CN104130860B - Method for enriching long-chain polyunsaturated fatty acid by utilizing immobilized thermomyces lanuginosus lipase - Google Patents
Method for enriching long-chain polyunsaturated fatty acid by utilizing immobilized thermomyces lanuginosus lipase Download PDFInfo
- Publication number
- CN104130860B CN104130860B CN201310161447.2A CN201310161447A CN104130860B CN 104130860 B CN104130860 B CN 104130860B CN 201310161447 A CN201310161447 A CN 201310161447A CN 104130860 B CN104130860 B CN 104130860B
- Authority
- CN
- China
- Prior art keywords
- immobilized
- lipase
- thermomyces lanuginosus
- fatty acid
- exchange resin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Abstract
The invention relates to a method for enriching long-chain polyunsaturated fatty acids by using immobilized thermomyces lanuginosus lipase, which comprises the following steps: (1) in the presence of immobilized thermomyces lanuginosus lipase under the condition of vacuum pumping or protective atmosphere, carrying out esterification reaction on free fatty acid and alcohol; (2) separating the enriched free long-chain polyunsaturated fatty acid from the product after the reaction. The method realizes a new method for enriching long-chain polyunsaturated fatty acid from deep-sea fish oil or microbial oil by utilizing the selective catalytic esterification of fatty acid with different chain lengths by immobilized lipase in the process of catalyzing the esterification of fatty acid and alcohol.
Description
Technical Field
The invention relates to a method for enriching long-chain polyunsaturated fatty acid by using immobilized Thermomyces lanuginosus lipase, which realizes a novel method for enriching long-chain polyunsaturated fatty acid from deep sea fish oil or microbial oil by using immobilized lipase to selectively catalyze esterification of fatty acid with different chain lengths in the process of catalyzing esterification of fatty acid and alcohol.
Technical Field
In recent years, long-chain polyunsaturated fatty acids (PUFAs) such as EPA, DHA, DPA and ARA have been the focus of research due to their unique physiological functions. Because of the low content of naturally occurring PUFAs, increasing research efforts have been directed to the enrichment and purification of PUFAs in order to increase their medical and health-care value.
At present, the PUFAs separation and purification method mainly adopts the traditional chemical methods, such as a low-temperature crystallization method, a urea inclusion method, a silver ion complexing separation method and the like. The method for purifying and enriching the PUFAs needs a large amount of organic reagents, causes great pollution to the environment, is complicated in process and time-consuming, and is easy to cause oxidation of the PUFAs.
The lipase catalysis method has high reaction efficiency, small enzyme consumption and mild reaction conditions, and the immobilized enzyme can be repeatedly utilized and is widely applied to reactions such as hydrolysis, alcoholysis, acidolysis, ester exchange, esterification and the like. In recent years, research work on the enrichment of PUFAs by using lipase has been advanced. CN101161819A prepares glyceride with total content of EPA and DHA reaching 50-80% by enzyme method; CN101348807B separates and purifies EPA and DHA by column chromatography method, and esterification reaction is carried out between EPA and DHA purified by lipase catalysis and glycerol to obtain glyceride rich in EPA and DHA. In the above patent methods and most reports in the literature, the hydrolyzed or alcoholyzed fatty acid (free or ethyl ester) is first subjected to chemical enrichment and purification of PUFAs, and then catalyzed esterification or ester exchange reaction is performed by lipase to obtain PUFAs glyceride with higher purity. The method has the disadvantages of complicated process, chemical purification, waste of a large amount of organic reagents due to the adoption of methods such as urea inclusion, low yield and residual of components such as urea.
There are some reports at home and abroad about research on enrichment of omega-3 PUFAs by selective hydrolysis of lipase, for example, the content of DHA in enriched omega-3 PUFAs in sardine oil is only increased from 13.62% to 29.94% by screening lipase by Okada (Tomoko Okada) and other people. The key of the lipase selective hydrolysis enrichment method is to screen out good selective lipase and control hydrolysis process parameters, and the currently reported lipases for catalyzing selective hydrolysis of fish oil in documents mainly comprise expensive lipases such as Pseudomonas (Pseudomonas sp.), Candida rugosa (Candida rugosa) and Candida cylindracea (Candida cylindracea). The main disadvantages of the lipase selective hydrolysis method are: the limitation of the source of the hydrolase causes higher production cost, difficult control of the hydrolysis reaction process and low enrichment purity, and cannot meet the market demand of high-content omega-3 PUFAs.
