CN113981018A - Method for preparing n-3 polyunsaturated fatty acid glyceride by enzyme method - Google Patents
Method for preparing n-3 polyunsaturated fatty acid glyceride by enzyme method Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
- A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
- A23D9/007—Other edible oils or fats, e.g. shortenings, cooking oils characterised by ingredients other than fatty acid triglycerides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6454—Glycerides by esterification
Abstract
The invention discloses a method for preparing n-3 polyunsaturated fatty acid glyceride by an enzyme method, and belongs to the technical field of oil processing. The invention firstly utilizes an adsorption method to reduce impurities such as peroxide value of reaction substrate n-3 polyunsaturated fatty acid non-glyceride and the like so as to achieve the purpose of reducing the influence of the impurities on the activity of lipase in the enzymatic reaction process, thereby improving the catalytic efficiency of the lipase in the enzymatic transesterification process and reducing the cost of preparing n-3 polyunsaturated fatty acid glyceride by an enzymatic method. The invention provides a method for improving the efficiency and yield of synthesizing n-3 polyunsaturated fatty glyceride by an enzyme method, which avoids the problem that the enzyme activity of lipase is easy to inactivate in the reaction process.
Description
Technical Field
The invention belongs to the technical field of grease processing, and particularly relates to a method for preparing n-3 polyunsaturated fatty glyceride by an enzyme method.
Background
n-3 polyunsaturated fatty acids (n-3PUFA) are polyunsaturated fatty acids in which the first unsaturated double bond is present between the third and fourth carbon atoms at the methyl end of the carbon chain. n-3PUFA is not only an important component of biological lipids, but also plays an important physiological role in the body due to its specific double bond structure.
The n-3PUFA has very important physiological activity function and health care function in human body, and plays an important role in regulating metabolism of human body. For example, EPA (Eicosapentaenoic Acid) mainly plays a role in the prevention and treatment of cardiovascular and cerebrovascular diseases and inflammation. EPA can reduce thromboxane formation by inhibiting platelet aggregation and increasing platelet cell membrane fluidity, thereby having the function of preventing myocardial infarction and cerebral infarction; EPA has functions of reducing blood lipid and cholesterol, and can be used for preventing and treating atherosclerosis of porridge; EPA has anti-inflammatory effect and can inhibit anaphylaxis. DHA (Docosahexaenoic Acid) can enhance the function of nervous system, and has good effects in protecting eyesight, preventing senile dementia, promoting infantile brain development and inhibiting tumor. DHA is the main component of retina (40-50%), and can prevent vision deterioration and activate weak retinal cells; is the main composition matter of human brain (accounting for 10% of human brain fat), can promote the growth and development of brain cells, prevent and treat senile dementia to a certain extent, and can promote intelligence development and improve brain function when being added into infant formula food; DHA can inhibit the growth and metastasis of cancer cells and reduce the growth rate of tumors.
There are three main forms of presence of n-3 PUFA: glyceride type, ethyl ester type and free fatty acid type. Researches indicate that the rate of ester bonds in ethyl ester hydrolyzed by the pancreatic lipase is 10-50 times lower than that of ester bonds in glyceride hydrolyzed, so that the ethyl ester is difficult to digest and absorb in vivo; meanwhile, ethyl ester type n-3PUFA can generate ethanol after being hydrolyzed in vivo, and certain potential safety hazard exists. The free n-3PUFA is fast in digestion and absorption, but is easy to oxidize to generate harmful substances and generate sour taste, so that the free n-3PUFA is not easy to accept by consumers. Compared with an ethyl ester type and a free fatty acid type, the glyceride type is not only easy to digest and absorb by human bodies and has good oxidation stability, but also has high consumer acceptance as a naturally existing form of n-3PUFA, so that the preparation of the high-purity glyceride type n-3PUFA is the key point of future research, and the development of a green and efficient novel method for enriching n-3PUFA glyceride has important practical significance.
Currently, the preparation method of n-3 polyunsaturated fatty acid glyceride comprises a direct esterification method of glycerin and n-3 free fatty acid ethyl ester or methyl ester and a partial hydrolysis method of oil rich in n-3 polyunsaturated fatty acid. In the former method, n-3 polyunsaturated fatty acid methyl ester or ethyl ester is purified in advance to obtain high-content n-3 polyunsaturated fatty acid non-glyceride, so that a glyceride product with higher n-3 polyunsaturated fatty acid content can be obtained, but the main obstacle in industrial application for restricting the enzymatic preparation of n-3 polyunsaturated fatty acid glyceride is that the lipase is very expensive, and if impurities in a reaction system have great influence on the enzyme activity of the lipase, the repeated use frequency of the lipase is low, the catalytic efficiency is reduced, and the production cost is correspondingly high.
