the system pressure is 8.5 Pa, and the distillation time is 60 minutes. The heavy phase component is collected, which is the flaxseed glycerol diglyceride oil product.

[0022] Through high performance liquid chromatography (HPLC) analysis, the content of triglyceride in the flaxseed glycerol diglyceride oil product is 13.9%, and the content of diglyceride is 84.9%.

[0023] Hypoglycemic effect verification of flaxseed glycerol diglyceride oil Experimental animals: Zebrafish were bred in fish water at 28°C (water quality: 200 mg of instant sea salt was added to each 1 L of reverse osmosis water, the conductivity was 450-550 μS / cm; the pH was 6.5-8.5; the hardness was 50-100 mg / L CaCO3), and were provided by the fish breeding center of the Ringta Biological Innovation Experimental Center. The experimental animal use license number was SYXK (Zhe) 2022-0004. The fish were bred and managed in accordance with the requirements of the international AAALAC accreditation (accreditation number: 001458), and the IACUC ethical review number was IACUC-2025-202509020007-01.

[0024] Experimental instruments, consumables and reagents: Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); precision electronic balance (CP214, OHAUS, USA); ultrasonic cleaner (JP-010T, Shenzhen Jiemeng Cleaning Equipment Co., Ltd., China); blood glucose meter (ACCU-CHEK Performa, Roche Diagnostics (Shanghai) Co., Ltd., China); blood glucose meter test paper (batch number 478829, Roche Diagnostics (Shanghai) Co., Ltd., China).

[0025] Egg yolk powder (batch number 20230203, Zhejiang Aige Biological Technology Co., Ltd., China); D-(+) -glucose (batch number A2425167, Shanghai Aladdin Bio-Chem Technology Co., Ltd., China); dimethyl sulfoxide (DMSO, batch number BCCD8942, Sigma, Switzerland); anhydrous ethanol (batch number 20230329, National Pharmaceutical Group Chemical Reagent Co., Ltd., China).

[0026] Experimental grouping Normal control group, model group, positive group and flaxseed glyceride oil group. The flaxseed glyceride oil group used the flaxseed glyceride oil obtained in Example 1, and the solvent was DMSO. The positive group used pioglitazone hydrochloride tablets (hereinafter referred to as pioglitazone), white tablets, batch number 2210085203, Jiangsu Deyuan Pharmaceutical Co., Ltd., and the solvent was DMSO; the positive group 2 used atorvastatin calcium, white powder, batch number F2121344, Shanghai Aladdin Bio-Chem Technology Co., Ltd., and the solvent was DMSO.

[0027] Experimental method Randomly selected 5 days after fertilization (5 dpf) wild type AB strain zebrafish in beaker, each beaker (each group) 30 zebrafish. Respectively, water soluble administration of samples, linseed oil diglyceride oil group 2000 μg / mL, positive group pioglitazone hydrochloride 20.0 μg / mL concentration, while setting normal control group and model group, each cup capacity is 25 mL. Except for the normal control group, the rest of the groups were water soluble administration of high-sugar high-fat feed to establish a high-sugar high-fat zebrafish model.

[0028] After 28℃ treatment for 2 days, the zebrafish were washed 3 times with standard dilution water, and the data were collected with a blood glucose meter. The glucose level of zebrafish was analyzed and statistically analyzed. The statistical analysis results of this index were used to evaluate the hypoglycemic efficacy of the samples. The statistical processing results were expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p<0.05 indicated that the difference was statistically significant. The experimental results are shown in Table 1.

[0029] Table 1. Hypoglycemic efficacy evaluation experiment results of samples (n=10)

[0030] ***p<0.001 compared with the model group According to the results in Table 1, the linseed diglyceride oil obtained in Example 1 can significantly reduce the blood glucose level of zebrafish.

[0031] Verification of the hypolipidemic efficacy of linseed oil diglyceride oil Experimental animals: Zebrafish were bred in water for fish at 28℃ (water quality: 200 mg of instant sea salt was added to 1 L of reverse osmosis water, the conductivity was 450-550 μS / cm; the pH was 6.5-8.5; the hardness was 50-100 mg / L CaCO3), and were provided by the fish center of the biological innovation center of ringte, the experimental animal use license number was SYXK (Zhe) 2022-0004, the feeding and management met the requirements of international AAALAC certification (certification number: 001458), and the IACUC ethical review number was IACUC-2025-202509020007-01.

[0032] Instruments, consumables and reagents: Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); precision electronic balance (CP214, OHAUS, USA); digital horizontal decolorization shaker (2D-9556-A, Taicang Huali Data Experimental Equipment Co., Ltd., China); 6-well plate (Zhejiang Belanber Biotechnology Co., Ltd., China).

[0033] D- (+) -glucose (batch number A2425167, Shanghai Aldrin Biochemical Technology Co., Ltd., China); egg yolk powder (batch number 20230203, Zhejiang Aige Biological Technology Co., Ltd., China); dimethyl sulfoxide (DMSO, batch number BCCD8942, Sigma, Switzerland); methyl cellulose (batch number B2006074, Shanghai Aldrin Biochemical Technology Co., Ltd., China); Oil Red O (batch number SHBN4926, Sigma, USA); 1,2-propanediol (batch number 20230614, China National Pharmaceutical Group Chemical Reagent Co., Ltd., China); 4% tissue cell fixative (batch number 20230401, Beijing Solaybao Technology Co., Ltd., China); PBS phosphate buffer (dry powder) (batch number: 23045283, Biosharp, China).

