A feed additive rich in organic trace elements and its preparation method

Abstract

This invention belongs to the field of feed additive technology, specifically a feed additive rich in organic trace elements and its preparation method; it includes the following steps: hydroxytyrosol is suspended in the trans isomer of ethyl eicosapentaenoic acid (EPA), and Candida antarcticis lipase B is added as a catalyst, and a transesterification reaction is carried out at 40-60°C under vacuum for 12-24 hours; after the reaction, the lipase is removed by filtration, and the filtrate is purified to obtain ethyl eicosapentaenoic acid modified hydroxytyrosol; the obtained ethyl eicosapentaenoic acid modified hydroxytyrosol is mixed with organic trace elements and a carrier in a certain proportion to prepare a feed additive rich in organic trace elements; this invention prepares a feed additive that can significantly improve the oxidative stability of EPA, improve the fat solubility of hydroxytyrosol, and eliminate its interference with organic trace elements.

Description

Technical Field

[0001] This invention belongs to the field of feed additive technology, specifically a feed additive rich in organic trace elements and its preparation method. Background Technology

[0002] Eicosapentaenoic acid (EPA) possesses various physiological functions, including anti-inflammatory effects, antioxidant stress relief, improved lipid metabolism, and promotion of neural development in young animals. In the feed industry, it is widely used as a functional fatty acid additive in livestock feed, aquatic feed, and pet food. Ethyl eicosapentaenoic acid (EPAee) is the ethyl esterified form of EPA. As a high-purity monomolecular form of EPA, it has advantages such as high purity and relatively good stability, making it one of the main forms of EPA added to feed.

[0003] Hydroxytyrosol, as a potential natural green feed additive, has broad application prospects. Existing studies have explored its effects in alleviating intestinal oxidative stress in piglets and improving broiler growth performance. However, hydroxytyrosol has strong hydrophilicity but poor fat solubility, making it difficult to effectively distribute in the oil phase and exert its antioxidant effect when directly added to oil-rich feeds. Furthermore, the ortho- and tho-dihydroxy groups in the hydroxytyrosol molecule give it a strong metal ion chelating ability.

[0004] In summary, the existing technology has the following shortcomings: EPAee has poor oxidative stability and is easily oxidized and rancid during feed processing and storage; hydroxytyrosol has poor fat solubility and is difficult to effectively exert its antioxidant effect in the oil phase; the ortho- and tho-dihydroxyl structure of hydroxytyrosol competitively chelates and interferes with organic trace elements. Therefore, it is necessary to develop a feed additive that can significantly improve the oxidative stability of EPA, improve the fat solubility of hydroxytyrosol, and eliminate its interference with organic trace elements, as well as its preparation method. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a feed additive rich in organic trace elements and its preparation method.

[0006] This invention adopts the following technical solution: a method for preparing a feed additive rich in organic trace elements, comprising the following steps: Ethyl cis-eicosapentaenoic acid with a purity greater than 95% was reacted under nitrogen protection at 230-300℃ and 4.0-5.5MPa for 3-5 hours, and then separated and purified to obtain the trans isomer of ethyl eicosapentaenoic acid. Hydroxytyrosol was suspended in the trans isomer of ethyl eicosapentaenoate, and Candida antarcticis lipase B was added as a catalyst to carry out transesterification. After the reaction was completed, the lipase was removed by filtration, and the filtrate was purified to obtain ethyl trans-eicosapentaenoic acid modified hydroxytyrosol. The obtained ethyl trans-eicosapentaenoic acid modified hydroxytyrosol was mixed with organic trace elements and a carrier in a certain proportion to prepare a feed additive rich in organic trace elements.

[0007] Preferably, the molar ratio of hydroxytyrosol to the trans isomer of eicosapentaenoic acid is 1:5 to 1:15.

[0008] Preferably, the amount of lipase added is 2%-10% of the total substrate mass.

