Synthetic method of high-content EPA / DHA glyceride with blood fat reducing function based on water-containing system
Through the combination method of batch addition of deionized water and enzymatic reaction, the problem of water molecules blocking effect in transesterification reaction was solved, and the synthesis of high content of EPA and DHA glycerides was achieved, which was gentle, efficient, green and environmentally friendly. , and reduces production costs.
Patent Information
- Application Number
- CN202411242803.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-05
- Publication Date
- 2024-12-27
AI Technical Summary
The synthesis of high content of EPA and DHA glycerides in the prior art is difficult to achieve in transesterification reactions, mainly because the presence of water molecules has a blocking effect on the transesterification reaction, which makes the reaction difficult to reach the end point.
The method of adding deionized water in batches is adopted to ensure that the water molecules in the reaction system exist, and combine with enzymatic reactions to selectively promote the transformation of fatty acid methyl ester, ethyl ester, isopropanol ester to triglycerides, and improve the reaction rate. and reduce costs.
The synthesis of high content of EPA and DHA glycerides is achieved, the reaction conditions are mild, efficient, green and environmentally friendly, and the reusability of lipase is high, which reduces production costs and is suitable for large-scale production.
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Figure CN119193723A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of functional glyceride synthesis, and specifically relates to a method for synthesizing high-content EPA / DHA glyceride with blood lipid lowering function based on an aqueous system. Background Art
[0002] As an important type of fatty acid, n-3 polyunsaturated fatty acids have multiple physiological functions in organisms. In particular, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) play a vital role in the health of the body. EPA, commonly known as the "blood vessel scavenger", has the function of regulating plasma lipid composition and reducing blood viscosity, thereby improving blood circulation and preventing cardiovascular and cerebrovascular diseases. DHA, commonly known as "brain gold", can enhance memory and promote children's brain and nerve development. In addition, EPA and DHA also have the functions of regulating bone metabolism, anti-tumor, preventing metabolic syndrome, anti-aging and beneficial to reproductive system health.
[0003] A large number of studies have shown that there are differences in the stability and bioavailability of glycerides, fatty acid ethyl esters and fatty acids. Among them, fatty acid ethyl esters have the highest stability and fatty acids have the lowest stability. In terms of bioavailability, glycerides have the highest bioavailability and fatty acid ethyl esters have the lowest bioavailability. After comprehensive analysis, glycerides are more suitable for consumers to eat than fatty acid ethyl esters and fatty acids.
[0004] The EPA and DHA glyceride products from natural sources generally have low EPA and DHA content. Marine organisms are the main source of EPA and DHA glycerides. The total content of EPA and DHA in processed marine fish oil products is less than 35%, such as 28% in tuna oil, 25% in sardine oil, and 31% in anchovies. Many studies have shown that products with high EPA and DHA content have more prominent physiological functions. Therefore, it is of great significance to obtain high-content EPA and DHA glycerides.
[0005] Transesterification is one of the mainstream processes for preparing high-content EPA and DHA glycerides. The molecular structure of EPA and DHA is complex and has large steric hindrance. Therefore, the biggest challenge in synthesizing high-content EPA and DHA glycerides is that EPA and DHA are difficult to combine with glycerol during the transesterification reaction. At present, studies have been conducted to remove the water produced by the esterification reaction, improve the reaction effect, promote the combination of EPA and DHA with glycerol, and increase the content of product glycerides by adding vacuum and nitrogen. However, under vacuum or nitrogen conditions, it usually takes a long time to reach the equilibrium of the reaction. The reason is that transesterification is composed of hydrolysis and esterification reactions. In the reaction, water molecules are the key hub in the transesterification reaction on the one hand, and on the other hand, they are crucial for the catalytic activity of lipase. During the hydrolysis reaction, the presence of water molecules is required to promote the breaking of ester bonds. However, under vacuum and nitrogen conditions, as the reaction proceeds, the water content in the system continues to decrease, which has a blocking effect on the hydrolysis reaction in the transesterification, destroying the continuous progress of the transesterification reaction and making it difficult to reach the reaction endpoint. At present, there is no relevant research on the combined application of vacuum and intermittent water addition technology in the production of structured fats and related products. Summary of the invention
[0006] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.
[0007] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.
[0008] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for synthesizing high-content EPA / DHA glycerides with lipid-lowering function based on an aqueous system.
