A method for reducing the glycidol content in enzymatic diglyceride

Abstract

The present invention discloses a method for reducing the glycidol content in enzymatic diglyceride, which belongs to the field of food safety. The method is to control the phosphorus content in the raw oil to 100-200 ppm, and the ratio of calcium and magnesium ion content to phosphorus content is ≤0.5. The present invention can greatly reduce the glycidol content in refined diglyceride finished oil by controlling the phosphorus ion content in the raw oil and controlling the ratio of calcium and magnesium ion content to phosphorus content, even if a conventional refining process is used, while not affecting the functionality of the diglyceride finished oil, the content of harmful substances can be reduced by more than 30%.

Description

Technical Field

[0001] The invention relates to a method for reducing the glycidol content in enzymatic diglyceride, and belongs to the field of food safety. Background Art

[0002] Diacylcerides (DAGs) are naturally occurring components of vegetable oils (especially rice oil and palm oil), with concentrations ranging from 2-15%. Unlike triglycerides, one of the hydroxyl groups on the glycerol backbone of diacylglycerol is not replaced by a fatty acid, resulting in distinct nutritional benefits. After absorption, diacylglycerols undergo a different metabolic pathway than triacylglycerols. Diacylglycerol digestion products are not resynthesized into fat in the small intestinal epithelial cells, but are directly oxidized for energy and do not accumulate in the body, thus contributing to weight loss. Numerous studies by researchers both domestically and internationally have shown that the consumption of diacylglycerol oil can significantly lower plasma lipid levels and inhibit body fat accumulation. Long-term consumption of diacylglycerol oil can reduce obesity, promote weight loss, and aid in the treatment of various metabolic diseases. Consequently, diacylglycerols are used as food and pharmaceutical additives.

[0003] Diaglycerides fall under the GRAS (Gradually Safe Assured) safety designation for food additives, a designation used by the US Food and Drug Administration (FDA), and are already used as cooking oils in Japan and the US. Conventional oils rich in diglycerides are similar to regular vegetable oils in taste, appearance, physical properties, and functionality. In 2009, the former Ministry of Health issued an announcement approving new resource foods, listing diglycerides as a new food ingredient. The requirements were a diglyceride content of ≥40%, a triglyceride content of ≤58%, and a daily intake of ≤30g. Because diglyceride content in vegetable oils is low, ranging from 2-15%, enzymatic methods are necessary to increase the diglyceride content. Currently, the more common methods include enzymatic hydrolysis, enzymatic glycerolysis, and enzymatic esterification.

[0004] Patent CN 101974575 B discloses a method for preparing diglyceride-rich rice bran oil. Using oleic acid monoglyceride (a more soluble substance in oils) and high-acidity rice bran oil as raw materials, the process utilizes the lipase Lipozyme RM IM to catalyze the reaction. Under vacuum and rotary conditions, factors such as reaction temperature, reaction time, and lipase dosage were optimized. Under optimal conditions, after a 6-hour reaction, the final diglyceride content in the product was 27.42%.

[0005] Patent CN101260417B discloses an enzymatic process for preparing 1,3-DAG in a petroleum ether medium at atmospheric pressure. This process involves adding pure glycerol and a fatty acid donor to petroleum ether, followed by the addition of molecular sieves, and reacting under the catalysis of lipase 435. After 2-5 hours of reaction, the diglyceride content in the system reaches 80%.

[0006] Patent CN100376541C discloses a method for preparing diglycerides. This method involves mixing and stirring an acyl acceptor containing glycerol, a fatty acid donor, and a hydrophilic carrier silica gel. The mixture then reacts with an immobilized enzyme to produce diglycerides. This patent primarily involves adsorbing glycerol onto the silica gel, with a reaction time of 8-24 hours.

[0007] Patent CN1208305C discloses a method for producing diglycerides. This method involves mixing a lower alcohol and triglycerides and then subjecting them to alcoholysis under the catalysis of immobilized lipase, converting the triglycerides into diglycerides. This method has a long reaction time of 1-10 hours.