Disclosure of Invention
[ problem ] to
Raw material n-3 polyunsaturated fatty acid methyl ester or ethyl ester (non-glyceride) for preparing n-3 polyunsaturated fatty acid glyceride by an enzyme method is easy to oxidize due to a plurality of double bonds, more impurities such as peroxide are generated, the existence of the impurities such as oxidation products possibly influences the catalytic effect of lipase, and the preparation cost of the n-3 polyunsaturated fatty acid glyceride is increased.
[ solution ]
The invention reduces the peroxide value of the adopted n-3 polyunsaturated fatty acid non-glyceride substrate, and then prepares the n-3 polyunsaturated fatty acid glyceride by using an enzymatic transesterification method, the enzymatic preparation process route obviously increases the repeated use times of the lipase, reduces the cost of large-scale preparation, and has wide market application prospect.
Specifically, the invention provides the following technical scheme: a process for the enzymatic preparation of n-3 polyunsaturated fatty acid glycerides, the process comprising the steps of:
s1, raw material treatment: subjecting the non-glyceride rich in n-3 polyunsaturated fatty acid to peroxide value reduction treatment, wherein the peroxide value of the non-glyceride rich in n-3 polyunsaturated fatty acid is reduced to not more than 4 mmol/kg;
s2, preparation of n-3 polyunsaturated fatty acid glyceride: mixing the non-glyceride which is obtained in the step S1 and is subjected to peroxide number reduction treatment and is rich in n-3 polyunsaturated fatty acid with an acyl acceptor, reacting under the catalysis of lipase, removing the lipase and the acyl acceptor after reacting for a certain time to obtain n-3 polyunsaturated fatty acid glyceride, wherein the acyl acceptor is glycerol and/or monoglyceride.
Preferably, in the step S1, the non-glyceride rich in n-3 polyunsaturated fatty acids includes one or more of fatty acid methyl esters and fatty acid ethyl esters.
Preferably, in the S1 step, the non-glyceride rich in n-3 polyunsaturated fatty acids includes one or more of ethyl alpha-linolenate, ethyl DHA, methyl DHA, ethyl EPA, and methyl EPA.
Preferably, the method for reducing the peroxide value of the non-glyceride rich in n-3 polyunsaturated fatty acids in step S1 includes an adsorption method.
Preferably, the adsorbent of the adsorption method comprises one or more of silica gel, activated clay, activated carbon, silicon dioxide and attapulgite, and preferably silica gel and activated carbon.
Preferably, the adsorption method comprises static adsorption or dynamic adsorption.
Preferably, the static adsorption means that the reaction substrate and the adsorbent are stirred and mixed.
Preferably, the dynamic adsorption is column chromatography adsorption.
Preferably, the adding amount of the adsorbent is 0.2-2% of the mass of the substrate, and preferably 0.5-1%; the adsorption time is 15min to 120min, preferably 20min to 60 min.
Preferably, the adsorption may be carried out at normal temperature, or may be carried out at elevated temperature without destroying the non-glyceride structure rich in n-3 polyunsaturated fatty acids.
Preferably, the adsorption is carried out under normal pressure, or under pressure or a vacuum without destroying the non-glyceride structure rich in the n-3 polyunsaturated fatty acid.
Preferably, the molar ratio of non-glyceride of n-3 polyunsaturated fatty acids to acyl acceptor is not less than 2: 1.
preferably, the lipase used in step S2 includes, but is not limited to, any one or more of lipases derived from Candida antarctica (Candida antarctica), Penicillium (Penicillium camembertii), burkholderia cepacia (burkholderia cepacia), Pseudomonas fluorescens (Pseudomonas fluorescens).
Preferably, the Lipase comprises Novozym435, Lipozyme435 and/or Lipase CL "Amano" IM from Candida antarctica (Candida antarctica); lipase AK derived from Pseudomonas fluorescens (Pseudomonas fluorescens); lipase G "Amano" 50 from Penicillium camembertii (Penicillium camembertii); any one or more of Lipase PS "Amano" SD lipases from Burkholderia cepacia (Burkholderia cepacia);
preferably, the preparation method of the n-3 polyunsaturated fatty acid glyceride is a batch or continuous preparation process.
Preferably, the addition amount of the lipase is 2 to 12% of the total mass of the reaction substrate.