[0009] In order to solve the above technical problems, the present invention provides the following technical solutions:
[0010] The non-glyceride, glycerol and lipase are mixed, and deionized water is added intermittently to carry out an enzymatic reaction;
[0011] After the reaction is finished, the lipase is removed to obtain a crude reaction product, which is then separated and purified to obtain a glyceride product.
[0012] As a preferred embodiment of the method for synthesizing high-content EPA / DHA glycerides with lipid-lowering function based on an aqueous system according to the present invention, the intermittent addition of deionized water is to add deionized water to the reaction system every 1 to 2.75 hours.
[0013] As a preferred solution of the method for synthesizing high-content EPA / DHA glycerides with lipid-lowering function based on an aqueous system described in the present invention, the amount of deionized water added each time is 3% to 7% of the mass of the non-glycerides.
[0014] As a preferred solution of the method for synthesizing high-content EPA / DHA glycerides with lipid-lowering function based on an aqueous system according to the present invention, the addition amount of the lipase is 6% to 8% of the mass of the non-glycerides.
[0015] As a preferred embodiment of the method for synthesizing high-content EPA / DHA glycerides with lipid-lowering function based on an aqueous system according to the present invention, the non-glycerides include one or more of fatty acids, fatty acid ethyl esters, fatty acid methyl esters and fatty acid isopropanol esters.
[0016] As a preferred embodiment of the method for synthesizing high-content EPA / DHA glyceride with lipid-lowering function based on an aqueous system according to the present invention, the molar ratio of the non-glyceride to glycerol is (2.8-5):1.
[0017] As a preferred embodiment of the method for synthesizing high-content EPA / DHA glyceride with lipid-lowering function based on an aqueous system according to the present invention, the temperature of the enzymatic reaction is 45-75°C.
[0018] As a preferred embodiment of the method for synthesizing high-content EPA / DHA glycerides with lipid-lowering function based on an aqueous system according to the present invention, the absolute pressure of the enzymatic reaction is 2.0-5.0 mbar.
[0019] As a preferred embodiment of the method for synthesizing high-content EPA / DHA glyceride with lipid-lowering function based on an aqueous system according to the present invention, the time of the enzymatic reaction is 4 to 16 hours.
[0020] As a preferred embodiment of the method for synthesizing high-content EPA / DHA glyceride with lipid-lowering function based on an aqueous system according to the present invention, the lipase comprises one or more strains selected from Candida antarctica, C. cylindracea, Rhizomucor miehei, Thermomyces lanuginosus, Pseudomonas cepacia, and Aspergilus oryzae.
[0021] Beneficial effects of the present invention:
[0022] (1) The enzymatic reaction system of the present invention uses an intermittent water addition method to ensure that the system has a certain water content, provide the required water molecules for the transesterification reaction, and promote the synthesis of glycerides;
[0023] (2) The intermittent addition of water in the present invention selectively promotes the conversion of fatty acid methyl esters, ethyl esters, isopropanol esters and fatty acids into triglycerides, which can accelerate the reaction rate, make the reaction reach equilibrium more quickly, and reduce the reaction cost;
[0024] (3) The present invention adopts an enzymatic method to prepare glycerides with high content of EPA and DHA, and the reaction conditions are mild, efficient, highly specific, and environmentally friendly;
[0025] (4) The lipase used in the present invention has a high degree of reusability, which can significantly reduce production costs and meet the requirements of large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:
[0027] Figure 1 It is a high performance liquid chromatogram of the sample of Example 1 of the present invention, Figure A is the composition and content of lipids in the fish oil ethyl ester raw material; Figure B is the composition and content of lipids in the crude reaction product; Figure C is the composition and content of lipids in the glyceride product.
[0028] Figure 2 This is a high performance liquid chromatogram of the crude product of the reaction of Comparative Example 1 of the present invention, and the data in the figure are the composition and content of lipids in the obtained crude product of the reaction. DETAILED DESCRIPTION
[0029] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.
[0030] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.
[0031] 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 term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.