CN200380106326.2 discloses esterifying a marine oil containing EPA and DHA in free acid or hexyl ester form with ethanol in the presence of a lipase catalyst and separating by distillation in the substantial absence of organic solvents. In which an immobilized MML enzyme (Rhizomucor miehei lipase) and a novel Novozyme lipase (Thermomyces lanuginosus lipase (TL lipase)) immobilized on silica gel particles were used. However, the conversion was not high, with only 43% conversion of the novel Novozyme lipase immobilized on silica gel particles. Furthermore, WO2008SE290A discloses that polyunsaturated fatty acid enriched marine petroleum can be used to form emulsions with water enriched components (for forming e.g. condiments and sauces) comprising eicosapentaenoic acid or docosahexaenoic acid as well as mono-and diglycerides. In this case, TL enzymes immobilized on polypropylene MP1000 were used. However, the enrichment effect on PUFAs is still not high, and is not as good as that of immobilized Rhizopus nigricans lipase (RM), PS (Pseudomonas sp.) and PF (Pseudomonas fluorescens).
Therefore, a new method for enriching PUFAs is urgently needed in the prior art, and has the characteristics of simple and convenient operation, low cost and higher enrichment purity.
Disclosure of Invention
In order to achieve the above objects, the present inventors have conducted intensive studies to provide a method for enriching long-chain polyunsaturated fatty acids (PUFAs) using immobilized thermomyces lanuginosus lipase (TL lipase), the method comprising:
(1) carrying out esterification reaction on free fatty acid and alcohol in the presence of immobilized thermomyces lanuginosus lipase under vacuum pumping or protective atmosphere, wherein the free lipase contains PUFAs;
(2) separating the enriched free PUFAs from the reacted product.
The present invention also provides a method for purifying long-chain polyunsaturated fatty acids using immobilized lipase, the method comprising:
(1) reacting free fatty acid with alcohol in the presence of immobilized thermomyces lanuginosus lipase under vacuum pumping or protective atmosphere;
(2) separating free fatty acids containing free PUFAs from the reacted product.
The invention also provides a method for reducing the content of short-chain fatty acids in free fatty acids by using the immobilized lipase, which comprises the following steps:
(1) reacting free fatty acid with alcohol in the presence of immobilized Thermomyces lanuginosus lipase under vacuum or protective atmosphere.
The invention also provides a method for improving the content of long-chain polyunsaturated fatty acid in free fatty acid by using the immobilized lipase, which comprises the following steps:
(1) reacting free fatty acid with alcohol in the presence of immobilized Thermomyces lanuginosus lipase under vacuum or protective atmosphere.
In one embodiment of the invention, the free fatty acids used are obtained by base-catalyzed hydrolysis, enzymatic hydrolysis or high-pressure high-temperature hydrolysis of fats and oils containing PUFAs.
In one embodiment of the present invention, the method of the present invention further comprises the step of providing free fatty acids:
(a) carrying out base catalytic hydrolysis on grease containing PUFAs under the condition of vacuum pumping or protective atmosphere;
(b) adding an extracting agent for extraction, acidifying the obtained water layer, and extracting again to obtain free fatty acid.
In the present invention, the fats and oils containing PUFAs include fish oils and microbial fats, preferably, the fish oils include sardine oil, tuna oil, herring oil, cod liver oil, salmon oil, tuna oil, salmon oil, eel oil, mackerel oil, and combinations thereof; the microbial oil comprises DHA algae oil, ARA oil and their combination.
In one embodiment of the present invention, the protective atmosphere of step (a) is selected from the group consisting of a nitrogen protective atmosphere, an argon protective atmosphere, and a helium protective atmosphere.
