Preparation method of mixed hydroxy fatty acid branched-chain fatty acid ester

Abstract

The invention discloses a preparation method of mixed type hydroxy fatty acid branched chain fatty acid ester, which comprises the following steps: catalyzing 12-hydroxy stearic acid and oleic acid to generate 12-oleic acid hydroxy stearate by adopting a chemical catalyst or lipase, catalyzing 12-hydroxy stearic acid and palmitic acid to generate 12-palmitic acid hydroxy stearate, and further catalyzing 12-hydroxy stearic acid and palmitic acid to generate mixed type hydroxy fatty acid branched chain fatty acid ester. And separating and purifying to obtain the high-purity hydroxy fatty acid branched chain fatty acid ester. Two high-purity hydroxy fatty acid branched-chain fatty acid esters are compounded to obtain mixed hydroxy fatty acid branched-chain fatty acid esters, and the mixed hydroxy fatty acid branched-chain fatty acid esters have the effects of preventing and treating diabetes and resisting inflammation. The synthesis process has the advantages of simple operation and low cost.

Description

Technical Field

[0001] The invention belongs to the technical field of oil processing, and particularly relates to a method for preparing a mixed hydroxy fatty acid branched-chain fatty acid ester. Background Art

[0002] Hydroxylated fatty acid branched-chain fatty acid esters are endogenous lipids found in mice. Scientific research has shown that 9-palmitoyl hydroxystearate (the hydroxyl-terminal fatty acid is stearic acid, the carboxyl-terminal fatty acid is palmitic acid, and the "9" represents the position of the ester bond, 9-PAHSA) within this lipid class has the potential to improve insulin resistance and combat inflammation. It was later discovered that different types of hydroxylated fatty acid branched-chain fatty acid esters have varying effects, such as 12-oleoyl hydroxystearate (12-OAHSA) and 12-palmitoyl hydroxystearate (12-PAHSA). The former can enhance glucose-stimulated insulin secretion, while the latter can reduce the expression of inflammatory factors.

[0003] The content of hydroxy fatty acid branched fatty acid esters from natural sources is extremely low, and the cost of enrichment and extraction is high, but the cost can be reduced through synthesis. Summary of the Invention

[0004] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

[0005] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0006] Therefore, the object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a mixed hydroxy fatty acid branched-chain fatty acid ester.

[0007] In order to solve the above technical problems, the present invention provides the following technical solution: a method for preparing a mixed hydroxy fatty acid branched-chain fatty acid ester, comprising:

[0008] Using lipase or other catalysts, hydroxyl-containing fatty acids and hydroxyl-free fatty acids as raw materials, different types of hydroxyl fatty acid branched-chain fatty acid esters (also known as fatty acid hydroxy fatty acid esters) are synthesized, and solvent crystallization combined with multi-stage molecular distillation are used for purification to obtain high-purity products;

[0009] Different types of high-purity hydroxy fatty acid branched-chain fatty acid esters are compounded to prepare mixed hydroxy fatty acid branched-chain fatty acid esters with multiple functions.

[0010] As a preferred embodiment of the preparation method of the present invention, the lipase is one or more of Palatase20000L, Novozym 435, Lipozyme TL IM, Lipozyme RMIM, Novozym 40086, Lipase AY, Lipase DF-15, IM-NE100, NE-10, NE-20, R-001, RIM-03, AP6, and Amano PS.

[0011] As a preferred embodiment of the preparation method of the present invention, the hydroxy fatty acid is selected from one or more of castor seed oil, shea butter, lesquerella seed oil, and hydrogenated and fractionated products;

[0012] The hydroxyl-free fatty acid is one or more of commercially available butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid, arachidonic acid, behenic acid, lignoic acid, EPA, DPA, DHA, and nervonic acid.

[0013] As a preferred embodiment of the preparation method of the present invention, the conditions for enzymatic synthesis of hydroxy fatty acid branched fatty acid esters are as follows: temperature 30-60°C, enzymatic hydrolysis time 2-12 hours, lipase addition amount 4-8% of the total mass of the substrate, solvent addition to aid dissolution, and molecular sieve addition to accelerate the reaction;

[0014] The solvent is n-hexane, and the amount added is 2 to 8 times the total mass of the substrate;

[0015] The added amount of the molecular sieve is 5-15% of the total mass of the system.

[0016] As a preferred embodiment of the preparation method of the present invention, the other catalyst is one or more of sulfuric acid, p-toluenesulfonic acid, and boron trifluoride;

[0017] The reaction conditions of other catalysts are 100-130°C, other catalysts require 4%-10% of the substrate mass, and the reaction time is 2-12 hours.

