Method for removing glycidyl ester in grease

Abstract

The invention discloses a method for removing glycidyl ester in grease, which creatively utilizes partial glyceride lipase to hydrolyze the glycidyl ester, epoxide hydrolase MdEH is used to remove an intermediate product obtained by hydrolysis, and the glycidyl ester is removed through cascade hydrolysis reaction, so that loss of triglyceride in the grease is not caused. No intermediate product is accumulated, and the removal rate of the glycidyl ester can reach 90% or above. The enzymatic hydrolysis method is mild in reaction condition, directly uses grease and water as raw materials, and has the advantages of being environment-friendly, green, safe, simple to operate, short in production period, low in energy consumption and the like. According to the method, the partial glyceride lipase and the epoxide hydrolase can be covalently fixed and combined on the epoxy resin and then added into the grease to carry out hydrolysis removal reaction, so that repeated utilization of the enzyme can be realized, and the content of the glycidyl ester is reduced to 1.0 mg / kg or below.

Description

Technical Field

[0001] The invention belongs to the technical field of oil processing, and particularly relates to a method for removing glycidyl ester from oil. Background Art

[0002] Glycidyl fatty acid esters (GE or GEs) are a class of epoxy compounds formed by the esterification of the hydroxyl groups on two adjacent carbon atoms of glycerol with free fatty acids after the removal of a water molecule. Glycidyl esters are commonly found in refined oils and fats and are a common hazardous substance in oils and fats. Their safety has recently attracted widespread attention. After ingestion, GEs undergo metabolic reactions in the body, hydrolyzing them through lipase to produce glycidol. Glycidol is genotoxic, reproductively toxic, and nephrotoxic, and is currently classified as a Class 2A carcinogen by the International Agency for Cancer (IARC). Research has shown that GEs often occur in combination with 3-chloropropanol esters, and the two can convert into each other in the presence of chloride ions. 3-Monochloropropane fatty acid esters (3-MCPDE or 3-MCPDEs) are a general term for a class of compounds formed when one or two hydroxyl groups on glycerol undergo esterification with free fatty acids, with the remaining hydroxyl groups replaced by chloride ions. 3-MCPDEs are also common contaminants in oils and fats. Once in the human body, 3-MCPDEs, which are hydrolyzed by lipase in the body, have been classified as a Class 2B carcinogen by the International Agency for Cancer (IARC). In 2018, the European Union first clearly defined the maximum limits for GEs and 3-MCPDEs in relevant foods. On January 1, 2021, the EU implemented Regulation (EU) 2020 / 1322, which clearly stipulates that the maximum limit for GEs in oils and fats is 1 mg / kg, and the maximum limit for oils and fats used in the production of baby food and cereal-based processed foods for infants and young children is 0.5 mg / kg.

[0003] The main causes of GEs are as follows: During the growth stage of oilseed crops, chloride ions in soil and irrigation water lead to the formation of chloropropanol esters, which are indirectly converted into GEs. Monoglycerides and diglycerides in oils and fats are precursors to GEs, which are generated through cyclic acyloxylium ion intermediates. The deodorization temperature during the refining process is typically 240-260°C, which introduces large amounts of GEs, posing a significant risk to the consumption of oils and fats. Since oils and fats are inevitably contaminated by GEs, the development of safe and effective methods for their removal is urgent.

[0004] Chinese invention patent application publication number CN 107090353 A discloses a method for reducing GEs in oils and fats by using glacial acetic acid instead of aqueous solution for steam distillation deodorization, alcohol washing, and organic solvent elution. This method requires the addition of excess alkali during the alkali refining and deacidification step, and separation and elution are performed using a reversed-phase C18 solid-phase extraction column with a volume ratio of n-hexane to ethyl acetate of 2:1. The addition of excess alkali can lead to material loss, and the use of organic solvents also increases costs.

[0005] Chinese invention patent application publication number CN 110257169A discloses a method for reducing the diacylglycerol content in deodorized oils and fats by first performing degumming and solvent fractionation, thereby reducing GE formation during refining. This method utilizes at least one solvent selected from n-hexane, acetone, and ethanol for fractionation, followed by solid-liquid separation of the oil and solvent mixture to produce a low-diacylglycerol oil. This method requires three cooling steps, resulting in a long production cycle and increased costs.