[0009] Preferably, the temperature of the transesterification reaction is 40-60℃, and the reaction time is 12-24 hours.

[0010] Preferably, the organic trace element is selected from one or more of amino acid chelated copper, amino acid chelated iron, and amino acid chelated zinc.

[0011] Preferably, the mass ratio of ethyl trans-eicosapentaenoic acid modified hydroxytyrosol to organic trace elements is 1:0.5 to 1:10.

[0012] Preferably, the carrier is selected from one or more of corn starch, maltodextrin, and silicon dioxide.

[0013] Preferably, the mass content of ethyl trans-eicosapentaenoic acid modified hydroxytyrosol in the feed additive is 1%-20%.

[0014] Preferably, the mass content of organic trace elements in the feed additive is 5%-50%.

[0015] A feed additive rich in organic trace elements, comprising ethyl trans-eicosapentaenoic acid modified hydroxytyrosol and organic trace elements. The ethyl trans-eicosapentaenoic acid modified hydroxytyrosol is prepared by transesterification reaction of hydroxytyrosol and the trans isomer of ethyl eicosapentaenoic acid.

[0016] The beneficial effects of this invention are as follows: This invention transforms ethyl cis-eicosapentaenoic acid (EPAee) into its trans isomer via thermal isomerization, thereby enhancing the antioxidant capacity of ethyl cis-eicosapentaenoic acid at the molecular configuration level. In the trans isomer, the substituents on both sides of the double bond are located in opposite directions, the molecular chain tends to be linear, and steric hindrance is increased, making it difficult for oxidizing agents to approach the double bond, thus significantly delaying the oxidative rancidity process of ethyl cis-eicosapentaenoic acid.

[0017] This invention utilizes enzymatic transesterification to covalently link hydrophilic hydroxytyrosol with ethyl trans-eicosapentaenoate (EDE), generating EDE-modified hydroxytyrosol, which significantly improves the lipophilicity of hydroxytyrosol. In an oil matrix rich in ω-3 fatty acids, EDE-modified hydroxytyrosol exhibits excellent oxidative stability. This invention enhances the lipophilicity of hydroxytyrosol while fully preserving its superior antioxidant activity, enabling it to effectively localize within the oil phase and exert its antioxidant protective effect.

[0018] This invention connects hydroxytyrosol and ethyl trans-eicosapentaenoate-modified hydroxytyrosol in the same molecule via covalent bonds, achieving intramolecular antioxidant coupling. The hydroxytyrosol moiety can scavenge free radicals generated by the oxidation of ethyl eicosapentaenoate in situ, providing continuous and efficient intramolecular antioxidant protection for ethyl eicosapentaenoate.

[0019] The ortho-dihydroxyl group in the hydroxytyrosol molecule of this invention endows it with a strong metal ion chelating ability, enabling it to form stable complexes with iron ions, copper ions, etc. When unmodified hydroxytyrosol coexists with feed rich in organic trace elements (such as amino acid chelates of copper, iron, and zinc), competitive chelation occurs, potentially disrupting the original chelating structure of the organic trace elements and interfering with the normal absorption and utilization of essential trace elements by animals. This invention addresses this by esterification, linking the hydroxyl group of hydroxytyrosol to the carboxyl group of ethyl eicosapenoate, altering the overall polarity distribution and spatial configuration of the molecule. This reduces the direct contact opportunity between the ortho-dihydroxyl group and metal ions, effectively minimizing competitive interference with the chelating structure of organic trace elements and ensuring that the availability of organic trace elements in the feed remains unaffected.

[0020] This invention utilizes an enzymatic catalytic transesterification reaction to synthesize ethyl trans-eicosapentaenoic acid modified hydroxytyrosol, using Candida antarcticis lipase B (CALB) as a catalyst, and completes the process in one step under mild conditions of 40-60℃ in a solvent-free system.

