Preparation method of medium-chain triglyceride, related product and application

Abstract

The invention provides a preparation method of medium-chain triglyceride, a related product and application, and relates to the technical field of grease manufacturing. The preparation method comprises the following steps: mixing anhydrous calcium hydrogen phosphate, hydroxyapatite, carboxymethyl chitosan, candida rugosa lipase and water, stirring, performing ultrasonic treatment, centrifuging to obtain immobilized lipase, and performing enzymolysis reaction on the immobilized lipase and coconut oil and / or palm kernel oil to obtain mixed fatty acid; and then catalyzing an esterification reaction between the mixed fatty acid and glycerol to obtain the medium-chain triglyceride. According to the method, the hydrolytic selectivity of the lipase to the medium-chain fatty acid is improved by adjusting the variety and the proportion of the components in the immobilized lipase, so that the yield of the medium-chain triglyceride is improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of oil manufacturing, and relates to a preparation method of medium-chain triglyceride, related products and application. BACKGROUND

[0002] Medium-chain triglyceride (MCT) is a kind of triglyceride containing C8, C10 and C12 fatty acids, which is an important food raw material. 10 At present, the preparation process of medium-chain triglyceride is to extract a mixture of caprylic acid (C7H 15 COOH) and capric acid (C9H 19 COOH) from coconut oil or palm kernel oil, and then bond them with glycerol through esterification reaction, so as to obtain medium-chain triglyceride.

[0003] Wang Yuanli et al. summarized the preparation methods of medium-chain fatty acid and its glyceride in the article “Development and Application Status of Medium-Chain Fatty Acid and Its Glyceride” (China Oils and Fats, 2024, 49 (04), pp.40-47). The preparation methods include alcoholysis method, esterification method, ester exchange method and biosynthesis method. Among them, the esterification method has a long development time, a relatively perfect process and a low cost, but the reaction conditions are relatively complex, there are many by-products, and the operation is relatively complicated. If high-efficiency production of MCT is to be realized, more optimization and exploration need to be carried out from technical improvement.

[0004] At present, the production of medium-chain triglyceride still faces a series of technical problems, especially for the plant raw materials commonly used in the production of medium-chain triglyceride. The enrichment of medium-chain fatty acid in the plant raw materials often needs additional processes, which consumes time and energy. The enzymatic hydrolysis method for preparing fatty acid from plant oil has a certain specificity, but the specificity cannot meet the need of improving the selectivity of medium-chain glyceric acid decomposition. The existing technology has not given a way to promote the enzymatic hydrolysis selectivity of lipase to medium-chain fatty acid by adjusting the components and structure of enzyme aid system, and then to synthesize MCT efficiently. SUMMARY

[0005] In view of the problems existing in the prior art, the purpose of the present application is to provide a preparation method of medium-chain triglyceride, products and application.

[0006] To achieve the above-mentioned purpose of the application, on the one hand, the present application provides a preparation method of medium-chain triglyceride, comprising the following steps: S1, mixing anhydrous calcium hydrogen phosphate, hydroxyapatite, carboxymethyl chitosan, Candida rugosa lipase and water, stirring, ultrasonic, centrifugation, to obtain immobilized lipase; S2, mixing the immobilized lipase obtained in step S1 with plant oil and water, carrying out enzymatic hydrolysis reaction, centrifugation, removing the water phase, to obtain hydrolysis product; S3, subjecting the hydrolysis product obtained in step S2 to molecular distillation to obtain mixed fatty acids; S4, mixing the mixed fatty acids obtained in step S3 with glycerol, adding lipase, esterification reaction to obtain medium-chain triglycerides; In step S1, the weight ratio of the anhydrous dicalcium phosphate, hydroxyapatite, carboxymethyl chitosan and the enzyme activity unit of the Candida rugosa lipase is 0.9-2.1:1.05-2.45:5.45-8.05:600000, unit g:g:g:U. In step S2, the plant oil is selected from at least one of coconut oil and palm kernel oil.

[0007] Further, in step S1, the weight ratio of the anhydrous dicalcium phosphate and the hydroxyapatite is 1-2:1.25-2.25.

[0008] Further, in step S1, the weight ratio of the anhydrous dicalcium phosphate and the hydroxyapatite is 1.25-1.75:1.5-2.

[0009] Further, in step S1, the weight ratio of the anhydrous dicalcium phosphate and the hydroxyapatite is 1.5:1.75.

[0010] Further, in step S1, the ratio of the sum of the weights of the anhydrous dicalcium phosphate and the hydroxyapatite to the weight of the carboxymethyl chitosan is 3.25:6.75.

[0011] Further, in step S1, the particle size D90 of the anhydrous dicalcium phosphate is <150μm, the average particle size of the hydroxyapatite is 2-80μm; and the degree of substitution of the carboxymethyl chitosan is ≥85%.

