A process for the preparation of a glyceride-rich artificial cream shortening base oil and products thereof

Abstract

This invention discloses a method for preparing a margarine / shortening base oil rich in diglycerides and the product thereof. The method includes: using palm stearin as raw material, preparing diglycerides through a lipase-catalyzed glycerolysis reaction; during the glycerolysis reaction, increasing the compatibility of glycerol with palm stearin by adding glycerol in stages, while simultaneously using monoglycerides and high-pressure homogenization for solubilization; after the reaction, separating unsaturated glycerides through low-temperature fractionation to obtain a highly saturated glyceride product; using a lipase (Lipase G50) to catalyze an esterification reaction between the highly saturated glyceride product and palmitic acid, esterifying the monoglycerides into diglycerides; wherein Lipase G50 is kept at an optimal pH using pH control to enhance its activity; after the esterification reaction, removing fatty acids from the product through molecular distillation to obtain a high-melting-point margarine / shortening base oil rich in diglycerides.

Description

Technical Field

[0001] This invention belongs to the field of oils and fats, specifically relating to a method for preparing a margarine / shortening base oil rich in diglycerides and the product thereof. Background Technology

[0002] Margarine / shortening are both pliable products made from saturated fats through rapid cooling and kneading, primarily used in baked goods such as cakes, bread, and cookies. As the demand for baked goods increases, so does the demand for margarine / shortening. However, the high fat content in these baked goods poses a health risk to consumers. Therefore, there is an urgent need to find an alternative fat that can improve the quality of baked goods while minimizing or even improving consumer health. Based on this situation, diglycerides are currently receiving considerable attention.

[0003] Diacylglycerols are a type of natural oil, present in relatively low amounts in natural oils, generally not exceeding 10% by mass. They are most abundant in cottonseed oil, palm oil, and olive oil, with their relative content depending on the specific oilseed from which they originate. Diacylglycerols can provide similar physical properties and taste to triglycerides, but due to their structural differences, their metabolic pathways differ significantly. When TAGs are ingested, due to the selectivity of lipases, they are hydrolyzed primarily to produce 2-monoacylglycerol esters (2-MAG) and free fatty acids. After absorption into the small intestinal epithelial cells, a portion (approximately 80%) is resynthesized into TAGs under the action of monoacylglycerol acyltransferases and diacylglycerol acyltransferases, and this reaction is rapid. The remaining portion (approximately 20%) undergoes TAG synthesis via the glycerol-3-phosphate pathway, and this process is slow. The resynthesized TAGs are then assembled into chylomicrons via microsomal triglyceride transport proteins, which are then transported into the bloodstream through the intestinal lymphatic system. Excessive TAG intake can cause a rapid increase in blood lipids in a short period, and long-term high blood lipids can lead to a series of chronic diseases. When DAG is ingested, 1,2-DAG is metabolized in the same way as TAG, mainly producing 2-MAG and free fatty acids under the action of lipase; while 1,3-DAG is metabolized to produce 1(3)-MAG and free fatty acids. 1(3)-MAG can hardly synthesize TAG, and most of the free fatty acids enter the liver for metabolism to produce energy, with a very small portion of the products entering the glycerol-3-phosphate pathway to synthesize TAG. Compared with TAG, after DAG ingestion, the body's postprandial blood lipid levels and the rate of increase are significantly reduced. Therefore, compared with ordinary cooking oils, DAG's unique metabolic pathway gives it physiological functions such as inhibiting the rise of postprandial blood lipids, reducing body fat accumulation, reducing weight, and regulating blood sugar.

[0004] Because diglycerides are present in low quantities in nature, most commercially available diglyceride products are obtained through artificial synthesis. The main methods for synthesizing diglycerides include chemical methods and enzymatic synthesis methods. Enzymatic methods are widely used in practical processing due to their mild reaction conditions, environmental friendliness, and high product safety.

[0005] The mainstream method for synthesizing diglycerides using enzymes is the enzymatic glycerol synthesis method, which uses edible oils and glycerol as raw materials and obtains diglyceride products through enzymatic glycerolysis. However, this reaction inevitably produces monoglycerides along with diglycerides. The removal of monoglycerides is mainly carried out using two-stage molecular distillation. Fatty acids are removed by primary molecular distillation, and monoglycerides are removed by secondary molecular distillation. The conditions for secondary molecular distillation are more stringent than those for primary distillation, which leads to the formation of harmful substances such as glycidyl esters and chloropropanol esters in the product. At the same time, in the glycerolysis reaction, the poor miscibility between glycerol and oils reduces the reaction efficiency, and excess glycerol adsorbs onto the enzyme surface, further inhibiting enzyme activity. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0007] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0008] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing a margarine / shortening base oil rich in diglycerides.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for preparing a margarine / shortening base oil rich in diglycerides, comprising,

[0010] Diglycerides were prepared from palm stearin via lipase-catalyzed glycerol hydrolysis. During the glycerol hydrolysis process, the compatibility between glycerol and palm stearin was increased by adding glycerol in stages, while simultaneously using monoglycerides and high-pressure homogenization for solubilization.