Preferably, the reaction temperature of the n-3 polyunsaturated fatty acid glyceride in the step S2 is 30-70 ℃, and the reaction time is 4-24 h.
More preferably, the reaction temperature of the n-3 polyunsaturated fatty acid glyceride in the step S2 is 50-70 ℃, and the reaction time is 6-20 h.
Preferably, in step S2, the pressure of the reaction system for preparing the n-3 polyunsaturated fatty acid glycerides is less than 500Pa, preferably less than 200 Pa.
Preferably, in step S2, the n-3 polyunsaturated fatty acid content of the non-glyceride n-3 polyunsaturated fatty acids is greater than 30%.
The invention also provides the application of the method in preparing diglyceride or concentrated fish oil.
The invention has the beneficial effects that:
the n-3 polyunsaturated fatty acid and the derivative thereof are easy to oxidize, and the invention firstly carries out peroxide value reduction treatment on a reaction substrate required by preparing the n-3 polyunsaturated fatty glyceride, thereby obviously increasing the use effect and the catalytic efficiency of lipase, reducing the cost of industrially preparing the n-3 polyunsaturated fatty glyceride and having important industrial value.
Detailed Description
The present invention is further described below with reference to examples, but the embodiments of the present invention are not limited thereto.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
1. Method for analysis of glycerides: methods and analysis conditions for HPLC analysis of diglycerides refer to the zhang yu paper (zhang yu. preparation of DHA and 2-DHA-monoglycerides purification and comparative study of regulation of lipid metabolism in HepG2 cells [ D ]. doctor academic thesis of south china university, 2019).
The glyceride yield calculation method is as follows:
the n-3 polyunsaturated fatty acid glycerides referred to in the present invention are diglycerides and triglycerides rich in n-3 polyunsaturated fatty acids.
2. Peroxide value analysis method: the peroxide value is analyzed by referring to a method of GB5009.227-2016 determination of peroxide value in food safety national standard food.
3. A method for removing ethyl ester, glycerol, monoglyceride and the like in a crude product of an enzymatic transesterification (molecular distillation method): reference is made to the method of the xundi master academic paper (xundi. enzymatic preparation of human milk substitute lipids enriched in medium-long carbon chain triglycerides [ D ]. south china master academic paper, 2019).
Example 1: (molar ratio of substrate)
(1) Treating the raw materials by reducing peroxide number: carrying out static adsorption treatment on fish oil fatty acid ethyl ester with peroxide value of 9.4mmol/kg (the total content of DHA and EPA is 71.3%), wherein the static adsorption conditions are as follows: fully mixing 20g of fish oil fatty acid ethyl ester and 1% of silica gel, stirring and adsorbing at normal temperature for 25min, and filtering to remove the adsorbent to obtain the fish oil fatty acid ethyl ester with the peroxide value reduced, wherein the peroxide value is 1.3 mmol/kg.
(2) Enzymatic synthesis of n-3 polyunsaturated fatty acid glycerides: adding glycerol into fatty acid ethyl ester subjected to peroxide reduction treatment according to a certain molar ratio, then adding lipase Lipozyme435 derived from Candida antarctica (Candida antarctica) with the enzyme addition amount of 10% (w/w, relative to the total mass of reactants), mixing the two materials in a 50mL reactor, quickly heating to 65 ℃, reacting for 8 hours, and controlling the pressure of a reaction system to be 300 Pa. After the ester exchange reaction is finished, recovering lipase, removing ethyl ester, monoglyceride and the like through molecular distillation to obtain n-3 polyunsaturated fatty glyceride, detecting the content of glyceride in the purified product by using HPLC, and calculating the yield of glyceride.
The influence of the molar ratio of the substrate in the step (2) on the yield of the n-3 polyunsaturated fatty acid glyceride is studied, and the result is shown in table 1, so that as the molar ratio of the fatty acid ethyl ester to the glycerol is gradually increased, the total yield of the diglyceride and the triglyceride obtained after the crude product is purified is increased, and when the molar ratio of the fatty acid ethyl ester to the glycerol is more than or equal to 2:1, the yield of the polyunsaturated fatty acid glyceride is higher.
TABLE 1 influence of the molar ratio of the reaction substrate in the step (2) on the yield of n-3 polyunsaturated fatty acid glycerides
Example 2: (reaction time optimization)
(1) Treating the raw materials by reducing peroxide number: carrying out static adsorption treatment on fish oil fatty acid ethyl ester with peroxide value of 9.4mmol/kg (the total content of DHA and EPA is 71.3%), wherein the static adsorption conditions are as follows: fully mixing 20g of fish oil fatty acid ethyl ester and 1% of silica gel, stirring and adsorbing at 40 ℃ for 25min, and filtering to remove the adsorbent to obtain the fish oil fatty acid ethyl ester with the peroxide value reduced, wherein the peroxide value is 1.1 mmol/kg.
