A method for preparing medium-chain triglycerides, related products and applications

Abstract

The application provides a preparation method of medium-chain triglyceride, related products and application, and relates to the technical field of oil manufacturing. The preparation method comprises the following steps: mixing anhydrous calcium hydrogen phosphate, hydroxyapatite, carboxymethyl chitosan, Candida rugosa lipase and water, stirring, ultrasonic treatment, centrifugation, and obtaining immobilized lipase; then, the immobilized lipase is used for enzymolysis reaction with coconut oil and / or palm kernel oil to obtain mixed fatty acids; and then, catalyzing esterification reaction between the mixed fatty acids and glycerol to obtain medium-chain triglyceride. Through adjusting the types and proportions of the components in the immobilized lipase, the hydrolysis selectivity of the lipase to medium-chain fatty acids is improved, and then the yield of the medium-chain triglyceride is improved.

Description

Technical Field

[0001] This invention belongs to the field of oil and fat manufacturing technology, and relates to a method for preparing medium-chain triglycerides, related products and applications. Background Technology

[0002] Medium-chain triglycerides (MCTs) are a type of glycerol containing C8, C96, C12, C23, C24 ... 10 Triglycerides of fatty acids are an important food ingredient. Currently, the preparation process of medium-chain triglycerides involves extracting caprylic acid (C7H4O3) from coconut oil or palm kernel oil. 15 COOH) and decanoic acid (C9H) 19 A mixture of COOH is then bonded to glycerol via an esterification reaction to obtain a medium-chain triglyceride.

[0003] In their article "Current Status of Development and Application of Medium-Chain Fatty Acids and Their Glycerides" (China Oils and Fats, 2024, 49(04), pp. 40-47), Wang Yuanli et al. reviewed the preparation methods of medium-chain fatty acids and their glycerides, including alcoholysis, esterification, transesterification, and biosynthesis. Among these, esterification has a long history of development, a relatively mature process, and low cost, but the reaction conditions are more complex, there are more byproducts, and the operation is more cumbersome. To achieve efficient production of MCTs, further optimization and exploration are needed, starting with technological improvements.

[0004] Currently, the production of medium-chain triglycerides (MCTs) faces a series of technical challenges. Particularly for the plant-based raw materials commonly used in MCT production, enriching medium-chain fatty acids often requires additional, time-consuming, and energy-intensive processes. While enzymatic hydrolysis of fatty acids from vegetable oils possesses a certain degree of specificity, this specificity is insufficient to meet the need for improved selectivity in the decomposition of medium-chain glyceric acid. Existing technologies have not yet provided a method to enhance the enzymatic selectivity of lipases for medium-chain fatty acids by regulating the composition and structure of the enzyme auxiliary system, thereby achieving efficient MCT synthesis. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of this invention is to provide a method for preparing medium-chain triglycerides, products and applications.

[0006] To achieve the above-mentioned objectives, the present invention provides a method for preparing medium-chain triglycerides, comprising the following steps:

[0007] S1. Mix anhydrous dicalcium phosphate, hydroxyapatite, carboxymethyl chitosan, Candida rhynchophylla lipase and water, stir, sonicate, and centrifuge to obtain immobilized lipase.

[0008] S2. The immobilized lipase obtained in step S1 is mixed with vegetable oil and water, and the enzymatic hydrolysis reaction is carried out. After centrifugation, the aqueous phase is removed to obtain the hydrolysis product.

[0009] S3. Perform molecular distillation on the hydrolysis products obtained in step S2 to obtain mixed fatty acids;

[0010] S4. Mix the mixed fatty acids obtained in step S3 with glycerol, add lipase, and perform esterification reaction to obtain medium-chain triglycerides.

[0011] In step S1, the ratio of the weights of the anhydrous dicalcium phosphate, hydroxyapatite, and carboxymethyl chitosan to the enzyme activity units of the Candida pleuropneumoniae lipase is 0.9-2.1:1.05-2.45:5.45-8.05:600000, in g:g:g:U.

[0012] In step S2, the vegetable oil is selected from at least one of coconut oil and palm kernel oil.

[0013] Further, in step S1, the weight ratio of the anhydrous dicalcium phosphate and the hydroxyapatite is 1-2:1.25-2.25.

[0014] Furthermore, in step S1, the weight ratio of the anhydrous dicalcium phosphate to the hydroxyapatite is 1.25-1.75:1.5-2.

[0015] Furthermore, in step S1, the weight ratio of the anhydrous dicalcium phosphate to the hydroxyapatite is 1.5:1.75.