[0008] Patent CN102965402A discloses a method for preparing diglycerides from camphor seed oil. The method uses 4-16% immobilized lipase (Lipozyme RM or TL) to hydrolyze camphor seed oil in the presence of 20-60% distilled water to prepare diglycerides.

[0009] The document "Study on the Synthesis of 1,3-DAG by Enzymatic Esterification Catalyzed Reaction in a Solvent-Free System" reports that 1,3-DAG was synthesized in a solvent-free system using pure oleic acid and glycerol as raw materials and specific lipase. The results showed that when the molar ratio of oleic acid to glycerol was 2:1, the enzyme amount was 6%, and the temperature was 65°C, the mass fraction of 1,3-DAG reached 62%.

[0010] The paper "Enzymatic Synthesis and Properties of High-Purity Diacylcerides" reports that lipase G50 catalyzes the synthesis of high-purity diacylglycerol from camellia oil fatty acids and glycerol. At a reaction temperature of 35°C, a 2% lipase addition, and a 4:1 molar ratio of glycerol to fatty acids, the diacylglycerol content in the esterified product reached 50.48% after 24 hours of reaction.

[0011] The paper "Biocatalyzed synthesis of sn-1,3-diacylglycerol oil from extravirgin olive oil" reports a two-step process for preparing diacylglycerol. First, excess ethanol was added to hydrolyze olive oil into ethyl esters using Novozymes 435 catalysis. The ethyl esters were then esterified to diacylglycerols using Lipozyme RM IM. After 24 hours of esterification at 40°C, the diacylglycerol content in the system reached nearly 40%.

[0012] The aforementioned documents and patents investigate the synthesis of diglycerides through various processes (alcoholysis, hydrolysis, and esterification) to produce diglyceride edible oils with varying diglyceride contents. However, these documents and patents focus solely on the synthesis process, neglecting subsequent processing steps and even less on the content of certain hazardous substances, such as glycidol, in refined edible oils.

[0013] While there's no consensus on the formation mechanism of glycidol, it's generally believed that it can form during the oil ester refining process. In the presence of a Lewis acid, glycerol esters first form an intermediate cyclic acyl oxide ion. This cyclic acyl oxide then reacts with chloride ions via nucleophilic substitution to form 3-chloropropanol ester. The cyclic acyl oxide deprotonates to form glycidol. The precursors for glycidol formation are primarily diglycerides and monoglycerides, and their content is directly related to the deodorization temperature. Glycidol begins to form at 200°C, and its formation rate increases significantly at 230°C.

[0014] Glycidol is genotoxic and carcinogenic, designated by IARC as a Class 2A carcinogen, meaning it is likely carcinogenic to humans. JECFA has calculated a benchmark dose (BMDL10) of 2.4 mg / kg / day to be carcinogenic to humans. A safe dose for this type of carcinogen cannot be established to protect human health. Internationally, it is generally required that its contamination in food be as low as technically feasible, known as the ALARA principle. In short, the glycidol content in edible oils should be controlled as low as possible.

[0015] Currently, many methods are available for reducing the glycidol content in oils and fats. The appropriate method can be selected based on the oil source, refining process, and processing equipment. Examples include low-temperature deodorization and short-path distillation as an alternative to deodorization. However, these methods have some drawbacks compared to traditional refining methods. For example, after low-temperature deodorization, the oil's color and odor may not meet the required standards. Short-path distillation equipment requires high investment, making it unsuitable for large-scale production.

[0016] Therefore, it is necessary to develop a method that can efficiently and simply reduce the glycidol content in enzymatic diacylglycerol. Summary of the Invention

[0017] In order to solve at least one of the above problems, the present application controls the phosphorus ion content in the raw oil during the enzymatic production of diglyceride and controls the ratio of calcium and magnesium ion content to phosphorus content. Even if conventional refining processes are used, the glycidol content in the refined diglyceride finished oil can be greatly reduced, and the content of harmful substances can be effectively reduced without affecting the functionality of the diglyceride finished oil.