(2) Enzymatic synthesis of n-3 polyunsaturated fatty acid glycerides: adding glycerol into fatty acid ethyl ester subjected to peroxide number reduction treatment in a molar ratio of 3:1, then adding lipase Novozym435 derived from Candida antarctica (Candida antarctica) in an amount of 5% (w/w, relative to the total mass of reactants), mixing the two in a 50mL reactor, rapidly heating to 65 ℃, and reacting for a period of time, wherein the pressure of a reaction system is controlled at 300 Pa. After the ester exchange reaction is finished, recovering lipase, removing ethyl ester, monoglyceride and the like through molecular distillation to obtain n-3 polyunsaturated fatty glyceride, detecting the content of glyceride in the purified product by using HPLC, and calculating the yield of glyceride.
The influence of the molar ratio of the substrate on the yield of the n-3 polyunsaturated fatty acid glyceride in the step (2) is studied, and the result is shown in table 2, so that the total yield of the diglyceride and the triglyceride obtained after purification is increased along with the extension of the transesterification reaction time, and the polyunsaturated fatty acid glyceride has higher yield when the reaction time is more than 4 hours.
TABLE 2 Effect of the reaction time of step (2) on the yield of n-3 polyunsaturated fatty acid glycerides
Example 3: (optimization of reaction temperature)
(1) Treating the raw materials by reducing peroxide number: carrying out static adsorption treatment on fish oil fatty acid ethyl ester with peroxide value of 9.4mmol/kg (the total content of DHA and EPA is 71.3%), wherein the static adsorption conditions are as follows: and (3) fully mixing 20g of fish oil fatty acid ethyl ester and 1% of silica gel, stirring and adsorbing at the temperature of 45 ℃ for 25min under the pressure of an adsorption system less than 4kPa, and filtering to remove the adsorbent to obtain the fish oil fatty acid ethyl ester with the peroxide value reduced, wherein the peroxide value is 0.92 mmol/kg.
(2) Enzymatic synthesis of n-3 polyunsaturated fatty acid glycerides: adding glycerol into the fatty acid ethyl ester subjected to peroxide number reduction treatment in a molar ratio of 3:1, then adding lipase Novozym435 derived from Candida antarctica (Candida antarctica) with the enzyme addition amount of 5% (w/w, relative to the total mass of reactants), mixing the two in a 50mL reactor, rapidly heating to a preset temperature, reacting for 8h, and controlling the pressure of the reaction system to be 300 Pa. After the ester exchange reaction is finished, recovering lipase, removing ethyl ester, monoglyceride and the like through molecular distillation to obtain n-3 polyunsaturated fatty glyceride, detecting the content of glyceride in the purified product by using HPLC, and calculating the yield of glyceride.
The influence of the reaction temperature in the step (2) on the yield of the n-3 polyunsaturated fatty acid glyceride is researched, the result is shown in table 3, it can be seen that the total yield of diglyceride and triglyceride tends to increase firstly and then decrease with the increase of the transesterification reaction temperature, and when the reaction temperature is higher than 70 ℃, the yield of the polyunsaturated fatty acid glyceride gradually decreases on the contrary, mainly because the stability of the lipase is influenced by high temperature, so that the enzyme activity is reduced.
TABLE 3 influence of the reaction temperature in the step (2) on the yield of n-3 polyunsaturated fatty acid glycerides
Example 4: (reaction System pressure optimization)
(1) Treating the raw materials by reducing peroxide number: carrying out static adsorption treatment on fish oil fatty acid ethyl ester with peroxide value of 9.4mmol/kg (the total content of DHA and EPA is 71.3%), wherein the static adsorption conditions are as follows: and (3) fully mixing 20g of fish oil fatty acid ethyl ester and 1% of silica gel, stirring and adsorbing at the temperature of 45 ℃ for 25min under the pressure of an adsorption system less than 4kPa, and filtering to remove the adsorbent to obtain the fish oil fatty acid ethyl ester with the peroxide value reduced, wherein the peroxide value is 0.92 mmol/kg.