In one embodiment of the invention, step (a) is base-catalyzed hydrolysis using a base solution selected from KOH alcohol-water solution, NaOH alcohol-water solution. Preferably, the base-catalyzed hydrolysis is refluxed at 40-80 ℃ for 0.5-2 hours with stirring.
In one embodiment of the present invention, in step (b), the extractant is selected from n-hexane, n-heptane, petroleum ether. The acidification is performed by using HCl and HNO3To proceed with. Wherein, the acidification is preferably carried out until the pH value is 1-3.
In the inventionIn an embodiment, the method further comprises drying the free fatty acids obtained in step (b) and removing the extractant. Wherein the drying agent is selected from anhydrous Na2SO4Anhydrous MgSO (MgSO)4Anhydrous CuSO4、P2O5. The method for removing the extracting agent comprises rotary evaporation, a nitrogen blowing method and vacuum drying.
In one embodiment of the present invention, the protective atmosphere in step (1) is selected from a nitrogen protective atmosphere, an argon protective atmosphere, and a helium protective atmosphere.
In one embodiment of the present invention, the alcohol includes monohydric alcohol, dihydric alcohol and trihydric alcohol having 1 to 12 carbon atoms. Preferably, the alcohol includes monohydric alcohol with 2-12 carbon atoms, ethylene glycol, propylene glycol and glycerol. More preferably, the alcohol includes monohydric alcohol having 3 to 12 carbon atoms and glycerin.
In one embodiment of the present invention, the molar ratio of the alcohol to the free fatty acid in step (1) is 10:1 to 1: 10.
In one embodiment of the invention, the immobilized Thermomyces lanuginosus lipase is a Thermomyces lanuginosus lipase immobilized on an ion exchange resin.
In one embodiment of the present invention, the Thermomyces lanuginosus lipase immobilized on an ion exchange resin is prepared by: (i) contacting ion exchange resin with Thermomyces lanuginosus lipase to obtain immobilized Thermomyces lanuginosus lipase; in a more preferred embodiment, the method further comprises the steps of: (ii) drying the immobilized thermomyces lanuginosus lipase.
In one embodiment of the invention, the ion exchange resin is contacted with Thermomyces lanuginosus lipase at 25-35 deg.C under 150-300 revolutions per minute (rpm) for 2-8 hours; in a more preferred embodiment, 0.1 to 5g of ion exchange resin, preferably 0.4 to 2.5g of ion exchange resin, is added per ml of the Thermomyces lanuginosus lipase liquor.
In one embodiment of the present invention, the ion exchange resin is a styrene resin, an acrylic resin, a cation resin or an anion resin, and preferably, the ion exchange resin is a weak base ion exchange resin.
In one embodiment of the present invention, the Thermomyces lanuginosus lipase immobilized on an ion exchange resin in step (1) is used in an amount of 1% to 20% based on the total mass of the alcohol and the fatty acid (hereinafter, the alcohol and the fatty acid are referred to as a reaction substrate).
In one embodiment of the invention, the pressure in the reaction vessel is 0.1 to 1KPa under the condition of vacuum pumping.
In one embodiment of the present invention, in the step (1), the fatty acid is reacted with the alcohol for 4 to 24 hours.
In one embodiment of the present invention, a water-carrying agent is added in the esterification reaction, and the preferred water-carrying agent is a molecular sieve.
In one embodiment of the present invention, the fatty acid and the alcohol are carried out at a temperature ranging from 20 to 80 ℃ for 2 to 24 hours in step (1). In the step (1), fatty acid with carbon number less than 20 reacts with alcohol to generate fatty acid ester, and PUFAs still exist in the form of free fatty acid, so that the separation and enrichment of PUFAs are realized.
Compared with the traditional physical and chemical method for enriching PUFAs, the method has a series of advantages of enzymatic reaction, greatly reduces the use of organic reagents and simplifies the process; compared with the method for enriching PUFAs by lipase selective hydrolysis reported in the existing literature, the principle of the enrichment method adopted by the invention is based on selective esterification rather than selective hydrolysis, the enrichment purity is higher, and the Thermomyces lanuginosus lipase is from industrial production, the price is lower, and the cost is greatly saved.