[0011] After the reaction is complete, unsaturated glycerides are separated by low-temperature fractionation to obtain highly saturated glycerides.

[0012] The esterification reaction of highly saturated glycerides with palmitic acid was catalyzed by Lipase G50, a dual-imprinted product using pH and nonionic surfactants.

[0013] After esterification, the product undergoes molecular distillation to remove fatty acids, resulting in a high-melting-point margarine / shortening base oil rich in diglycerides.

[0014] As a preferred embodiment of the preparation method described in this invention, the enzyme-catalyzed glycerol hydrolysis reaction includes using palm stearin and edible glycerol as raw materials, the reaction temperature is 60~80℃, and the molar ratio of glycerol to palm stearin is 3:1~1:5.

[0015] The amount of lipase added is 8-15 wt% of the weight of palm stearin, and the stirring speed is 600-800 rpm.

[0016] As a preferred embodiment of the preparation method described in this invention, the method of segmented addition of glycerol synergistically with monoglycerides and high-pressure homogenization to increase the compatibility of glycerol with palm stearin includes:

[0017] Add 0.5-1.5% monoglyceride by mass, add 20-40% glycerol in the first stage, homogenize at 20-40 MPa for 5-15 min, add lipase, react for 0.5-1.5 h, and filter out the lipase.

[0018] In the second stage, add 30-40% glycerol, homogenize at 20-40 MPa for 5-15 minutes, then reintroduce lipase for 0.5-1.5 hours, and filter out the lipase.

[0019] In the third stage, 20-45% glycerol is added, and the mixture is homogenized at 20-40 MPa for 5-15 minutes, followed by lipase reaction for 6-10 hours.

[0020] In a preferred embodiment of the preparation method described in this invention, the melting point of the palm stearin is 44~58℃.

[0021] The lipase is a commercially available lipase, including but not limited to Lipozyme RM IM, Novozyme 435, and NS40086.

[0022] As a preferred embodiment of the preparation method described in this invention, the step of obtaining a highly saturated glycerol product by low-temperature fractionation of unsaturated glycerol esters includes,

[0023] After glycerolysis, the temperature is reduced to 50-60℃ at a rate of 5-10℃ / h, and the mixture is stirred slowly at a rate of 30-50 rpm for 8-24 hours to allow the saturated glycerides to crystallize.

[0024] Highly saturated glycerol products obtained by centrifugation to separate unsaturated glycerol esters.

[0025] As a preferred embodiment of the preparation method described in this invention, the method for preparing the double-blotted Lipase G50 lipase includes,

[0026] Prepare an acetate-phosphate buffer solution with a pH of 5-6, add 10-30% Lipase G50 to the buffer solution, stir at 25°C for 30-60 min, filter the lipase, and dehydrate under vacuum at room temperature to obtain pH-blotted lipase Lipase G50.

[0027] The nonionic surfactant Tween 20-60 was dissolved in isopropanol solution at a concentration of 20-60 mg / mL. 10-30% of pH-imprinted lipase Lipase G50 was added to the solution. The mixture was stirred at 25°C for 30-60 min, filtered, and freeze-dried for 12-24 h to obtain pH and nonionic surfactant-imprinted lipase Lipase G50.

[0028] As a preferred embodiment of the preparation method described in this invention, the esterification reaction includes a molar ratio of free palmitic acid to glycerol ester backbone of 2~5:1, a stirring speed of 500~800 rpm, a temperature of 70~80℃, an amount of double-imprinted Lipase G50 added of 10~14wt%, a vacuum degree of 10~30mbar, and a reaction time of 14~18h.

[0029] As a preferred embodiment of the preparation method described in this invention, the conditions for removing fatty acids by molecular distillation are: distillation temperature of 160°C, pressure of 3Pa, and condenser temperature of 30°C.

[0030] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for preparing a margarine / shortening base oil rich in diglycerides, wherein the product has a diglyceride content greater than 80%, a glycidyl ester content less than 1.6 mg / Kg, and a chloropropanol ester content of 0.5 mg / Kg.

[0031] Beneficial effects of this invention:

[0032] (1) This invention prepares margarine / shortening base oil rich in diglycerides by enzymatic glycerolysis of palm stearin. During the glycerolysis process, the compatibility between glycerol and palm stearin is increased by adding glycerol in stages and using high-pressure homogenization, thereby improving the reaction efficiency. After the glycerolysis reaction is completed, the glycerolysis product is separated by cooling to remove unsaturated glycerides and obtain highly saturated glycerides, thereby increasing the melting point of the final product.