[0018] After extensive research, the inventors discovered that the phosphorus ion content in the feedstock oil represents the phospholipid content in the system. Phospholipids are strong emulsifiers and easily form an emulsified system in the presence of water, making oil-water separation more difficult. During the preparation of diglycerides, the presence of phospholipids can cause glycerol (produced by hydrolysis or added) to form an emulsified system with the oil, complicating separation and causing some glycerol to remain in the oil system. Glycerol is also a precursor to glycidol, so increasing glycerol levels increases the amount of glycidol. Too little phospholipids can create a reverse micelle system, causing more glycerol to dissolve in the oil. However, through extensive experiments, we discovered that if the ratio of calcium and magnesium ions to phosphate ions in the system is within a certain range, the emulsification of the system is significantly weakened, resulting in a corresponding decrease in the amount of residual glycerol, thereby reducing the glycidol content.

[0019] The first object of the present invention is to provide a method for reducing the glycidol content in the process of enzymatic production of diglyceride, wherein the method is to control the phosphorus content in the raw oil to 100-200 ppm and the ratio of calcium and magnesium ion content to phosphorus content is ≤0.5.

[0020] In one embodiment, the ratio of the calcium and magnesium ion content to the phosphorus content is:

[0021] .

[0022] In one embodiment, the phosphorus content in the feedstock oil is controlled by adding water to the system if the phosphorus content in the system is too high, removing excess phosphorus ions through hydration degumming, and reducing the phosphorus content to within the range of 100-200 ppm.

[0023] In one embodiment, the phosphorus content in the feedstock oil is controlled by adding phospholipids to the system to bring the phosphorus content to within the range of 100-200 ppm if the phosphorus content in the system is too low.

[0024] In one embodiment, the phospholipid can be soybean lecithin, rapeseed lecithin, sunflower oil lecithin, peanut lecithin, etc.

[0025] In one embodiment, the ratio of the calcium and magnesium ion content to the phosphorus content is controlled by adding citric acid or phosphoric acid, preferably citric acid, to the system to chelate the calcium and magnesium ions if the ratio is greater than 0.5, so that the ratio of the calcium and magnesium ion content to the phosphorus content is ≤0.5; the heavy phase is removed by centrifugation at a speed of 8000 rpm for 10 minutes, and the upper oil phase after centrifugation is reacted.

[0026] A diglyceride produced by an enzymatic method, wherein the glycidol content of the diglyceride is reduced by the above method.

[0027] In one embodiment, the enzymatic production of diglycerides comprises:

[0028] Step 1: Use the second method of GB 5009.268-2016 to detect the phosphorus, calcium, and magnesium ion contents in the feedstock oil. If the phosphorus content in the system is not within the range of 100-200 ppm or the ratio of calcium and magnesium ion content to phosphorus content is ≤0.5, adjust it by any of the above methods.

[0029] Step 2: preparing diglyceride by enzymatic method to obtain crude diglyceride;

[0030] Step 3: Water washing: add 50% deionized water to the crude diglyceride prepared by the enzymatic method, heat to 80°C and stir and wash for 30 minutes. After the washing is completed, the oil and water are separated, and the oil phase is dried to obtain the washed diglyceride;

[0031] Step 4: Molecular distillation: Separate the washed diglyceride into fatty acids and monoglycerides in a separation distillation, with a separation temperature of ≥200°C and a vacuum degree of ≤5 Pa;

[0032] Step 5: Decolorization: The diglyceride after molecular distillation is heated to 105°C under vacuum, and then 1% white clay is added for decolorization. After decolorization, the white clay is removed by filtration to obtain decolorized diglyceride.

[0033] Step 6: Deodorization: The decolorized diglyceride is deodorized at a temperature of 240°C for 2 hours and a vacuum degree of ≤5 mbar. The deodorized diglyceride is the finished product.