Drawings
FIG. 1 is a schematic diagram showing an example of the method for enriching a long-chain polyunsaturated fatty acid using immobilized Thermomyces lanuginosus lipase according to the present invention.
Detailed Description
Hereinafter, the present inventors will describe in detail the method for enriching long-chain polyunsaturated fatty acids (PUFAs) by using immobilized Thermomyces lanuginosus lipase (Thermomyces lanuginosus) according to the present invention with reference to the drawings, but it should be noted that these descriptions are not intended to limit the scope of the present application.
Unlike the prior art methods for enriching long chain polyunsaturated fatty acids (PUFAs) involving selective hydrolysis, the method of the present invention actually employs methods for enriching long chain polyunsaturated fatty acids (PUFAs) involving selective esterification. Specifically, the invention provides a method for enriching long-chain polyunsaturated fatty acids (PUFAs) by using immobilized thermomyces lanuginosus lipase. Free fatty acid containing PUFAs is subjected to esterification reaction with alcohols under the catalysis of lipase, short-carbon-chain fatty acid (the carbon number is less than 20) preferentially reacts to generate esters, and the PUFAs still exist in the form of the free fatty acid, so that the enrichment effect is achieved. According to the invention, different immobilized lipases (including Thermomyces lanuginosus lipase (TL lipase) immobilized on ion exchange resin and commercial enzyme) are screened and compared, so that the immobilized TL lipase is most suitable for the process method, the esterification selectivity is good, and the enrichment purity of PUFAs is high.
The present invention also provides a method for purifying long-chain polyunsaturated fatty acids using immobilized lipase, the method comprising: (1) reacting free fatty acid with alcohol in the presence of immobilized thermomyces lanuginosus lipase under vacuum pumping or protective atmosphere; (2) separating free fatty acids containing free PUFAs from the reacted product. In the method, free fatty acid containing PUFAs is subjected to esterification reaction with alcohols under the catalysis of lipase, short-carbon-chain fatty acid (the carbon number is less than 20) preferentially reacts to generate esters, and the PUFAs still exist in the form of the free fatty acid, so that the purity of the PUFAs is improved, and the purification effect is further achieved.
The invention also provides a method for reducing the content of short-chain fatty acids in free fatty acids by using the immobilized lipase, which comprises the following steps: (1) reacting free fatty acid with alcohol in the presence of immobilized Thermomyces lanuginosus lipase under vacuum or protective atmosphere. The method comprises the step of carrying out esterification reaction on free fatty acid containing PUFAs and alcohols under the catalysis of lipase, wherein short-carbon-chain fatty acid (the carbon number is less than 20) preferentially reacts to generate esters, and further the effect of reducing the content of the short-carbon-chain fatty acid in the free fatty acid is achieved.
The invention also provides a method for improving the content of long-chain polyunsaturated fatty acid in free fatty acid by using the immobilized lipase, which comprises the following steps: (1) reacting free fatty acid with alcohol in the presence of immobilized Thermomyces lanuginosus lipase under vacuum or protective atmosphere. In the method, free fatty acid containing PUFAs is subjected to esterification reaction with alcohols under the catalysis of lipase, short-carbon fatty acid (the carbon number is less than 20) is preferentially reacted to generate esters, and the PUFAs still exist in the form of the free fatty acid, so that the effect of improving the content of long-chain polyunsaturated fatty acid in the free fatty acid is achieved.
Referring to fig. 1, in this example, the method for enriching long-chain polyunsaturated fatty acids (PUFAs) using immobilized thermomyces lanuginosus lipase (thermomyces lanuginosus) according to the invention may comprise the following steps:
1) base-catalyzed hydrolysis: under the protection of nitrogen, a certain amount of KOH alcohol-water solution and PUFA-containing grease are refluxed for 1 hour under the condition of magnetic stirring at 60 ℃ so as to hydrolyze the grease. After the reaction, adding a certain amount of water, extracting unsaponifiable matter by using normal hexane, acidifying a hydration layer by using HCl until the pH is =1, extracting free fatty acid by using normal hexane, and then extracting anhydrous Na2SO4After drying, the solvent is removed by rotary evaporation or nitrogen blow to obtain free fatty acid.
