Preparation method and product of breast milk substitute fat with controllable OPL / OPO / LPL ratio

Abstract

The present invention discloses a preparation method of a breast milk substitute fat with controllable OPL / OPO / LPL ratio, and the steps are as follows: Step 1, using palm stearin as a raw material, separating out palm stearin with a tripalmitin content of more than 85 wt%; Step 2, the separated palm stearin is alcoholyzed under the catalysis of a regioselective lipase to obtain 2-monopalmitin with a purity of more than 85 wt%; Step 3, in a solvent or solvent-free system, after 2-monopalmitin is heated and mixed with a certain proportion of oleic acid / linoleic acid mixture, a lipase is added for enzymatic catalytic esterification reaction to obtain a crude breast milk substitute fat rich in OPL / OPO / LPL: Step 4, separating free fatty acids in the reaction system from the above-mentioned crude product to obtain a breast milk substitute fat rich in OPL / OPO / LPL. In the breast milk substitute fat product of the present invention, the total content of OPL, OPO and LPL can reach 75 wt% to 91 wt%, and OPL / OPO is in the range of 0.2 to 2.9 and LPL / OPO is in the range of 0 to 1.0, which can be regulated, and problems such as acyl migration, many by-products and low yield in the preparation of OPL / OPO / LPL by acidolysis reaction in the prior art are avoided.

Description

Technical Field

[0001] The present invention relates to a preparation method and product of a breast milk substitute fat with controllable OPL / OPO / LPL ratio, belonging to the technical field of oil synthesis. Background Art

[0002] Breast milk is the most ideal food for infants. As the most important energy-providing substance, fat provides more than half of the energy and essential fatty acids, fat-soluble vitamins and other lipid components for infants and young children with a mass of about 3-5% of breast milk. Among them, the content of 1,3-dioleoyl-2-palmitoyl glycerol (hereinafter referred to as OPO), 1-oleoyl-2-palmitoyl-3-linoleoyl glycerol (hereinafter referred to as OPL), and 1,3-dilinoleoyl-2-palmitoyl glycerol (hereinafter referred to as LPL) reaches 40%-50% of the total amount of human milk fat. In recent years, many scholars at home and abroad have studied the composition of human milk fat in different regions of the world and found that the content and ratio of OPL, OPO and LPL in breast milk fat of different regions vary greatly. This may be related to the eating habits of vegetable oils rich in oleic acid or linoleic acid consumed in each region. In European countries such as Italy, Finland, Spain and other countries, the OPO content in breast milk fat is the highest, the OPL / OPO ratio is 0.2-0.9 (mass ratio), and the LPL content is relatively low. In the composition of human milk fat in China, the OPL content is the highest, followed by OPO and LPL, which are the three most abundant structural lipids, and the mass ratio of OPL / OPO / LPL is about 1.5:1:0.6 (assuming the mass content of OPO is 1, calculating OPL / OPO and LPL / OPO to obtain the relative content ratio of OPL / OPO / LPL).

[0003] In the context of the continuous decline in the exclusive breastfeeding rate, formula milk has become the main supplementary food for infant growth and development. Breast milk esters are widely used in formula milk because of their composition and structure similar to breast milk fat. CN 109566769 A reported the preparation of oils rich in OPO and OPL by the catalytic transesterification reaction of triglyceride palmitate and acyl donors oleic acid and linoleic acid with lipase as the catalyst. However, the content of the product by this method is low. CN 111593046 A adopted the same strategy, using magnetic carrier-immobilized lipase as the catalyst, and obtained OPL oil with a content of 35-50 wt% after a long reaction time (8-12 hours). CN109402186 A introduced the reaction of tripalmitin (PPP) with oleic acid-based and linoleic acid-based acyl donors, and after separation and purification, acyl donors were added again for a secondary reaction to obtain an oil mixture rich in OPL. This method can obtain OPL oil with a relatively high content, but it has the problems of complex operation, high reaction temperature (40-90 °C), and high energy consumption for two molecular distillations. Currently, the reports on OPO and OPL mainly use the method of enzymatic acidolysis of tripalmitin, which has problems such as long reaction time, high temperature, inevitable acyl migration, and generally low product purity. Therefore, providing a simple and efficient preparation method with adjustable OPL / OPO / LPL ratio is of great significance for promoting the breast milkization process of infant formula milk. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a preparation method of a breast milk substitute fat with controllable OPL / OPO / LPL ratio in view of the deficiencies of the above-mentioned prior art. The total content of OPL, OPO, and LPL in the prepared breast milk substitute can reach 75 wt% - 91 wt%, and the OPL / OPO is in the range of 0.2 - 2.9 (mass ratio) and the LPL / OPO is in the range of 0 - 1.0 (mass ratio), which can be accurately regulated. At the same time, it avoids the problems of acyl migration, many by-products, and low yield in the preparation of OPL / OPO / LPL by acidolysis reaction in the prior art.