[0032] The raw materials and reagents used in the present invention are as follows: the fatty acid ester rich in EPA and DHA is fish oil ethyl ester, purchased from Xi'an Mixianer Biotechnology Co., Ltd., and the total content of EPA and DHA is greater than 70%; glycerol is purchased from Sinopharm Chemical Reagent Co., Ltd.; fatty acid methyl ester, fatty acid isopropanol ester and fatty acid are purchased from Sigma-Aldrich; lipase Candida antarctica, Rhizomucor miehei, Thermomyces lanuginosus are purchased from Wuxi Youpuke Biotechnology Co., Ltd., and lipase C. cylindracea, Pseudomonas cepacia, Aspergilus oryzae are purchased from Xibao Biotechnology (Shanghai) Co., Ltd.
[0033] Example 1
[0034] This embodiment provides a method for synthesizing a high-content EPA / DHA glyceride product with lipid-lowering function using lipase Candida antarctica:
[0035] The non-glyceride rich in EPA / DHA, glycerol and lipase are mixed, and deionized water is added intermittently to carry out an enzymatic reaction;
[0036] After the reaction is completed, the lipase is removed to obtain a crude reaction product, which is then separated and purified to obtain a glyceride product.
[0037] In a 250 mL conical flask, 20 g of fish oil ethyl ester and 1.73 g of glycerol (glycerol: ethyl ester = 1:3.5, molar ratio) were added, and then lipase Candida antarctica was added, and the enzyme addition amount was 6% (based on the mass fraction of fish oil). After mixing evenly, under the conditions of reaction temperature of 65 ° C and absolute pressure of 2.0 mbar, magnetic stirring was performed at 300 r / min, and 7% (based on the mass fraction of fish oil ethyl ester) of deionized water was added to the system at 2 h and 4 h of reaction, respectively, and the total reaction time was 6 h;
[0038] After the reaction, the supernatant was taken by centrifugation at 1000 r / min for 5 min to remove the lipase to obtain a crude reaction product. The crude reaction product was subjected to molecular distillation (temperature 160°C, pressure 1Pa, feed flow rate 10mL / min, scraper speed 300r / min) to obtain a purified glyceride product.
[0039] The lipid composition was detected by HPLC-RID and the triglyceride content was calculated.
[0040] The EPA and DHA contents were tested by CG.
[0041] Figure 1 The HPLC chromatogram of the sample. Figure 1 A is the composition and content of lipids in the fish oil ethyl ester raw material; Figure 1 B is the composition and content of lipids in the crude reaction product; Figure 1 C is the composition and content of lipids in the glyceride product. Table 1 shows the composition and content of lipids in the fish oil ethyl ester raw material, the crude reaction product and the purified glyceride product.
[0042] Table 1 Comparison of lipid content
[0043] Triglycerides Diacylceride Monoglyceride fatty acid Ethyl ester Fish Oil Ethyl Esters Raw Materials — — — 0.23％ 99.77％ Reaction crude product 84.34％ 5.55％ 0.52％ 3.82％ 5.77％ Purified glyceride products 94.12％ 5.88％ 0.00％ 0.00％ 0.00％
[0044] It can be seen from Table 1 that the content of triglyceride in the glyceride product is as high as 94.12%.
[0045] Table 2 shows the EPA and DHA content in fish oil ethyl esters and purified glycerides.
[0046] Table 2 Comparison of EPA and DHA content
[0047] EPA DHA EPA+DHA Fish Oil Ethyl Esters Raw Materials 6.31％ 73.65％ 79.96％ Purified glyceride products 7.10％ 79.56％ 86.66％
[0048] As can be seen from Table 2, the EPA and DHA in the product are slightly increased, and the total content of EPA and DHA in the purified glyceride product is as high as 86.66%.
[0049] Example 2
[0050] This example investigates the effect of different amounts of deionized water added on the glyceride content in the crude reaction product (without molecular distillation treatment). This example includes 6 groups of experiments, each of which differs in the amount of water added but has the same interval.
[0051] In a 25 mL conical flask, 2 g of fish oil ethyl ester and 0.173 g of glycerol (glycerol: ethyl ester = 1:3.5, molar ratio) were added, and then lipase Candida antarctica was added, and the enzyme addition amount was 6% (based on the mass fraction of fish oil ethyl ester). After mixing evenly, under the conditions of reaction temperature of 65 ° C and absolute pressure of 2.0 mbar, magnetic stirring was carried out at 300 r / min. At 2 h and 4 h of reaction, an equal amount of deionized water was added to the system, and the addition amount was set to 0, 1%, 3%, 5%, 7%, and 9%, and the total reaction time was 6 h.