[0012] Further, in step S1, the stirring speed is 300-500rpm, the stirring time is 20-60min, and the ultrasonic time is 10-30min; the centrifugal speed is 14000-15000rpm, and the centrifugal time is 10-15min.

[0013] Further, in step S2, the weight ratio of the immobilized lipase, plant oil and water is 1-5:8-40:1-5; the enzymatic reaction temperature is 50-60℃, the enzymatic reaction time is 3-4h, the centrifugal speed is 5000-10000rpm, and the centrifugal time is 5-15min.

[0014] Further, 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 by the second-stage molecular distillation is the mixed fatty acid.

[0015] Further, in step S4, the molar ratio of the mixed fatty acid 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 1-2h.

[0016] In another aspect, the present application provides the immobilized lipase prepared by the above preparation method.

[0017] In particular, the present application provides the immobilized lipase prepared in step S1 of the above preparation method.

[0018] In still another aspect, the present application provides the use of the above preparation method or the above immobilized lipase in the production of medium-chain triglyceride.

[0019] In the last aspect, the present application provides the medium-chain triglyceride prepared by the above preparation method or the above immobilized lipase.

[0020] Compared with the prior art, the present application has the following beneficial effects: 1. The present application provides a method for preparing medium-chain triglyceride by using a new type of immobilized lipase. By adjusting the types and proportions of components in the immobilized lipase, the selectivity of the lipase for the hydrolysis of medium-chain fatty acids in the raw material vegetable oil is improved, and thus the content of octanoic acid fatty acid and decanoic acid fatty acid in the medium-chain triglyceride is improved.

[0021] 2. The medium-chain triglyceride preparation process of the present application uses less chlorine-containing compounds compared with the prior art, and produces less 3-chloropropanol fatty acid ester. DETAILED DESCRIPTION

[0022] Terms and statements of the present application: 1. The articles "a", "an", and "the": unless otherwise expressly specified to one (kind) of object, include plural objects.

[0023] 2. Numerical ranges: Unless otherwise expressly specified, all ranges or ratios disclosed herein are to be understood to be inclusive of any and all sub-ranges or sub-ratios subsumed therein. For example, a stated range or ratio of 1 to 30 should be considered to include any and all sub-ranges between (and inclusive of) the minimum value of 1 and the maximum value of 30; to include any and all integer values between 1 and 30; to include any and all fractional values between 1 and 30; and to include any and all sub-ranges or sub-ratios consisting of an integer or fraction of 1 or 30, with both endpoints being inclusive.

[0024] The following non-limiting examples can provide a more complete understanding of the application to one of ordinary skill in the art, but are not intended to limit the application in any way. The following examples merely provide further illustration of the scope of the application claimed herein, and many modifications and variations of the application can be made by those of ordinary skill in the art based on the teachings of the present disclosure, which are to be within the scope of the application claimed.

[0025] The application is further described in the following detailed examples. The various chemical reagents used in the examples are obtained from commercial sources unless otherwise stated. The amounts stated below are amounts by mass unless otherwise stated. The procedures are understood to be carried out at room temperature unless otherwise stated.

[0026] In the following examples, some reagents are obtained as follows in Table 1.

[0027] Table 1

[0028] Example 1 A method for preparing a medium-chain triglyceride.

[0029] The procedure is as follows: S1, 1.5 g of anhydrous calcium hydrogen phosphate, 1.75 g of hydroxyapatite, 6.75 g of carboxymethyl chitosan (degree of substitution ≥ 90%), 600,000 U (400 U / mg, 1.5 g by weight) of Candida rugosa lipase, and 1 L of water are mixed, stirred at 400 rpm for 30 min at room temperature, then ultrasonicated for 20 min, and centrifuged at 14,000 rpm for 10 min to obtain immobilized lipase. The anhydrous calcium hydrogen phosphate has a particle size D90 < 120 μm and a particle size D50 of 50-90 μm; the hydroxyapatite has a spherical shape and an average particle size of 20 μm.

[0030] S2, the immobilized lipase obtained in step S1 is mixed with 81.6 g of coconut oil and 20 g of water, and subjected to enzymatic reaction at 55°C for 3.5 h, then centrifuged at 7,700 rpm for 10 min to remove the water phase to obtain a hydrolysis product.

[0031] S3, the hydrolysis product obtained in step S2 is subjected to molecular distillation to obtain a mixed fatty acid; The molecular distillation consists of a first molecular distillation, a second molecular distillation and a third molecular distillation; The temperature of the first molecular distillation is 80-120℃, and the pressure is 50-150Pa; The temperature of the second molecular distillation is 160-180℃, and the pressure is 10-50Pa; The temperature of the third molecular distillation is 200-240℃, and the pressure is less than 10Pa; The fraction obtained from the second molecular distillation is the mixed fatty acid.