[0016] Further, in step S1, the weight ratio of the sum of the anhydrous dicalcium phosphate and the hydroxyapatite to the weight of the carboxymethyl chitosan is 3.25:6.75.

[0017] Further, in step S1, the particle size D90 of the anhydrous dicalcium phosphate is <150 μm, the average particle size of the hydroxyapatite is 2-80 μm, and the degree of substitution of the carboxymethyl chitosan is ≥85%.

[0018] Further, in step S1, the stirring speed is 300-500 rpm, the stirring time is 20-60 min, the ultrasonic time is 10-30 min; the centrifugation speed is 14000-15000 rpm, and the centrifugation time is 10-15 min.

[0019] Further, in step S2, the weight ratio of the immobilized lipase, vegetable oil, and water is 1-5:8-40:1-5; the temperature of the enzymatic hydrolysis reaction is 50-60℃; the time of the enzymatic hydrolysis reaction is 3-4 hours; the centrifugation speed is 5000-10000 rpm; and the centrifugation time is 5-15 minutes.

[0020] Furthermore, in step S3, the molecular distillation includes first-stage molecular distillation, second-stage molecular distillation, and third-stage molecular distillation;

[0021] The temperature of the first-stage molecular distillation is 80-120℃, and the pressure is 50-150Pa;

[0022] The temperature of the second-stage molecular distillation is 160-180℃, and the pressure is 10-50Pa;

[0023] The temperature of the third-stage molecular distillation is 200-240℃, and the pressure is less than 10Pa.

[0024] The fraction obtained from the second-stage molecular distillation is the mixed fatty acid.

[0025] Further, in step S4, the molar ratio of the mixed fatty acids to glycerol is 2.5-3.5:1; the lipase is Novozym 435; the temperature of the esterification reaction is 60-70℃; and the time of the esterification reaction is 1-2 hours.

[0026] On the other hand, the present invention provides an immobilized lipase prepared by the above-described preparation method.

[0027] Specifically, the present invention provides the immobilized lipase obtained in step S1 of the above preparation method.

[0028] In another aspect, the present invention provides the above-described preparation method or the application of the above-described immobilized lipase in the production of medium-chain triglycerides.

[0029] Finally, the present invention provides medium-chain triglycerides prepared by the above-described preparation method or by the above-described immobilized lipase.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. This invention provides a method for preparing medium-chain triglycerides using a novel immobilized lipase. By adjusting the types and ratios of components in the immobilized lipase, the hydrolytic selectivity of the lipase for medium-chain fatty acids in the raw vegetable oil is improved, thereby increasing the content of caprylic and capric fatty acids in the medium-chain triglycerides.

[0032] 2. The medium-chain triglyceride preparation process of the present invention uses fewer chlorine-containing compounds and produces a lower content of 3-chloropropanol fatty acid esters compared with the prior art. Detailed Implementation

[0033] Terminology and Declarations of this Invention:

[0034] 1. Articles “a,” “a kind,” and “the”: These include plural objects unless otherwise explicitly specified as a single (kind) object.

[0035] 2. Numerical Range: Unless otherwise expressly stated, all ranges or ratios disclosed herein shall be construed as including any and all subranges or subratios contained herein. For example, a stated range or ratio of 1 to 30 shall be considered to be included between the minimum value of 1 and the maximum value of 30, and includes any subranges or subratios, integers, decimals, or subranges or subratios consisting of integers or decimals, including endpoints.

[0036] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0037] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention were obtained through conventional commercial means. Unless otherwise specified, all contents mentioned below are mass contents. Unless otherwise specified, it is understood that the process was carried out at room temperature.

[0038] In the following examples, some of the reagents were sourced from the sources shown in Table 1.

[0039] Table 1

[0040]

[0041] Example 1

[0042] A method for preparing medium-chain triglycerides.

[0043] The steps are as follows:

[0044] S1. Mix 1.5g anhydrous dicalcium phosphate, 1.75g ​​hydroxyapatite, 6.75g carboxymethyl chitosan (substitution degree ≥90%), 600,000U (1.5g by weight, based on 400U / mg) of *Candida pleuropsii* lipase, and 1L of water. Stir at 400rpm for 30min at room temperature, then sonicate for 20min, and centrifuge at 14,000rpm for 10min to obtain immobilized lipase. The anhydrous dicalcium phosphate has a particle size D90 < 120μm and a particle size D50 of 50-90μm; the hydroxyapatite is spherical with an average particle size of 20μm.

