CN115232842A - Preparation method of breast milk substitute fat with controllable OPL/OPO/LPL ratio and product thereof - Google Patents

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

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CN115232842A
CN115232842A CN202210895481.1A CN202210895481A CN115232842A CN 115232842 A CN115232842 A CN 115232842A CN 202210895481 A CN202210895481 A CN 202210895481A CN 115232842 A CN115232842 A CN 115232842A
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郑明明
张羽飞
李雅丽
张逸
龚阳敏
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Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
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Abstract

The invention discloses a preparation method of breast milk substitute fat with controllable OPL/OPO/LPL ratio, which comprises the following steps: step one, taking palm stearin as a raw material, and extracting the palm stearin with the tripalmitin content of more than 85 wt%; step two, carrying out alcoholysis on the separated palm stearin under the catalysis of regioselective lipase to obtain 2-palmitic acid monoglyceride with the purity of more than 85 wt%; step three, in a solvent or solvent-free system, heating and uniformly mixing 2-monoglyceride palmitate and a mixture of oleic acid and linoleic acid in a certain proportion, adding lipase to perform an enzyme-catalyzed esterification reaction, and obtaining a crude product of the breast milk substitute fat rich in OPL/OPO/LPL: and step four, separating free fatty acid in the reaction system from the crude product to obtain the breast milk substitute fat rich in OPL/OPO/LPL. The total content of OPL, OPO and LPL in the breast milk substitute fat product can reach 75wt% -91 wt%, OPL/OPO is within the range of 0.2-2.9, and LPL/OPO is within the range of 0-1.0, so that regulation and control can be realized, and the problems of acyl migration, more byproducts, low yield and the like in the preparation of OPL/OPO/LPL by acidolysis reaction in the prior art are avoided.

Description

Preparation method of breast milk substitute fat with controllable OPL/OPO/LPL ratio and product thereof
Technical Field
The invention relates to a preparation method of breast milk substitute fat with controllable OPL/OPO/LPL ratio and a product thereof, belonging to the technical field of fat synthesis.
Background art:
the breast milk is the most ideal food for infants, and fat is used as the most important energy supply substance, and provides more than half of energy, fat-soluble vitamins and other lipid components necessary for growth and development for infants by the mass of about 3-5% of the breast milk. Wherein, the content of 1,3-dioleic acid-2-palmitic acid triglyceride (OPO), 1-oleic acid-2-palmitic acid-3-linoleic acid triglyceride (OPL) and 1,3-dilinoleic acid-2-palmitic acid triglyceride (LPL) reaches 40% -50% of the total amount of human milk fat. In recent years, a plurality of scholars at home and abroad research the human milk fat composition of different regional populations in the world, and find that the content and the proportion of OPL, OPO and LPL in the milk fat of different regional populations are greatly different. This may be related to the dietary habits of vegetable oils rich in oleic acid or linoleic acid consumed in various regions. The OPO content in mother milk fat is highest in European countries such as Italy, finland, spain, etc., the ratio of OPL/OPO is 0.2-0.9 (mass ratio), and the content of LPL is lower. And in the composition of human milk fat in China, the content of OPL is the highest, next, OPO and LPL are three structural lipids with the highest content, and the mass ratio of OPL/OPO/LPL is about 1.5.
On the background of a continuous decline in pure breast feeding rates, formula milk powder becomes a major adjunct to infant growth and development. Because its composition and structure are similar to breast milk fat, breast milk esters are widely used in formula milk powders. CN 109566769A reports that triglyceride palmitate, acyl donor oleic acid and linoleic acid are used as raw materials, and the grease rich in OPO and OPL is prepared by the catalytic transesterification reaction of lipase. But the method has low product content. CN 111593046A adopts the same strategy, uses magnetic carrier immobilized lipase as a catalyst, and obtains 35-50 wt% of OPL grease after long-time reaction (8-12 hours). CN 109402186A describes the preparation of OPL-rich oil mixtures by reacting tripalmitin (PPP) with oleic and linoleic acid acyl donors, separating and purifying, and then adding acyl donors again to carry out a secondary reaction. The method can obtain the OPL grease with higher content, but has the defects of complex operation, high reaction temperature (40-90 ℃) and higher energy consumption of twice molecular distillation. Currently, regarding OPO and OPL reports, the method of acidolysis of tripalmitin mainly by an enzyme method has the problems of long reaction time, high temperature, unavoidable acyl migration, low product purity and the like. Therefore, the simple and efficient preparation method with the adjustable OPL/OPO/LPL ratio is provided, and has important significance for promoting the maternal emulsification process of the infant formula milk powder.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for preparing the human milk substitute fat with controllable OPL/OPO/LPL ratio aiming at the defects in the prior art, wherein the total content of OPL, OPO and LPL in the prepared human milk substitute can reach 75wt% -91 wt%, 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), the method can be accurately regulated and controlled, and simultaneously, the problems of acyl migration, more byproducts and low yield in the preparation of OPL/OPO/LPL by acidolysis reaction in the prior art are avoided.