[0021] Hydroxytyrosol possesses various biological activities, including strong antioxidant, anti-inflammatory, antibacterial, lipid-lowering, and gut health-promoting functions. As a natural green feed additive, hydroxytyrosol has high bioavailability. By combining these physiological functions of hydroxytyrosol with its antioxidant protective effect on ethyl eicosapentaenoic acid (EEA), the feed additive obtained by this invention provides stable ω-3 fatty acid nutrition while also leveraging the comprehensive benefits of hydroxytyrosol, such as anti-inflammatory effects and improved gut health, thus achieving an organic unity of nutritional supplementation and health promotion.

[0022] This invention combines ethyl eicosapentaenoic acid (EEA) modified with hydroxytyrosol and organic trace elements (such as amino acid-chelated copper, iron, zinc, and manganese) to create a feed additive rich in organic trace elements. This product provides a source of ω-3 fatty acids with significantly improved oxidative stability and efficient intramolecular antioxidant protection, while avoiding interference with the availability of organic trace elements. It also overcomes the dual shortcomings of existing technologies, namely the easy oxidation of EEA and the interference of hydroxytyrosol with trace element absorption, and has promising prospects for industrial application. Detailed Implementation

[0023] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0024] Example 1 A method for preparing a feed additive rich in organic trace elements according to an embodiment of the present invention includes the following steps: Step 1: Thermal isomerization reaction Ethyl cis-eicosapentaenoic acid with a purity greater than 95% was placed in a high-pressure reactor and reacted for 3 hours at 230°C and 4.0 MPa under nitrogen protection to obtain a crude product containing the trans isomer of ethyl eicosapentaenoic acid.

[0025] Step 2: Isolation and purification of the trans isomer of ethyl eicosapride The crude product obtained in step 1 was separated by reversed-phase preparative liquid chromatography, using a mixture of silver nitrate aqueous solution and acetonitrile as the mobile phase.

[0026] Preferably, the volume ratio of silver nitrate aqueous solution to acetonitrile is 20:80. The detection wavelength is 210 nm.

[0027] The fractions corresponding to the chromatographic peaks of various trans isomers in the chromatogram were collected and extracted with organic solvents. The organic phase was then removed by rotary evaporation to obtain the trans isomer of ethyl eicosapentaenoate. The organic solvent was selected as n-hexane. The purity of the trans isomer of ethyl eicosapentaenoate was greater than 90%.

[0028] Step 3: Enzymatic transesterification reaction Hydroxytyrosol was suspended in the trans isomer of ethyl eicosapentaenoic acid obtained in step 2, and Candida antarcticis lipase B (Novozym 435) was added as a catalyst to carry out transesterification under vacuum conditions; the reaction was carried out in a solvent-free system under nitrogen protection throughout.

[0029] The molar ratio of hydroxytyrosol to the trans isomer of eicosapentaenoic acid (EPA) was 1:5. The amount of lipase added was 2% of the total substrate mass. The transesterification reaction was carried out at 50°C for 12 hours. The vacuum level was -0.08 MPa.

[0030] Step 4: Separation and purification of the product After the reaction was completed, the lipase was removed by filtration, yielding a reaction mixture containing hydroxytyrosol-trans eicosapentaenoic acid ester. The filtrate was purified to obtain ethyl trans eicosapentaenoic acid ester-modified hydroxytyrosol.

[0031] Purification was performed by molecular distillation at 160°C and a vacuum of 1 Pa. Further purification was conducted by silica gel column chromatography with gradient elution using a hexane / ethyl acetate mixture.

[0032] Step 5: Preparation of feed additives The ethyl trans-eicosapentaenoic acid modified hydroxytyrosol obtained in step 4 is mixed with organic trace elements and feed-acceptable carriers in a certain proportion to prepare a feed additive rich in organic trace elements.

[0033] Preferably, the mixing time is 5 minutes.

[0034] The organic trace elements were selected from amino acid chelated copper.