[0045] S2. The immobilized lipase obtained in step S1 is mixed with 81.6g of coconut oil and 20g of water, and the mixture is enzymatically hydrolyzed at 55℃ for 3.5h. After centrifugation at 7700rpm for 10min, the aqueous phase is removed to obtain the hydrolysis product.

[0046] S3. Perform molecular distillation on the hydrolysis products obtained in step S2 to obtain mixed fatty acids;

[0047] Molecular distillation consists of first-stage molecular distillation, second-stage molecular distillation, and third-stage molecular distillation.

[0048] The temperature of the first-stage molecular distillation is 80-120℃, and the pressure is 50-150Pa;

[0049] The temperature of the second-stage molecular distillation is 160-180℃, and the pressure is 10-50Pa;

[0050] The temperature of the third-stage molecular distillation is 200-240℃, and the pressure is less than 10Pa.

[0051] The fraction obtained from the second-stage molecular distillation is the mixed fatty acid.

[0052] S4. Mix the mixed fatty acids obtained in step S3 with glycerol at a molar ratio of 3:1, add lipase Novozym435 (10wt%), molecular sieve (0.12wt%), and phosphoric acid (0.02wt%), and esterify at 65℃ for 2 hours. After the esterification reaction is complete, centrifuge at 7500 rpm while hot to remove the lower layer of glycerol, and the upper product is medium-chain triglycerides.

[0053] Example 2

[0054] Compared with Example 1, the only difference is that in step S1, the amount of anhydrous dicalcium phosphate is changed to 1.75g ​​and the amount of hydroxyapatite is changed to 1.5g, while the rest are the same.

[0055] Example 3

[0056] Compared with Example 1, the only difference is that in step S1, the amount of anhydrous dicalcium phosphate is changed to 1.25g and the amount of hydroxyapatite is changed to 2g, while the rest are the same.

[0057] Example 4

[0058] Compared with Example 1, the only difference is that in step S1, the amount of anhydrous dicalcium phosphate is changed to 2g and the amount of hydroxyapatite is changed to 1.25g, while the rest are the same.

[0059] Example 5

[0060] Compared with Example 1, the only difference is that in step S1, the amount of anhydrous dicalcium phosphate is changed to 1g and the amount of hydroxyapatite is changed to 2.25g, while the rest are the same.

[0061] Comparative Example 1

[0062] Compared with Example 1, the only difference is that in step S1, the amount of anhydrous dicalcium phosphate is changed to 0.75g and the amount of hydroxyapatite is changed to 2.5g, while the rest are the same.

[0063] Comparative Example 2

[0064] Compared with Example 1, the only difference is that in step S1, the amount of anhydrous dicalcium phosphate is changed to 2.25g and the amount of hydroxyapatite is changed to 1g, while the rest are the same.

[0065] The amounts of anhydrous dicalcium phosphate and hydroxyapatite used in Examples 1-5, Comparative Examples 1 and 2 are summarized in Table 2.

[0066] Table 2

[0067]

[0068] Example 6

[0069] Compared with Example 1, the only difference is that in step S1, the amount of anhydrous dicalcium phosphate is changed to 0.9g, the amount of hydroxyapatite is changed to 1.05g, and the amount of carboxymethyl chitosan is changed to 8.05g, while the rest are the same.

[0070] Example 7

[0071] Compared with Example 1, the only difference is that in step S1, the amount of anhydrous dicalcium phosphate is changed to 2.1g, the amount of hydroxyapatite is changed to 2.45g, and the amount of carboxymethyl chitosan is changed to 5.45g; all other steps are the same.

[0072] Comparative Example 3

[0073] Compared with Example 1, the only difference is that in step S1, the amount of anhydrous dicalcium phosphate is changed to 0.6g, the amount of hydroxyapatite is changed to 0.7g, and the amount of carboxymethyl chitosan is changed to 8.7g; all other steps are the same.

[0074] Comparative Example 4

[0075] Compared with Example 1, the only difference is that in step S1, the amount of anhydrous dicalcium phosphate is changed to 2.4g, the amount of hydroxyapatite is changed to 2.8g, and the amount of carboxymethyl chitosan is changed to 4.8g; all other steps are the same.

[0076] The amounts of anhydrous dicalcium phosphate, hydroxyapatite, and carboxymethyl chitosan used in Examples 1, 6, 7, Comparative Example 3, and Comparative Example 4 are summarized in Table 3 below.

[0077] Table 3

[0078]

[0079] Example 8

[0080] Compared with Example 1, the only difference is that in step S1, the specifications of the anhydrous dicalcium phosphate are changed to a particle size D90 < 90 μm and a particle size D50 of 35-55 μm, while the rest are the same.