[0005] The technical solution adopted by the present invention to solve the above-mentioned problems is as follows:

[0006] A preparation method of a breast milk substitute fat with controllable OPL / OPO / LPL ratio mainly includes the following four steps:

[0007] Step 1, fractionation of palm stearin raw material: Using palm stearin with a triglyceride palmitate content greater than 60 wt% as the raw material, adding a solvent, adjusting the temperature to dissolve it completely, and then slowly cooling to precipitate triglyceride palmitate crystals, and filtering and drying to obtain palm stearin with a triglyceride palmitate content greater than 85 wt% after fractionation;

[0008] Step 2: Preparation of 2-monopalmitin: After the fractionated palm stearin is mixed with ethanol, it is subjected to alcoholysis under the catalysis of regioselective lipase to obtain a mixture of 2-monopalmitin and fatty acid ethyl ester, etc. The fatty acid ethyl ester is removed by separation and purification to obtain 2-monopalmitin with a purity greater than 85 wt%.

[0009] Step 3: Preparation of breast milk substitute fat with controllable OPL / OPO / LPL ratio: In a solvent or solvent-free system, 2-monopalmitin is heated and mixed with a certain proportion of oleic acid / linoleic acid mixture, and then lipase is added. At 30-50 °C, an enzymatic catalytic esterification reaction is carried out to obtain a crude product of breast milk substitute fat rich in OPL / OPO / LPL:

[0010] Step 4: Separation, purification of the product and recycling of fatty acids: The above-mentioned crude product is subjected to molecular distillation at a certain vacuum degree and temperature with a certain scraping film speed and flow rate to separate the free fatty acids in the reaction system and obtain breast milk substitute fat rich in OPL / OPO / LPL. Among them, the free fatty acids are further used to remove saturated fatty acids such as palmitic acid by solvent crystallization, and the oleic acid and linoleic acid mixture is recycled and can be used as a raw material for recycling.

[0011] According to the above scheme, in Step 1, the fractionation solvent is one or more of ethyl acetate, acetone, etc., preferably ethyl acetate. The palm stearin raw material is completely dissolved in the fractionation solvent at 50-60 °C, and slowly cooled to 20-30 °C to precipitate tripalmitin crystals. The solvent is removed by filtration to obtain fractionated palm stearin with a content greater than 85 wt%.

[0012] According to the above scheme, in Step 1, the content of tripalmitin in the palm stearin raw material used is greater than 60%, the content of palmitic acid is greater than 70 wt%, and the content of palmitic acid at the sn-2 position is greater than 70 wt%; the content of tripalmitin in the fractionated palm stearin is 80 wt% - 90 wt%, the content of palmitic acid is 85 wt% - 95 wt%, and the content of palmitic acid at the sn-2 position is 85 wt% - 95 wt%.

[0013] According to the above scheme, in Step 2, the regioselective lipase is Sn-1,3 lipase, including: lipase from Rhizopus oryzae, lipase from Aspergillus niger, lipase from Burkholderia cepacia, lipase from porcine pancreas, lipase from Thermomyces lanuginosus, etc.; in Step 3, the lipase includes: lipase from Candida lipolytica, lipase from Candida cylindracea, lipase from Candida rugosa, lipase from Penicillium camemberti, and hydrophobic mesoporous carrier immobilized enzyme (such as Candida sp.99-125@OMS-C8, OMS-C8 represents octyl-modified mesoporous carrier), etc.

[0014] According to the above scheme, in step two, the substrates used in the alcoholysis reaction are fractionated palm stearin and ethanol (wherein, the molar ratio of palm stearin to ethanol is 1:8 - 1:14), and the solvent is one or more of acetone, n-hexane, cyclohexane, n-octane, etc. (the solvent dosage is: 5 - 10 mL of solvent is added per 1 g of tripalmitin); the addition amount of the regioselective lipase is 5 wt% - 20 wt% of the mass of palm stearin; the reaction temperature is 20 - 40 °C, and the reaction is stirred for 1.5 - 4 h.

[0015] According to the above scheme, in step two, the reaction product of the alcoholysis is filtered to remove the lipase, and the solvent is removed by rotary evaporation under reduced pressure to obtain a mixture of 2-monopalmitin and fatty acid ethyl ester, etc. Then, separation and purification are carried out to obtain 2-monopalmitin with a purity greater than 85 wt%. The separation and purification methods include but are not limited to molecular distillation and solvent low-temperature crystallization. When using the molecular distillation method, the vacuum degree is 0.1 - 2 Pa, the distillation temperature is 60 - 90 °C, the feeding rate is 0.5 - 1 L / h, and the rotation speed of the scraping film device is 200 - 400 r / min. When using the solvent low-temperature crystallization method, at room temperature, a certain solvent is used to extract ethyl palmitate, and at the same time, 2-monopalmitin crystallizes out, and then after filtration and drying, 2-monopalmitin with a content greater than 85 wt% is obtained. Among them, the solvent includes but is not limited to one or more of acetone, n-hexane, cyclohexane, n-octane, etc.