[0035] The mass ratio of ethyl trans-eicosapentaenoic acid modified hydroxytyrosol to organic trace elements is 1:0.5.

[0036] The carrier is corn starch.

[0037] The mass content of ethyl trans-eicosapentaenoic acid modified hydroxytyrosol in the feed additive is 1%.

[0038] The mass content of organic trace elements in feed additives is 5%.

[0039] Example 2 A method for preparing a feed additive rich in organic trace elements according to an embodiment of the present invention includes the following steps: Step 1: Thermal isomerization reaction Ethyl cis-eicosapentaenoic acid with a purity greater than 95% was placed in a high-pressure reactor and reacted for 4 hours at 260°C and 4.8 MPa under nitrogen protection to obtain a crude product containing the trans isomer of ethyl eicosapentaenoic acid.

[0040] Step 2: Isolation and purification of the trans isomer of ethyl eicosapride The crude product obtained in step 1 was separated by reversed-phase preparative liquid chromatography, using a mixture of silver nitrate aqueous solution and acetonitrile as the mobile phase.

[0041] Preferably, the volume ratio of silver nitrate aqueous solution to acetonitrile is 30:70. The detection wavelength is 210 nm.

[0042] The fractions corresponding to the chromatographic peaks of various trans isomers in the chromatogram were collected and extracted with organic solvents. The solvent was removed by rotary evaporation of the organic phase to obtain the trans isomer of ethyl eicosapenoate. The organic solvent was selected from petroleum ether. The purity of the trans isomer of ethyl eicosapenoate was greater than 90%.

[0043] Step 3: Enzymatic transesterification reaction Hydroxytyrosol was suspended in the trans isomer of ethyl eicosapentaenoic acid obtained in step 2, and Candida antarcticis lipase B (Novozym 435) was added as a catalyst to carry out transesterification under vacuum conditions; the reaction was carried out in a solvent-free system under nitrogen protection throughout.

[0044] The molar ratio of hydroxytyrosol to the trans isomer of ethyl eicosapentaenoic acid was 1:10. The amount of lipase added was 6% of the total substrate mass. The transesterification reaction was carried out at 50°C for 18 hours. The vacuum level was -0.090 MPa.

[0045] Step 4: Separation and purification of the product After the reaction was completed, the lipase was removed by filtration, yielding a reaction mixture containing hydroxytyrosol-trans eicosapentaenoic acid ester. The filtrate was purified to obtain ethyl trans eicosapentaenoic acid ester-modified hydroxytyrosol.

[0046] Purification was performed by molecular distillation at 180°C and a vacuum of 3 Pa. Further purification was conducted by silica gel column chromatography with gradient elution using a hexane / ethyl acetate mixture.

[0047] Step 5: Preparation of feed additives The ethyl trans-eicosapentaenoic acid modified hydroxytyrosol obtained in step 4 is mixed with organic trace elements and feed-acceptable carriers in a certain proportion to prepare a feed additive rich in organic trace elements.

[0048] Preferably, the mixing time is 8 minutes.

[0049] The organic trace elements are selected from amino acid chelated iron.

[0050] The mass ratio of ethyl trans-eicosapentaenoic acid modified hydroxytyrosol to organic trace elements is 1:5.

[0051] The carrier was selected from maltodextrin.

[0052] The mass content of the trans isomer of eicosapentaenoic acid ethyl ester in the feed additive is 10%.

[0053] The mass content of organic trace elements in the feed additive is 27%.

[0054] Example 3 A method for preparing a feed additive rich in organic trace elements according to an embodiment of the present invention includes the following steps: Step 1: Thermal isomerization reaction Ethyl cis-eicosapentaenoic acid with a purity greater than 95% was placed in a high-pressure reactor and reacted for 5 hours at 300°C and 5.5 MPa under nitrogen protection to obtain a crude product containing the trans isomer of ethyl eicosapentaenoic acid.