[0032] S4, the mixed fatty acid obtained from step S3 is mixed with glycerol at a molar ratio of 3:1, and lipase Novozym435 (10wt%), molecular sieve (0.12wt%), phosphoric acid (0.02wt%) are added, and esterification reaction is carried out at 65℃ for 2h. After the esterification reaction is completed, centrifugation is carried out at 7500rpm while hot, and the lower layer glycerol is removed. The upper layer product is medium-chain triglyceride.

[0033] Example 2 Compared with Example 1, the only difference is that in step S1, the amount of anhydrous calcium hydrogen phosphate is changed to 1.75g, and the amount of hydroxyapatite is changed to 1.5g, and the rest is the same.

[0034] Example 3 Compared with Example 1, the only difference is that in step S1, the amount of anhydrous calcium hydrogen phosphate is changed to 1.25g, and the amount of hydroxyapatite is changed to 2g, and the rest is the same.

[0035] Example 4 Compared with Example 1, the only difference is that in step S1, the amount of anhydrous calcium hydrogen phosphate is changed to 2g, and the amount of hydroxyapatite is changed to 1.25g, and the rest is the same.

[0036] Example 5 Compared with Example 1, the only difference is that in step S1, the amount of anhydrous calcium hydrogen phosphate is changed to 1g, and the amount of hydroxyapatite is changed to 2.25g, and the rest is the same.

[0037] Comparative Example 1 Compared with Example 1, the only difference is that in step S1, the amount of anhydrous calcium hydrogen phosphate is changed to 0.75g, and the amount of hydroxyapatite is changed to 2.5g, and the rest is the same.

[0038] Comparative Example 2 Compared with Example 1, the only difference is that in step S1, the amount of anhydrous calcium hydrogen phosphate is changed to 2.25g, and the amount of hydroxyapatite is changed to 1g, and the rest is the same.

[0039] The amounts of anhydrous dibasic calcium phosphate, hydroxyapatite of Examples 1 to 5, Comparative Example 1 and Comparative Example 2 are summarized in Table 2.

[0040] Table 2

[0041] Example 6 Comparative Example 1 differs from Example 1 only in that in Step S1, the amount of anhydrous dibasic calcium phosphate is changed to 0.9 g, the amount of hydroxyapatite is changed to 1.05 g, and the amount of carboxymethyl chitosan is changed to 8.05 g, and the rest are the same.

[0042] Example 7 Comparative Example 2 differs from Example 1 only in that in Step S1, the amount of anhydrous dibasic calcium phosphate is changed to 2.1 g, the amount of hydroxyapatite is changed to 2.45 g, and the amount of carboxymethyl chitosan is changed to 5.45 g, and the rest are the same.

[0043] Comparative Example 3 Comparative Example 3 differs from Example 1 only in that in Step S1, the amount of anhydrous dibasic calcium phosphate is changed to 0.6 g, the amount of hydroxyapatite is changed to 0.7 g, and the amount of carboxymethyl chitosan is changed to 8.7 g, and the rest are the same.

[0044] Comparative Example 4 Comparative Example 4 differs from Example 1 only in that in Step S1, the amount of anhydrous dibasic calcium phosphate is changed to 2.4 g, the amount of hydroxyapatite is changed to 2.8 g, and the amount of carboxymethyl chitosan is changed to 4.8 g, and the rest are the same.

[0045] The amounts of anhydrous dibasic calcium phosphate, hydroxyapatite and carboxymethyl chitosan of Examples 1, 6, 7, Comparative Example 3 and Comparative Example 4 are summarized in Table 3 below.

[0046] Table 3

[0047] Example 8 Comparative Example 5 differs from Example 1 only in that in Step S1, the specification of the anhydrous dibasic calcium phosphate is changed to a particle size D90 < 90 μm and a particle size D50 of 35-55 μm, and the rest are the same.

[0048] Example 9 Comparative Example 6 differs from Example 1 only in that in Step S1, the specification of the anhydrous dibasic calcium phosphate is changed to a particle size D90 < 150 μm and a particle size D50 > 90 μm, and the rest are the same.

[0049] Comparative Example 5 The difference compared with Example 1 is only that in step S1, the specification of the anhydrous dibasic calcium phosphate is changed to a particle size D90 < 425 μm, a particle size D50 > 150 μm, and a particle size D10 > 75 μm, and the rest is the same.

[0050] The specifications of the anhydrous dibasic calcium phosphate in Example 1, Example 8, Example 9, and Comparative Example 5 are summarized in Table 4 below.

[0051] Table 4

[0052] Example 10 The difference compared with Example 1 is only that in step S1, the specification of the hydroxyapatite is changed to spherical shape with an average particle size of 80 μm, and the rest is the same.