(2) Enzymatic synthesis of n-3 polyunsaturated fatty acid glycerides: adding glycerol into the fatty acid ethyl ester subjected to peroxide number reduction treatment in a molar ratio of 3:1, then adding lipase Novozym435 derived from Candida antarctica (Candida antarctica) with the enzyme addition amount of 5% (w/w, relative to the total mass of reactants), mixing the two in a 50mL reactor, rapidly heating to 65 ℃, and reacting for 8 hours, wherein the pressure of the reaction system is controlled within a preset range. After the ester exchange reaction is finished, recovering lipase, removing ethyl ester, monoglyceride and the like through molecular distillation to obtain n-3 polyunsaturated fatty glyceride, detecting the content of glyceride in the purified product by using HPLC, and calculating the yield of glyceride.
The influence of the pressure of the reaction system in the step (2) on the yield of the n-3 polyunsaturated fatty acid glyceride is researched, and the result is shown in table 4, so that the total yield of the diglyceride and the triglyceride is gradually increased along with the reduction of the pressure of the transesterification reaction system, when the reaction pressure is less than 500Pa, the purified n-3 polyunsaturated fatty acid glyceride has higher yield, and according to the result in the table, the pressure of the reaction system can be selected to be not higher than 500Pa, preferably not higher than 200 Pa.
TABLE 4 influence of the pressure of the reaction system in the step (2) on the yield of n-3 polyunsaturated fatty acid glycerides
Example 5: (optimization of adsorption type)
(1) The method for reducing peroxide number of raw materials comprises the following steps: performing static adsorption or dynamic adsorption treatment on fish oil fatty acid ethyl ester with peroxide value of 9.4mmol/kg (total content of DHA and EPA is 71.3%), wherein the static adsorption conditions are as follows: fully mixing 20g of fish oil fatty acid ethyl ester and 1% of silica gel, stirring and adsorbing at the temperature of 45 ℃ for 25min under the pressure of an adsorption system less than 4kPa, and filtering to remove an adsorbent to obtain the fish oil fatty acid ethyl ester with the peroxide value reduced, wherein the peroxide value is 0.92 mmol/kg; the dynamic adsorption conditions were: loading the column by a wet method, washing with dichloromethane with 2 times volume to remove impurities in the column when the interface of the silica gel is not reduced, balancing for 2h, and standing for later use; under the condition of room temperature, 40g of silica gel in the column, the loading amount of 3.5g, and isocratic elution of n-hexane/anhydrous ether (volume is 5:1), the elution flow rate is 2mL/min, 4 column volume sample effluents are collected, and after a solvent is removed, the fish oil fatty acid ethyl ester with the peroxide value reduced treatment is obtained, wherein the peroxide value is 0.67 mmol/kg. The contrast group is n-3 polyunsaturated fatty glyceride prepared by directly carrying out enzyme transesterification reaction with glycerol without reducing peroxide value treatment.
(2) Enzymatic synthesis of n-3 polyunsaturated fatty acid glycerides: glycerol was added to the peroxide number-reduced (or control) fatty acid ethyl ester at a molar ratio of 3:1, then lipase Novozym435 derived from Candida antarctica (Candida antarctica) was added in an amount of 5% (w/w, relative to the total mass of the reactants), and the mixture was mixed in a 50mL reactor, and the temperature was rapidly raised to 65 ℃ for 8 hours, with the pressure of the reaction system being controlled to 300Pa or less. After the ester exchange reaction is finished, recovering lipase, removing ethyl ester, monoglyceride and the like through molecular distillation to obtain n-3 polyunsaturated fatty glyceride, detecting the content of glyceride in the purified product by using HPLC, and calculating the yield of glyceride.
The influence of the adsorption method in the step (1) on the yield of the n-3 polyunsaturated fatty acid glyceride is researched, and the result is shown in table 5, and the results in the table show that the impurities such as peroxide value and the like in the reaction substrate can be well reduced and the reaction activity of the lipase is improved no matter static adsorption or dynamic adsorption is adopted; for reaction substrates which are not subjected to peroxide reduction treatment, the enzyme has low catalytic efficiency in catalyzing the reaction of the substrates.