[0006] The above chemical methods for removing GEs from oils and fats are complicated, costly, and not environmentally friendly, and have certain limitations. It is necessary to improve the existing technologies and methods. Summary of the Invention

[0007] Based on this, the purpose of the present invention is to provide a method for removing glycidyl esters from oils and fats, so as to improve the problems of existing chemical removal methods such as material loss, high production costs, and environmental friendliness.

[0008] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions.

[0009] The present invention provides a method for removing glycidyl esters from oils and fats by enzymatic decomposition:

[0010] Partial glyceride lipase PCL, epoxide hydrolase MdEH and buffer solution are added to the oil and fat, and a hydrolysis reaction is carried out at a pH of 5.0-8.0 and a temperature of 20°C-40°C.

[0011] In some embodiments, the pH of the hydrolysis reaction is 6.0-7.0, the temperature is 30° C.-40° C., and the time of the hydrolysis reaction is 2-10 hours.

[0012] In some embodiments, the addition amount of the partial glyceride lipase PCL is not less than 0.02% by weight of the oil, and the addition amount of the epoxide hydrolase MdEH is not less than 0.03% by weight of the oil.

[0013] In some preferred embodiments, the addition amount of the partial glyceride lipase PCL is not less than 0.20% by weight of the oil, and the addition amount of the epoxide hydrolase MdEH is not less than 0.10% by weight of the oil.

[0014] In some more preferred embodiments, the addition amount of the partial glyceride lipase PCL is 0.20%-0.25% of the mass of the oil, preferably 0.21%-0.23%; the addition amount of the epoxide hydrolase MdEH is 0.10%-0.16% of the mass of the oil, preferably 0.12%-0.14%.

[0015] In some embodiments, the partial glyceride lipase PCL and the epoxide hydrolase MdEH are co-immobilized enzymes, and the added amount of the co-immobilized enzymes is not less than 0.1% of the mass of the oil.

[0016] In some preferred embodiments, the added amount of the dual-enzyme co-immobilized enzyme is not less than 1% of the mass of the oil.

[0017] In some more preferred embodiments, the added amount of the dual-enzyme co-immobilized enzyme is 1%-3% of the mass of the oil.

[0018] In some embodiments, the hydrolysis reaction is carried out under inert gas protection conditions. The inert gas can be helium, argon, neon or nitrogen.

[0019] In some embodiments, the buffer solution is KH2PO4-K2HPO4 or NaH2PO4-Na2HPO4; preferably, the concentration of the buffer solution is 50mM-200mM, and the amount of the buffer solution added is 10%-50% of the mass of the oil, preferably 20%-50%.

[0020] The above technical solution can be used to remove glycidyl esters from the following oils and fats: vegetable oils and fats such as olive oil, rice oil, rapeseed oil, peanut oil, corn oil, palm oil, soybean oil, walnut oil, almond oil, linseed oil, camellia oil, sunflower oil, peony seed oil, safflower seed oil, and perilla seed oil, or animal oils and fats such as lard, fish oil, and krill oil.

[0021] The inventors of the present invention have creatively discovered that by using a specific partial glycerol lipase PCL and a specific epoxide hydrolase MdEH to hydrolyze oils and fats while controlling the reaction conditions of the hydrolysis reaction, glycidyl esters in oils and fats can be efficiently removed through a cascade hydrolysis reaction. The glycidyl ester removal rate can reach over 90%, without causing the loss of triglycerides in the oils and fats and without accumulating intermediate products.

[0022] In addition, partial glyceride lipase PCL and epoxide hydrolase MdEH can also perform hydrolysis reactions in the form of dual-enzyme co-immobilization, thereby achieving enzyme reuse.

[0023] The enzymatic hydrolysis method of the present invention for removing glycidyl esters from oils and fats directly uses oils and water as raw materials, has mild reaction conditions, and has the advantages of being environmentally friendly, green and safe, simple to operate, short production cycle, and not consuming a lot of energy. DETAILED DESCRIPTION

[0024] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to enable those skilled in the art to have a more thorough and comprehensive understanding of the disclosure of the present invention.

[0025] In the following examples, the experimental methods without specific conditions are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0027] The present invention is further described in detail below with reference to specific embodiments.

[0028] Example 1

[0029] Reaction vessel 1: olive oil, glycidyl ester standards, 100 mM KH2PO4-K2HPO4 buffer solution, partial glyceride lipase PCL and epoxide hydrolase MdEH.

[0030] Reaction vessel 2: olive oil, glycidyl ester standards, 100 mM KH2PO4-K2HPO4 buffer solution and partial glyceride lipase PCL.