[0016] According to the above scheme, in step three, the molar ratio of oleic acid to linoleic acid is (6 - 1):1. Further, the mixture of a certain proportion of oleic acid / linoleic acid is prepared by blending the hydrolysis product of vegetable oil rich in oleic acid and the hydrolysis product of vegetable oil rich in linoleic acid. Among them, oleic acid comes from the hydrolysis products of high-oleic rapeseed oil, peanut oil, sunflower oil, olive oil, etc. The main fatty acid composition: the oleic acid content is 50 wt% - 85 wt%, the linoleic acid content is 5 wt% - 20 wt%, and the palmitic acid content is 2 wt% - 15 wt%; linoleic acid comes from the hydrolysis products of sunflower oil, soybean oil, corn oil, cottonseed oil, etc. The main fatty acid composition: the linoleic acid content is 50 wt% - 70 wt%, the oleic acid content is 10 wt% - 35 wt%, and the palmitic acid content is 2 wt% - 20 wt%.

[0017] According to the above scheme, in step three, the molar ratio of the 2-monopalmitin to the oleic acid / linoleic acid mixture is 1:4 - 1:9, the addition amount of the lipase is 3% - 8% of the total mass of the reactants (here the total mass refers to the total mass of the 2-monopalmitin and the oleic acid / linoleic acid mixture), the reaction temperature is 30 - 50 °C, and the reaction is stirred for 20 - 60 min.

[0018] According to the above scheme, in step three, when a solvent system is adopted, a certain amount of solvent needs to be added, which is selected from one or more of n-hexane, cyclohexane, n-octane, isooctane, etc. When there is no solvent, an oleic acid / linoleic acid mixture is used as the solvent.

[0019] According to the above scheme, in step four, excess oleic acid, linoleic acid and a small amount of palmitic acid are separated from the breast milk fat product by molecular distillation; the vacuum degree of the molecular distillation is less than 4 Pa, the temperature is 180 - 220 °C, the scraping film speed is 200 - 400 rpm, and the flow rate is 0.5 - 1 L / h.

[0020] According to the above scheme, in step four, the mixed fatty acids distilled by molecular distillation can be used as reaction raw materials for recycling after treatment: saturated fatty acids (stearic acid and palmitic acid) are removed by crystallization using a solvent (n-hexane, petroleum ether, etc.), and oleic acid and linoleic acid are obtained after the solvent after crystallization separation is distilled.

[0021] The route for preparing the breast milk substitute fat by the above method is shown in Formula 1. The total content of OPL, OPO and LPL in the prepared breast milk substitute fat can reach 75 wt% - 91 wt%, the palmitic acid at the 2-position accounts for 83 wt% - 92 wt% of all palmitic acid contents, OPL / OPO fluctuates within the range of 0.2 - 2.9, and LPL / OPO fluctuates within the range of 0 - 1.0.

[0022]

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] 1. The present invention prepares the OPL / OPO / LPL composite oil and fat through the esterification reaction of monoglyceryl 2-palmitate, which has the advantages of mild reaction conditions, strong selectivity, high reaction activity, fast reaction speed, high product purity, good quality, etc. The total content of the target products OPL, OPO and LPL can reach 75 wt% - 91 wt%, solving the problems such as acyl migration caused by high temperature, many by-products, and low yield in the traditional method, resulting in low purity of the breast milk substitute fat.

[0025] 2. The present invention controls the addition ratio of oleic acid / linoleic acid and reaction parameters such as temperature and time, regulates the esterification activity difference between the two fatty acids of oleic acid and linoleic acid on the glycerol backbone, and can accurately control the ratio of OPL, OPO and LPL in the product, providing a reliable method for the development of breast milk fat substitutes with different compositions.

[0026] 3. The present invention can realize the recycling of fatty acids through product separation and purification and fatty acid recovery, which is green and efficient without generating waste; moreover, compared with the column chromatography and solvent fractionation reported in previous literature for the separation and purification method of monoglyceride in the preparation process, it is more simple, green and uses less solvent. Description of the Drawings

[0027] Figure 1 It is the liquid chromatogram of the triglyceride composition of palm stearin before and after advancing step one in Example 1;

[0028] Figure 2 It is the gas chromatogram of the glycolysis product in step two and 2-monopalmitin after crystallization and purification in Example 1;

[0029] Figure 3 It is the high performance liquid chromatogram of the products of Examples 1 - 6; among which, for 6 different oleic acid / linoleic acid mixtures, by molar ratio, oleic acid:linoleic acid = 6:1, 4:1, 3:1, 2:1, 1.5:1, 1:1;

[0030] Figure 4 It is the relationship diagram of the OPL / OPO / LPL content, ratio and the oleic acid / linoleic acid ratio in the substrate in the products of Examples 1 - 6; among which, for 6 different oleic acid / linoleic acid mixtures, by molar ratio, oleic acid:linoleic acid = 6:1, 4:1, 3:1, 2:1, 1.5:1, 1:1. Detailed implementation manners

[0031] To better understand the present invention, the content of the present invention will be further clarified below in conjunction with specific embodiments, but the present invention is not limited only to the following embodiments.