[0033] (2) This invention utilizes the principle that the macromolecular structure of biological enzymes exhibits flexible characteristics in an aqueous environment and rigid characteristics in a non-aqueous system. By adjusting the pH value of Lipase G50 to achieve the optimal catalytic pH state, and dissolving nonionic surfactants with isopropanol to coat the lipase surface, the lipase surface changes from hydrophilic to hydrophobic. The catalytic activity and stability of lipase are improved through the dual imprinting of pH and nonionic surfactants. Using palmitic acid as an acyl donor and Lipase G50 enhanced by dual imprinting as a catalyst, monoglycerides in hypersaturated glycerides are esterified. After molecular distillation and deacidification, a high-melting-point margarine / shortening base oil rich in diglycerides and low in harmful substances is obtained. Detailed Implementation

[0034] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0035] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0036] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0037] Determination of Lipase G50 esterification activity:

[0038] Glycerol and oleic acid were added to a batch reactor at a molar ratio of 1:1, along with 4% Lipase G50. The reaction was carried out at 30°C and a stirring rate of 600 rpm for 1 hour.

[0039] The initial activity of the enzyme was assessed based on the esterification rate (%) of fatty acids in the system after 1 hour of reaction.

[0040] Example 1

[0041] (1) Add palm stearin with a melting point of 44°C to the batch reactor, raise the temperature to 60°C to fully melt the palm stearin, add monoglyceride with a mass fraction of 1%, stir to fully dissolve the monoglyceride in the palm stearin, and add glycerin in three stages according to the molar ratio of glycerol to palm stearin of 1:1.

[0042] In the first stage, 20% glycerol was added and homogenized at 40 MPa for 5 minutes to ensure that the glycerol and palm stearin were fully mixed. Lipozyme RM IM was then added at a rate of 10 wt% of the weight of the palm stearin, and stirring was started at 600 rpm. The reaction was carried out for 0.5 hours, and the lipase was then filtered out.

[0043] In the second stage, 35% glycerol was added, and the mixture was homogenized at 40 MPa for 5 minutes. The lipase was then added back in and reacted for 0.5 hours with a stirring speed of 600 rpm. The lipase was then filtered out.

[0044] In the third stage, 45% glycerol was added, and the mixture was homogenized at 40 MPa for 5 minutes. Lipase was then added and reacted for 6 hours with a stirring rate of 600 rpm. The content of monoglycerides in the reactants was 35.9%, diglycerides was 46.5%, and triglycerides was 17.6%.

[0045] After the reaction was completed, the temperature was reduced to 50℃ at a rate of 5℃ / h, and the mixture was stirred slowly at a rate of 30 rpm for 12 hours to allow the saturated glycerides to crystallize. The high-saturated glyceride product was obtained by centrifugation to separate the unsaturated glycerides.

[0046] (2) Using Lipase G50 as a catalyst, the monoglyceride in the glycerol hydrolysis product is esterified into diglyceride through esterification reaction. Before the reaction, Lipase G50 is first double-imprinted with pH and nonionic surfactant to improve its catalytic activity and stability, so that it is in the best catalytic state.

[0047] The optimal catalytic pH for Lipase G50 is 5-6. Prepare an acetate-phosphate buffer solution with a pH of 5, add 20% (w / w) of Lipase G50 to the buffer solution, stir at 25°C for 45 min to obtain a mixed solution, filter the lipase, and dehydrate under vacuum at room temperature to obtain pH-blotted lipase Lipase G50.

[0048] The nonionic surfactant Tween 20 was dissolved in isopropanol solution at a concentration of 40 mg / mL. 20% pH-imprinted lipase Lipase G50 was added to the solution. The mixture was stirred at 25 °C for 45 min, filtered, and freeze-dried for 12 h to obtain pH and nonionic surfactant-imprinted lipase Lipase G50.

[0049] The esterification activity of pH-blotted Lipase G50 was 25.3%, the activity of double-blotted Lipase G50 was 34.2%, and the enzyme activity of unregulated Lipase G50 was 15.8%.

[0050] (3) The obtained high saturated glycerol ester product was added to a batch reactor, and free palmitic acid was added based on the amount of glycerol ester glycerol skeleton. The molar ratio of free palmitic acid to glycerol ester glycerol skeleton was 3:1. Stirring was started at 600 rpm, the temperature was raised to 70℃, and pH-controlled lipase Lipase G50 was added at 10 wt%. Vacuum was started at 10 mbar and the reaction was carried out for 14 h to obtain the glycerol ester product.

[0051] The product contained 0.9% monoglyceride, 80.9% diglyceride, and 18.2% triglyceride, with a diglyceride yield of 80.9%.

[0052] (4) Removal of fatty acids by molecular distillation

[0053] The conditions for removing fatty acids by molecular distillation were: distillation temperature of 160℃, pressure of 3Pa, and condenser temperature of 30℃. The product contained 81.2% diglycerides, 1.47 mg / Kg glycidyl esters, and 0.35 mg / Kg chloropropanol esters. The melting point of the product was determined to be 56℃.

[0054] Example 2

[0055] (1) Add palm stearin with a melting point of 52°C to the batch reactor, raise the temperature to 70°C to fully melt the palm stearin, add monoglyceride with a mass fraction of 1.5%, stir to fully dissolve the monoglyceride in the palm stearin, and add glycerin in three stages according to the molar ratio of glycerol to palm stearin of 3:1.