TABLE 5 Effect of the adsorption method of step (1) on the yield of n-3 polyunsaturated fatty acid glycerides
Example 6: (optimization of adsorbent)
(1) Treating the raw materials by reducing peroxide number: carrying out static adsorption treatment on fish oil fatty acid ethyl ester with peroxide value of 9.4mmol/kg (the total content of DHA and EPA is 71.3%), wherein the static adsorption conditions are as follows: fully mixing 20g of fish oil fatty acid ethyl ester and 1% of adsorbent, wherein the pressure of an adsorption system is less than 4kPa, and stirring and adsorbing at 45 ℃ for 25min to obtain the fish oil fatty acid ethyl ester with the peroxide value reduced;
(2) enzymatic synthesis of n-3 polyunsaturated fatty acid glycerides: adding glycerol into fatty acid ethyl ester subjected to peroxide number reduction treatment in a molar ratio of 3:1, then adding lipase Novozym435 derived from Candida antarctica (Candida antarctica) with the enzyme addition amount of 5% (w/w, relative to the total mass of reactants), mixing the two in a 50mL reactor, rapidly heating to 65 ℃, reacting for 8 hours, and controlling the pressure of a reaction system to be less than 300 Pa. After the ester exchange reaction is finished, recovering lipase, removing ethyl ester, monoglyceride and the like through molecular distillation to obtain n-3 polyunsaturated fatty glyceride, detecting the content of glyceride in the purified product by using HPLC, and calculating the yield of glyceride.
The influence of the types of the adsorbents on the yield of the n-3 polyunsaturated fatty acid glyceride in the step (1) is researched, and the results are shown in table 6, and the results in the table show that different adsorbent materials have slightly different adsorption effects on impurities in fatty acid ethyl ester, but the peroxide value of the fish oil fatty acid ethyl ester can be well reduced, and the lipase catalysis effect is improved; however, when the fatty acid ethyl ester is not subjected to peroxide value reduction treatment (control group), the yield of glyceride obtained after enzymatic reaction and purification is significantly lower than that of the experimental group, which also indicates that the catalytic efficiency and the use times of the lipase can be improved by performing enzymatic transesterification after the reaction substrate is subjected to peroxide value reduction treatment.
TABLE 6 influence of adsorbent species in step (1) on the yield of n-3 polyunsaturated fatty acid glycerides
Example 7: (amount of adsorbent added)
(1) Treating the raw materials by reducing peroxide number: carrying out static adsorption treatment on fish oil fatty acid ethyl ester with peroxide value of 9.4mmol/kg (the total content of DHA and EPA is 71.3%), wherein the static adsorption conditions are as follows: and (3) fully mixing 20g of fish oil fatty acid ethyl ester and a certain amount of silica gel, stirring and adsorbing at the temperature of 45 ℃ for 25min under the pressure of an adsorption system less than 4kPa to obtain the fish oil fatty acid ethyl ester with the peroxide number reduced treatment.
(2) Enzymatic synthesis of n-3 polyunsaturated fatty acid glycerides: adding glycerol into fatty acid ethyl ester subjected to peroxide number reduction treatment in a molar ratio of 3:1, then adding lipase Novozym435 derived from Candida antarctica (Candida antarctica) with the enzyme addition amount of 5% (w/w, relative to the total mass of reactants), mixing the two in a 50mL reactor, rapidly heating to 65 ℃, reacting for 8 hours, and controlling the pressure of a reaction system to be less than 300 Pa. After the ester exchange reaction is finished, recovering lipase, removing ethyl ester, monoglyceride and the like through molecular distillation to obtain n-3 polyunsaturated fatty glyceride, detecting the content of glyceride in the purified product by using HPLC, and calculating the yield of glyceride.
The influence of the addition of the adsorbent in the step (1) on the yield of the n-3 polyunsaturated fatty acid glyceride is researched, and the results are shown in table 7, and the results in the table show that different addition of silica gel can well remove impurities in the fish oil fatty acid ethyl ester, so that the catalytic effect of the lipase is improved.
TABLE 7 influence of adsorbent addition in step (1) on the yield of enzymatically transesterified glycerides
Example 8: (optimization of Lipase)
(1) Treating the raw materials by reducing peroxide number: carrying out static adsorption treatment on fish oil fatty acid ethyl ester with peroxide value of 9.4mmol/kg (the total content of DHA and EPA is 71.3%), wherein the static adsorption conditions are as follows: fully mixing 20g of fish oil fatty acid ethyl ester and 1% of silica gel, stirring and adsorbing at 45 ℃ for 25min under the pressure of an adsorption system less than 4kPa to obtain the fish oil fatty acid ethyl ester with the peroxide value reduced;
(2) enzymatic synthesis of n-3 polyunsaturated fatty acid glycerides: glycerol was added to the peroxide value-reduced fatty acid ethyl ester in a molar ratio of 3:1, then immobilized or free Lipase from different species was added in an amount of 5% (w/w, relative to the total mass of the reactants), mixed together in a 50mL reactor under the following reaction conditions for the lipases Lipozyme435, Lipozyme RM and Lipase DF IM: the reaction temperature is 65 ℃, the reaction is carried out for 8 hours, and the pressure of the reaction system is controlled to be below 200 Pa. For Lipase G50 and Lipase AK, the optimum temperature of the Lipase is about 40 ℃, and the Lipase is inactivated by high temperature, so the reaction conditions are as follows: the reaction temperature is 40 ℃, the reaction time is 24 hours, and the pressure of the reaction system is controlled below 200 Pa. After the enzymatic transesterification reaction is finished, recovering lipase, removing ethyl ester, monoglyceride and the like through molecular distillation to obtain n-3 polyunsaturated fatty glyceride, detecting the content of glyceride in a purified product by using HPLC, and calculating the yield of glyceride.