[0018] As a preferred embodiment of the preparation method of the present invention, the solvent crystallization comprises:

[0019] The volume ratio of solvent to product is 1:0.6-1.8, the extraction temperature is -20--15°C, the extraction time is 4-24 hours, and after extraction, the solid is filtered and then molecular distilled;

[0020] The solvents are divided into two categories according to polarity: one category is pentane, n-hexane, octane, heptane, and isohexane; the other category is acetone, acetonitrile, and ethanol; the solvent composition can be one of the two categories or a mixture of different polarities, and the volume ratio of the mixed solvent is 1:0.5~1.5.

[0021] As a preferred embodiment of the preparation method of the present invention, the molecular distillation parameters are:

[0022] The primary distillation is 80-100°C, the secondary distillation is 100-150°C, the scraping speed is 100-150r / min, the feed rate is 0.5-3.5mL / min, the absolute pressure is 2-10Pa, and after the primary distillation, the heavy phase is taken for secondary distillation. The heavy phase after the secondary distillation is high-purity hydroxy fatty acid branched fatty acid ester.

[0023] Another object of the present invention is to overcome the deficiencies in the prior art and provide a mixed hydroxy fatty acid branched-chain fatty acid ester for use in edible oils and their products, health foods, and special dietary foods.

[0024] Beneficial effects of the present invention:

[0025] (1) The present invention develops a mixed hydroxy fatty acid branched-chain fatty acid ester, which has both the efficacy of preventing and treating diabetes and anti-inflammatory effects, and the efficacy can be changed by adjusting the compounding ratio, which can meet consumers' needs for two different effects.

[0026] (2) The present invention provides a method for preparing a mixed hydroxy fatty acid branched fatty acid ester. Compared with conventional column chromatography, solvent crystallization combined with molecular distillation can greatly increase production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0028] Figure 1 The liquid chromatograms of the raw material and product (12-oleyl hydroxystearate) before and after enzymatic synthesis in Example 1 are shown.

[0029] Figure 2 This is a liquid chromatogram of the product (12-oleyl hydroxystearate) synthesized using other catalysts in Example 2.

[0030] Figure 3 This is the secondary mass spectrum of 12-oleyl hydroxystearate in Example 2, wherein the instrument used is ultra performance liquid chromatography-quadrupole tandem time-of-flight mass spectrometry. DETAILED DESCRIPTION

[0031] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0032] Unless otherwise specified, the raw materials used in the examples of the present invention are all commercially available.

[0033] Example 1

[0034] This embodiment provides a method for preparing a mixed hydroxy fatty acid branched-chain fatty acid ester, comprising the following steps:

[0035] (1) Take 500 g of hydrogenated castor oil, add 100 g of potassium hydroxide, 400 g of water, and 400 g of ethanol, and reflux with stirring at 95°C for 2 h. Then add 11% aqueous hydrochloric acid. After separation, remove the upper layer and wash with boiling water until the pH of the lower aqueous phase is neutral. The upper layer is the hydroxy fatty acid.

[0036] (2) The prepared hydroxy fatty acid and commercially available oleic acid were mixed in a mass ratio of 1:6, and n-hexane accounting for 5 times the total mass of the substrate was added, and stirred at 60°C. After the hydroxy fatty acid was dissolved, Lipozyme TLIM accounting for 6% of the total mass of the substrate was added, and then 10% of 3A molecular sieves (accounting for the total mass of the system) was added. The reaction was stirred at 60°C for 8 hours to obtain a crude product of 12-oleic acid hydroxystearate. The crude product of 12-palmitic acid hydroxystearate was synthesized by replacing the substrate with 12-hydroxystearic acid and palmitic acid in the same way.

[0037] See the liquid chromatograms of the raw material and product (12-oleyl hydroxystearate) before and after enzymatic synthesis. Figure 1 .

[0038] (3) Prepare a 1:1 n-hexane-acetone solvent, take the crude enzymatic hydrolysis product and the n-hexane-acetone solvent, mix them in a volume ratio of 1:1, place them at -5°C for 10 hours, and then filter out the solid to obtain preliminarily purified 12-oleic acid hydroxystearate and 12-palmitic acid hydroxystearate.

[0039] (4) The product obtained in (3) is subjected to molecular distillation separation, with a feed temperature of 30°C; a feed rate of 1 mL / min; a scraping speed of 150 r / min; a first-stage evaporation surface temperature of 90°C, a condensation surface temperature of 30°C; a second-stage evaporation surface temperature of 110°C, a condensation surface temperature of 30°C, and an absolute pressure of 2 to 10 Pa. After the first-stage separation, the heavy phase is taken for second-stage separation. The heavy phase taken after the second-stage separation is high-purity 12-oleic acid hydroxystearate and 12-palmitic acid hydroxystearate.

[0040] (5) High-purity 12-oleic acid hydroxystearate and 12-palmitic acid hydroxystearate are compounded in a mass ratio of 1:1 to obtain a mixed hydroxy fatty acid branched-chain fatty acid ester-I.