The technical scheme adopted by the invention for solving the problems is as follows:
a preparation method of human milk substitute fat with controllable OPL/OPO/LPL ratio mainly comprises the following four steps:
step one, fractionation of palm hard ester raw materials: taking palm stearin with the tripalmitin content of more than 60wt% as a raw material, adding a solvent, adjusting the temperature to completely dissolve the raw material, slowly cooling to separate out the tripalmitin crystals, filtering and drying to obtain the palm stearin with the tripalmitin content of more than 85 wt%;
step two, preparation of 2-palmitic acid monoglyceride: mixing the separated palm stearin and ethanol, carrying out alcoholysis under the catalysis of regioselective lipase to obtain a mixture of 2-monoglyceride palmitate and fatty acid ethyl ester, and the like, and removing the fatty acid ethyl ester through separation and purification to obtain 2-monoglyceride palmitate with the purity of more than 85 wt%;
step three, preparing the breast milk substitute fat with controllable OPL/OPO/LPL ratio: in a solvent or solvent-free system, heating and uniformly mixing 2-palmitic acid monoglyceride and a mixture of oleic acid and linoleic acid in a certain proportion, adding lipase, and carrying out enzyme catalytic esterification reaction at 30-50 ℃ to obtain a crude product of the breast milk substitute fat rich in OPL/OPO/LPL:
step four, separating and purifying the product and recycling the fatty acid: and (3) performing molecular distillation on the crude product at a certain vacuum degree and temperature at a certain film scraping speed and flow rate, and separating free fatty acid in a reaction system to obtain the breast milk substitute fat rich in OPL/OPO/LPL. Wherein, the free fatty acid is crystallized by a solvent to remove saturated fatty acid such as palmitic acid, and the like, and the mixture of oleic acid and linoleic acid is recycled and can be used as a raw material for recycling.
According to the scheme, in the step one, the separation solvent is one or more of ethyl acetate, acetone and the like, and ethyl acetate is preferred. Completely dissolving the palm hard ester raw material in a separating solvent at 50-60 ℃, slowly cooling to 20-30 ℃ to separate out tripalmitin crystals, and removing the solvent by suction filtration to obtain the palm hard ester with the content of more than 85wt% after separation.
According to the scheme, in the first step, the content of the tripalmitin in the raw material of the palm stearin is more than 60%, the content of the palmitic acid is more than 70wt%, and the content of the sn-2-position palmitic acid is more than 70wt%; the content of tripalmitin in the fractionated palm stearin is 80-90 wt%, the content of palmitic acid is 85-95 wt%, and the content of sn-2-position palmitic acid is 85-95 wt%.
According to the scheme, in the second step, the regioselective lipase is Sn-1,3 lipase, and comprises the following steps: a lipase derived from rhizopus oryzae, a lipase derived from aspergillus niger, a lipase derived from burkholderia cepacia, a lipase derived from porcine pancreas, a lipase derived from thermomyces lanuginosus, and the like; in step three, the lipase comprises: lipase derived from Candida lipolytica, lipase derived from Candida cylindracea, lipase derived from Candida rugosa, lipase derived from Penicillium camembertii, and hydrophobic mesoporous carrier immobilized enzyme (such as Candida sp.99-125@ OMS-C) 8 OMS-C8 represents an octyl-modified mesoporous support), and the like.
According to the scheme, in the second step, the substrate used in the alcoholysis reaction is the palm stearin and the ethanol after fractionation (wherein, the molar ratio of the palm stearin to the ethanol is 1:8-1: 14), and the solvent is one or more of acetone, normal hexane, cyclohexane, normal octane and the like (the dosage of the solvent is that 5-10mL of solvent is added into every 1g of tripalmitin); the addition amount of the regioselective lipase is 5-20 wt% of the mass of the palm stearin; the reaction temperature is 20-40 ℃, and the stirring reaction is carried out for 1.5-4 h.
According to the scheme, in the second step, the reaction product of alcoholysis is filtered to remove lipase, and the solvent is removed through reduced pressure rotary evaporation to obtain a mixture of 2-monoglyceride palmitate and fatty acid ethyl ester, and then the mixture is separated and purified to obtain 2-monoglyceride palmitate with the purity of more than 85 wt%. Separation and purification methods include, but are not limited to, molecular distillation and solvent low temperature crystallization. When the molecular distillation method is used, the vacuum degree is 0.1-2 Pa, the distillation temperature is 60-90 ℃, the feeding speed is 0.5-1L/h, and the rotating speed of a film scraper is 200-400 r/min. When a solvent low-temperature crystallization method is used, ethyl palmitate is extracted by using a certain solvent at room temperature, meanwhile, 2-monoglyceride palmitate is crystallized and precipitated, and then the 2-monoglyceride palmitate with the content of more than 85wt% is obtained by filtering and drying, wherein the solvent comprises one or more of acetone, n-hexane, cyclohexane, n-octane and the like.