TABLE 8 influence of the kind of lipase in the reaction of step (2) on the yield of n-3 polyunsaturated fatty acid glycerides
The influence of the lipase species on the yield of n-3 polyunsaturated fatty acid glycerides in step (2) was investigated, and the results are shown in Table 8, from which it can be seen that lipases from Candida antarctica, Burkholderiacepacia, Penicilliumcamembertii, Pseudomonas fluorescens have a certain catalytic activity during the enzymatic transesterification, whereas lipases from Rhizomucormieihei, Rhizopus oryzae have almost no activity during the enzymatic transesterification and are not able to catalyze the reaction of N-3 polyunsaturated fatty acid ethyl esters rich in DHA and EPA with glycerol.
When other lipase is adopted, the method of the invention can also effectively maintain the activity of the lipase and increase the catalytic activity of the lipase in an enzymatic ester exchange reaction system, thereby realizing the improvement of the utilization rate of the enzyme.
Example 9: (optimization of acyl Acceptor type)
(1) Treating the raw materials by reducing peroxide number: carrying out static adsorption treatment on fish oil fatty acid ethyl ester with peroxide value of 9.4mmol/kg (the total content of DHA and EPA is 71.3%), wherein the static adsorption conditions are as follows: fully mixing 20g of fish oil fatty acid ethyl ester and 1% of silica gel, stirring and adsorbing at 45 ℃ for 25min under the pressure of an adsorption system less than 4kPa to obtain the fish oil fatty acid ethyl ester with the peroxide value reduced;
(2) enzymatic synthesis of n-3 polyunsaturated fatty acid glycerides: adding an acyl acceptor (the molar ratio of fatty acid ethyl ester to glycerol is 3:1 when the acyl acceptor is glycerol, and the molar ratio of fatty acid ethyl ester to monoglyceride is 2:1 when the acyl acceptor is monoglyceride) into fatty acid ethyl ester subjected to peroxide number reduction treatment in a certain molar ratio, then adding lipase Novozym435 derived from Candida antarctica (Candida antarctica) with the enzyme addition amount of 5% (w/w, relative to the total mass of reactants), mixing the materials in a 50mL reactor, rapidly heating to 65 ℃, reacting for 8 hours, and controlling the pressure of the reaction system to be below 300 Pa. After the enzymatic transesterification reaction is finished, recovering lipase, removing ethyl ester, monoglyceride and the like through molecular distillation to obtain n-3 polyunsaturated fatty glyceride, detecting the content of glyceride in a purified product by using HPLC, and calculating the yield of glyceride.
The influence of the acyl acceptor type in the step (2) on the yield of n-3 polyunsaturated fatty acid glycerides is explored, and the results are shown in Table 9, wherein the results show that glycerol or monoglyceride can be used for reacting with fatty acid ethyl ester to prepare diglyceride and triglyceride rich in n-3 polyunsaturated fatty acid.
TABLE 9 influence of acyl acceptor species on yield of enzymatically transesterified glycerides in step (2) of the reaction
Example 10: (repeated use)
(1) Treating the raw materials by reducing peroxide number: carrying out static adsorption treatment on fish oil fatty acid ethyl ester with peroxide value of 9.4mmol/kg (the total content of DHA and EPA is 71.3%), wherein the static adsorption conditions are as follows: fully mixing 20g of fish oil fatty acid ethyl ester with 1% of silica gel or 1% of activated carbon, stirring and adsorbing at 45 ℃ for 25min under the pressure of an adsorption system less than 4kPa to obtain the fish oil fatty acid ethyl ester with the peroxide value reduced;
(2) enzymatic synthesis of n-3 polyunsaturated fatty acid glycerides: adding glycerol into the fatty acid ethyl ester subjected to peroxide number reduction treatment in a molar ratio of 3:1, then adding lipase Novozym435 derived from Candida antarctica (Candida antarctica) with the enzyme addition amount of 5% (w/w, relative to the total mass of reactants), mixing the two in a 50mL reactor, rapidly heating to 65 ℃, and reacting for 8 hours, wherein the pressure of the reaction system is controlled at 300 Pa. After the ester exchange reaction is finished and lipase is recovered, removing ethyl ester, monoglyceride and the like from the crude product through molecular distillation to obtain n-3 polyunsaturated fatty glyceride, detecting the content of glyceride in the purified product by using HPLC (high performance liquid chromatography) and calculating the yield of glyceride.