[0053] Comparative Example 6 The difference compared with Example 2 is only that in step S1, the specification of the hydroxyapatite is changed to needle shape with an average particle size of 20 nm, and the rest is the same.

[0054] The specifications of the hydroxyapatite in Example 1, Example 10, and Comparative Example 6 are summarized in Table 5 below.

[0055] Table 5

[0056] Example 11 The difference compared with Example 1 is only that in step S1, carboxymethyl chitosan (substitution degree ≥ 90%) is replaced by an equal weight of carboxymethyl chitosan (substitution degree > 85%), and the rest is the same.

[0057] Comparative Example 7 The difference compared with Example 1 is only that in step S1, the Candida rugosa lipase is replaced by porcine pancreatic lipase with the same enzyme activity unit (U), and the rest is the same.

[0058] Comparative Example 8 The difference compared with Example 1 is only that in step S1, carboxymethyl chitosan is replaced by an equal weight of alginic acid, and the rest is the same.

[0059] Effect Evaluation 1, Detection of the Content of Each Fatty Acid in Medium-Chain Triglyceride

[0060] The medium-chain triglyceride obtained in step S4 of each example and comparative example is sampled, and the proportion of each fatty acid in the medium-chain triglyceride is detected according to the method recorded in the standard GB5009.257-2016 “National Food Safety Standard Determination of Trans Fatty Acids in Food”. The specific process is as follows: (1) Methyl esterification. Take 60 mg of the sample to be tested (raw material coconut oil and medium-chain triglyceride prepared by each example and comparative example) and place it in a 10 mL test tube with a stopper. Add 4 mL of isooctane to dissolve thoroughly, add 0.2 mL of potassium hydroxide-methanol solution, vortex for 1 min, and let the mixture in the test tube clarify. Add 1 g of sodium bisulfate to neutralize the excess potassium hydroxide, vortex for 30 s, centrifuge at 4000 rpm for 5 min, and filter the supernatant through a 0.45 μm filter membrane. The filtrate is used as the sample to be tested.

[0061] (2) Gas chromatography instrument conditions. The detection conditions are the same as those described in 5.3 of the standard GB 5009.257-2016 "National Food Safety Standard Determination of Trans Fatty Acids in Food".

[0062] (3) Quantitative determination: inject the standard working solution and the sample to be tested into the gas chromatograph, and determine the content of each fatty acid methyl ester compound according to the chromatographic peak response area of the standard solution using the normalization method.

[0063] The compound names and molecular formulas of different types of fatty acid methyl esters are shown in Table 6.

[0064] Table 6

[0065] The experimental results are shown in Table 7.

[0066] Table 7

[0067] Among them, the proportions of C8:0+C10:0, C12:0+C14:0+C16:0+C18:0+C18:1+C18:2 and other components are calculated and shown in Table 8.

[0068] Table 8

[0069] As can be seen from Tables 7 and 8, the medium-chain triglyceride synthesis method provided by each example of the present application can significantly increase the proportion of caprylic acid fatty acids and capric acid fatty acids contained in the medium-chain triglyceride, from a total of 16.8% in the raw material to a maximum of 95.0%, and in particular, the content of capric acid fatty acids can be significantly increased from 8.0% in the raw material to 42.9%-46.2%.

[0070] Effect evaluation 2, detection of the content of 3-chloropropanol fatty acid ester (calculated as 3-chloropropanol capric acid glycerol diester) in medium-chain triglyceride.

[0071] The content of 3-chloropropanol fatty acid ester (calculated as 3-chloropropanol decanoic acid glycerol diester) in the medium-chain triglyceride prepared in Examples 1-11 was detected by the method recorded in the standard GB / T 44621-2024 "Grain and oil testing GC / MS method for determination of 3-chloropropanol fatty acid ester and glycidyl fatty acid ester" Part 4 (difference method). The medium-chain triglyceride was prepared by the preparation method of the medium-chain triglyceride recorded in Example 1 of the prior art CN118222539A, and served as a control group. Each group of experiments was repeated 3 times, and the average value was taken. The weight content of 3-chloropropanol fatty acid ester (calculated as 3-chloropropanol decanoic acid glycerol diester) in each group is shown in Table 9.

[0072] Table 9

[0073] It can be seen that, compared with the prior art, the content of 3-chloropropanol fatty acid ester in the medium-chain triglyceride product obtained by the preparation method of the medium-chain triglyceride provided by the present application is lower.

[0074] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

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, add lipase, and perform esterification reaction to obtain medium-chain triglycerides. 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. In step S2, the vegetable oil is selected from at least one of coconut oil and palm kernel oil.

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 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%.

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 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; 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 esterification reaction temperature is 60-70℃; and the esterification reaction time is 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.

10. Medium-chain triglycerides prepared by the preparation method according to any one of claims 1-7 or by the immobilized lipase according to claim 8.

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