[0032] In the following examples, the preparation method of the mesoporous support-immobilized enzyme (Lipase@OMS-C8): At room temperature, 5.0 g of F127 and 12.5 g of KCl were dissolved in 500 mL of HCl (1.0 M), 6.0 g of 1,3,5-trimethylbenzene was added, and the mixture was mechanically stirred at 750 rpm for 24 h. 20.8 g of tetraethyl orthosilicate was added and stirred for 15 min, then left standing for 24 h. Then, the obtained mixture was poured into a reaction kettle and heated at 200 °C for 24 h, taken out, filtered, and dried to obtain a powdered solid. The powdered solid was then placed in a muffle furnace and calcined at 550 °C for 6 h to obtain mesoporous silica (particle size 8 μm, mesoporous pore diameter 22 nm). 1.5 mL of a hydrophobic modifier (octyltrimethoxysilane, C8), 150 μL of triethylamine, 10.0 g of mesoporous silica, and 100 mL of anhydrous toluene were added to the reaction kettle for hydrothermal reaction. The hydrothermal reaction conditions were 130 °C for 20 h. After the reaction was completed, the mixture was taken out, filtered, and dried to obtain hydrophobic mesoporous silica. The free lipase Candida sp. 99-125 was dissolved in a phosphate buffer (50 mM, pH = 9) to prepare an enzyme solution with a concentration of 60 mg / mL. After ice-bathing for 30 min, it was centrifuged at 10,000 rpm for 10 min, and the supernatant was taken. The hydrophobic mesoporous silica and the supernatant were added to a ground conical flask at a ratio of 10 mg / mL, reacted on a shaker (200 rpm, 30 °C) for 30 min, then centrifuged, and the precipitate was freeze-dried to obtain the immobilized enzyme Candida sp. 99-125@OMS-C8.

[0033] In the following examples, the preparation method of a mixture of oleic acid / linoleic acid in a certain proportion, taking the fatty acids obtained by hydrolyzing high-linoleic sunflower oil and common sunflower oil as an example, 1000 mL of 6 different oleic acid / linoleic acid mixtures (in terms of molar ratio, oleic acid: linoleic acid = 6:1, 4:1, 3:1, 2:1, 1.5:1, 1:1) were prepared as shown in Table 1. First, the hydrolyzed fatty acids were methyl esterified and their fatty acid compositions were detected by gas chromatography. Among the fatty acids obtained by hydrolyzing high-linoleic sunflower oil, the content of oleic acid was 86.5 wt%, the content of linoleic acid was 8.0 wt%, the content of palmitic acid was 3.6 wt%, and the content of other fatty acids such as stearic acid was 1.9 wt%. Among the fatty acids obtained by hydrolyzing common sunflower oil, the content of linoleic acid was 65.0 wt%, the content of oleic acid was 25.0 wt%, the content of palmitic acid was 6.4 wt%, and the content of other fatty acids such as stearic acid was 3.6 wt%. Then, the above fatty acids were prepared into oleic acid / linoleic acid mixtures in the corresponding proportions as acyl donors, with the serial numbers being 1-6 respectively.

[0034] Table 1 Preparation of acyl donors

[0035]

[0036] In the following examples, the palm stearin raw material is 58°C palm stearin, in which the tristearin content is greater than 60%, the palmitic acid content is greater than 70 wt%, and the palmitic acid content at the sn-2 position is greater than 70 wt%.

[0037] Example 1

[0038] A method for preparing a breast milk substitute fat with a controllable OPL / OPO / LPL ratio and its product, specifically including the following steps:

[0039] (1) Preparation of tristearin: Place 200 g of palm stearin raw material in a reactor containing 1 L of ethyl acetate, adjust the temperature to 50°C, completely dissolve the palm stearin, then cool it to 20°C to precipitate tristearin crystals. After filtration and drying, 178.4 g of palm stearin with a tristearin content of 87.1% is obtained, the palmitic acid content is 93.2 wt%, the palmitic acid content at the sn-2 position is 91.9 wt%, and the yield is 89.2%.