And (3) adding the lipase recovered in the step (2) into the next reaction for enzymatically synthesizing the glyceride, wherein the rest conditions are consistent with those of the first reaction, and after 8 times of repeated use, detecting the content of the glyceride in the purified product by using HPLC-ELSD and calculating the yield of the glyceride.
TABLE 10 influence of adsorbent species in step (1) on the yield of n-3 polyunsaturated fatty acid glycerides
The influence of the adsorption treatment on the repeated use times of the enzyme in the step (2) is researched, the result is shown in table 10, and the result is shown in the table, compared with a control group, the raw material is subjected to adsorption treatment by different adsorbents, whether in the first reaction or after being repeatedly used for 8 times, and then is used for catalyzing and synthesizing the n-3 polyunsaturated fatty acid glyceride, the yield of the glyceride is obviously higher than that of the control group, and the peroxide reduction value treatment of the raw material can obviously improve the catalytic effect of the lipase and the repeated use times of the lipase.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (10)
1. A method for preparing n-3 polyunsaturated fatty acid glyceride by an enzyme method, which is characterized by comprising the following steps:
s1, raw material treatment: subjecting the non-glyceride rich in n-3 polyunsaturated fatty acid to peroxide value reduction treatment, wherein the peroxide value of the non-glyceride rich in n-3 polyunsaturated fatty acid is reduced to not more than 4 mmol/kg;
s2, preparation of n-3 polyunsaturated fatty acid glyceride: mixing the non-glyceride which is obtained in the step S1 and is subjected to peroxide number reduction treatment and is rich in n-3 polyunsaturated fatty acid with an acyl acceptor, reacting under the catalysis of lipase, removing the lipase and the acyl acceptor after reacting for a certain time to obtain n-3 polyunsaturated fatty acid glyceride, wherein the acyl acceptor is glycerol and/or monoglyceride.
2. The method according to claim 1, wherein the step of reducing the peroxide value of the non-glyceride rich in n-3 polyunsaturated fatty acids comprises an adsorption process in the step of S1.
3. The method according to claim 2, wherein the adsorbent for the adsorption method comprises one or more of silica gel, activated clay, activated carbon, silica and attapulgite, preferably silica gel and activated carbon.
4. A process according to any one of claims 1 to 3, wherein the adsorption process comprises static adsorption or dynamic adsorption.
5. A method according to any one of claims 1 to 4, wherein the adsorbent is added in an amount of 0.2% to 2%, preferably 0.5% to 1%, by mass of the substrate; the adsorption time is 15min to 120min, preferably 20min to 60 min.
6. A process according to any one of claims 1 to 5, wherein the molar ratio of non-glyceride esters of n-3 polyunsaturated fatty acids to acyl acceptors is not less than 2: 1.
7. the method according to any one of claims 1 to 6, wherein the lipase used in step S2 includes, but is not limited to, any one or more of lipases derived from Candida antarctica (Candida antarctica), Penicillium (Penicillium camembertii), Burkholderia cepacia (Burkholderia cepacia), Pseudomonas fluorescens (Pseudomonas fluorescens).
8. The method according to any one of claims 1 to 7, wherein the amount of the lipase added is 2% to 12% based on the total mass of the reaction substrate.
9. The method according to any one of claims 1 to 8, wherein the reaction temperature of the n-3 polyunsaturated fatty acid glycerides in the S2 step is 30-70 ℃ and the reaction time is 4-24 h.
10. Use of the method of any one of claims 1 to 9 for the preparation of diglycerides or concentrated fish oil.
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Inventor after: Chen Ye Inventor after: Wang Xiaosan Inventor after: Wang Yifan Inventor after: Li Caiming Inventor before: Wang Xiaosan Inventor before: Chen Ye Inventor before: Wang Yifan Inventor before: Li Caiming |