[0034] In one embodiment, the enzymatic method comprises a hydrolysis method and a glycerolysis method; wherein:

[0035] 1) Hydrolysis: Mix oil and water in a 1:1 ratio, then add 0.3% liquid lipase (Lipozyme TL, G50, ROL, Lipozeme RM, CalB, etc.) and react at 35°C for 8 hours. After the reaction, separate the oil and water. The separated oil phase is crude diglyceride.

[0036] 2) Glycerol hydrolysis: Add 5.2% glycerol (based on the mass of the oil) to the oil, followed by 3% immobilized lipase (lipase from Alcaligenes and / or from Candida, such as Lipozyme TL IM, Lopozyme RM, Lipase-30SD, Lipozyme 435, G50 immobilized enzyme, etc.). Stir the reaction at 70°C for 8 hours. After the reaction is complete, filter and remove the immobilized enzyme to obtain crude diacylglycerol.

[0037] Application of the above method for reducing the glycidol content in the process of producing diglyceride by enzymatic method in the production of diglyceride.

[0038] The beneficial effects of the present invention are:

[0039] By controlling the phosphorus ion content in the raw oil and the ratio of the calcium and magnesium ion content to the phosphorus content, the present invention can greatly reduce the glycidol content in the refined diglyceride finished oil even if a conventional refining process is adopted. While not affecting the functionality of the diglyceride finished oil, the content of harmful substances can be reduced by more than 30%. DETAILED DESCRIPTION

[0040] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention are described in further detail below.

[0041] The present invention relates to a method:

[0042] 1. Detection method: The phosphorus, calcium and magnesium ion contents were detected using the second method of GB 5009.268-2016; the glycidol content was determined according to the standard recommended by the German Oils and Fats Association DGF C-VI 18 (10).

[0043] 2. There are many methods for enzymatically preparing diglycerides. The present invention adopts hydrolysis and glycerol decomposition:

[0044] 1) Hydrolysis: Mix oil and water in a 1:1 ratio, then add 0.3% liquid lipase (Lipozyme TL, G50, ROL, Lipozeme RM, CalB, etc.) and incubate at 35°C for 8 hours. After the reaction, separate the oil and water, and the separated oil phase is crude diglyceride.

[0045] 2) Glycerol hydrolysis method: Add 5.2% glycerol (based on the mass of the oil) to the oil, followed by 3% immobilized lipase (lipase from Alcaligenes and / or lipase from Candida, such as Lipozyme TL IM, Lopozyme RM, Lipase-30SD, Lipozyme 435, G50 immobilized enzyme, etc.). Stir the reaction at 70°C for 8 hours. After the reaction is complete, filter and remove the immobilized enzyme to obtain crude diacylglycerol.

[0046] 3. Hydration degumming: Heat the oil to 80°C, then add 3% deionized water according to the mass ratio, stir and react for 30 minutes, and centrifuge after the reaction (separation conditions 8000rpm 10min). The upper oil phase after separation can be used as raw material for subsequent reactions.

[0047] Example 1

[0048] 1. Determine the phosphorus, calcium and magnesium ion contents in the raw soybean oil. The phosphorus content is 137 ppm, and the ratio of calcium and magnesium ion content to phosphorus content is 0.46.

[0049] 2. Prepare diglyceride by hydrolysis to obtain crude diglyceride.

[0050] 3. Water washing: Add 50% deionized water to the crude diglyceride prepared by the enzymatic method, heat to 80°C and stir to wash for 30 minutes. After washing, separate the oil and water, and dry the oil phase to obtain the washed diglyceride.

[0051] 4. Molecular distillation: Separate the fatty acids and monoglycerides from the washed diglycerides in a separation distillation with a separation temperature of ≥200°C and a vacuum degree of ≤5 Pa.

[0052] 5. Decolorization: The diglyceride after molecular distillation is heated to 105°C under vacuum, and then 1% white clay is added for decolorization. After decolorization, the white clay is removed by filtration to obtain the decolorized diglyceride.

[0053] 6. Deodorization: Deodorize the decolorized diglyceride. The deodorization temperature is 240℃, the time is 2h, and the vacuum degree is ≤5mbar. The finished diglyceride is obtained after deodorization.