[0031] Reaction vessel 3: olive oil, glycidyl ester standards, 100 mM KH2PO4-K2HPO4 buffer solution and epoxide hydrolase MdEH.

[0032] In the above reaction vessel, the added amount of glycidyl ester standard is 90 mg / kg, the added amount of buffer solution is 30% of the mass of olive oil, the added amount of partial glyceride lipase PCL is 0.5% of the mass of olive oil, and the added amount of epoxide hydrolase MdEH is 0.21% of the mass of olive oil.

[0033] Stir thoroughly to thoroughly mix the contents of each reaction vessel. Purge nitrogen to protect the reaction product. Seal the reaction vessel. Maintain the reaction system temperature at 20°C, pH 7.0, and stirring at 400 rpm. After 4 hours, cease stirring to terminate the hydrolysis reaction. Centrifuge the hydrolyzate at 10,000 rpm for 5 minutes. Remove the upper oil phase and perform glycidyl ester determination according to the method in AOCS Cd 29a-13.

[0034] After treatment with partial glyceride lipase PCL and epoxide hydrolase MdEH, the glycidyl ester content of the olive oil in reaction vessel 1 was 4.65±0.63 mg / kg, with a glycidyl ester removal rate of 94.83±0.70%. After treatment with partial glyceride lipase PCL, the glycidyl ester content of the olive oil in reaction vessel 2 was 13.20±0.79 mg / kg, with a glycidyl ester removal rate of 85.33±0.88%, far lower than the glycidyl ester removal efficiency achieved by the cascade of lipase PCL and MdEH in reaction vessel 1. After treatment with epoxide hydrolase MdEH, the glycidyl ester content of the olive oil in reaction vessel 3 remained as high as 88.28±0.35 mg / kg, indicating that the use of epoxide hydrolase MdEH alone is ineffective in removing glycidyl esters from oils and fats.

[0035] Example 2

[0036] Reaction vessel 1: olive oil, glycidyl ester standards, 100 mM KH2PO4-K2HPO4 buffer solution, partial glyceride lipase PCL and epoxide hydrolase MdEH.

[0037] Reaction vessel 2: olive oil, glycidyl ester standards, 100 mM KH2PO4-K2HPO4 buffer solution, triglyceride lipase Palatase 20000L and epoxide hydrolase MdEH.

[0038] Reaction vessel 3: olive oil, glycidyl ester standards, 100 mM KH2PO4-K2HPO4 buffer solution, partial glyceride lipase AOL and epoxide hydrolase MdEH.

[0039] Reaction vessel 4: olive oil, glycidyl ester standards, 100 mM KH2PO4-K2HPO4 buffer solution, partial glyceride lipase GMGL and epoxide hydrolase MdEH.

[0040] In the above reaction vessel, the addition amount of glycidyl ester standard is 90 mg / kg, the addition amount of buffer solution is 30% of the mass of olive oil, the addition amount of partial glyceride lipase PCL, triglyceride lipase Palatase20000L, partial glyceride lipase AOL and partial glyceride lipase GMGL is 0.5% of the mass of olive oil respectively, and the addition amount of epoxide hydrolase MdEH is 0.21% of the mass of olive oil.

[0041] The contents of each reaction vessel were thoroughly mixed by stirring. Nitrogen was introduced to protect the reaction product. The reaction vessel was then sealed. The reaction system temperature was maintained at 30°C, pH 7.0, and stirring was continued at 400 rpm. After 4 hours of reaction, stirring was stopped to terminate the hydrolysis reaction. The hydrolyzed product was centrifuged at 10,000 rpm for 5 minutes. The upper oil phase was collected, dissolved in n-hexane-isopropanol, thoroughly mixed, dried to remove water, filtered through a 0.22 μm filter, and analyzed for glyceride content by high-performance liquid chromatography. Separate oil samples were also collected and analyzed for glycidyl esters according to the method described in AOCS Cd 29a-13. The results are shown in Table 1.

[0042] Table 1 Effects of different lipases

[0043]

[0044]

[0045] Note: The unit of glycidyl ester is mg / kg.