[0055] Step 2: Isolation and purification of the trans isomer of ethyl eicosapride The crude product obtained in step 1 was separated by reversed-phase preparative liquid chromatography, using a mixture of silver nitrate aqueous solution and acetonitrile as the mobile phase.

[0056] Preferably, the volume ratio of silver nitrate aqueous solution to acetonitrile is 40:60. The detection wavelength is 210 nm.

[0057] The fractions corresponding to the chromatographic peaks of various trans isomers in the chromatogram were collected and extracted with organic solvents. The solvent was removed by rotary evaporation of the organic phase to obtain the trans isomer of ethyl eicosapentaenoate. Ethyl acetate was selected as the organic solvent. The purity of the trans isomer of ethyl eicosapentaenoate was greater than 90%.

[0058] Step 3: Enzymatic transesterification reaction Hydroxytyrosol was suspended in the trans isomer of ethyl eicosapentaenoic acid obtained in step 2, and Candida antarcticis lipase B (Novozym 435) was added as a catalyst to carry out transesterification under vacuum conditions; the reaction was carried out in a solvent-free system under nitrogen protection throughout.

[0059] The molar ratio of hydroxytyrosol to the trans isomer of eicosapentaenoic acid (EPA) was 1:15. The amount of lipase added was 10% of the total substrate mass. The transesterification reaction was carried out at 60°C for 24 hours. The vacuum level was -0.095 MPa.

[0060] Step 4: Separation and purification of the product After the reaction was completed, the lipase was removed by filtration, yielding a reaction mixture containing hydroxytyrosol-trans eicosapentaenoic acid ester. The filtrate was purified to obtain ethyl trans eicosapentaenoic acid ester-modified hydroxytyrosol.

[0061] Purification was performed by molecular distillation at 200°C and a vacuum of 5 Pa. Further purification was conducted by silica gel column chromatography with gradient elution using a hexane / ethyl acetate mixture.

[0062] Step 5: Preparation of feed additives The ethyl trans-eicosapentaenoic acid modified hydroxytyrosol obtained in step 4 is mixed with organic trace elements and feed-acceptable carriers in a certain proportion to prepare a feed additive rich in organic trace elements.

[0063] Preferably, the mixing time is 10 minutes.

[0064] The organic trace elements are selected from one or more of the amino acid chelated zinc.

[0065] The mass ratio of ethyl trans-eicosapentaenoic acid modified hydroxytyrosol to organic trace elements is 1:10.

[0066] The carrier is selected from one or more of silicon dioxide.

[0067] The mass content of ethyl trans-eicosapentaenoic acid modified hydroxytyrosol in the feed additive is 20%.

[0068] The mass content of organic trace elements in feed additives is 50%.

[0069] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that: ethyl trans-eicosapentaenoic acid modified hydroxytyrosol is not added, and ethyl trans-eicosapentaenoic acid modified hydroxytyrosol is not added during the formulation of the feed additive.

[0070] Performance testing The feed additives rich in organic trace elements obtained in Examples 1-3 and the sample of Comparative Example 1 were placed in a constant temperature oven at 60℃ for accelerated oxidation, and the peroxide value (POV) was measured periodically. The results are shown in Table 1.

[0071] Table 1. Changes in peroxide value of different samples under accelerated oxidation conditions at 60℃ As shown in Table 1, the feed additives rich in organic trace elements obtained in Examples 1-3 exhibited a significantly lower rate of peroxide value increase under accelerated oxidation conditions at 60℃ compared to Comparative Example 1. This indicates that the present invention effectively enhances the antioxidant capacity of EPA at the molecular configuration level by converting ethyl cis-eicosapentaenoic acid (EPA) to its trans isomer through thermal isomerization, significantly delaying the oxidative rancidity process. Comparative Example 2 Comparative Example 2 directly used hydroxytyrosol; no ethyl trans-eicosapentaenoic acid (EEA) modification of hydroxytyrosol was required. The total antioxidant capacity of the ethyl trans-eicosapentaenoic acid (EEA) modified hydroxytyrosol obtained in Examples 1-3 and Comparative Example 2 was tested using FRAP. The results are shown in Table 2.