[0052] After the reaction was completed, the supernatant was taken by centrifugation at 1000 r / min for 5 min to remove the lipase and obtain the crude reaction product.
[0053] The lipid composition and content in the crude reaction product were detected by HPLC-RID.
[0054] The effect of the amount of deionized water added in the enzymatic reaction on the glyceride content in the crude reaction product (without molecular distillation treatment) is shown in Table 3.
[0055] Table 3 Comparison of lipid content in crude reaction products at different ion water addition amounts
[0056] Water addition / % Triglycerides Diacylceride Monoglyceride fatty acid Ethyl ester 0 72.27％ 12.14％ 0.88％ 1.72％ 13.01％ 1％ 78.20％ 9.24％ 0.22％ 1.52％ 8.82％ 3％ 82.11％ 7.51％ 0.34％ 2.27％ 7.77％ 5％ 83.10％ 6.01％ 0.67％ 3.35％ 6.87％ 7％ 84.34％ 5.55％ 0.52％ 3.82％ 5.77％ 9％ 73.27％ 4.87％ 0.77％ 13.13％ 7.96％
[0057] It can be seen from Table 3 that when 7% deionized water is added every 2 hours, the catalytic effect is the best, and the triglyceride content is as high as 84.34%.
[0058] Example 3
[0059] This example explores the effect of different addition times of deionized water on the glyceride content in the crude reaction product. This example includes 12 groups of experiments, each of which differs in that the time intervals for adding water to the reaction system are different, but the content of water added each time is the same.
[0060] In a 25 mL conical flask, 2 g of fish oil ethyl ester and 0.173 g of glycerol (glycerol: ethyl ester = 1:3.5, molar ratio) were added, and then lipase Candida antarctica was added at an enzyme addition amount of 6% (based on the mass fraction of fish oil ethyl ester). After mixing evenly, the reaction temperature was 65°C and the absolute pressure was 2.0 mbar. The magnetic stirring was 300 r / min, and 7% deionized water was added to the system at different times during the reaction. The total reaction time was 6 h, and the times were set to 0.25 h, 0.5 h, 0.75 h, 1 h, 1.25 h, 1.5 h, 1.75 h, 2 h, 2.25 h, 2.5 h, 2.75 h, and 1.5 h, respectively.
[0061] The effect of the addition time of deionized water in the enzymatic reaction on the triglyceride content in the crude reaction product (without molecular distillation treatment) is shown in Table 4.
[0062] Table 4 Comparison of lipid content in crude reaction products at different addition times
[0063]
[0064]
[0065] As shown in Table 4, under the condition of adding water every 1 to 2.75 hours, the triglyceride content in the obtained crude product was higher than 80%, and the highest triglyceride content in the crude product was 84.34% when the interval was 2 hours.
[0066] Example 4
[0067] This example explores the effects of different deionized water addition methods on the glyceride content in the crude reaction product. This example includes two groups of experiments, the difference between the two groups of experiments is that the interval between water addition and the amount of water added are different, but the total amount of water added is the same.
[0068] In a 25 mL conical flask, 2 g of fish oil ethyl ester and 0.173 g of glycerol (glycerol: ethyl ester = 1:3.5, molar ratio) were added, and then lipase Candida antarctica was added at an enzyme addition amount of 6% (based on the mass fraction of fish oil ethyl ester). After mixing evenly, the reaction temperature was 65 ° C, the absolute pressure was 2.0 mbar, and magnetic stirring was performed at 300 r / min. During the reaction, deionized water was added to the system in a total addition amount of 10%, and the total reaction time was 6 h.
[0069] There are two schemes for adding deionized water: scheme 1: adding 2% deionized water at 1h, 2h, 3h, 4h, and 5h respectively; scheme 2: adding 5% deionized water at 2h and 4h respectively.
[0070] After the reaction was completed, the supernatant was taken by centrifugation at 1000 r / min for 5 min to remove the lipase and obtain the crude reaction product.
[0071] The experiment without adding deionized water was used as the control group, and the lipid composition and content in the crude reaction product were detected by HPLC-RID.
[0072] The effect of the addition method of deionized water in the enzymatic reaction on the glyceride content in the crude reaction product is shown in Table 5.