[0056] In the first stage, add 30% glycerol and homogenize at 30 MPa for 10 min to fully mix the glycerol with palm stearin. Add lipase Novozym 435 at a weight of 8 wt% of palm stearin and start stirring at 800 rpm. React for 1 h and then filter out the lipase.

[0057] In the second stage, 30% glycerol was added, and the mixture was homogenized at 30 MPa for 10 minutes. The lipase was then added back in and reacted for 1 hour with the stirring speed at 800 rpm. The lipase was then filtered out.

[0058] In the third stage, 40% glycerol was added, homogenized at 30 MPa for 10 min, and lipase was added for 4 h of reaction at a stirring speed of 800 rpm.

[0059] The reactants contained 57.3% monoglycerides, 37.6% diglycerides, and 5.1% triglycerides.

[0060] After the reaction was completed, the temperature was reduced to 55℃ at a rate of 8℃ / h, and the mixture was stirred slowly at a rate of 40 rpm for 24 hours to allow the saturated glycerides to crystallize. The high-saturated glyceride product was obtained by centrifugation to separate the unsaturated glycerides.

[0061] (2) Using Lipase G50 as a catalyst, the monoglyceride in the glycerol hydrolysis product is esterified into diglyceride through esterification reaction. Before the reaction, Lipase G50 is first double-imprinted with pH and nonionic surfactant to improve its catalytic activity and stability, so that it is in the best catalytic state.

[0062] The optimal catalytic pH for Lipase G50 is 5-6. Prepare an acetate-phosphate buffer solution with a pH of 5.5, add 30% (w / w) of Lipase G50 to the buffer solution, stir at 25°C for 30 min to obtain a mixed solution, filter the lipase, and dehydrate under vacuum at room temperature to obtain pH-blotted lipase Lipase G50.

[0063] The nonionic surfactant Tween 40 was dissolved in isopropanol solution at a concentration of 60 mg / mL. 30% pH-imprinted lipase Lipase G50 was added to the solution. The mixture was stirred at 25 °C for 30 min, filtered, and freeze-dried for 18 h to obtain pH and nonionic surfactant-imprinted lipase Lipase G50.

[0064] The esterification activity of pH-blotted Lipase G50 was 26.7%, the activity of double-blotted Lipase G50 was 35.1%, and the enzyme activity of unregulated Lipase G50 was 15.8%.

[0065] (3) The obtained high saturated glycerol product was added to a batch reactor, and free palmitic acid was added based on the amount of glycerol skeleton of glycerol ester. The molar ratio of free palmitic acid to glycerol skeleton of glycerol ester was 5:1. Stirring was started at 800 rpm. The temperature was raised to 75°C. Lipase G50 was added at 14 wt%. Vacuum was started at 20 mbar. The reaction was carried out for 16 h to obtain the glycerol ester product.

[0066] The product contained 0.6% monoglyceride, 93.6% diglyceride, and 5.8% triglyceride, with a diglyceride yield of 93.6%.

[0067] (3) Removal of fatty acids by molecular distillation

[0068] The conditions for removing fatty acids by molecular distillation were: distillation temperature of 160℃, pressure of 3Pa, and condenser temperature of 30℃. The product contained 93.8% diglycerides, 1.57 mg / Kg glycidyl esters, and 0.39 mg / Kg chloropropanol esters. The melting point of the product was determined to be 63℃.

[0069] Example 3

[0070] (1) Add palm stearin with a melting point of 58°C to the batch reactor, raise the temperature to 80°C to fully melt the palm stearin, add monoglyceride with a mass fraction of 0.5%, stir to fully dissolve the monoglyceride in the palm stearin, and add glycerin in three stages according to the molar ratio of glycerol to palm stearin of 1:3.

[0071] In the first stage, 40% glycerol was added and homogenized at 20 MPa for 15 min to ensure that the glycerol and palm stearin were fully mixed. Lipase NS 40086 was then added at a rate of 15 wt% of the weight of the palm stearin. The mixture was then stirred at 700 rpm for 1.5 h, and the lipase was filtered out.

[0072] In the second stage, 40% glycerol was added, and the mixture was homogenized at 20 MPa for 15 minutes. The lipase was then added back in and reacted for 1.5 hours with a stirring speed of 700 rpm. The lipase was then filtered out.

[0073] In the third stage, 20% glycerol was added, and the mixture was homogenized at 20 MPa for 15 min. Lipase was then added and reacted for 7 h with a stirring rate of 700 rpm. The content of monoglycerides in the reactants was 15.8%, diglycerides was 43.4%, and triglycerides was 40.8%.

[0074] After the reaction was completed, the temperature was reduced to 60℃ at a rate of 10℃ / h, and the mixture was stirred slowly at a rate of 50 rpm for 8 hours to allow the saturated glycerides to crystallize. The high-saturated glyceride product was obtained by centrifugation to separate the unsaturated glycerides.