[0081] Example 9

[0082] Compared with Example 1, the only difference is that in step S1, the specifications of the anhydrous dicalcium phosphate are changed to particle size D90 < 150 μm and particle size D50 > 90 μm, while the rest are the same.

[0083] Comparative Example 5

[0084] Compared with Example 1, the only difference is that in step S1, the specifications of the anhydrous dicalcium phosphate are changed to particle size D90 < 425 μm, particle size D50 > 150 μm, and particle size D10 > 75 μm, while the rest are the same.

[0085] The specifications of anhydrous dicalcium phosphate in Examples 1, 8, 9 and Comparative Example 5 are summarized in Table 4 below.

[0086] Table 4

[0087]

[0088] Example 10

[0089] Compared with Example 1, the only difference is that in step S1, the hydroxyapatite is changed to a spherical shape with an average particle size of 80 μm, while the rest are the same.

[0090] Comparative Example 6

[0091] Compared with Example 2, the only difference is that in step S1, the hydroxyapatite is changed to needle-shaped with an average particle size of 20 nm, while the rest are the same.

[0092] The hydroxyapatite specifications of Examples 1, 10, and 6 are summarized in Table 5 below.

[0093] Table 5

[0094]

[0095] Example 11

[0096] Compared with Example 1, the only difference is that in step S1, carboxymethyl chitosan (substitution degree ≥90%) is replaced with an equal weight of carboxymethyl chitosan (substitution degree >85%), and all other steps are the same.

[0097] Comparative Example 7

[0098] Compared with Example 1, the only difference is that in step S1, the Candida lipase is replaced with porcine pancreatic lipase with the same enzyme activity unit (U), and all other steps are the same.

[0099] Comparative Example 8

[0100] Compared with Example 1, the only difference is that in step S1, carboxymethyl chitosan is replaced with an equal weight of alginate, and all other steps are the same.

[0101] Effect evaluation 1. Detection of the content of each fatty acid in medium-chain triglycerides.

[0102] The medium-chain triglycerides obtained in step S4 of each embodiment and comparative example were sampled, and the proportions of various fatty acids in the medium-chain triglycerides were detected according to the method described in standard GB5009.257-2016 "National Food Safety Standard - Determination of Trans Fatty Acids in Food". The specific process is as follows:

[0103] (1) Methyl esterification. Take 60 mg of the sample to be tested (raw material coconut oil and medium-chain triglycerides prepared in each example and comparative example) and place it in a 10 mL stoppered test tube. Add 4 mL of isooctane to dissolve it completely, add 0.2 mL of potassium hydroxide-methanol solution, vortex and mix for 1 min, and let the mixture in the test tube become clear. Add 1 g of sodium bisulfate to neutralize the excess potassium hydroxide, vortex and mix for 30 s, centrifuge at 4000 rpm for 5 min, filter the supernatant through a 0.45 μm filter membrane, and use the filtrate as the sample test solution.

[0104] (2) Gas chromatography instrument conditions. Same as the detection conditions described in section 5.3 of the national standard GB 5009.257-2016 "National Food Safety Standard - Determination of Trans Fatty Acids in Food".

[0105] (3) Quantitative determination: The standard working solution and the sample solution to be tested are injected into the gas chromatograph respectively. The normalization method is used to determine the quantitative determination based on the response surface area of ​​the chromatographic peak of the standard solution.

[0106] The compound names and molecular formulas of different types of fatty acid methyl esters are shown in Table 6 below.

[0107] Table 6

[0108]

[0109] The experimental results are shown in Table 7 below.

[0110] Table 7

[0111]

[0112] The proportions of C8:0+C10:0, C12:0+C14:0+C16:0+C18:0+C18:1+C18:2 and other components are shown in Table 8 below.

[0113] Table 8

[0114]

[0115] As shown in Tables 7 and 8, the medium-chain triglyceride synthesis methods provided in the embodiments of the present invention can significantly increase the proportion of caprylic acid fatty acid and decanoic acid fatty acid in medium-chain triglycerides, from a total of 16.8% in the raw materials to a maximum of 95.0%, and in particular, can significantly increase the content of decanoic acid fatty acid, from 8.0% in the raw materials to 42.9%-46.2%.

[0116] 2. Evaluation of efficacy: Detection of the content of 3-chloropropanol fatty acid ester (calculated as 3-chloropropanol decanoic acid diglyceride) in medium-chain triglycerides.