[0073] Table 5 Comparison of lipid content in crude reaction products under different deionized water addition methods
[0074] Triglycerides Diacylceride Monoglyceride fatty acid Ethyl ester No deionized water added 74.27％ 12.14％ 0.88％ 1.72％ 11.01％ Solution 1 78.84％ 8.53％ 0.59％ 2.21％ 9.83％ Solution 2 83.10％ 6.01％ 0.67％ 3.35％ 6.87％
[0075] It can be seen from Table 5 that although the total amount of water added in Scheme 1 and Scheme 2 is the same, the frequency of water addition is different, and the effect on improving the triglyceride content is limited; adding deionized water in the manner of Scheme 2, that is, adding 5% deionized water at 2h and 4h respectively, the ester exchange effect is the best, and the ester exchange effect of adding deionized water is significantly higher than that without adding water.
[0076] Example 5
[0077] This example explores the effects of different non-glycerides on the glyceride content in the crude reaction product. This example includes 4 groups of experiments, and the difference between the 4 groups of experiments is that the types of non-glycerides are different.
[0078] In a 25 mL conical flask, 2 g of non-glyceride was added, and the non-glyceride esters were fatty acid methyl ester, fatty acid ethyl ester, fatty acid isopropanol ester and fatty acid, and 0.173 g of glycerol was added (glycerol: non-glyceride molar ratio = 1:3.5), and then lipase Candida antarctica was added, and the enzyme addition amount was 8% (based on the mass fraction of fish oil ethyl ester). After mixing evenly, under the conditions of reaction temperature of 65 ° C and absolute pressure of 2.0 mbar, magnetic stirring was carried out at 300 r / min, and 5% (based on the mass fraction of fish oil ethyl ester) of deionized water was added to the system at 2h and 4h of the reaction, respectively, and the total reaction time was 6h.
[0079] After the reaction was completed, the supernatant was taken by centrifugation at 1000 r / min for 5 min, and the lipase was removed to obtain a crude reaction product. The lipid composition and content in the crude reaction product were detected by HPLC-RID.
[0080] The experiment without adding deionized water was used as the control group.
[0081] The effect of the types of non-glycerides in the enzymatic reaction on the triglyceride content in the crude reaction product (without molecular distillation treatment) is shown in Table 6.
[0082] Table 6 Effect of different non-glycerides on the content of triglycerides in the crude reaction product
[0083] Non-glycerides Methyl ester Ethyl ester Isopropyl alcohol ester fatty acid No deionized water added 76.51％ 74.27％ 54.21％ 78.33％ Add deionized water 83.68％ 83.10％ 67.39％ 84.72％
[0084] It can be seen from Table 6 that when methyl ester is selected as non-glycerol lipid, the triglyceride content in the crude product is the highest; the intermittent addition of deionized water selectively promotes the conversion of fatty acid methyl ester, ethyl ester, isopropanol ester and fatty acid into triglycerides, among which the content of methyl ester, ethyl ester and fatty acid group triglyceride exceeds 83%; when fatty acids are used as raw materials, the intermittent addition of water significantly increases the triglyceride content in the crude reaction product. In the esterification reaction of fatty acids and glycerol, many studies have adopted the method of vacuuming or passing inert gas to remove the water produced by the reaction to promote the reaction. In the present invention, we found an unexpected effect by intermittently adding water to the reaction system. This method of adding water not only has no inhibitory effect on the esterification reaction, but can promote the reaction.
[0085] Example 6
[0086] This example explores the effect of different lipases on the glyceride content in the crude reaction product. This example includes 6 groups of experiments, and the difference between the 6 groups of experiments is that the types of lipases are different.
[0087] In a 25 mL conical flask, 2 g of fish oil ethyl ester, 0.173 g of glycerol (glycerol: ethyl ester = 1:3, molar ratio) and lipase were added. The lipase was Candida antarctica, C. cylindracea, Rhizomucor miehei, Thermomyces lanuginosus, Pseudomonas cepacia or Aspergilus oryzae. The enzyme addition amount was 8% (based on the mass fraction of fish oil ethyl ester). After mixing evenly, the reaction was carried out at a reaction temperature of 65°C and an absolute pressure of 2.0 mbar with magnetic stirring at 300 r / min for 10 h.
[0088] After the reaction was completed, the supernatant was taken by centrifugation at 1000 r / min for 5 min to remove the lipase and obtain the crude reaction product.
[0089] The lipid composition and content in the crude reaction product were detected by HPLC-RID.