According to the scheme, in the third step, the molar ratio of the oleic acid to the linoleic acid is (6-1) to 1. Furthermore, the oleic acid/linoleic acid mixture with a certain proportion is prepared by mixing a vegetable oil hydrolysate rich in oleic acid and a vegetable oil hydrolysate rich in linoleic acid. Wherein, the oleic acid is from hydrolysis products of high oleic rapeseed oil, peanut oil, sunflower oil, olive oil, and the like, and the main fatty acid composition is as follows: oleic acid content is 50-85 wt%, linoleic acid content is 5-20 wt%, and palmitic acid content is 2-15 wt%; the linoleic acid is from hydrolysate of sunflower seed oil, soybean oil, corn oil and cottonseed oil, and the like, and mainly comprises the following fatty acids: the content of linoleic acid is 50-70 wt%, the content of oleic acid is 10-35 wt%, and the content of palmitic acid is 2-20 wt%.
According to the scheme, in the third step, the mass ratio of the 2-palmitic acid monoglyceride 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 reactants (the total mass refers to the total mass of the 2-palmitic acid monoglyceride and the oleic acid/linoleic acid mixture), the reaction temperature is 30-50 ℃, and the stirring reaction is carried out for 20-60 min.
According to the scheme, when a solvent system is adopted in the third step, a certain amount of solvent is required to be added, and the solvent is selected from one or more of n-hexane, cyclohexane, n-octane, isooctane and the like. In the absence of a solvent, an oleic acid/linoleic acid mixture is used as the solvent.
According to the scheme, in the fourth step, excessive oleic acid, linoleic acid and a small amount of palmitic acid are separated from the breast milk fat product through molecular distillation; the vacuum degree of molecular distillation is less than 4Pa, the temperature is 180-220 ℃, the film scraping speed is 200-400 rpm, and the flow is 0.5-1L/h.
According to the scheme, in the step four, the mixed fatty acid distilled from the molecules can be recycled as a reaction raw material through treatment: removing saturated fatty acid (stearic acid and palmitic acid) by crystallization with solvent (n-hexane, petroleum ether, etc.), and distilling the separated solvent to obtain oleic acid and linoleic acid.
The route for preparing the breast milk substitute fat by the method is shown as formula 1, the total content of OPL, OPO and LPL in the prepared breast milk substitute fat can reach 75wt% -91 wt%, the content of 2-position palmitic acid accounts for 83wt% -92 wt% of all palmitic acid, OPL/OPO is within the range of 0.2-2.9, and LPL/OPO fluctuates within the range of 0-1.0.
Figure BDA0003766779440000041
Compared with the prior art, the invention has the beneficial effects that:
1. the method for preparing the OPL/OPO/LPL composite grease through the esterification reaction of the 2-palmitic acid monoglyceride has the advantages of mild reaction conditions, strong selectivity, high reaction activity, high reaction speed, high product purity, good quality and the like, and the total content of target products OPL, OPO and LPL can reach 75-91 wt%, so that the problems of low purity of breast milk substitute grease and the like caused by acyl migration, more byproducts and low yield at high temperature in the traditional method are solved.
2. According to the invention, the addition ratio of oleic acid/linoleic acid and reaction parameters such as temperature and time are controlled, the esterification activity difference of oleic acid and linoleic acid on the glycerol skeleton is regulated, the ratio of OPL, OPO and LPL in the product can be accurately controlled, and a reliable method is provided for the development of breast milk fat substitutes with different compositions.
3. According to the invention, the product is separated and purified and the fatty acid is recovered, so that the cyclic utilization of the fatty acid can be realized, the environment is protected, the efficiency is high, and no waste is generated; in addition, compared with column chromatography and solvent fractionation reported in the prior literature, the method for separating and purifying monoglyceride in the preparation process is simpler, more convenient and more green, and uses less solvent.
Drawings
FIG. 1 is a liquid chromatogram of the triglyceride composition of palm stearin after one minute advance of the procedure in example 1;
FIG. 2 is a gas chromatogram of 2-palmitic acid monoglyceride after purification by crystallization and glycolysis product of the step in example 1;
FIG. 3 is a high performance liquid chromatogram of the products of examples 1-6; wherein the molar ratio of the 6 different oleic acid/linoleic acid mixtures is oleic acid: linoleic acid =6:1, 4:1, 3:1, 2:1, 1.5, 1:1;
FIG. 4 is a graph of the OPL/OPO/LPL content, ratio in the products of examples 1-6 as a function of oleic acid/linoleic acid ratio in the substrate; wherein the molar ratio of the 6 different oleic acid/linoleic acid mixtures is oleic acid: linoleic acid =6:1, 4:1, 3:1, 2:1, 1.5, 1:1.