[0075] (2) Using Lipase G50 as a catalyst, the monoglyceride in the glycerol hydrolysis product is esterified into diglyceride through esterification reaction. Before the reaction, Lipase G50 is first double-imprinted with pH and nonionic surfactant to improve its catalytic activity and stability, so that it is in the best catalytic state.

[0076] The optimal catalytic pH for Lipase G50 is 5-6. Prepare an acetate-phosphate buffer solution with a pH of 6, add 10% (w / w) of Lipase G50 to the buffer solution, stir at 25°C for 60 min to obtain a mixed solution, filter the lipase, and dehydrate under vacuum at room temperature to obtain pH-blotted lipase Lipase G50.

[0077] The nonionic surfactant Tween 60 was dissolved in isopropanol solution at a concentration of 20 mg / mL. 10% pH-imprinted lipase Lipase G50 was added to the solution. The mixture was stirred at 25 °C for 60 min, filtered, and freeze-dried for 24 h to obtain pH and nonionic surfactant-imprinted lipase Lipase G50.

[0078] The esterification activity of pH-blotted Lipase G50 was 25.1%, the activity of double-blotted Lipase G50 was 33.7%, and the enzyme activity of unregulated Lipase G50 was 15.8%.

[0079] (3) The obtained high-saturated glycerol product was added to a batch reactor, and free palmitic acid was added based on the amount of glycerol backbone of the glycerol ester. The molar ratio of free palmitic acid to glycerol backbone of glycerol ester was 2:1. Stirring was started at 500 rpm, the temperature was raised to 80℃, and double-imprinted lipase Lipase G50 was added at 12 wt%. Vacuum was started at 30 mbar and the reaction was carried out for 18 h to obtain the glycerol ester product. The content of monoglyceride in the product was 0.7%, the content of diglyceride was 58.0%, the content of triglyceride was 41.3%, and the yield of diglyceride was 58.0%.

[0080] (4) Removal of fatty acids by molecular distillation

[0081] The conditions for removing fatty acids by molecular distillation were: distillation temperature of 160℃, pressure of 3Pa, and condenser temperature of 30℃. The product contained 58.8% diglycerides, 1.33 mg / Kg glycidyl esters, and 0.27 mg / Kg chloropropanol esters. The melting point of the product was determined to be 71℃.

[0082] Comparative Example 1

[0083] The glycerol hydrolysis reaction was carried out according to Example 1, without the addition of monoglycerides, without the addition of glycerol in batches, without homogenization, and with all other reaction conditions being the same.

[0084] Palm stearin with a melting point of 44°C is added to an intermittent reactor, and the temperature is raised to 60°C to fully melt the palm stearin.

[0085] Glycerin was added at a molar ratio of 1:1 to palm stearin. Lipozyme RM IM was added at a concentration of 10 wt% of the palm stearin. Stirring was started at 600 rpm for 7 hours. The content of monoglycerides in the reactants was 24.3%, diglycerides was 46.9%, and triglycerides was 28.8%.

[0086] Compared to Example 1, the triglyceride content in the product of Comparative Example 1 was 28.8%, significantly higher than that in Example 1, indicating that the reaction in Comparative Example 1 was far from reaching equilibrium. Therefore, a longer reaction time is required to reach equilibrium, thereby reducing reaction efficiency.

[0087] Comparative Example 2

[0088] Referring to Example 2, after the glycerol hydrolysis is completed, instead of using a programmed cooling method to separate unsaturated glycerides, conventional methods are used to remove glycerol by high-temperature layering centrifugation.

[0089] Palm stearin with a melting point of 52℃ was added to an intermittent reactor, and the temperature was raised to 70℃ to fully melt the palm stearin. 1.5% monoglyceride was added and stirred to fully dissolve the monoglyceride in the palm stearin. Glycerin was added in three stages according to a molar ratio of glycerol to palm stearin of 3:1.

[0090] In the first stage, add 30% glycerol and homogenize at 30 MPa for 10 min to fully mix the glycerol with palm stearin. Add lipase at 8 wt% of the weight of palm stearin and start stirring at 800 rpm. React for 1 h and then filter out the lipase.

[0091] In the second stage, 30% glycerol was added, and the mixture was homogenized at 30 MPa for 10 minutes. The lipase was then added back in and reacted for 1 hour with the stirring speed at 800 rpm. The lipase was then filtered out.

[0092] In the third stage, 40% glycerol was added, homogenized at 30 MPa for 10 min, and lipase was added for 4 h of reaction at a stirring speed of 800 rpm.

[0093] The reactants contained 57.3% monoglycerides, 37.6% diglycerides, and 5.2% triglycerides.

[0094] After the reaction was completed, the temperature was raised to 90°C, and the mixture was stirred slowly at a speed of 40 rpm for 1 hour to separate glycerol from glycerol esters and obtain the glycerolysis product.

[0095] The obtained glycerol hydrolysis product was transferred to a batch reactor, and free palmitic acid was added based on the amount of glycerol backbone in the glycerol ester, with a molar ratio of free palmitic acid to glycerol backbone of 5:1. Stirring was initiated at 800 rpm, and the temperature was raised to 80°C. Double-imprinted lipase Lipase G50 was added at a concentration of 14 wt%, and a vacuum of 20 mbar was applied. The reaction was carried out for 16 h to obtain the glycerol ester product. The product was then subjected to molecular distillation to obtain a diglyceride product with a diglyceride content of 92.7% and a melting point of 57°C.