[0046] As shown in Table 1, the hydrolysis of oils using partial glyceride lipase PCL and epoxide hydrolase MdEH, partial glyceride lipase AOL and epoxide hydrolase MdEH, and partial glyceride lipase GMGL and epoxide hydrolase MdEH did not result in hydrolysis of triglycerides in the oils. However, the hydrolysis of oils using triglyceride lipase Palatase 20000L and epoxide hydrolase MdEH resulted in severe hydrolysis of triglycerides. However, compared with the hydrolysis using partial glyceride lipase AOL and epoxide hydrolase MdEH, partial glyceride lipase GMGL and epoxide hydrolase MdEH, the hydrolysis using partial glyceride lipase PCL and epoxide hydrolase MdEH achieved significantly better glycidyl ester removal.

[0047] Example 3

[0048] Reaction vessel 1: olive oil, glycidyl ester standards, 100 mM KH2PO4-K2HPO4 buffer solution, partial glyceride lipase PCL and epoxide hydrolase MdEH.

[0049] Reaction vessel 2: olive oil, glycidyl ester standards, 100 mM KH2PO4-K2HPO4 buffer solution, partial glyceride lipase PCL and epoxide hydrolase SbEH.

[0050] In the above reaction vessel, the amount of glycidyl ester standard added is 90 mg / kg, the amount of buffer solution added is 20% of the mass of olive oil, the amount of partial glyceride lipase PCL added is 0.5% of the mass of olive oil, and the amount of epoxide hydrolase MdEH and epoxide hydrolase SbEH added is 0.21% of the mass of olive oil.

[0051] Stir thoroughly to thoroughly mix the contents of each reaction vessel. Nitrogen was introduced to protect the reaction product. The reaction vessel was then sealed. The reaction system was maintained at a temperature of 20°C, a pH of 7.0, and a stirring rate of 400 rpm. Stirring was stopped after 4 hours to terminate the hydrolysis reaction. The hydrolyzed product was centrifuged at 10,000 rpm for 5 minutes. The upper oil phase was collected and analyzed for glycidyl esters according to the method in AOCS Cd 29a-13. The lower aqueous phase was collected and analyzed for glycidol by gas chromatography using a CP-Chirasil Dex CB column (25 m × 0.32 mm, 0.25 μm). The heating program was as follows: initial temperature 90°C, hold for 6 minutes, then increase the temperature at 7°C / min to 170°C, hold for 2 minutes; the detection port temperature was 220°C.

[0052] After treatment with partial glyceride lipase PCL and epoxide hydrolase MdEH, the olive oil in reaction vessel 1 had a glycidyl ester content of 3.78±0.16 mg / kg, a removal efficiency of 95.80±0.18%, and a final concentration of the intermediate glycidol of 13.21±0.13 mg / kg. After treatment with partial glyceride lipase PCL and epoxide hydrolase SbEH, the olive oil in reaction vessel 2 had a glycidyl ester content of 11.69±0.37 mg / kg, a glycidyl ester removal efficiency of 87.01±0.41%, and a final concentration of the intermediate glycidol of 52.81±0.11 mg / kg. These results indicate that the catalytic effect of epoxide hydrolase SbEH on the intermediate glycidol was much lower than that of MdEH. This may be due to the lower enzyme activity and stability of SbEH, which is far less catalytic than MdEH.

[0053] Example 4

[0054] Olive oil, glycidyl ester standard, 100mM KH2PO4-K2HPO4 buffer solution, partial glyceride lipase PCL and epoxide hydrolase MdEH were added to reaction vessels 1, 2 and 3, wherein the amount of glycidyl ester standard added was 90 mg / kg, the amount of buffer solution added was 30% of the mass of olive oil, the amount of partial glyceride lipase PCL added to reaction vessel 1 was 0.15% of the mass of olive oil, and the amount of epoxide hydrolase MdEH added was 0.05% of the mass of olive oil, the amount of partial glyceride lipase PCL added to reaction vessel 2 was 0.23% of the mass of olive oil, and the amount of epoxide hydrolase MdEH added was 0.14% of the mass of olive oil, and the amount of partial glyceride lipase PCL added to reaction vessel 3 was 0.5% of the mass of olive oil, and the amount of epoxide hydrolase MdEH added was 0.20% of the mass of olive oil.

[0055] Stir thoroughly to thoroughly mix the contents of each reaction vessel. Purge nitrogen to protect the reaction product. Seal the reaction vessel. Maintain the reaction system temperature at 30°C, pH 7.0, and stirring at 400 rpm. After 4 hours, cease stirring to terminate the hydrolysis reaction. Centrifuge the hydrolyzate at 10,000 rpm for 5 minutes. The upper oil phase was collected and analyzed for glycidyl esters according to the method described in AOCS Cd 29a-13.