2) Enzyme-catalyzed selective esterification: according to a certain proportion, taking alcohols and the free fatty acid obtained in the step 1) as substrates, adding immobilized TL lipase, and reacting in a vacuum or nitrogen-filled environment, so that fatty acid with the carbon number less than 20 reacts with the alcohols to generate fatty acid ester, and PUFAs still exist in the form of free fatty acid, thereby achieving the effect of separating the PUFAs from other fatty acids.
In the present invention, the method for providing free fatty acid is well known to those skilled in the art, and may include, but is not limited to, enzymatic hydrolysis, high pressure high temperature hydrolysis, etc. in addition to the base-catalyzed hydrolysis shown in FIG. 1.
In the present invention, "base-catalyzed hydrolysis" refers to a method for preparing free fatty acids and glycerin, which comprises adding an alkali solution, such as a NaOH-alcohol solution, to raw fats and oils to catalyze the hydrolysis of the fats and oils.
In the present invention, "enzymatic hydrolysis" refers to a process for producing free fatty acids and glycerin, which comprises adding water to a raw oil or fat, and reacting the mixture under low temperature conditions using an enzyme (including but not limited to an immobilized enzyme), such as lipase, phospholipase, etc., as a catalyst. It is clear to a person skilled in the art that an immobilized enzyme refers to an enzyme immobilized on a carrier, such as an immobilized lipase or the like.
In the present invention, "high-pressure high-temperature hydrolysis" refers to a method for producing fatty acids and glycerin, which comprises adding water to a raw material fat and oil and allowing the mixture to react under high-temperature high-pressure conditions.
In the present invention, the raw material oil to be hydrolyzed is an oil rich in long-chain polyunsaturated fatty acids, including but not limited to deep sea fish oil, microbial oil or a mixture thereof, and the fish oil containing PUFAs includes but not limited to: sardine oil, tuna oil, herring oil, cod liver oil, salmon oil, tuna oil, salmon oil, eel oil, and mackerel oil, and the microbial oil comprises protein limited to DHA algae oil and ARA oil.
In the present invention, the alcohols used include: monohydric alcohols having 1 to 12 carbon atoms, ethylene glycol, propylene glycol, glycerin, and the like.
In the invention, the ratio of the fatty acid to the alcohol is 10:1 to 1:10 by mole.
In the present invention, the immobilized enzyme can be prepared by the following method: weighing 40-100g of weak base ion exchange resin in a triangular flask (for example, 500ml with the capacity of 250-.
In the present invention, the amount of immobilized TL lipase used is 1% to 20% of the mass of the reaction substrate (mass of alcohol and fatty acid).
In the invention, the pressure in the reaction vessel is 0.1-1 KPa under the condition of vacuum pumping.
In the invention, under the condition of nitrogen filling, a molecular sieve is added for removing water after 1-3 hours of reaction.
In the invention, the reaction temperature is 20-80 ℃.
In the invention, the reaction time is 2-24 hours.
In the present invention, the ion exchange resin includes a styrene-based resin, an acrylic-based resin, a cationic resin (a strongly acidic cationic resin and a weakly acidic cationic resin), an anionic resin (a strongly basic anionic resin and a weakly basic anionic resin), and the like. Preferably, the ion exchange resin of the present invention is a weakly basic anion exchange resin. In the invention, the weak base anion exchange resin mainly exchanges anion exchange resin with primary, secondary and tertiary amine groups. Such resins contain weakly basic groups, such as primary amino groups (also known as primary amino groups) -NH2Secondary amino (secondary amino) -NHR, or tertiary amino (tertiary amino) -NR2They are weakly alkaline in water by dissociation of OH-. The positively charged groups of the resin can be adsorbed to and combined with anions in solution, thereby generating anion exchange action. Such resins in most cases adsorb all other acid molecules in solution. It can only work under neutral or acidic conditions (such as pH 1-9). It can be Na2CO3、NH4OH is regenerated. The weakly basic anion exchange resin is commercially available from Shanghai resin Co., Ltd, south-developing chemical plant, Zhejiang dispute industries, Ltd, Cheng-Guang chemical research institute resin plant, Jiangsu Coleis resin Co., U.S. Rohm&Available from Hass corporation, Success corporation, Dow chemical corporation, etc.