[0096] It is evident that in Comparative Example 2, the unsaturated glycerides were not removed by low-temperature crystallization, and the melting point of the resulting product was much lower than that of the glycerides in Example 2.

[0097] Comparative Example 3

[0098] Following the reaction conditions of Example 3, esterification was performed without the addition of double-imprinted lipase Lipase G50, and the product was directly subjected to molecular distillation.

[0099] Palm stearin with a melting point of 58°C was added to an intermittent reactor, and the temperature was raised to 80°C to fully melt the palm stearin. 0.5% monoglyceride was added and stirred to fully dissolve the monoglyceride in the palm stearin. Glycerin was added in three stages according to a molar ratio of glycerol to palm stearin of 1:3.

[0100] In the first stage, 40% glycerol was added and homogenized at 20 MPa for 15 minutes to ensure that the glycerol and palm stearin were fully mixed. Lipase was then added at a rate of 15 wt% of the weight of the palm stearin, and stirring was started at 700 rpm. The reaction was carried out for 1.5 hours, and the lipase was then filtered out.

[0101] In the second stage, 40% glycerol was added, and the mixture was homogenized at 20 MPa for 15 minutes. The lipase was then added back in and reacted for 1.5 hours with a stirring speed of 700 rpm. The lipase was then filtered out.

[0102] In the third stage, 20% glycerol was added, and the mixture was homogenized at 20 MPa for 15 min. Lipase was then added and reacted for 7 h with a stirring rate of 700 rpm. The content of monoglycerides in the reactants was 15.8%, diglycerides was 43.4%, and triglycerides was 40.8%.

[0103] After the reaction is complete, the temperature is lowered to 60℃ at a rate of 10℃ / h, and the mixture is stirred slowly at a rate of 50 rpm for 8 hours to allow the saturated glycerides to crystallize. The high-saturated glyceride product is obtained by centrifugation to separate the unsaturated glycerides.

[0104] The obtained hypersaturated glycerol product was subjected to molecular distillation under the following conditions: distillation temperature of 160℃, pressure of 3Pa, and condenser temperature of 30℃, to obtain glycerol ester products. The product contained 14.4% monoglyceride, 43.5% diglyceride, and 42.1% triglyceride, with a diglyceride yield of 43.5%.

[0105] According to the traditional method, monoglycerides were removed by two-stage molecular distillation. The molecular distillation conditions were: distillation temperature of 210℃, pressure of 2Pa, and condenser temperature of 30℃. The product obtained was a glyceride product with a diglyceride content of 50.4%. Since monoglycerides were removed by molecular distillation, the yield of diglycerides after two-stage molecular distillation was much lower than that of the product obtained by esterification reaction in Example 3. The content of glycidyl ester was 6.22 mg / Kg, and the content of chloropropanol ester was 1.27 mg / Kg.

[0106] Therefore, it can be seen that by using the double-imprinted Lipase G50 lipase esterification process, monoglycerides in glycerol hydrolysis products are esterified into diglycerides, resulting in a higher yield of diglycerides and higher purity of the product. At the same time, milder conditions can be used in the molecular distillation stage, resulting in fewer harmful substances.

[0107] Because enzymes and other protein macromolecules exhibit flexible characteristics in aqueous solutions and rigid characteristics in non-aqueous solutions, and also possess memory properties, by adjusting the pH value of the enzyme in an aqueous solution to bring it to the optimal pH state, and by having a nonionic surfactant interact with the active site of the lipase to open the cap of its activity, and then removing the water by freeze-drying, the enzyme will retain its active catalytic structural characteristics.

[0108] Comparative Example 4

[0109] Referring to Example 1, only the glycerol was added in stages, without homogenization, and the other reaction conditions were the same as in Example 1.

[0110] Palm stearin with a melting point of 44℃ was added to an intermittent reactor, and the temperature was raised to 60℃ to fully melt the palm stearin. 1% monoglyceride was added and stirred to fully dissolve the monoglyceride in the palm stearin. Glycerin was added in three stages according to a glycerol to palm stearin molar ratio of 1:1.

[0111] In the first stage, add 20% glycerol, add lipase Lipozyme RM IM, the amount of lipase added is 10 wt% of the weight of palm stearin, and start stirring at 600 rpm. React for 0.5 h, and then filter out the lipase.

[0112] In the second stage, 35% glycerol was added, and the lipase was added again and reacted for 0.5 hours with a stirring speed of 600 rpm. The lipase was then filtered out.

[0113] In the third stage, 45% glycerol was added, and lipase was added to react for 6 hours with a stirring rate of 600 rpm. The content of monoglycerides in the reactants was 32.1%, the content of diglycerides was 39.6%, and the content of triglycerides was 28.3%.