[0040] (2) Preparation of 2-monopalmitin: Add 0.12 mol of fractionated palm stearin (100 g) and ethanol in a molar ratio of 1:8, that is, add 0.99 mol of ethanol (57.9 mL), mix well in 500 mL of n-hexane, add 20.0 g of Lipozyme TL IM (20% of the mass of palm stearin), and the reaction is carried out in a three-necked flask equipped with a condenser reflux device. The reaction temperature is 35°C, and the stirring reaction is carried out for 1.5 h. After the reaction is completed, filter to remove the lipase, and remove the solvent by rotary evaporation under reduced pressure. Then, a mixture of 2-monopalmitin and ethyl fatty acid is obtained. Add 500 mL of n-hexane to precipitate 2-monopalmitin crystals at room temperature, filter and dry to obtain 32.4 g of 2-monopalmitin product with a purity of 91.5 wt% and a yield of 79.2%.

[0041] (3) Preparation of breast milk fat with a controllable OPL / OPO / LPL ratio: Mix 2-monopalmitin and acyl donor 6 (see Table 1) in a molar ratio of 1:4, heat and stir to fully dissolve the substrate, add 8% of the total substrate mass of mesoporous immobilized enzyme Candidasp.99-125@OMS-C8, and react at a temperature of 45°C with stirring for 20 min to obtain a crude product rich in OPL / OPO / LPL;

[0042] (4) Separation, purification of the product and recovery of fatty acids: The crude product rich in OPL / OPO / LPL is subjected to molecular distillation to remove free fatty acids under the conditions of a vacuum of 3.5 Pa, a temperature of 220 °C, a scraping film speed of 400 rpm, and a flow rate of 1 L / h, to obtain a breast milk substitute fat product rich in OPL / OPO / LPL; among them, the free fatty acids are added with five times the volume of n-hexane for crystallization to remove saturated acids (stearic acid and palmitic acid), and the solvent after crystallization separation is distilled to obtain oleic acid and linoleic acid.

[0043] It was determined that the total content of OPL, OPO and LPL in the obtained product was 75.7 wt%, among which the content of OPL was 44.5 wt%, the content of OPO was 15.5 wt%, the content of LPL was 15.7 wt%, and OPL:OPO:LPL = 2.87:1:1.01.

[0044] Example 2

[0045] A preparation method and product of a breast milk substitute fat with a controllable OPL / OPO / LPL ratio, specifically including the following steps:

[0046] (1) Preparation of tripalmitin: 200 g of palm stearin raw material was placed in a reactor containing 1 L of ethyl acetate, the temperature was adjusted to 60 °C, the palm stearin was completely dissolved, and then the temperature was lowered to 30 °C to precipitate tripalmitin crystals. After filtration and drying, 176.2 g of palm stearin with a tripalmitin content of 86% was obtained, the palmitic acid content was 91.9 wt%, the palmitic acid content at the sn-2 position was 91.5 wt%, and the yield was 88.1%.

[0047] (2) Preparation of 2-monopalmitin: 0.12 mol of fractionated palm stearin (100 g) was added to 1.23 mol of ethanol (72.3 mL) at a molar ratio of palm stearin to ethanol of 1:10 and mixed evenly in 500 mL of n-hexane. 8.0 g of Lipozyme RM IM (8% of the mass of palm stearin) was added, and the reaction was carried out in a three-necked flask equipped with a condensing reflux device at a reaction temperature of 40 °C and stirred for 3 h. After the reaction, the lipase was removed by filtration, and after removing the solvent by rotary evaporation under reduced pressure, a mixture of 2-monopalmitin and ethyl fatty acid was obtained. The ethyl fatty acid was removed by molecular distillation, and the process conditions were a vacuum of 2 Pa, a distillation temperature of 90 °C, a feed rate of 1 L / h, and a scraping film speed of 200 r / min. 32.6 g of 2-monopalmitin product with a purity of 92.2 wt% and a yield of 79.7% was obtained.

[0048] (3) Preparation of breast milk fat with controllable OPL / OPO / LPL ratio: Mix monopalmitin and acyl donor 5 (see Table 1) at a molar ratio of 1:5, heat and stir to fully dissolve the substrate, add 7% of Lipozyme RM IM based on the total substrate mass, react at a temperature of 40 °C with stirring for 30 min to obtain a crude product rich in OPL / OPO / LPL;

[0049] (4) Separation, purification of the product and recovery of fatty acids: Under the conditions of a vacuum of 1 Pa, a temperature of 190 °C, a wiping film speed of 200 rpm, and a flow rate of 0.5 L / h, the crude product rich in OPL / OPO / LPL is subjected to molecular distillation to remove free fatty acids, obtaining a breast milk fat product rich in OPL / OPO / LPL. The recovery of fatty acids is the same as in step (4) of Example 1.

[0050] It was determined that the total content of OPL, OPO, and LPL in the obtained product was 77.8 wt%, among which the content of OPL was 42.6 wt%, the content of OPO was 28.2 wt%, the content of LPL was 7.0 wt%, and OPL:OPO:LPL = 1.51:1:0.25.

[0051] Example 3

[0052] A preparation method and product of a breast milk substitute fat with controllable OPL / OPO / LPL ratio, specifically including the following steps:

[0053] (1) Preparation of tripalmitin: The same as step (1) in Example 2.