[0090] The effect of the type of lipase in the enzymatic reaction on the glyceride content in the crude reaction product (without molecular distillation treatment) is shown in Table 7.
[0091] Table 7 Comparison of glyceride content in crude reaction products under different lipase types
[0092] Lipase Type Triglycerides Diacylceride Monoglyceride fatty acid Ethyl ester Candida antarctica 75.41％ 16.34％ 0.46％ 2.25％ 5.55％ C. cylindracea 73.59％ 16.45％ 0.83％ 1.74％ 7.39％ Rhizomucormiehei 4.21％ 21.10％ 2.72％ 1.74％ 70.23％ Thermomyces lanuginosus 1.87％ 0.74％ 0.34％ 0.00％ 97.06％ Pseudomonas cepacia 10.28％ 16.54％ 1.03％ 0.97％ 71.18％ Aspergillus oryzae 1.16％ 0.64％ 0.36％ 0.64％ 97.20％
[0093] As shown in Table 7, the transesterification activity of lipases Candida antarctica and C. cylindracea is relatively high. The transesterification activity of lipases Rhizomucormiehei, Thermomyces lanuginosus, Pseudomonas cepacia and Aspergilus oryzae is significantly weaker than that of lipases Candida antarctica and C. cylindracea. Among them, the crude product catalyzed by lipase Candida antarctica has the highest triglyceride content of 75.41%.
[0094] Example 7
[0095] This example explores the effect of different absolute pressures of enzymatic reactions on the glyceride content in the crude reaction product. This example includes 6 groups of experiments, and the difference between the 6 groups of experiments is that the absolute pressures of the reactions are different.
[0096] In a 25 mL conical flask, 2 g of fish oil ethyl ester and 0.173 g of glycerol (glycerol: ethyl ester = 1:3, molar ratio) were added, and then lipase Candida antarctica was added, and the enzyme addition amount was 8% (based on the mass fraction of fish oil ethyl ester). After mixing evenly, the reaction temperature was 65 ° C, and the reaction was carried out at different absolute pressure conditions with magnetic stirring at 300 r / min for 10 h. The absolute pressures were set to 2.0, 3.0, 4.5, 5.0, 10.0, and 20.0 mbar respectively;
[0097] After the reaction was completed, the supernatant was taken by centrifugation at 1000 r / min for 5 min to remove the lipase and obtain the crude reaction product.
[0098] The effect of absolute pressure in the enzymatic reaction on the glyceride content in the crude reaction product is shown in Table 8.
[0099] Table 8 Comparison of glyceride content in crude reaction products at different absolute pressures
[0100] Absolute pressure / mbar Triglycerides Diacylceride Monoglyceride fatty acid Ethyl ester 2.0 75.41％ 16.34％ 0.46％ 2.25％ 5.55％ 3.0 72.56％ 15.40％ 1.55％ 2.59％ 7.90％ 4.0 72.45％ 15.15％ 0.94％ 2.26％ 9.21％ 5.0 73.17％ 18.19％ 0.84％ 2.60％ 5.11％ 10.0 64.01％ 20.78％ 0.43％ 2.37％ 12.40％ 20.0 51.64％ 15..71％ 3.31％ 5.28％ 24.06％
[0101] It can be seen from Table 8 that when the absolute pressure is 2.0-5.0 mbar, the effect of transesterification is more significant, and the triglyceride content in the obtained crude reaction product exceeds 70%. Among them, when the absolute pressure is 2.0 mbar, the triglyceride content is the highest, which is 75.41%.
[0102] Example 8
[0103] This example explores the effect of different molar ratios of fish oil ethyl ester and glycerol on the glyceride content in the crude reaction product. This example includes 5 groups of experiments, and the difference between the 5 groups of experiments is that the substrate molar ratio is different.
[0104] 2 g of fish oil ethyl ester and different contents of glycerol were added to a 25 mL conical flask, with the molar ratio of glycerol:ethyl ester being 2.8, 3.0, 3.5, 4.0, and 5.0, and then lipase Candida antarctica was added, with the enzyme addition amount being 8% (based on the mass fraction of fish oil ethyl ester). After mixing evenly, the reaction was carried out at a reaction temperature of 65° C. and an absolute pressure of 2.0 mbar with magnetic stirring at 300 r / min for 10 h.