[0114] The triglyceride content in Comparative Example 4 was significantly higher than that in Example 1, indicating that the reaction efficiency of the homogenized reaction system was much higher than that of the simple staged addition reaction system. Homogenization allows for thorough mixing of glycerol and oils, increasing their solubility and preventing glycerol from coating the lipase surface, thereby improving reaction efficiency.

[0115] Comparative Example 5

[0116] Referring to Example 1, the amount of glycerol added in stages was changed, while other reaction conditions remained the same as in Example 1.

[0117] Palm stearin with a melting point of 44℃ was added to an intermittent reactor, and the temperature was raised to 60℃ to fully melt the palm stearin. 1% monoglyceride was added and stirred to fully dissolve the monoglyceride in the palm stearin. Glycerin was added in three stages according to a glycerol to palm stearin molar ratio of 1:1.

[0118] In the first stage, 10% glycerol was added and homogenized at 40 MPa for 5 minutes to ensure that the glycerol and palm stearin were fully mixed. Lipozyme RM IM was then added at a rate of 10 wt% of the weight of the palm stearin, and stirring was started at 600 rpm. The reaction was carried out for 0.5 hours, and the lipase was then filtered out.

[0119] In the second stage, 50% glycerol was added, and the mixture was homogenized at 40 MPa for 5 minutes. The lipase was then added back in and reacted for 0.5 hours with a stirring speed of 600 rpm. The lipase was then filtered out.

[0120] In the third stage, 40% glycerol was added, and the mixture was homogenized at 40 MPa for 5 min. Lipase was then added and reacted for 6 h with a stirring rate of 600 rpm. The content of monoglycerides in the reactants was 33.7%, diglycerides was 43.4%, and triglycerides was 22.9%.

[0121] The triglyceride content in Comparative Example 5 was higher than that in Example 1, indicating that under the same conditions, the amount of glycerol added in stages will affect the reaction efficiency of the system. In Comparative Example 5, less glycerol was added in the first stage, resulting in fewer monoglycerides and diglycerides generated in the first stage. In the second stage, more glycerol was added, which prevented glycerol from completely mixing with the fats, leaving more glycerol in a free state. Some glycerol coated the lipase surface, ultimately leading to a decrease in reaction efficiency.

[0122] Comparative Example 6

[0123] Referring to Example 2, without the addition of monoglycerides, other reaction conditions were the same as in Example 2.

[0124] Palm stearin with a melting point of 52°C was added to an intermittent reactor, and the temperature was raised to 70°C to fully melt the palm stearin. Glycerin was added in three stages according to a molar ratio of glycerin to palm stearin of 3:1.

[0125] In the first stage, add 30% glycerol and homogenize at 30 MPa for 10 min to fully mix the glycerol with palm stearin. Add lipase Novozym 435 at a weight of 8 wt% of palm stearin and start stirring at 800 rpm. React for 1 h and then filter out the lipase.

[0126] In the second stage, 30% glycerol was added, and the mixture was homogenized at 30 MPa for 10 minutes. The lipase was then added back in and reacted for 1 hour with the stirring speed at 800 rpm. The lipase was then filtered out.

[0127] In the third stage, 40% glycerol was added, homogenized at 30 MPa for 10 min, and lipase was added for 4 h of reaction at a stirring speed of 800 rpm.

[0128] The reactants contained 48.8% monoglycerides, 36.8% diglycerides, and 14.4% triglycerides.

[0129] The triglyceride content in Comparative Example 6 was much higher than that in Example 2, indicating that the reaction in Comparative Example 6 was not as complete as that in Example 2. Adding monoglycerides in the early stage helps the glycerol mix with the oil, improving reaction efficiency. Within a limited time, the reaction produces sufficient monoglycerides and diglycerides, which facilitates thorough mixing of glycerol and oil after subsequent addition of glycerol, further improving reaction efficiency.

[0130] Comparative Example 7

[0131] Following the conditions of Example 3, esterification reactions were carried out in successive batches using double-imprinted lipase Lipase G50 and unimprinted lipase Lipase G50, with other conditions consistent with Example 3.

[0132] After five consecutive reactions, the activity of the double-imprinted lipase Lipase G50 was measured to be 27.2%, with a loss rate of 19.3%, while the activity of the unimprinted lipase Lipase G50 was 9.3%, with a loss rate of 41.1%. Therefore, the activity stability of the double-imprinted lipase Lipase G50 was significantly higher than that of the unimprinted lipase.

[0133] Because Lipase G50 lipase is water-soluble, its surface has a higher proportion of hydrophilic groups than hydrophobic groups. A nonionic surfactant interacts with the lipase through hydrophobic forces; its polar head interacts with the hydrophilic groups of the lipase, while its nonpolar head interacts with the hydrophobic groups, thus changing the lipase surface from hydrophilic to hydrophobic. This increases the lipase's solubility in the hydrophobic system, thereby enhancing its catalytic effect. Simultaneously, it prevents excessive interaction between the lipase and polar substances in the reaction system, preventing the lipase from losing water and causing structural damage and inactivation, thus protecting the lipase's structure. Furthermore, the nonionic surfactant interacts with the active site of the lipase, opening the cap and removing water to maintain the lipase's active catalytic conformation, thereby increasing its activity.