Detailed Description
For a better understanding of the present invention, the following examples are given to further illustrate the present invention, but the present invention is not limited to the following examples.
In the following examples, preparation of mesoporous support immobilized enzyme (Lipase @ OMS-C) 8 ): 5.0g of F127 and 12.5g of KCl were dissolved in 500mL of HCl (1.0M) at room temperature, and 6.0g of 1,3, 5-trimethylbenzene was added thereto and mechanically stirred at 750rpm for 24 hours. Adding 20.8g of tetraethyl silicate, stirring for 15min, and standing for 24h. Then, the obtained mixture was poured into a reaction kettle, heated at 200 ℃ for 24 hours, taken out, filtered and dried to obtain a powdery solid. And then putting the powder solid into a muffle furnace to be calcined for 6 hours at 550 ℃ to obtain mesoporous silica (the particle size is 8 mu m, and the mesoporous aperture is 22 nm). Adding 1.5mL of hydrophobic modifier (octyltrimethoxysilane, C8), 150 mu L of triethylamine, 10.0g of mesoporous silica and 100mL of anhydrous toluene into a reaction kettle for hydrothermal reaction under the hydrothermal reaction condition of 130 ℃ for 20h, taking out the mixture after the hydrothermal reaction is finished, filtering and drying to obtain the hydrophobic mesoporous silica. Free lipase Candida sp.99-125 was dissolved in a phosphate buffer (50mM, pH = 9) to prepare a 60mg/mL enzyme solution, which was then centrifuged at 10,000rpm for 10min after 30min in an ice bath, and the supernatant was collected. Adding hydrophobic mesoporous silica and supernatant into ground conical flask according to the ratio of 10mg/mL, reacting in shaker (200rpm, 30 deg.C) for 30min, centrifuging, precipitating, and lyophilizing to obtain immobilized enzyme Candida sp.99-125@ OMS-C 8 。
In the following examples, a method for preparing a mixture of oleic acid and linoleic acid in a certain ratio, using fatty acid obtained by hydrolyzing high linoleic acid sunflower seed oil and normal sunflower seed oil as an example, 1000mL of 6 different mixtures of oleic acid and linoleic acid (in terms of molar ratio, oleic acid: linoleic acid =6:1, 4:1, 3:1, 2:1, 1.5, 1:1) are shown in table 1. First, hydrolyzed fatty acids were methyl esterified and then the fatty acid composition was examined by gas phase analysis. The fatty acid obtained by hydrolyzing the high linoleic acid sunflower seed oil contains 86.5wt% of oleic acid, 8.0wt% of linoleic acid, 3.6wt% of palmitic acid and 1.9wt% of stearic acid and other fatty acids. The fatty acid obtained by hydrolyzing common sunflower seed oil contains 65.0wt% of linoleic acid, 25.0wt% of oleic acid, 6.4wt% of palmitic acid and 3.6wt% of stearic acid and other fatty acids. Then, the fatty acids were blended according to Table 1 to give oleic acid/linoleic acid mixtures of the corresponding proportions as acyl donors, numbered 1-6, respectively.
TABLE 1 formulation of acyl donors
Figure BDA0003766779440000051
In the following examples, the raw material of palm stearin is 58 ℃ palm stearin, in which the tripalmitin content is more than 60%, the palmitic acid content is more than 70wt%, and the sn-2 position palmitic acid content is more than 70wt%.
Example 1
A preparation method of breast milk substitute fat with controllable OPL/OPO/LPL ratio and a product thereof specifically comprise the following steps:
(1) Preparing tripalmitin: 200g of palm hard ester raw material is placed in a reactor containing 1L of ethyl acetate, the temperature is adjusted to 50 ℃, palm hard ester is completely dissolved, then the temperature is reduced to 20 ℃, tripalmitin is crystallized and separated out, and 178.4g of palm hard ester with the tripalmitin content of 87.1 percent is obtained after filtration and drying, the palmitic acid content is 93.2 percent by weight, the sn-2 position palmitic acid content is 91.9 percent by weight, and the yield is 89.2 percent.