[0041] (6) Mouse pancreatic β-cell clones and mouse bone marrow-derived dendritic cells were used to test the hypoglycemic and anti-inflammatory effects of 12-oleoyl hydroxystearate, 12-palmitoyl hydroxystearate, and mixed hydroxy fatty acid branched-chain fatty acid ester-I. The specific procedures are as follows:

[0042] Mouse pancreatic β-cell clones were cultured at 37°C in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) supplemented with 100 IU / mL penicillin, 100 IU / mL streptomycin, 1 mM sodium pyruvate, 50 mM 2-mercaptoethanol, and 10 mM Hepes buffer (pH 7.4).

[0043] Before the experiment, the cells were serum starved for 3 h using KRB buffer and treated with hydroxy fatty acid branched chain fatty acid ester (20 μM, experimental group) or dimethyl sulfoxide (0.01%, blank group) for the last h. The insulin content in the culture medium was then detected using a kit.

[0044] Bone marrow-derived dendritic cells (BMDCs) were differentiated by stimulating cells with 20 ng / mL granulocyte-macrophage colony-stimulating factor (GM-CSF). Ten minutes before lipopolysaccharide (100 ng / mL) stimulation, cells were incubated with various branched-chain fatty acid esters of hydroxy fatty acids (20 μM, experimental group) or dimethyl sulfoxide (0.01%, blank group). Twenty-four hours after lipopolysaccharide stimulation, cell culture fluid was collected for cytokine analysis. Tnf-α and Il-6 levels were measured using ELISA kits.

[0045] The results showed that 12-oleyl hydroxystearate can enhance insulin secretion (under glucose stimulation); 12-palmityl hydroxystearate can reduce the expression of cellular TNF-α and IL-6, but cannot enhance insulin secretion under glucose stimulation; and mixed hydroxy fatty acid branched-chain fatty acid ester-I has both effects.

[0046] The lipid compositions of the products from the three stages of enzymatic synthesis, solvent crystallization, and molecular distillation are shown in Table 1 , and the hypoglycemic and anti-inflammatory efficacy tests of the hydroxy fatty acid branched-chain fatty acid esters are shown in Table 2 .

[0047] Table 1 Lipid composition at each stage of enzymatic synthesis and separation and purification

[0048]

[0049] Table 2 Hypoglycemic and anti-inflammatory efficacy tests of hydroxy fatty acid branched-chain fatty acid esters

[0050]

[0051] Example 2

[0052] The difference between this embodiment and embodiment 1 is that the catalyst in (2) is changed to p-toluenesulfonic acid, no n-hexane is added, and no molecular sieve is added. The remaining steps are the same as those in embodiment 1 to prepare mixed hydroxy fatty acid branched-chain fatty acid ester-II.

[0053] The liquid chromatogram of the product (12-oleyl hydroxystearate) synthesized by other catalysts in Example 2 is shown in FIG. Figure 2 The mass spectrum of the product (12-oleyl hydroxystearate) is shown in Figure 3 .

[0054] Example 3

[0055] The difference between this embodiment and embodiment 1 is that the catalyst in (2) is replaced with Novozym 435, and the remaining steps are the same as those in embodiment 1 to prepare mixed hydroxy fatty acid branched fatty acid ester-III.

[0056] Comparative Example 1

[0057] The difference between this comparative example and Example 1 is that the reaction time in step (2) is changed to 6 hours, and the remaining steps are the same as in Example 1 to prepare a comparative mixed hydroxy fatty acid branched fatty acid ester-I.

[0058] Comparative Example 2

[0059] The difference between this comparative example and Example 1 is that the temperature of the secondary evaporation surface of the molecular distillation in step (4) is changed to 100° C., and the remaining steps are the same as those in Example 1 to prepare a comparative mixed hydroxy fatty acid branched fatty acid ester-II.

[0060] Comparative Example 3

[0061] The difference between this comparative example and Example 1 is that the compounding ratio in step (5) is changed to a mass ratio of 1:0.5, and the remaining steps are the same as Example 1 to prepare a comparative mixed hydroxy fatty acid branched fatty acid ester-III.

[0062] Comparative Example 4

[0063] The difference between this comparative example and Example 1 is that the compounding ratio in step (5) is changed to a mass ratio of 1:0, and the remaining steps are the same as those in Example 1 to prepare a single 12-oleic acid hydroxystearate.

[0064] Comparative Example 5

[0065] The difference between this comparative example and Example 1 is that the compounding ratio in step (5) is changed to a mass ratio of 0:0.5, and the remaining steps are the same as those in Example 1 to prepare a single 12-palmitoyl hydroxystearate.

[0066] The lipid compositions at each stage of synthesis, separation and purification of Example 1, Example 2 and Example 3 were compared with those of Comparative Examples 1, Comparative Example 2 and Comparative Example 3, as shown in Table 3.