[0134] Since isopropanol has good solubility for nonionic surfactants, and contains trace amounts of water which keeps lipase in a soft state, and isopropanol is relatively mild and will not have a significant impact on the conformation of lipase, using isopropanol as a solvent to dissolve surfactants and lipases can enable nonionic surfactants to interact better with lipases, thereby improving the imprinting effect and further enhancing the activity of lipases.

[0135] Therefore, the lipases obtained by dual imprinting with pH and non-standard surfactants can not only greatly enhance the activity of lipases, but also greatly improve the reaction stability of lipases, thereby increasing reaction efficiency and greatly reducing reaction costs.

[0136] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A method for preparing a margarine / shortening base oil rich in diglycerides, characterized in that: include, Diglycerides were prepared from palm stearin via lipase-catalyzed glycerol hydrolysis. During the glycerol hydrolysis process, the compatibility between glycerol and palm stearin was increased by adding glycerol in stages, while simultaneously using monoglycerides and high-pressure homogenization for solubilization. After the reaction is complete, unsaturated glycerides are separated by low-temperature fractionation to obtain highly saturated glycerides. The esterification reaction of highly saturated glycerides with palmitic acid was catalyzed by Lipase G50, a dual-imprinted product using pH and nonionic surfactants. After esterification, the product undergoes molecular distillation to remove fatty acids, resulting in a high-melting-point margarine / shortening base oil rich in diglycerides. Among them, the methods for increasing the compatibility of glycerol with palm stearin include segmented addition of glycerol synergistically with monoglycerides and high-pressure homogenization. Add 0.5-1.5% monoglyceride by mass, add 20-40% glycerol in the first stage, homogenize at 20-40 MPa for 5-15 min, add lipase, react for 0.5-1.5 h, and filter out the lipase. In the second stage, add 30-40% glycerol, homogenize at 20-40 MPa for 5-15 min, then reintroduce lipase for 0.5-1.5 h, and filter out the lipase. In the third stage, 20-45% glycerol is added, homogenized at 20-40 MPa for 5-15 minutes, and then lipase is added for 6-10 hours of reaction. The method for preparing the double-blotted Lipase G50 lipase includes, Prepare an acetate-phosphate buffer solution with a pH of 5-6, add 10-30% (w / w) of Lipase G50 to the buffer solution, stir at 25°C for 30-60 min, filter the lipase, and dehydrate under vacuum at room temperature to obtain pH-blotted lipase Lipase G50. Nonionic surfactants Tween 20, Tween 40, or Tween 60 were dissolved in isopropanol solution at a concentration of 20-60 mg / mL. 10-30% of pH-imprinted lipase Lipase G50 was added to the solution. The mixture was stirred at 25°C for 30-60 min, filtered, and freeze-dried for 12-24 h to obtain pH- and nonionic surfactant-imprinted lipase Lipase G50.

2. The preparation method according to claim 1, characterized in that: The enzyme-catalyzed glycerol hydrolysis reaction includes using palm stearin and edible glycerol as raw materials, with a reaction temperature of 60-80℃ and a molar ratio of glycerol to palm stearin of 3:1-1:

5. The amount of lipase added is 8-15 wt% of the weight of palm stearin, and the stirring speed is 600-800 rpm.

3. The preparation method according to claim 1, characterized in that: The melting point of the palm stearin is 44~58℃; The lipases are commercially available lipases, including Lipozyme RM IM, Novozyme 435, and NS 40086.

4. The preparation method according to claim 1, characterized in that: The method of separating unsaturated glycerides through low-temperature fractionation to obtain highly saturated glycerides includes, After glycerol hydrolysis, the temperature is lowered to 50-60℃ at a rate of 5-10℃ / h, and the mixture is stirred slowly at a rate of 30-50 rpm for 8-24 hours to allow the saturated glycerides to crystallize. Highly saturated glycerides are obtained by centrifugation to separate unsaturated glycerides.

5. The preparation method according to claim 1, characterized in that: The esterification reaction includes a molar ratio of free palmitic acid to glycerol ester backbone of 2~5:1, a stirring speed of 500~800 rpm, a temperature of 70~80℃, an amount of double-imprinted Lipase G50 added of 10~14wt%, a vacuum degree of 10~30mbar, and a reaction time of 14~18h.

6. The preparation method according to claim 1, characterized in that: The conditions for removing fatty acids by molecular distillation are: distillation temperature of 160℃, pressure of 3Pa, and condenser temperature of 30℃.

7. A margarine / shortening base oil product rich in diglycerides prepared by any of the preparation methods described in claims 1 to 6.

8. The product as described in claim 7, characterized in that: The product has a diglyceride content greater than 80%, a glycidyl ester content less than 1.6 mg / kg, and a chloropropanol ester content of 0.5 mg / kg.