[0054] (2) Preparation of monopalmitin: Add 0.12 mol of fractionated palm stearin (100 g) to 1.49 mol of ethanol (86.8 mL) at a molar ratio of palm stearin to ethanol of 1:12, mix well in 500 mL of n-hexane, add 6.0 g of Candida lipolytica lipase (CLL, accounting for 6% of the mass of palm stearin), and the reaction is carried out in a three-necked flask equipped with a condensing reflux device at a reaction temperature of 45 °C with stirring for 4 h. After the reaction is completed, filter to remove the lipase, and after removing the solvent by rotary evaporation under reduced pressure, a mixture of monopalmitin and ethyl fatty acid is obtained. Use molecular distillation to remove ethyl fatty acid, and the process conditions are a vacuum of 1 Pa, a distillation temperature of 60 °C, a feeding rate of 0.5 L / h, and a wiping film speed of 400 r / min to obtain 33.3 g of monopalmitin product with a purity of 91.3 wt% and a yield of 81.5%.

[0055] (3) Preparation of breast milk fat with controllable OPL / OPO / LPL ratio: Mix monopalmitin and acyl donor 4 (see Table 1) at a molar ratio of 1:6, heat and stir to fully dissolve the substrate, add Candida sp. 99-125@OMS-C8 accounting for 6% of the total substrate mass, react at 50 °C with stirring for 30 min to obtain a crude product rich in OPL / OPO / LPL;

[0056] (4) Separation, purification of the product and recovery of fatty acids: The same as step (4) in Example 1.

[0057] It was determined that the total content of OPL, OPO and LPL in the obtained product was 79.9 wt%, among which the content of OPL was 38.5 wt%, the content of OPO was 37.4 wt%, the content of LPL was 4.0 wt%, and OPL:OPO:LPL = 1.03:1:0.11.

[0058] Example 4

[0059] A preparation method and product of breast milk substitute fat with controllable OPL / OPO / LPL ratio, specifically including the following steps:

[0060] (1) Preparation of tripalmitin: The same as step (1) in Example 2.

[0061] (2) Preparation of monopalmitin: Add 0.12 mol of fractionated palm stearin (100 g) and 1.73 mol of ethanol (101.3 mL) at a molar ratio of palm stearin to ethanol of 1:14, mix well in 500 mL of n-hexane, add 10.0 g of Candida sp. 99-125@OMS-C8 (accounting for 10% of the mass of palm stearin), react in a three-necked flask equipped with a condensing reflux device, react at 30 °C with stirring for 4 h. After the reaction, remove ethyl fatty acid by solvent crystallization method, and the conditions are the same as step (2) in Example 1.

[0062] (3) Preparation of breast milk fat with controllable OPL / OPO / LPL ratio: Mix monopalmitin and acyl donor 3 (see Table 1) at a molar ratio of 1:7, heat and stir to fully dissolve the substrate, add Candida lipolytica enzyme (CLL) accounting for 5% of the total substrate mass, react at 35 °C with stirring for 40 min to obtain a crude product rich in OPL / OPO / LPL;

[0063] (4) Separation, purification of the product and recovery of fatty acids: The same as step (4) in Example 1.

[0064] It was determined that the total content of OPL, OPO, and LPL in the obtained product was 80.6 wt%, among which the content of OPL was 30.3 wt%, the content of OPO was 48.7 wt%, the content of LPL was 1.6 wt%, and OPL:OPO:LPL = 0.62:1:0.03.

[0065] Example 5

[0066] A preparation method and product of a breast milk substitute fat with a controllable OPL / OPO / LPL ratio specifically include the following steps:

[0067] (1) Preparation of tripalmitin: The same as step (1) in Example 2.

[0068] (2) Preparation of 2-monopalmitin: 0.12 mol of fractionated palm stearin (100 g) was added to 1.23 mol of ethanol (72.3 mL) at a molar ratio of palm stearin to ethanol of 1:10 and mixed well in 500 mL of n-hexane. 12.0 g of Lipozyme TL IM (12% of the mass of palm stearin) was added, and the reaction was carried out in a three-necked flask equipped with a condenser reflux device at a reaction temperature of 35 °C with stirring for 2.5 h. The purification method was the same as step (2) in Example 1.

[0069] (3) Preparation of breast milk fat with a controllable OPL / OPO / LPL ratio: 2-monopalmitin and acyl donor 2 (see Table 1) were mixed at a molar ratio of 1:8, heated and stirred to fully dissolve the substrate, 4% of Lipozyme TL IM based on the total substrate mass was added, the reaction temperature was 30 °C, and the reaction was stirred for 50 min to obtain a crude product rich in OPL / OPO / LPL;

[0070] (4) Separation, purification of the product and recovery of fatty acids: The same as step (4) in Example 1.