[0034] Experimental method: 30 wild type AB strain zebrafish at 5 days post fertilization (5 dpf) were randomly selected in a beaker. Each group was treated with 2000 μg / mL of sample, 11.6 μg / mL of positive group atorvastatin calcium, and 25 mL of normal control group and model group. Except for the normal control group, the rest of the groups were given high-sugar high-fat feed to establish a high-sugar high-fat zebrafish model. After 2 days of treatment at 28°C, Oil Red O was given for whole body fat staining. After staining, 10 zebrafish were randomly selected from each experimental group for photography under a dissecting microscope, and the data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software. The tail blood vessel staining intensity of zebrafish was analyzed, and the statistical analysis results of the index were used to evaluate the blood lipid-lowering effect of the sample. The statistical processing results were expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p<0.05 indicated that the difference was statistically significant. The experimental results are shown in Table 2 and Figure 1 Figure 1 (a) is the normal control group, Figure 1 (b) is the model group, Figure 1 (c) is the positive group (atorvastatin calcium), Figure 1 (d) is the flaxseed oil glyceride oil group.

[0035] Table 2. Blood lipid-lowering efficacy evaluation of the sample (n=10)

[0036] *** p<0.001 compared with the model control group According to Table 2 and Figure 1 ​As a result, the linseed glyceride oil obtained in Example 1 can significantly reduce the blood lipid level of zebra fish.

[0037] Example 2 The difference from Example 1 is only that 80 g of linseed oil, 20 g of glycerol, and 20 g of a composite lipase (a mixture of lipase TL 100L and lipase CALB in a weight ratio of 9:8) are taken in the flask.

[0038] The content of diglyceride in the product is 82.1% analyzed by high performance liquid chromatography (HPLC).

[0039] Example 3 The difference from Example 1 is only that 86 g of linseed oil, 14 g of glycerol, and 20 g of a composite lipase (a mixture of lipase TL 100L and lipase CALB in a weight ratio of 9:8) are taken in the flask.

[0040] The content of diglyceride in the product is 85.8% analyzed by high performance liquid chromatography (HPLC).

[0041] Example 4 The difference from Example 1 is only that 83 g of linseed oil, 17 g of glycerol, and 10 g of a composite lipase (a mixture of lipase TL 100L and lipase CALB in a weight ratio of 9:8) are taken in the flask.

[0042] The content of diglyceride in the product is 81.3% analyzed by high performance liquid chromatography (HPLC).

[0043] Example 5 The difference from Example 1 is only that 83 g of linseed oil, 17 g of glycerol, and 30 g of a composite lipase (a mixture of lipase TL 100L and lipase CALB in a weight ratio of 9:8) are taken in the flask.

[0044] The content of diglyceride in the product is 84.9% analyzed by high performance liquid chromatography (HPLC).

[0045] Comparative Example 1 The difference from Example 1 is only that a single lipase TL 100L is used in step S1, and the total amount of addition is the same as the total amount of composite enzyme in Example 1 (20 g).

[0046] The content of diglyceride in the product is 73.6% analyzed by high performance liquid chromatography (HPLC).

[0047] Comparative Example 2 The difference from Example 1 is only that a single lipase CALB is used in step S1, and the total amount of addition is the same as the total amount of composite enzyme in Example 1 (20 g).

[0048] High performance liquid chromatography (HPLC) analysis showed that the diglyceride content in the product was 60.2%.

[0049] Comparative Example 3 Compared to Example 1, the only difference is that the complex lipase used in step S1 is TL 100L and CALB, but in a weight ratio of 1:1 (4.25g each). The remaining steps and parameters are exactly the same.

[0050] The final product contains 78.3% diglycerides.

[0051] The basic principles, main features, and advantages of this invention in the explored field have been described in detail above, and some usage examples have been detailed. Finally, it should be noted that the examples given above are only for illustrative purposes and are not intended to limit the invention. Although we have described this invention in detail with reference to the examples, those skilled in the art can still modify the described examples and solutions, or replace related technical parts. Therefore, any modifications or equivalent substitutions made within the spirit and principles of this invention are within the protection scope of the claims of this patent.

Claims

1. The application of flaxseed diglyceride oil in the preparation of lipid-lowering and / or blood sugar-lowering products, characterized in that, The preparation method of the flaxseed diglyceride oil includes the following steps: Step S1: Flaxseed oil, glycerol and complex lipase are mixed to undergo an enzyme-catalyzed glycerolysis reaction to obtain a mixture rich in metaglycerides; Step S2: Centrifuge the mixture containing diglycerides and monoglycerides prepared in step S1 and collect the upper oil layer. Step S3: Perform a first molecular distillation on the upper oil described in step S2 to collect the heavy phase component; Step S4: Perform a second molecular distillation on the heavy phase component described in step S3, and collect the heavy phase component, which is flaxseed diglyceride oil. The compound lipase is a mixture of lipase TL 100L and lipase CALB in a weight ratio of 9:

8.

2. The application according to claim 1, characterized in that, In step S1, the weight ratio of flaxseed oil to glycerin is 4-6:

1.

3. The application according to claim 1, characterized in that, In step S2, the centrifugation speed is 10000-15000 rpm / min.

4. The application according to claim 1, characterized in that, In step S3, the temperature of the first molecular distillation is 180-190℃, the pressure is 8-9Pa, and the time is 150-180 minutes.

5. The application according to claim 1, characterized in that, In step S4, the temperature of the second molecular distillation is 210-220℃, the pressure is 8-9 Pa, and the time is 40-60 minutes.

6. The application according to any one of claims 1-5, characterized in that, The flaxseed diglyceride oil contains more than 80% diglycerides.

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