In the present invention, "long-chain polyunsaturated fatty acid" means: fatty acids with carbon chain lengths above 20 carbons and unsaturation greater than 3, including but not limited to docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA).
The present invention will be described in further detail with reference to specific embodiments. The fatty acid content data in this specification are mass percentages unless otherwise indicated.
In the following examples of the invention, the immobilized TL lipase used was prepared by the following method:
weighing 10-50g of weakly basic ion exchange resin in a 250ml triangular flask, adding 10-50ml of TL enzyme solution, shaking in a shaking table at 4-60 ℃ at the speed of 10-200 revolutions per minute (rpm) for 0.5-16 hours, taking out the enzyme, putting the enzyme into a clean culture dish, and putting the culture dish into a fume hood for drying.
As the weakly basic ion exchange resin, there may be used, but not limited to, Amberlite IRA900C1, Amberlite FPA91C1, Amberlite FPA54, D301R, D392, D380, D382, D284, D280, JK206, NKX-8, DuoliteA-161, LX1000HA, Dowex1 x 2.
In the following examples of the invention, the methyl esterification process is described in reference to GB-T17376-2008; the gas chromatographic analysis conditions refer to GB-T17377-2008;
in the following examples of the invention tuna oil, herring oil, cod liver oil and sardine oil were purchased from NorskHydro A/S; DHA algal oil was purchased from motak.
Example 1 enrichment purification of PUFAs in DHA algal oil
(1) Base-catalyzed hydrolysis: firstly, 36.8g of KOH is dissolved in 70.4mL of water and 422.4mL of 95% ethanol to prepare KOH alcohol-water solution, 160g of DHA algae oil is added under the protection of nitrogen, and the mixture is refluxed for 1 hour under the condition of magnetic stirring at 50 ℃ to hydrolyze the oil. After the reaction was complete, 381mL of water was added, unsaponifiable material was extracted with 500mL of 2N-hexane, the hydrated layer was acidified to pH =1 with 3N HCl, free fatty acids were extracted with 300mL of 2N-hexane, and anhydrous Na was added2SO4After drying, the solvent was removed by rotary evaporation to obtain free fatty acids.
(2) 9.57g of the free fatty acid prepared in step (1) and 3g of glycerol (molar ratio of free fatty acid to glycerol is about 1: 1) were weighed into a 50mL reactor, 10% of the mass of immobilized TL lipase (based on the mass of the reaction substrate, i.e., the total mass of free fatty acid and glycerol) was added, and the reaction was carried out under vacuum conditions of 60 deg.C (gauge pressure reading of 0.2 kPa) at a stirring rate of 250 rpm. The reaction was sampled every 4 hours, separated by Thin Layer Chromatography (TLC) method (developer: n-hexane: ether: formic acid =80:20:2), scraped free fatty acid band, methyl esterified, and Gas Chromatography (GC) analysis was performed to determine change in the content of PUFAs in the free acid.
TABLE 1 Effect of immobilized TL Lipase on enrichment of PUFAs at different reaction times
The self-made immobilized TL lipase is used for catalyzing the reaction for about 8 hours, and the content of DHA and DPA is respectively improved to more than 60 percent and more than 21 percent from 36.87 percent and 14.38 percent of the original DHA algae oil.
Using commercially available enzymes
TL IM (available from Novixin Co.),
RM IM (available from Novixin Co.) and
435 (from Novixin) was subjected to a control experiment and reacted for 24 hours to analyze the change of the content of PUFAs in the free fatty acid.