(2) Preparation of 2-palmitic acid monoglyceride: 0.12mol of the separated palm stearin (100 g) is added with 0.99mol of ethanol (57.9 mL) according to the molar ratio of the palm stearin to the ethanol of 1:8, evenly mixed in 500mL of normal hexane, added with 20.0g of Lipozyme TL IM (accounting for 20 percent of the mass of the palm stearin), reacted in a three-neck flask provided with a condensation reflux device, the reaction temperature is 35 ℃, and stirred for reaction for 1.5h. After the reaction, the lipase was removed by filtration, and after removing the solvent by rotary evaporation under reduced pressure, a mixture of 2-monoglyceryl palmitate and ethyl fatty acid ester was obtained, 500mL of n-hexane was added to crystallize and precipitate 2-monoglyceryl palmitate at room temperature, and the mixture was filtered and dried to obtain 32.4g of a 2-monoglyceryl palmitate product with a purity of 91.5wt% and a yield of 79.2%.
(3) Preparing mother cream with controllable OPL/OPO/LPL ratio: 2-monoglyceride palmitate and acyl donor 6 (shown in Table 1) are mixed according to the molar ratio of 1:4, heated and stirred to fully dissolve the substrate, mesoporous immobilized enzyme Candida sp.99-125@ OMS-C with the total mass of the substrate being 8 percent is added 8 The reaction temperature is 45 ℃, and the mixture is stirred to react for 20min to obtain a crude product rich in OPL/OPO/LPL;
(4) Separation and purification of the product and recovery of fatty acids: removing free fatty acid from the crude product rich in OPL/OPO/LPL by molecular distillation under the conditions of vacuum degree of 3.5Pa, temperature of 220 ℃, film scraping speed of 400rpm and flow rate of 1L/h to obtain a breast milk substitute fat product rich in OPL/OPO/LPL; wherein, the free fatty acid is added with n-hexane with five times volume for crystallization to remove saturated acid (stearic acid and palmitic acid), and the solvent after crystallization separation is distilled to obtain oleic acid and linoleic acid.
The total content of OPL, OPO and LPL in the resulting product was determined to be 75.7wt%, with OPL content of 44.5wt%, OPO content of 15.5wt%, LPL content of 15.7wt%, OPL: LPL = 2.87.
Example 2
A preparation method of human milk substitute fat with controllable OPL/OPO/LPL ratio and a product thereof specifically comprise the following steps:
(1) Preparing tripalmitin: 200g of palm stearate raw material is placed in a reactor containing 1L of ethyl acetate, the temperature is adjusted to 60 ℃, palm stearate is completely dissolved, then the temperature is reduced to 30 ℃, tripalmitin is crystallized and separated out, and filtration and drying are carried out, thus obtaining 176.2g of palm stearate with tripalmitin content of 86%, palmitic acid content of 91.9wt%, sn-2 position palmitic acid content of 91.5wt% and yield of 88.1%.
(2) Preparation of 2-palmitic acid monoglyceride: 0.12mol of the palm stearin (100 g) after extraction is added with 1.23mol of ethanol (72.3 mL) according to the molar ratio of the palm stearin to the ethanol being 1. After the reaction is finished, filtering to remove lipase, performing reduced pressure rotary evaporation to remove the solvent to obtain a mixture of 2-monoglyceride palmitate and fatty acid ethyl ester, and removing the fatty acid ethyl ester by using a molecular distillation method under the process conditions of vacuum degree of 2Pa, distillation temperature of 90 ℃, feeding speed of 1L/h and rotating speed of a film scraper of 200r/min to obtain 32.6g of 2-monoglyceride palmitate product with the purity of 92.2wt% and the yield of 79.7%.
(3) Preparing mother cream with controllable OPL/OPO/LPL ratio: mixing 2-monoglyceride palmitate with acyl donor 5 (shown in Table 1) according to a molar ratio of 1:5, heating, stirring and fully dissolving a substrate, adding Lipozyme RM IM accounting for 7% of the total mass of the substrate, reacting at 40 ℃, and stirring for 30min to obtain a crude product rich in OPL/OPO/LPL;
(4) Separation and purification of products and recovery of fatty acids: and removing free fatty acid from the crude product rich in OPL/OPO/LPL by molecular distillation under the conditions of vacuum degree of 1Pa, temperature of 190 ℃, film scraping speed of 200rpm and flow rate of 0.5L/h to obtain the breast milk fat product rich in OPL/OPO/LPL. The fatty acid was recovered in the same manner as in step (4) of example 1.
The total content of OPL, OPO and LPL in the resulting product was determined to be 77.8wt%, with an OPL content of 42.6wt%, an OPO content of 28.2wt%, an LPL content of 7.0wt%, an OPL: OPO: LPL = 1.51.
Example 3
A preparation method of human milk substitute fat with controllable OPL/OPO/LPL ratio and a product thereof specifically comprise the following steps:
(1) Preparing tripalmitin: the same procedure as in (1) of example 2.