[0105] After the reaction was completed, the supernatant was taken by centrifugation at 1000 r / min for 5 min to remove the lipase and obtain the crude reaction product.
[0106] The lipid composition and content in the crude reaction product were detected by HPLC-RID.
[0107] The effect of the molar ratio of fish oil ethyl ester to glycerol (fish oil ethyl ester: glycerol) in the enzymatic reaction on the glyceride content in the crude reaction product (without molecular distillation treatment) is shown in Table 9.
[0108] Table 9 Comparison of glyceride content in crude reaction products at different molar ratios of fish oil ethyl ester and glycerol
[0109]
[0110]
[0111] It can be seen from Table 9 that when the molar ratio of fish oil ethyl ester to glycerol is 3.5, the effect of transesterification is most significant, and the content of triglyceride in the obtained crude reaction product is as high as 84.58%.
[0112] Example 9
[0113] This example explores the effect of different enzymatic reaction temperatures on the glyceride content in the crude reaction product. This example includes 4 groups of experiments, and the difference between the 4 groups of experiments is: different reaction temperatures.
[0114] In a 25 mL conical flask, 2 g of fish oil ethyl ester and 0.173 g of glycerol (glycerol: ethyl ester = 1:3.5, molar ratio) were added, and then lipase Candida antarctica was added, and the enzyme addition amount was 8% (based on the mass fraction of fish oil ethyl ester). After mixing evenly, the absolute pressure was 2.0 mbar, the reaction temperature was set to 45, 55, 65, and 75 ° C, and the magnetic stirring was 300 r / min for 10 hours;
[0115] After the reaction was completed, the supernatant was taken by centrifugation at 1000 r / min for 5 min to remove the lipase and obtain the crude reaction product.
[0116] The lipid composition and content in the crude reaction product were detected by HPLC-RID.
[0117] The effect of temperature on the glyceride content in the crude product of the enzymatic reaction is shown in Table 10.
[0118] Table 10 Comparison of glyceride content in crude reaction products at different temperatures
[0119] Temperature / ℃ Triglycerides Diacylceride Monoglyceride fatty acid Ethyl ester 45 58.72％ 26.78％ 1.02％ 0.50％ 12.38％ 55 63.66％ 22.79％ 0.82％ 0.44％ 12.69％ 65 84.58％ 4.87％ 0.00％ 1.69％ 8.86％ 75 85.08％ 9.54％ 0.40％ 0.28％ 5.09％
[0120] As shown in Table 10, under the conditions of 65°C and 75°C, the triglyceride content in the crude product exceeded 84.5%, but under the condition of 65°C, the total content of diglyceride and monoglyceride was lower, so the triglyceride content in the product obtained after molecular distillation was higher. And under the condition of 65°C, the stability of lipase was higher.
[0121] Example 10
[0122] This example explores the changes in the glyceride content in the crude product at different reaction times during the enzymatic reaction.
[0123] In a 25 mL conical flask, 2 g of fish oil ethyl ester and 0.173 g of glycerol (glycerol: ethyl ester = 1:3.5, molar ratio) were added, and then lipase Candida antarctica was added at an enzyme addition amount of 6%. After mixing evenly, the reaction temperature was 65 ° C, the absolute pressure was 2.0 mbar, the magnetic stirring speed was 300 r / min, and samples were taken at different reaction times;
[0124] After the reaction was completed, the supernatant was taken by centrifugation at 1000 r / min for 5 min to remove the lipase and obtain the crude reaction product.
[0125] The lipid composition and content in the crude reaction product were detected by HPLC-RID.
[0126] The effect of enzymatic reaction time on the triglyceride content in the crude reaction product (without molecular distillation treatment) is shown in Table 11.
[0127] Table 11 Comparison of triglyceride content in crude reaction products at different reaction times
[0128] Reaction time 2h 4h 6h 8h 10h 12h 14h 16h Triglycerides 35.67％ 62.21％ 74.28％ 79.75％ 84.13％ 84.66％ 85.21％ 85.74％
[0129] It can be seen from Table 11 that the longer the reaction time, the higher the triglyceride content, but the 6-h reaction basically reached an equilibrium state, so 6 h is the optimal reaction time.
[0130] Embodiment 11
[0131] This example is used to evaluate the reusability of lipase in the method of enzymatically synthesizing a high-content EPA / DHA glyceride product with lipid-lowering function.