[0072] Table 2. Total antioxidant capacity of FRAP in different samples (mmol Fe)2+ / g) As shown in Table 2, the total FRAP antioxidant capacity of the ethyl trans-eicosapentaenoic acid modified hydroxytyrosol obtained in Examples 1-3 is comparable to that of the unmodified hydroxytyrosol, further confirming that the present invention fully retains its excellent total antioxidant capacity while improving the lipid solubility of hydroxytyrosol.

[0073] The metal ion chelating ability of the ethyl trans-eicosapentaenoic acid modified hydroxytyrosol obtained in Examples 1-3 and Comparative Example 2 was tested, and the results are shown in Table 3.

[0074] Table 3. Metal ion chelation rate (%) of different samples As shown in Table 3, the metal ion chelation rate of ethyl trans-eicosapentaenoic acid modified hydroxytyrosol obtained in Examples 1-3 was significantly lower than that of unmodified hydroxytyrosol. The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a feed additive rich in organic trace elements, characterized in that: Includes the following steps: Ethyl cis-eicosapentaenoic acid with a purity greater than 95% was reacted under nitrogen protection at 230-300℃ and 4.0-5.5MPa for 3-5 hours, and then separated and purified to obtain the trans isomer of ethyl eicosapentaenoic acid. Hydroxytyrosol was suspended in the trans isomer of ethyl eicosapentaenoate, and Candida antarcticis lipase B was added as a catalyst to carry out transesterification. After the reaction was completed, the lipase was removed by filtration, and the filtrate was purified to obtain ethyl trans-eicosapentaenoic acid modified hydroxytyrosol. The obtained ethyl trans-eicosapentaenoic acid modified hydroxytyrosol was mixed with organic trace elements and a carrier in a certain proportion to prepare a feed additive rich in organic trace elements.

2. The method for preparing a feed additive rich in organic trace elements according to claim 1, characterized in that: The molar ratio of hydroxytyrosol to the trans isomer of eicosapentaenoic acid is 1:5 to 1:

15.

3. The method for preparing a feed additive rich in organic trace elements according to claim 2, characterized in that: The amount of lipase added is 2%-10% of the total substrate mass.

4. The method for preparing a feed additive rich in organic trace elements according to claim 3, characterized in that: The temperature for transesterification is 40-60℃, and the time for transesterification is 12-24 hours.

5. The method for preparing a feed additive rich in organic trace elements according to claim 4, characterized in that: The organic trace elements are selected from one or more of amino acid chelated copper, amino acid chelated iron, and amino acid chelated zinc.

6. The method for preparing a feed additive rich in organic trace elements according to claim 5, characterized in that: The mass ratio of ethyl trans-eicosapentaenoic acid modified hydroxytyrosol to organic trace elements is 1:0.5 to 1:

10.

7. The method for preparing a feed additive rich in organic trace elements according to claim 6, characterized in that: The carrier is selected from one or more of corn starch, maltodextrin, and silicon dioxide.

8. The method for preparing a feed additive rich in organic trace elements according to claim 7, characterized in that: The mass content of ethyl trans-eicosapentaenoic acid modified hydroxytyrosol in feed additives is 1%-20%.

9. The method for preparing a feed additive rich in organic trace elements according to claim 8, characterized in that: The mass content of organic trace elements in feed additives is 5%-50%.

10. A feed additive rich in organic trace elements prepared by the method according to any one of claims 1-9, characterized in that: It contains ethyl trans-eicosapentaenoic acid modified hydroxytyrosol and organic trace elements; The ethyl trans-eicosapentaenoic acid modified hydroxytyrosol is prepared by transesterification reaction of hydroxytyrosol and the trans isomer of ethyl eicosapentaenoic acid.

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