[0054] 7. The glycidol content was detected to be 1780 ppb.

[0055] Example 2

[0056] Different from Example 1, the method for preparing crude diglyceride is glycerolysis, and the glycidol content is detected to be 1277 ppb.

[0057] Example 3

[0058] Different from Example 1, the phosphorus content in the raw soybean oil was 189 ppm, the ratio of calcium and magnesium ion content to phosphorus content was 0.23, and the glycidol content was detected to be 1062 ppb.

[0059] Example 4

[0060] Different from Example 3, the method for preparing crude diglyceride is glycerolysis, and the glycidol content is detected to be 1876 ppb.

[0061] Example 5

[0062] Different from Example 1, the phosphorus content in the raw soybean oil was 153 ppm, the ratio of calcium and magnesium ion content to phosphorus content was 0.31, and the glycidol content was detected to be 1291 ppb.

[0063] Example 6

[0064] Different from Example 5, the method for preparing crude diglyceride was glycerolysis, and the glycidol content was detected to be 1027 ppb.

[0065] Example 7

[0066] Different from Example 1, the phosphorus content in the raw soybean oil was 103 ppm, the ratio of calcium and magnesium ion content to phosphorus content was 0.42, and the glycidol content was detected to be 1792 ppb.

[0067] Example 8

[0068] Different from Example 7, the method for preparing crude diglyceride is glycerol hydrolysis, and the glycidol content is detected to be 1366 ppb.

[0069] Example 9

[0070] Different from Example 1, the phosphorus content in the raw soybean oil is 279 ppm, the ratio of calcium and magnesium ion content to phosphorus content is 0.73, and the detected glycidol content is 3219 ppb.

[0071] Taking the same raw soybean oil with a phosphorus content of 279 ppm and a calcium and magnesium ion to phosphorus ratio of 0.73, approximately 20 ppm of citric acid and 0.1% water were added to the system, adjusting the phosphorus content in the raw soybean oil to 192 ppm and the calcium and magnesium ion to phosphorus ratio to 0.37. The oil was then subjected to the same enzymatic hydrolysis method, followed by water washing, molecular distillation, decolorization, and deodorization. The glycidol content was found to be 1947 ppb. Compared to a solution without adjusting the phosphorus content, calcium and magnesium ion content, and phosphorus ratio, the glycidol content was reduced by 39.51%.

[0072] Example 10

[0073] Different from Example 1, the phosphorus content in the raw soybean oil is 436 ppm, the ratio of calcium and magnesium ion content to phosphorus content is 0.11, and the detected glycidol content is 3462 ppb.

[0074] The same soybean oil with a phosphorus content of 436 ppm and a calcium and magnesium ion to phosphorus ratio of 0.11 was added to 1% water and stirred at 80°C for 30 minutes. Excess phospholipids were removed by centrifugation after the reaction. This reaction adjusted the phosphorus content of the soybean oil to 157 ppm and the calcium and magnesium ion to phosphorus ratio to 0.39. The oil was then subjected to the same enzymatic hydrolysis method, followed by washing, molecular distillation, decolorization, and deodorization. The glycidol content was found to be 1588 ppb. Compared to a solution without adjusting the phosphorus content, calcium and magnesium ion content, and phosphorus ratio, the glycidol content was reduced by 54.13%.

[0075] Comparative Example 1

[0076] Different from Example 1, the phosphorus content in the raw soybean oil is 125 ppm, the ratio of calcium and magnesium ion content to phosphorus content is 0.81, and the detected glycidol content is 3920 ppb.

[0077] Comparative Example 2

[0078] Different from Example 1, the phosphorus content in the raw soybean oil is 321 ppm, the ratio of calcium and magnesium ion content to phosphorus content is 0.64, the method for preparing crude diglyceride is glycerol hydrolysis, and the glycidol content is detected to be 4688 ppb.