[0067] Table 3

[0068]

[0069]

[0070]

[0071] Table 4 Comparison of hypoglycemic and anti-inflammatory effects of mixed hydroxy fatty acid branched fatty acid esters I in different proportions

[0072]

[0073] The following conclusions can be drawn: the catalytic effect of using other catalysts such as p-toluenesulfonic acid is better than that of the enzymatic method. The reason may be that the melting point of 12-hydroxystearic acid is about 80℃. Although n-hexane is added to the enzymatic method to help dissolve it, the reaction is still not as complete as after melting. The longer the reaction time, the more products there are, but there is little difference between 6h and 8h.

[0074] The greater the proportion of catalyst, the more products are generated;

[0075] Solvent fractionation is more suitable for removing oleic acid from the system. The reason may be that palmitic acid is a saturated acid with a higher melting point and is easier to crystallize in the solvent, resulting in the inability to separate it. The evaporation surface temperature of molecular distillation has a great influence on the separation effect.

[0076] The efficacy of the two mixed hydroxy fatty acid branched-chain fatty acid esters of Comparative Example 3 and Example 1 was compared and analyzed. It was found that the insulin secretion of Comparative Example 3 was not as good as that of Example 1. The experimental data are shown in Table 4, which shows that the efficacy of mixed hydroxy fatty acid branched-chain fatty acid esters can be adjusted by physical compounding.

[0077] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, and all of these should be included in the scope of the present invention.

Claims

1. A method for preparing a mixed hydroxy fatty acid branched-chain fatty acid ester, characterized in that: include, Using lipase or other catalysts, hydroxyl-containing fatty acids and hydroxyl-free fatty acids as raw materials, different types of hydroxyl fatty acid branched-chain fatty acid esters are synthesized, and solvent crystallization combined with multi-stage molecular distillation are used for purification to obtain high-purity products; Different types of high-purity hydroxy fatty acid branched-chain fatty acid esters are compounded to prepare mixed hydroxy fatty acid branched-chain fatty acid esters with multiple functions.

2. The preparation method according to claim 1, wherein: The lipase is one or more of Palatase 20000L, Novozym 435, Lipozyme TL IM, Lipozyme RMIM, Novozym 40086, Lipase AY, Lipase DF-15, IM-NE100, NE-10, NE-20, R-001, RIM-03, AP6, and Amano PS.

3. The preparation method according to claim 1 or 2, wherein: The hydroxy fatty acid is selected from one or more of castor seed oil, shea butter, lesquerella seed oil, and hydrogenated or fractionated products; The hydroxyl-free fatty acid is one or more of commercially available butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid, arachidonic acid, behenic acid, lignoic acid, EPA, DPA, DHA, and nervonic acid.

4. The preparation method according to claim 1, wherein: The conditions for enzymatic synthesis of hydroxy fatty acid branched fatty acid esters are as follows: temperature of 30-60°C, enzymatic hydrolysis time of 2-12 hours, lipase addition amount of 4-8% of the total mass of the substrate, solvent addition to aid dissolution, and molecular sieve addition to accelerate the reaction; The solvent is n-hexane, and the amount added is 2 to 8 times the total mass of the substrate; The added amount of the molecular sieve is 5-15% of the total mass of the system.

5. The preparation method according to claim 1, wherein: The other catalyst is one or more of sulfuric acid, p-toluenesulfonic acid, and boron trifluoride; The reaction conditions of other catalysts are 100-130°C, other catalysts require 4%-10% of the substrate mass, and the reaction time is 2-12 hours.

6. The preparation method according to claim 1, wherein: The solvent crystallization comprises: The volume ratio of solvent to product is 1:0.6-1.8, the extraction temperature is -20--15°C, the extraction time is 4-24 hours, and after extraction, the solid is filtered and then molecular distilled; The solvents are divided into two categories according to polarity: one category is pentane, n-hexane, octane, heptane, and isohexane; the other category is acetone, acetonitrile, and ethanol; the solvent composition can be one of the two categories or a mixture of different polarities, and the volume ratio of the mixed solvent is 1:0.5-1.

5.

7. The preparation method according to claim 1 or 6, wherein: The molecular distillation parameters are: The primary distillation is 80-100°C, the secondary distillation is 100-150°C, the scraping speed is 100-150r / min, the feed rate is 0.5-3.5mL / min, the absolute pressure is 2-10Pa, and after the primary distillation, the heavy phase is taken for secondary distillation. The heavy phase after the secondary distillation is high-purity hydroxy fatty acid branched fatty acid ester.

8. The mixed hydroxy fatty acid branched fatty acid ester prepared by the preparation method according to any one of claims 1 to 7 can be used in edible oils and their products, health foods, and special dietary foods.

Images