[0056] After treatment with partial glycerol lipase PCL and epoxide hydrolase MdEH, the glycidyl ester content of the olive oil in reaction vessel 1 was 29.86±0.22 mg / kg, and the glycidyl ester removal rate reached 66.82±0.24%; after treatment with partial glycerol lipase PCL and epoxide hydrolase MdEH, the glycidyl ester content of the olive oil in reaction vessel 2 was 4.83±0.19 mg / kg, and the glycidyl ester removal rate was 94.63±0.21%; after treatment with partial glycerol lipase PCL and epoxide hydrolase MdEH, the glycidyl ester content of the olive oil in reaction vessel 3 was 5.02±0.04 mg / kg, and the glycidyl ester removal rate was 94.42±0.04%.

[0057] When partial glyceride lipase PCL and epoxide hydrolase MdEH are added to the reaction system, glycidyl esters can be removed from oils and fats through a catalytic cascade hydrolysis reaction. The hydrolysis reaction rate and glycidyl ester removal rate increase with increasing PCL and MdEH addition levels. Experimental results show that when the partial glyceride lipase PCL and epoxide hydrolase MdEH addition levels reach 0.23% and 0.14% of the oil mass, respectively, the hydrolysis reaction rate and glycidyl ester removal rate remain essentially stable, and even with further increases in enzyme addition, these values remain essentially unchanged.

[0058] Example 5

[0059] Olive oil, glycidyl ester standard, 100 mM KH2PO4-K2HPO4 buffer solution, partial glyceride lipase PCL and epoxide hydrolase MdEH were added to reaction vessels 1 and 2, wherein the addition amount of glycidyl ester standard was 90 mg / kg, the addition amount of buffer solution was 30% of the mass of olive oil, the addition amount of partial glyceride lipase PCL was 0.5% of the mass of olive oil, and the addition amount of epoxide hydrolase MdEH was 0.21% of the mass of olive oil.

[0060] Stir thoroughly to thoroughly mix the contents of each reaction vessel. Nitrogen was introduced to protect the reaction product. The reaction vessels were then sealed. The reaction temperature was maintained at 30°C, the pH of reaction vessels 1 and 2 were maintained at 6.0 and 9.0, respectively, and the stirring rate was maintained at 400 rpm. After 4 hours of reaction, stirring was stopped to terminate the hydrolysis reaction. The hydrolyzate was centrifuged at 10,000 rpm for 5 minutes. The upper oil phase was collected and glycidyl esters were determined according to the method described in AOCS Cd 29a-13.

[0061] After treatment with partial glyceride lipase PCL and epoxide hydrolase MdEH, the glycidyl ester content of the olive oil in reaction vessel 1 was 3.11±0.18 mg / kg, with a glycidyl ester removal rate of 96.54±0.20%. In contrast, the glycidyl ester content of the olive oil in reaction vessel 2 was 83.72±0.09 mg / kg, with a glycidyl ester removal rate of only 6.98±0.10%. This suggests that a highly alkaline reaction system may affect the active center of the enzyme protein, leading to reduced enzyme activity and even denaturation and inactivation, making it less conducive to glycidyl ester removal.

[0062] Example 6

[0063] Olive oil, glycidyl ester standard, 100 mM KH2PO4-K2HPO4 buffer solution, partial glyceride lipase PCL and epoxide hydrolase MdEH were added to reaction vessels 1 and 2, wherein the amount of glycidyl ester standard added was 90 mg / kg, the amount of buffer solution added to reaction vessels 1 and 2 was 50% and 5% of the mass of olive oil, respectively, the amount of partial glyceride lipase PCL added was 0.5% of the mass of olive oil, and the amount of epoxide hydrolase MdEH added was 0.21% of the mass of olive oil.

[0064] Stir thoroughly to thoroughly mix the contents of each reaction vessel. Purge nitrogen to protect the reaction product. Seal the reaction vessel. Maintain the reaction system temperature at 30°C, pH 7.0, and stirring at 400 rpm. After 4 hours, cease stirring to terminate the hydrolysis reaction. Centrifuge the hydrolyzate at 10,000 rpm for 5 minutes. The upper oil phase was collected and analyzed for glycidyl esters according to the method described in AOCS Cd 29a-13.