[0079] Comparative Example 3

[0080] Different from Example 1, the phosphorus content in the raw soybean oil is 118 ppm, the ratio of calcium and magnesium ion content to phosphorus content is 0.89, the method for preparing crude diglyceride is glycerol hydrolysis, and the glycidol content is detected to be 2981 ppb.

[0081] The enzymatic methods for preparing diglycerides in various examples and comparative examples, the phosphorus content, the ratio of calcium and magnesium ion content to phosphorus content, and the glycidol content in the prepared diglycerides are shown in Table 1:

[0082] Table 1: Enzymatic method for preparing diglycerides in various examples and comparative examples, phosphorus content, ratio of calcium and magnesium ion content to phosphorus content, and glycidol content in the prepared diglycerides

[0083]

[0084] As can be seen from the table, when the phosphorus content in the feedstock oil is between 100-200 ppm and the ratio of calcium and magnesium ion content to phosphorus content is ≤0.5, the glycidol content of the prepared diglyceride is significantly lower than that of diglyceride with a phosphorus content outside the range of 100-200 ppm or a ratio of calcium and magnesium ion content to phosphorus content greater than 0.5. Furthermore, after adjusting the phosphorus content to 100-200 ppm and the ratio of calcium and magnesium ion content to phosphorus content to ≤0.5, the glycidol content of the prepared diglyceride is significantly reduced.

Claims

1. A method for reducing the glycidol content in enzymatic diglycerides, characterized in that: The method is to control the phosphorus content in the raw oil to be 100-200 ppm, and the ratio of the calcium and magnesium ion content to the phosphorus content is in the range of 0.23-0.5; The ratio of calcium and magnesium ion content to phosphorus content is: ； The enzymatic method is a hydrolysis method or a glycerolysis method; If the phosphorus content in the crude oil is too high, water is added to the crude oil to remove excess phosphorus ions through hydration degumming, so that the content is within the range of 100-200ppm; If the phosphorus content in the feedstock oil is too low, add phospholipids to the feedstock oil to bring its content within the range of 100-200 ppm; If the ratio of calcium and magnesium ion content to phosphorus content in the raw oil is greater than 0.5, citric acid is added to the raw oil to chelate the calcium and magnesium ions so that the ratio of calcium and magnesium ion content to phosphorus content is 0.23~0.

5.

2. The method according to claim 1, characterized in that The lecithin is soybean lecithin, rapeseed lecithin and peanut lecithin.

3. A method for preparing diglyceride, characterized in that: The method includes: Step 1: Detect the phosphorus, calcium, and magnesium ion contents in the feed oil, control the phosphorus content in the feed oil to be 100-200 ppm, and the ratio of the calcium and magnesium ion content to the phosphorus content to be in the range of 0.23-0.5; if the phosphorus content in the feed oil is not in the range of 100-200 ppm and the ratio of the calcium and magnesium ion content to the phosphorus content is greater than 0.5, adjust the phosphorus, calcium, and magnesium ion content according to claim 1; Step 2: preparing diglyceride by enzymatic method to obtain crude diglyceride; Step 3: washing with water, adding deionized water to the crude diglyceride prepared by the enzymatic method, heating and stirring, washing with water, separating the oil and water after washing, and drying the oil phase to obtain washed diglyceride; Step 4: molecular distillation, separating the washed diglyceride into fatty acids and monoglycerides in a separation distillation kettle, with a separation temperature of ≥200°C and a vacuum degree of ≤5Pa; Step 5: Decolorization: the diglyceride after molecular distillation is heated under vacuum, and then white clay is added for decolorization. After decolorization, the white clay is removed by filtration to obtain decolorized diglyceride. Step 6: Deodorization: The decolorized diglyceride is deodorized at a vacuum degree of ≤5mbar. The deodorized diglyceride is the finished product.

4. The method according to claim 3, characterized in that The enzymatic method is a hydrolysis method and a glycerol decomposition method.

5. A low-glycidyl diglyceride, characterized in that The method according to claim 3 or 4 is used for preparation.

6. Use of the method according to any one of claims 1 to 4 in enzymatic production of diglycerides.

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