(2) Preparation of 2-palmitic acid monoglyceride: 0.12mol of fractionated palm stearin (100 g) was prepared as follows: 1.49mol of ethanol (86.8 mL) is added into 500mL of n-hexane according to the molar ratio of 1 to 12, 6.0g of Candida lipolytica enzyme (CLL, accounting for 6 percent of the mass of the palm stearin) is added, the reaction is carried out in a three-neck flask provided with a condensation reflux device, the reaction temperature is 45 ℃, and the reaction is stirred for 4 hours. After the reaction is finished, filtering to remove lipase, performing reduced pressure rotary evaporation to remove the solvent to obtain a mixture of 2-monoglyceride palmitate and fatty acid ethyl ester, and removing the fatty acid ethyl ester by using a molecular distillation method under the process conditions of vacuum degree of 1Pa, distillation temperature of 60 ℃, feeding speed of 0.5L/h and rotating speed of a film scraper of 400r/min to obtain 33.3g of 2-monoglyceride palmitate product with purity of 91.3wt% and yield of 81.5%.
(3) Preparing the mother milk fat with controllable OPL/OPO/LPL ratio: mixing 2-monoglyceride palmitate and acyl donor 4 (shown in Table 1) at a molar ratio of 1:6, heating, stirring to dissolve substrate, adding Candida sp.99-125@ OMS-C with total substrate mass of 6% 8 Stirring and reacting for 30min at the reaction temperature of 50 ℃ to obtain a crude product rich in OPL/OPO/LPL;
(4) Separation and purification of the product and recovery of fatty acids: the same procedure as in step (4) of example 1.
The total content of OPL, OPO and LPL in the obtained product was determined to be 79.9wt%, wherein the OPL content was 38.5wt%, the OPO content was 37.4wt%, the LPL content was 4.0wt%, the OPL: OPO: LPL = 1.03.
Example 4
A preparation method of human milk substitute fat with controllable OPL/OPO/LPL ratio and a product thereof specifically comprise the following steps:
(1) Preparing tripalmitin: the same procedure as in (1) of example 2.
(2) Preparation of 2-monoglyceride palmitate: 0.12mol of fractionated palm stearin (100 g) was prepared as follows: ethanol molar ratio of 1 8 (accounting for 10 percent of the mass of the palm stearin), the reaction is carried out in a three-neck flask provided with a condensation reflux device, the reaction temperature is 30 ℃, and the reaction is stirred for 4 hours. After the reaction was completed, the fatty acid ethyl ester was removed by solvent crystallization under the same conditions as in the step (2) of example 1.
(3) Preparing the mother milk fat with controllable OPL/OPO/LPL ratio: mixing 2-palmitic acid monoglyceride and acyl donor 3 (shown in Table 1) at a molar ratio of 1:7, heating, stirring to dissolve the substrate completely, adding Candida lipolytica (CLL) with a mass of 5% of the total substrate, reacting at 35 deg.C, and stirring for 40min to obtain crude product rich in OPL/OPO/LPL;
(4) Separation and purification of products and recovery of fatty acids: the same procedure as in step (4) of example 1.
The total content of OPL, OPO and LPL in the obtained product was determined to be 80.6wt%, wherein the OPL content was 30.3wt%, the OPO content was 48.7wt%, the LPL content was 1.6wt%, the OPL: OPO: LPL = 0.62.
Example 5
A preparation method of human milk substitute fat with controllable OPL/OPO/LPL ratio and a product thereof specifically comprise the following steps:
(1) Preparing tripalmitin: the same procedure as in (1) of example 2.
(2) Preparation of 2-palmitic acid monoglyceride: 0.12mol of fractionated palm stearin (100 g) was prepared as follows: adding 1.23mol of ethanol (72.3 mL) into 500mL of normal hexane according to the molar ratio of ethanol being 1. The purification method was the same as in step (2) of example 1.
(3) Preparing the mother milk fat with controllable OPL/OPO/LPL ratio: mixing 2-palmitic acid monoglyceride and acyl donor 2 (shown in Table 1) at a molar ratio of 1:8, heating and stirring to dissolve the substrate sufficiently, adding lipozyme TL IM accounting for 4% of the total substrate mass, reacting at 30 deg.C, and stirring for 50min to obtain crude product rich in OPL/OPO/LPL;
(4) Separation and purification of products and recovery of fatty acids: the same procedure as in step (4) of example 1.
The total content of OPL, OPO and LPL in the obtained product was determined to be 82.1wt%, wherein the OPL content was 24.3wt%, the OPO content was 57.1wt%, the LPL content was 0.7wt%, the OPL: OPO: LPL = 0.43.
Example 6
A preparation method of human milk substitute fat with controllable OPL/OPO/LPL ratio and a product thereof specifically comprise the following steps:
(1) Preparing tripalmitin: the same procedure as in (1) of example 2.
(2) Preparation of 2-palmitic acid monoglyceride: 0.12mol of fractionated palm stearin (100 g) was prepared as follows: adding 1.23mol of ethanol (72.3 mL) into 500mL of normal hexane according to the molar ratio of ethanol being 1. The purification method was the same as in step (2) of example 1.