[0071] It was determined that the total content of OPL, OPO, and LPL in the obtained product was 82.1 wt%, among which the content of OPL was 24.3 wt%, the content of OPO was 57.1 wt%, the content of LPL was 0.7 wt%, and OPL:OPO:LPL = 0.43:1:0.01.

[0072] Example 6

[0073] A preparation method and product of a breast milk substitute fat with a controllable OPL / OPO / LPL ratio specifically include the following steps:

[0074] (1) Preparation of tripalmitin: The same as step (1) in Example 2.

[0075] (2) Preparation of monoglyceryl 2-palmitate: 0.12 mol of fractionated palm stearin (100 g) was added to 1.23 mol of ethanol (72.3 mL) at a molar ratio of palm stearin to ethanol of 1:10 and mixed well in 500 mL of n-hexane. 15.0 g of Lipozyme TL IM (15% of the mass of palm stearin) was added. The reaction was carried out in a three-necked flask equipped with a condenser reflux device at a reaction temperature of 35 °C and stirred for 2 h. The purification method was the same as step (2) in Example 1.

[0076] (3) Preparation of breast milk fat with controllable OPL / OPO / LPL ratio: Monoglyceryl 2-palmitate and acyl donor 1 (see Table 1) were mixed at a molar ratio of 1:8, heated and stirred to dissolve thoroughly in an equal volume of n-hexane. 3% of Candidasp. 99-125@OMS-C8 based on the total substrate mass was added. The reaction temperature was 40 °C and the reaction was stirred for 60 min to obtain a crude product rich in OPL / OPO / LPL.

[0077] (4) Separation and purification of the product and recovery of fatty acids: The same as step (4) in Example 1.

[0078] It was determined that the total content of OPL, OPO and LPL in the obtained product was 91.0 wt%, among which the content of OPL was 18.1 wt%, the content of OPO was 72.3 wt%, the content of LPL was 0.1 wt%, and OPL:OPO:LPL = 0.25:1:0.

[0079] Table 2 shows the mass content of OPL / OPO / LPL, the ratio of OPL / OPO / LPL and the fatty acid composition at the sn-2 position in the reaction products prepared in Examples 1-6.

[0080] Table 2

[0081]

[0082] 1 Taking the content of OPO as 1, calculate OPL / OPO and LPL / OPO to obtain the relative content ratio of OPL / OPO / LPL.

[0083] 2 The mass fraction (wt%) of palmitic acid at the sn-2 position in all palmitic acids was carried out according to the provisions of GB 30604-2015.

[0084] It can be seen that the present invention provides a preparation method of triglyceride rich in 1-oleoyl-2-palmitoyl-3-linoleoyl glycerol (OPL), 1,3-dioleoyl-2-palmitoyl glycerol (OPO) and 1,3-dilinoleoyl-2-palmitoyl glycerol (LPL). The technical route of this method is proposed for the first time. The total content of OPL, OPO and LPL in the obtained product can reach 75wt% - 91wt%, and the ratio of OPL / OPO / LPL can be adjusted, which can be widely applied to infant formula suitable for different regions. When the ratio of oleic acid / linoleic acid in the acyl donor is reduced from 6:1 to 1:1, the content of OPL in the product increases from 18.1wt% to 44.5wt%, the content of OPO decreases from 72.3wt% to 15.5wt%, and the content of LPL increases from 0.1wt% to 15.7wt%. When the ratio of oleic acid / linoleic acid is 6:1, 4:1, 3:1, 2:1, the range of OPL / OPO is (0.2 - 1):1, and the content of LPL is relatively low, which conforms to the characteristics of breast milk fat structure in regions such as Europe. When the ratio of oleic acid / linoleic acid is 1.5:1 (Example 2), OPL:OPO:LPL = 1.51:1:0.25, which conforms to the characteristics of Chinese breast milk fat. Therefore, Example 2 is preferably used as the raw material for Chinese infant milk powder.

[0085] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several improvements and transformations can be made, and these all belong to the protection scope of the present invention.