[0065] After treatment with partial glyceride lipase PCL and epoxide hydrolase MdEH, the glycidyl ester content of the olive oil in reaction vessel 1 was 6.91±0.32 mg / kg, with a glycidyl ester removal rate of 92.32±0.36%. In contrast, the glycidyl ester content of the olive oil in reaction vessel 2 was 37.41±1.18 mg / kg, with a glycidyl ester removal rate of only 58.43±1.13%. When too little buffer solution is added, the water-oil contact area is reduced, resulting in a smaller contact area between the enzyme and the substrate, hindering the hydrolysis reaction and reducing the removal efficiency.

[0066] Example 7

[0067] Preparation of immobilized enzyme with an enzyme activity ratio of 2.5:1 U / g: dissolve partial glyceride lipase PCL and epoxide hydrolase MdEH in 0.5 M NaH2PO4-Na2HPO4 buffer solution, calculate the addition amount according to the enzyme activity ratio, and prepare an enzyme solution with a total enzyme concentration of 10 mg / mL. Mix the epoxy resin and the enzyme solution at a ratio of 1 g:15 mL, immobilize at 10°C and pH 7.0 for 8 h, filter, and wash to obtain the dual-enzyme co-immobilized enzyme PCL&MdEH.

[0068] Commercially available palm oil (glycidyl ester content 4.08 mg / kg), 100 mM NaH2PO4-Na2HPO4 buffer solution, and dual-enzyme co-immobilized enzyme PCL&MdEH were added to reaction vessels 1 and 2. The amount of buffer solution added was 10% of the mass of the palm oil, and the amount of the dual-enzyme co-immobilized enzyme PCL&MdEH added was 2% of the mass of the palm oil.

[0069] Stir thoroughly to thoroughly mix the contents of each reaction vessel. Purge nitrogen to protect the reaction product. Seal the reaction vessels. Maintain the reaction temperature at 30°C, the pH of reaction vessels 1 and 2 at 7.0 and 4.0, respectively, and stir at 400 rpm. After 6 hours of reaction, cease stirring to terminate the hydrolysis reaction. Centrifuge the hydrolyzate at 10,000 rpm for 5 minutes, and collect the upper oil phase for glycidyl ester determination according to the method described in AOCS Cd 29a-13.

[0070] After treatment with PCL, a partial glyceride lipase, and MdEH, the palm oil in reaction vessel 1 had a glycidyl ester content of 0.33 ± 0.07 mg / kg, achieving a glycidyl ester removal rate of 91.91 ± 0.07%. In contrast, the palm oil in reaction vessel 2 had a glycidyl ester content of 3.01 ± 0.02 mg / kg, achieving a glycidyl ester removal rate of only 26.23 ± 0.49%. A strongly acidic reaction system also hinders the binding of the enzyme's active center to the substrate. Under these conditions, the enzyme protein cannot achieve its maximum enzymatic activity, hindering the hydrolysis reaction and reducing the removal efficiency.

[0071] Example 8

[0072] Preparation of immobilized enzyme with an enzyme activity ratio of 2.5:1 U / g: dissolve partial glyceride lipase PCL and epoxide hydrolase MdEH in 0.1 M NaH2PO4-Na2HPO4 buffer solution, calculate the addition amount according to the enzyme activity ratio, and prepare an enzyme solution with a total enzyme concentration of 15 mg / mL. Mix the epoxy resin and the enzyme solution at a ratio of 1 g:20 mL, immobilize at 15°C and pH 5.0 for 6 h, filter, and wash to obtain the dual-enzyme co-immobilized enzyme PCL&MdEH.

[0073] Commercially available palm oil (glycidyl ester content 4.08 mg / kg), 100 mM KH2PO4-K2HPO4 buffer solution, and co-immobilized enzymes PCL&MdEH were added to reaction vessels 1 and 2. The amount of buffer solution added to reaction vessels 1 and 2 was 20% and 3% of the mass of the palm oil, respectively, and the amount of the co-immobilized enzymes PCL&MdEH added was 2% of the mass of the palm oil.

[0074] Stir thoroughly to thoroughly mix the contents of each reaction vessel. Purge nitrogen to protect the reaction product. Seal the reaction vessel. Maintain the reaction system temperature at 30°C, pH 7.0, and stirring at 400 rpm. After 6 hours of reaction, discontinue stirring to terminate the hydrolysis reaction. Centrifuge the hydrolyzate at 10,000 rpm for 5 minutes. The upper oil phase was collected and analyzed for glycidyl esters according to the method described in AOCS Cd 29a-13.