(3) Preparing the mother milk fat with controllable OPL/OPO/LPL ratio: mixing 2-monoglyceryl palmitate with acyl donor 1 (shown in Table 1) at a molar ratio of 1:8, heating and stirring to dissolve in n-hexane, adding 3% of total substrate mass 8 Stirring and reacting for 60min at the reaction temperature of 40 ℃ to obtain a crude product rich in OPL/OPO/LPL;
(4) Separation and purification of products and recovery of fatty acids: the same procedure as in step (4) of example 1.
The total content of OPL, OPO and LPL in the obtained product was determined to be 91.0wt%, wherein the OPL content was 18.1wt%, the OPO content was 72.3wt%, the LPL content was 0.1wt%, OPL: OPO: LPL = 0.25.
Table 2 shows the OPL/OPO/LPL mass contents, OPL/OPO/LPL ratios and the fatty acid compositions at the sn-2 positions in the reaction products obtained in examples 1 to 6.
TABLE 2
Figure BDA0003766779440000091
1 And setting the content of OPO as 1, and calculating OPL/OPO and LPL/OPO to obtain the relative content ratio of OPL/OPO/LPL.
2 The mass fraction (wt%) of the palmitic acid in the sn-2 position to all palmitic acid was determined as specified in GB 30604-2015.
Therefore, the invention provides a preparation method rich in 1-oleic acid-2-palmitic acid-3-linoleic acid triglyceride (OPL), 1,3-dioleic acid-2-palmitic acid triglyceride (OPO) and 1,3-dilinoleic acid-2-palmitic acid triglyceride (LPL), the technical route of the method is firstly proposed, the total content of the OPL, the OPO and the LPL of the obtained product can reach 75wt% -91 wt%, and the proportion of the OPL/OPO/LPL can be adjusted, so that the method can be widely applied to infant formula milk powder suitable for different regions. When the proportion of oleic acid/linoleic acid in the acyl donor is reduced from 6:1 to 1:1, the content of OPL in the product is increased from 18.1wt% to 44.5wt%, the content of OPO is reduced from 72.3wt% to 15.5wt%, and the content of LPL is increased from 0.1wt% to 15.7wt%, wherein when the proportion of oleic acid/linoleic acid is 6:1, 4:1, 3:1 and 2:1, the content of OPL/OPO is in the range of (0.2-1): 1, the content of LPL is lower, and the milk fat structure characteristics of Europe and other regions are met; when the ratio of oleic acid/linoleic acid is 1.5 (example 2), OPL: OPO: LPL = 1.51.
The above description is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make various modifications and changes without departing from the inventive concept, and these modifications and changes are all within the scope of the present invention.

Claims (11)

1. A preparation method of breast milk substitute fat with controllable OPL/OPO/LPL ratio is characterized by mainly comprising the following steps:
step one, fractionation of a palm stearin raw material: taking palm stearin with the tripalmitin content of more than 60wt% as a raw material, adding a solvent, adjusting the temperature to completely dissolve the palm stearin, cooling to separate out tripalmitin crystals, filtering and drying to obtain the palm stearin with the tripalmitin content of more than 85 wt%;
step two, preparation of 2-palmitic acid monoglyceride: mixing the palm stearin obtained in the step one with ethanol, carrying out alcoholysis under the catalysis of regioselective lipase, and then separating and purifying to remove fatty acid ethyl ester to obtain 2-palmitic acid monoglyceride with the purity of more than 85 wt%;
step three, preparing the breast milk substitute fat with controllable OPL/OPO/LPL ratio: in a solvent or solvent-free system, heating and uniformly mixing the mixture of the 2-monoglyceride palmitate and the oleic acid/linoleic acid obtained in the step two, adding lipase, and carrying out enzyme catalytic esterification reaction at 30-50 ℃ to obtain a crude product of the breast milk substitute fat rich in OPL/OPO/LPL:
step four, separating and purifying the product and recycling the fatty acid: and (4) performing molecular distillation on the crude product obtained in the step three, and separating free fatty acid in a reaction system to obtain the breast milk substitute fat rich in OPL/OPO/LPL.
2. The method for preparing human milk substitute lipid with controllable OPL/OPO/LPL ratio as claimed in claim 1, wherein in the step one, the separation solvent is one or two of ethyl acetate and acetone; completely dissolving a palm stearin raw material in a separation solvent at 50-60 ℃, cooling to 20-30 ℃ to separate out tripalmitin crystals, and filtering to remove the solvent to obtain the palm stearin with the content of more than 85wt% after separation.