Claims

1. A preparation method of a breast milk substitute fat with controllable OPL / OPO / LPL ratio, characterized in that, It mainly includes the following steps: Step 1: Fractionation of palm stearin raw material: Using palm stearin with a tristearin content greater than 60 wt% as the raw material, adding a solvent, adjusting the temperature to completely dissolve it, then cooling down to precipitate tristearin crystals, and filtering and drying to obtain palm stearin with a tristearin content greater than 85 wt% after fractionation; Step 2: Preparation of 2-monopalmitin: After mixing the palm stearin obtained in Step 1 with ethanol, alcoholysis is carried out under the catalysis of a regioselective lipase, and then fatty acid ethyl ester is removed by separation and purification to obtain 2-monopalmitin with a purity greater than 85 wt%; Step 3: Preparation of breast milk substitute fat with controllable OPL / OPO / LPL ratio: In a solvent or solvent-free system, after heating and mixing the 2-monopalmitin obtained in Step 2 with a mixture of oleic acid / linoleic acid, adding a lipase, and carrying out an enzymatic catalytic esterification reaction at 30 - 50 °C to obtain a crude breast milk substitute fat rich in OPL / OPO / LPL: Among them, the molar ratio of the 2-monopalmitin to the oleic acid / linoleic acid mixture is 1:4 - 1:9, the addition amount of the lipase is 3% - 8% of the total mass of the reactants in this step, and the oleic acid / linoleic acid mixture is composed of oleic acid and linoleic acid in a molar ratio of (6 - 1):1; Step 4: Separation, purification of the product and recovery and utilization of fatty acids: Subjecting the crude product obtained in Step 3 to molecular distillation to separate the free fatty acids in the reaction system and obtain a breast milk substitute fat rich in OPL / OPO / LPL; The total content of OPL, OPO and LPL in the breast milk substitute fat is 75 wt% - 91 wt%, the content of palmitic acid at the sn-2 position accounts for 83 wt% - 92% of all palmitic acid contents, the mass ratio of OPL / OPO is in the range of 0.2 - 2.9, and the mass ratio of LPL / OPO is in the range of 0 - 1.

0.

2. The preparation method of a breast milk substitute fat with controllable OPL / OPO / LPL ratio according to claim 1, characterized in that, In Step 1, the fractionation solvent is one or two of ethyl acetate and acetone; the palm stearin raw material is completely dissolved in the fractionation solvent at 50 - 60 °C, cooled to 20 - 30 °C to precipitate tristearin crystals, and the solvent is removed by suction filtration to obtain palm stearin with a content greater than 85 wt% after fractionation.

3. The preparation method of a breast milk substitute fat with controllable OPL / OPO / LPL ratio according to claim 1, characterized in that, In Step 1, the tristearin content in the used palm stearin raw material is greater than 60%, the palmitic acid content is greater than 70 wt%, and the palmitic acid content at the sn-2 position is greater than 70 wt%; the tristearin content in the palm stearin after fractionation is 85 wt% - 90 wt%, the palmitic acid content is 85 wt% - 95 wt%, and the palmitic acid content at the sn-2 position is 85 wt% - 95 wt%.

4. The preparation method of a breast milk substitute fat with controllable OPL / OPO / LPL ratio according to claim 1, characterized in that In Step 2, the regioselective lipase is a Sn-1,3 lipase, selected from lipases derived from Rhizopus oryzae, Aspergillus niger, Burkholderia cepacia, porcine pancreas, Thermomyces lanuginosus; in Step 3, the lipase is selected from lipases derived from Candida lipolytica, Candida cylindracea, Candida rugosa, Penicillium camemberti, and hydrophobic mesoporous support immobilized enzymes.

5. The preparation method of a breast milk substitute fat with controllable OPL / OPO / LPL ratio according to claim 1, characterized in that, In step two, the molar ratio of the fractionated palm stearin to ethanol is 1:8 to 1:14; a solvent may also be included, and the solvent is one or more of acetone, n-hexane, cyclohexane, and n-octane. The dosage of the solvent is: 5 to 10 mL of the solvent is added per 1 g of tripalmitin; the addition amount of the regioselective lipase is 5 wt% to 20 wt% of the mass of the palm stearin; the reaction temperature is 20 to 40 °C, and the stirring reaction is carried out for 1.5 to 4 h.

6. The preparation method of a breast milk substitute fat with controllable OPL / OPO / LPL ratio according to claim 1, characterized in that, In step two, the reaction product of alcoholysis is filtered to remove the lipase, the solvent is removed by rotary evaporation under reduced pressure, and then separation and purification are carried out to obtain 2-monopalmitin with a purity greater than 85 wt%; among them, the separation and purification methods include, but are not limited to, molecular distillation and low-temperature solvent crystallization.

7. The preparation method of a breast milk substitute fat with a controllable OPL / OPO / LPL ratio according to claim 1, characterized in that The oleic acid / linoleic acid mixture is prepared by blending the hydrolysis product of a vegetable oil rich in oleic acid with the hydrolysis product of a vegetable oil rich in linoleic acid; among them, oleic acid comes from the hydrolysis products of rapeseed oil, peanut oil, sunflower oil, and olive oil; linoleic acid comes from the hydrolysis products of sunflower seed oil, soybean oil, corn oil, and cottonseed oil.

8. The preparation method of a breast milk substitute fat with controllable OPL / OPO / LPL ratio according to claim 1, characterized in that, In step three, the reaction temperature is 30 to 50 °C, and the stirring reaction is carried out for 20 to 60 min.

9. The preparation method of a breast milk substitute fat with controllable OPL / OPO / LPL ratio according to claim 1, wherein, In step four, the vacuum degree of molecular distillation is less than 4 Pa, the temperature is 180 to 220 °C, the scraping film speed is 200 to 400 rpm, and the flow rate is 0.5 to 1 L / h.

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