[0132] In a 25 mL conical flask, 2 g of fish oil ethyl ester and 0.173 g of glycerol (glycerol: ethyl ester = 1:3.5, molar ratio) were added, and then lipase Candida antarctica or C. cylindracea was added, and the enzyme addition amount was 6% (based on the mass fraction of fish oil ethyl ester). After mixing evenly, the reaction temperature was 65 ° C, the absolute pressure was 2.0 mbar, and the magnetic stirring was 300 r / min. During the reaction process, 7% deionized water was added to the system every 2 hours, and the total reaction time was 6 hours;
[0133] After the reaction was completed, the supernatant was taken by centrifugation at 1000 r / min for 5 min to remove the lipase and obtain the crude reaction product.
[0134] The isolated lipase was used for the next set of transesterification experiments, and the other conditions were consistent with the previous set of experimental conditions.
[0135] The lipid composition and content in the crude reaction product were detected by HPLC-RID.
[0136] The reuse of different lipases is shown in Table 12.
[0137] Table 12 Comparison of triglyceride content in crude products of lipase-catalyzed reactions at different repetition times
[0138]
[0139] It can be seen from Table 12 that after being reused 10 times, the content of triglyceride was still higher than 80% under the catalysis of lipase, indicating that the lipase Candida antarctica and C. cylindracea have good reusability.
[0140] Comparative Example 1
[0141] The difference between this comparative example and Example 7 is that the enzymatic reaction is carried out under normal pressure, and the other conditions are consistent with Example 7.
[0142] Figure 2 The composition and content of lipids in the crude product of this comparative example reaction, the triglyceride content in the obtained crude lipid product is only 5.54%. Therefore, the enzymatic reaction must be carried out under vacuum conditions to achieve a high yield of triglycerides in the product.
[0143] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.
Claims
1. A method for synthesizing high-content EPA / DHA glycerides with lipid-lowering function based on an aqueous system, characterized in that: include, The non-glyceride, glycerol and lipase are mixed, and deionized water is added intermittently to carry out an enzymatic reaction; After the reaction is completed, the lipase is removed to obtain a crude reaction product, which is then separated and purified to obtain a glyceride product.
2. The method for synthesizing high-content EPA / DHA glycerides with lipid-lowering function based on an aqueous system as claimed in claim 1, characterized in that: The intermittent addition of deionized water is to add deionized water to the reaction system every 1 to 2.75 hours.
3. The method for synthesizing high-content EPA / DHA glyceride with blood lipid lowering function based on an aqueous system as claimed in claim 2, characterized in that: The amount of deionized water added each time is 3% to 7% of the mass of the non-glyceride.
4. The method for synthesizing high-content EPA / DHA glyceride with lipid-lowering function based on an aqueous system as claimed in claim 1, characterized in that: The added amount of the lipase is 6% to 8% of the mass of the non-glyceride.
5. The method for synthesizing high-content EPA / DHA glyceride with blood lipid lowering function based on an aqueous system as claimed in claim 4, characterized in that: The non-glyceride includes one or more of fatty acids, fatty acid ethyl esters, fatty acid methyl esters and fatty acid isopropanol esters.
6. The method for synthesizing high-content EPA / DHA glyceride with blood lipid lowering function based on an aqueous system as claimed in claim 1, characterized in that: The molar ratio of the non-glyceride to glycerol is (2.8-5):
1.
7. The method for synthesizing high-content EPA / DHA glyceride with blood lipid lowering function based on an aqueous system as claimed in claim 1, characterized in that: The temperature of the enzymatic reaction is 45-75°C.
8. The method for synthesizing high-content EPA / DHA glyceride with lipid-lowering function based on an aqueous system as claimed in claim 7, characterized in that: The absolute pressure of the enzymatic reaction is 2.0-5.0 mbar.
9. The method for synthesizing high-content EPA / DHA glyceride with blood lipid lowering function based on an aqueous system as claimed in claim 8, characterized in that: The enzymatic reaction time is 4 to 16 hours.
10. The method for synthesizing high-content EPA / DHA glyceride with blood lipid lowering function based on an aqueous system according to claim 4, characterized in that: The lipase is derived from one or more strains of Candida antarctica, C. cylindracea, Rhizomucormiehei, Thermomyces lanuginosus, Pseudomonas cepacia, and Aspergilus oryzae.