[0117] The content of 3-chloropropanol fatty acid esters (calculated as 3-chloropropanol decanoic acid diglyceride) in the medium-chain triglycerides obtained in Examples 1-11 was determined using the method described in Part 4 (Differential Method) of GB / T 44621-2024 "Grain and Oil Inspection - GC / MS Method for Determination of 3-Chloropropanol Fatty Acid Esters". Medium-chain triglycerides were prepared using the method described in Example 1 of the prior art CN118222539A and used as a control group. Each experiment was repeated three times, and the average value was taken. The weight content of 3-chloropropanol fatty acid esters (calculated as 3-chloropropanol decanoic acid diglyceride) in each group is shown in Table 9.

[0118] Table 9

[0119]

[0120] It is evident that, compared to existing technologies, the medium-chain triglyceride preparation method provided by this invention yields a medium-chain triglyceride product with a lower content of 3-chloropropanol fatty acid esters.

[0121] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a medium-chain triglyceride, characterized in that, Includes the following steps: S1. Mix anhydrous dicalcium phosphate, hydroxyapatite, carboxymethyl chitosan, Candida rhynchophylla lipase and water, stir, sonicate, and centrifuge to obtain immobilized lipase. S2. The immobilized lipase obtained in step S1 is mixed with vegetable oil and water, and the enzymatic hydrolysis reaction is carried out. After centrifugation, the aqueous phase is removed to obtain the hydrolysis product. S3. Perform molecular distillation on the hydrolysis products obtained in step S2 to obtain mixed fatty acids; S4. Mix the mixed fatty acids obtained in step S3 with glycerol at a molar ratio of 3:1, add 10wt% lipase Novozym435, 0.12wt% molecular sieve, and 0.02wt% phosphoric acid, and carry out esterification reaction to obtain medium-chain triglycerides. In step S1, the weight ratio of the anhydrous dicalcium phosphate, hydroxyapatite, and carboxymethyl chitosan to the enzyme activity units of the Candida albicans lipase is 0.9-2.1:1.05-2.45:5.45-8.05:600000, in g:g:g:U; in step S1, the particle size D90 of the anhydrous dicalcium phosphate is <150μm, and the average particle size of the hydroxyapatite is 2-80μm. In step S2, the vegetable oil is selected from at least one of coconut oil and palm kernel oil; In step S3, the molecular distillation includes first-stage molecular distillation, second-stage molecular distillation, and third-stage molecular distillation; The temperature of the first-stage molecular distillation is 80-120℃, and the pressure is 50-150Pa; The temperature of the second-stage molecular distillation is 160-180℃, and the pressure is 10-50Pa; The temperature of the third-stage molecular distillation is 200-240℃, and the pressure is less than 10Pa. The fraction obtained from the second-stage molecular distillation is the mixed fatty acid.

2. The preparation method according to claim 1, characterized in that, In step S1, the weight ratio of the anhydrous dicalcium phosphate and the hydroxyapatite is 1-2:1.25-2.

25.

3. The preparation method according to claim 2, characterized in that, In step S1, the weight ratio of the anhydrous dicalcium phosphate and the hydroxyapatite is 1.25-1.75:1.5-2.

4. The preparation method according to claim 3, characterized in that, In step S1, the weight ratio of the anhydrous dicalcium phosphate and the hydroxyapatite is 1.5:1.

75.

5. The preparation method according to claim 1, characterized in that, In step S1, the weight ratio of the sum of the anhydrous dicalcium phosphate and the hydroxyapatite to the weight of the carboxymethyl chitosan is 3.25:6.

75.

6. The preparation method according to claim 1, characterized in that, In step S1, the degree of substitution of the carboxymethyl chitosan is ≥85%.

7. The preparation method according to claim 1, characterized in that, In step S1, the stirring speed is 300-500 rpm, the stirring time is 20-60 min, the ultrasonic time is 10-30 min; the centrifugation speed is 14000-15000 rpm, and the centrifugation time is 10-15 min. In step S2, the weight ratio of the immobilized lipase, vegetable oil, and water is 1-5:8-40:1-5; the temperature of the enzymatic hydrolysis reaction is 50-60℃; the time of the enzymatic hydrolysis reaction is 3-4 hours; the centrifugation speed is 5000-10000 rpm; and the centrifugation time is 5-15 minutes. In step S4, the esterification reaction is carried out at a temperature of 60-70°C for 1-2 hours.

8. The immobilized lipase prepared by the preparation method according to any one of claims 1-7.

9. The preparation method according to any one of claims 1-7 or the immobilized lipase according to claim 8 in the production of medium-chain triglycerides.

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