[0075] After treatment with PCL and MdEH, the glycidyl ester content of the palm oil in reaction vessel 1 was 0.35 ± 0.02 mg / kg, achieving a glycidyl ester removal rate of 91.42 ± 0.02%. In contrast, the glycidyl ester content of the palm oil in reaction vessel 2 was 2.25 ± 0.00 mg / kg, achieving a glycidyl ester removal rate of only 44.81 ± 0.06%. Adding too little buffer solution hinders enzyme-substrate contact, thus affecting the hydrolysis reaction and reducing the removal efficiency.

[0076] Example 9

[0077] Preparation of immobilized enzyme with an enzyme activity ratio of 2.5:1 U / g: dissolve partial glyceride lipase PCL and epoxide hydrolase MdEH in 0.8 M NaH2PO4-Na2HPO4 buffer solution, calculate the addition amount according to the enzyme activity ratio, and prepare an enzyme solution with a total enzyme concentration of 5 mg / mL. Mix the epoxy resin and the enzyme solution at a ratio of 1 g:10 mL, immobilize at 20°C and pH 7.0 for 10 h, filter, and wash to obtain the dual-enzyme co-immobilized enzyme PCL&MdEH.

[0078] Commercially available palm oil (glycidyl ester content 4.08 mg / kg), 100 mM NaH2PO4-Na2HPO4 buffer solution, and co-immobilized enzymes PCL&MdEH were added to reaction vessels 1 and 2. The amount of buffer solution added was 15% of the mass of the palm oil, and the amount of the co-immobilized enzymes PCL&MdEH added was 2% of the mass of the palm oil.

[0079] Stir thoroughly to thoroughly mix the contents of each reaction vessel. Nitrogen was introduced to protect the reaction product. The reaction vessels were then sealed. The reaction system temperature in vessels 1 and 2 was maintained at 30°C and 50°C, respectively, the pH was maintained at 7.0, and the stirring speed was 400 rpm. After 6 hours of reaction, stirring was stopped to terminate the hydrolysis reaction. The hydrolyzate was centrifuged at 10,000 rpm for 5 minutes. The upper oil phase was collected and glycidyl esters were determined according to the method in AOCS Cd 29a-13.

[0080] After treatment with PCL and MdEH, the palm oil in reaction vessel 1 had a glycidyl ester content of 0.37 ± 0.00 mg / kg, achieving a glycidyl ester removal rate of 90.93 ± 0.00%. In contrast, the palm oil in reaction vessel 2 had a glycidyl ester content of 2.46 ± 0.19 mg / kg, achieving a glycidyl ester removal rate of only 39.80 ± 4.76%. When the reaction system is at a high temperature, the active sites of the protein are destroyed, reducing the catalytic activity of the enzyme.

[0081] Example 10

[0082] Preparation of immobilized enzyme with an enzyme activity ratio of 2.5:1 U / g: dissolve partial glyceride lipase PCL and epoxide hydrolase MdEH in 0.3 M NaH2PO4-Na2HPO4 buffer solution, calculate their addition amounts according to the enzyme activity ratio, and prepare an enzyme solution with a total enzyme concentration of 10 mg / mL. Mix epoxy resin and enzyme solution at a ratio of 1 g:10 mL, immobilize at 10°C and pH 6.0 for 8 h, filter, and wash to obtain dual-enzyme co-immobilized enzyme PCL&MdEH.

[0083] Commercially available palm oil (glycidyl ester content 4.08 mg / kg), 100 mM KH2PO4-K2HPO4 buffer solution, and co-immobilized enzyme PCL&MdEH were added to reaction vessels 1, 2, and 3. The amount of buffer solution added was 15% of the mass of the palm oil. The amount of co-immobilized enzyme PCL&MdEH added to containers 1, 2, and 3 was controlled to be 1%, 3%, and 12% of the mass of the palm oil, respectively.

[0084] Stir thoroughly to thoroughly mix the contents of each reaction vessel. Nitrogen was introduced to protect the reaction product. The reaction vessels were then sealed. The reaction system temperature in vessels 1, 2, and 3 was maintained at 30°C, pH 7.0, and stirring at 400 rpm. After 6 hours of reaction, stirring was stopped to terminate the hydrolysis reaction. The hydrolyzate was centrifuged at 10,000 rpm for 5 minutes. The upper oil phase was collected and glycidyl esters were determined according to the method described in AOCS Cd29a-13.