3. The method for preparing human milk substitute fat with controllable OPL/OPO/LPL ratio as claimed in claim 1, wherein in the first step, the tripalmitin content in the raw material of palm stearin is more than 60%, the palmitic acid content is more than 70wt%,sn-palmitic acid content in position 2 is greater than 70wt%; the tripalmitin content in the fractionated palm stearin is 85-90 wt%, the palmitic acid content is 85-95 wt%,snthe content of the-2-position palmitic acid is 85wt% -95 wt%.
4. The method of claim 1, wherein in step two, the regioselective lipase is selected from the group consisting ofSn-1,3The lipase is selected from one or more of lipase derived from rhizopus oryzae, lipase derived from aspergillus niger, lipase derived from burkholderia cepacia, lipase derived from porcine pancreas and lipase derived from thermomyces lanuginosus; in the third step, the lipase is selected from one or more of lipase derived from candida lipolytica, lipase derived from candida cylindracea, lipase derived from candida rugosa, lipase derived from penicillium camembertii and hydrophobic mesoporous carrier immobilized enzyme.
5. The method for preparing the human milk fat substitute with the controllable OPL/OPO/LPL ratio according to claim 1 is characterized in that in the second step, the molar ratio of the palm stearin to the ethanol after fractionation is 1 to 8 to 1; the solvent can also be included, the solvent is one or more of acetone, normal hexane, cyclohexane and normal octane, and the dosage of the solvent is as follows: adding 5 to 10mL of solvent into 1g tripalmitin; the addition amount of the regioselective lipase is 5-20 wt% of the mass of the palm stearin; the reaction temperature is 20 to 40 ℃, and the reaction is stirred for 1.5 to 4 hours.
6. The method for preparing the human milk substitute fat with the controllable OPL/OPO/LPL ratio as claimed in claim 1, wherein in the second step, the reaction product of alcoholysis is filtered to remove lipase, and is subjected to reduced pressure rotary evaporation to remove solvent, and then is subjected to separation and purification to obtain 2-palmitic acid monoglyceride with purity of more than 85 wt%; wherein, the separation and purification method includes but is not limited to molecular distillation and solvent low-temperature crystallization.
7. The method for preparing human milk substitute fat with controllable ratio of OPL/OPO/LPL as claimed in claim 1, wherein in step three, the oleic acid/linoleic acid mixture is composed of oleic acid and linoleic acid in molar ratio (6~1): 1.
8. The method for preparing human milk substitute fat with controllable OPL/OPO/LPL ratio as claimed in claim 7, wherein said oleic acid/linoleic acid mixture is prepared by blending vegetable oil hydrolysate rich in oleic acid and vegetable oil hydrolysate rich in linoleic acid; wherein the oleic acid is from hydrolysate of oleum Rapae, oleum Arachidis Hypogaeae, oleum Helianthi, and oleum Olivarum; the linoleic acid is hydrolysate of oleum Helianthi, soybean oil, oleum Maydis, and oleum gossypii semen.
9. The preparation method of the breast milk substitute fat with the controllable OPL/OPO/LPL ratio according to claim 1, characterized in that in the third step, the mass ratio of the 2-monoglyceride palmitate to the oleic acid/linoleic acid mixture is 1 to 1, the addition amount of lipase is 3 to 8% of the total mass of the reactants in the step, the reaction temperature is 30 to 50 ℃, and the stirring reaction is carried out for 20 to 60min.
10. The method for preparing the human milk substitute fat with the controllable OPL/OPO/LPL ratio according to claim 1, wherein in the fourth step, the vacuum degree of molecular distillation is less than 4Pa, the temperature is 180 to 220 ℃, the film scraping speed is 200 to 400rpm, and the flow rate is 0.5 to 1L/h.
11. The breast milk substitute fat prepared by the method of any one of claims 1~9, wherein the total content of OPL, OPO and LPL in the breast milk substitute fat is 75wt% to 91wt%, the content of palmitic acid in the 2-position accounts for 83wt% to 92wt% of all palmitic acid, the mass ratio of OPL/OPO is in the range of 0.2 to 2.9, and the mass ratio of LPL/OPO is in the range of 0 to 1.0.
CN202210895481.1A 2022-07-27 2022-07-27 Preparation method of breast milk substitute fat with controllable OPL/OPO/LPL ratio and product thereof Pending CN115232842A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116406709A (en) * 2021-12-31 2023-07-11 内蒙古伊利实业集团股份有限公司 Grease composition and application thereof
CN117625586A (en) * 2023-11-23 2024-03-01 广东惠尔泰生物科技有限公司 Sn-1,3 specific immobilized lipase, and preparation method and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116406709A (en) * 2021-12-31 2023-07-11 内蒙古伊利实业集团股份有限公司 Grease composition and application thereof
CN117625586A (en) * 2023-11-23 2024-03-01 广东惠尔泰生物科技有限公司 Sn-1,3 specific immobilized lipase, and preparation method and application thereof

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