[0085] After 0.5 h of treatment, the glycidyl ester content of the palm oil in reaction vessel 1 was 1.39 ± 0.14 mg / kg, and the glycidyl ester removal rate reached 65.98 ± 3.29%. After 6 h of treatment, the glycidyl ester content was 0.72 ± 0.02 mg / kg, and the glycidyl ester removal rate reached 88.97 ± 0.25%. After 0.5 h of treatment, the glycidyl ester content of the palm oil in reaction vessel 2 was 0.66 ± 0.05 mg / kg, and the glycidyl ester removal rate reached 83.75 ± 1.32%, and the glycidyl ester content after treatment for 6 hours was 0.67±0.02 mg / kg, and the glycidyl ester removal rate reached 89.22±0.25%; the glycidyl ester content of the palm oil in reaction vessel 3 was 0.69±0.03 mg / kg after treatment for 0.5 hours, and the glycidyl ester removal rate reached 83.01±0.77%. After treatment for 6 hours, the glycidyl ester content was 0.66±0.00 mg / kg, and the glycidyl ester removal rate reached 88.85±0.37%. The results showed that when co-immobilized enzyme was added, the hydrolysis reaction rate and glycidyl ester removal rate would also increase with the increase of the amount of co-immobilized enzyme added. When the addition amount of co-immobilized enzyme PCL&MdEH was 1% of the mass of palm oil, the glycidyl ester content could be removed to below 1 mg / kg; with the increase of the addition amount of co-immobilized enzyme, the rate of its catalytic reaction also increased. When the addition amount exceeded 3% of the mass of palm oil, the hydrolysis rate of glycidyl ester no longer increased with the increase of enzyme addition, indicating that the enzyme addition amount had reached saturation.

[0086] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for removing glycidyl esters from oils and fats, characterized in that: Partial glyceride lipase PCL, epoxide hydrolase MdEH and buffer solution are added to the oil and fat, and a hydrolysis reaction is carried out at a pH of 5.0-8.0 and a temperature of 20°C-40°C.

2. The method for removing glycidyl esters from grease as claimed in claim 1, wherein: The pH of the hydrolysis reaction is 6.0-7.0, the temperature is 30° C.-40° C., and the time of the hydrolysis reaction is 2-10 hours.

3. The method for removing glycidyl esters from grease as claimed in claim 1, wherein: The addition amount of the partial glyceride lipase PCL is not less than 0.02% of the mass of the oil, and the addition amount of the epoxide hydrolase MdEH is not less than 0.03% of the mass of the oil.

4. The method for removing glycidyl esters from grease as claimed in claim 3, wherein: The addition amount of the partial glyceride lipase PCL is not less than 0.20% of the mass of the oil, and the addition amount of the epoxide hydrolase MdEH is not less than 0.10% of the mass of the oil.

5. The method for removing glycidyl esters from grease as claimed in claim 4, wherein: The addition amount of the partial glyceride lipase PCL is 0.20%-0.25% of the mass of the oil, preferably 0.21%-0.23%; the addition amount of the epoxide hydrolase MdEH is 0.10%-0.16% of the mass of the oil, preferably 0.12%-0.14%.

6. The method for removing glycidyl esters from grease as claimed in claim 1, wherein: The partial glyceride lipase PCL and the epoxide hydrolase MdEH are dual-enzyme co-immobilized enzymes, and the added amount of the dual-enzyme co-immobilized enzymes is not less than 0.1% of the mass of the oil.

7. The method for removing glycidyl esters from grease as claimed in claim 6, wherein: The added amount of the dual-enzyme co-immobilized enzyme is not less than 1% of the mass of the oil.

8. The method for removing glycidyl esters from grease as claimed in claim 7, wherein: The added amount of the dual-enzyme co-immobilized enzyme is 1%-3% of the mass of the oil.

9. The method for removing glycidyl esters from grease as claimed in claim 1, wherein: The hydrolysis reaction is carried out under inert gas protection conditions.

10. The method for removing glycidyl esters from oils and fats according to any one of claims 1 to 9, wherein: The buffer solution is KH2PO4-K2HPO4 or NaH2PO4-Na2HPO4; Preferably, the concentration of the buffer solution is 50 mM-200 mM, and the amount of the buffer solution added is 10%-50% of the mass of the oil, preferably 20%-50%.

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