TABLE 2 enrichment Effect of different commercial lipase catalyzed reactions for 24 hours
As can be seen from the comparison of the data, the PUFAs content has not changed significantly even when the reaction time is as long as 24 hours under the catalysis of the commercial lipase. This is probably because the above lipases have no esterification selectivity under the conditions of the present invention, and cannot achieve the enrichment effect of the process of the present invention.
Example 2 enrichment purification of PUFAs from tuna oil
(1) Base-catalyzed hydrolysis: firstly, 13.2g of NaOH is dissolved in 35mL of water and 211mL of 95% ethanol to prepare NaOH alcohol-water solution, 80g of tuna oil is added under the protection of helium, and the mixture is refluxed for 1.5 hours under the condition of magnetic stirring at 55 ℃ so as to hydrolyze the grease. After the reaction, 190mL of water was added, 250mL of 2N-hexane was used to extract unsaponifiable matter, and 3N HNO was used for the hydration layer3Acidification to pH =2, extraction of free fatty acids with 200mL × 2 n-hexane, followed by anhydrous MgSO4After drying, the solvent was removed by rotary evaporation to give free fatty acids (23.16% and 6.04% DHA and EPA, respectively, by gas phase analysis).
(2) Weighing 8 parts of 9.82g of free fatty acid prepared in the step (1), respectively adding alcohol (the molar ratio of acid to alcohol is 1: 1) shown in the table 3 into a 50mL reactor, adding 10% of immobilized TL lipase (based on the mass of a reaction substrate), charging nitrogen for protection, reacting at the temperature of 60 ℃, stirring at the speed of 250rpm, reacting for 1 hour, adding 3g of 4A molecular sieve, and continuing to react for 11 hours. After the reaction, the reaction mixture was separated by TLC (developer: n-hexane: ether: formic acid =80:20:2), and then a free fatty acid band was scraped off, and methyl esterification treatment was carried out, and the change in the content of PUFAs in the free acid was measured by GC analysis, and the results are shown in Table 3.
TABLE 3 Effect of immobilized TL Lipase on enrichment of PUFAs in different reaction substrates
The data in Table 3 show that different reaction substrates have an effect on the enrichment effect of PUFAs. Under the above reaction conditions, the content of DHA and EPA in tuna oil was increased from 23.16% and 6.04% to 72.53% and 9.88%, respectively, using glycerol as a reaction substrate.
Example 3 enrichment purification of PUFAs in sardine oil
(1) Base-catalyzed hydrolysis: firstly, 18g of KOH is dissolved in 35mL of water and 210mL of 95% ethanol to prepare KOH alcohol-water solution, 70g of sardine oil is added under the protection of argon, and the mixture is refluxed for 0.5 hour under the condition of magnetic stirring at 60 ℃ to hydrolyze the oil. After the reaction, 200mL of water was added, unsaponifiable matter was extracted with 200mL of 2N-hexane, and the hydrated layer was treated with 3N HNO3Acidification to pH =1, extraction of free fatty acids with 200mL × 2 n-hexane, followed by anhydrous MgSO4After drying, the solvent was removed by rotary evaporation to obtain free fatty acids.
(2) 9.75g of 7 parts of the free fatty acid prepared in step (1) were weighed out, and each of them was reacted with lauryl alcohol (molar ratio of the free acid to the lauryl alcohol is shown in Table 4) in a 50mL reactor, 10% of immobilized TL lipase (based on the mass of the reaction substrate) was added, and the reaction was carried out at a stirring rate of 250rpm under vacuum pumping conditions at 60 ℃ (gauge pressure reading of 0.7 kPa) for 12 hours. After the reaction, the reaction mixture was separated by TLC (developer: n-hexane: ether: formic acid =80:20:2), and then a free fatty acid band was scraped off, and methyl esterification treatment was carried out, and the change in the content of PUFAs in the free acid was measured by GC analysis, and the results are shown in Table 4. The analysis result shows that when the molar ratio of the free acid to the lauryl alcohol is 1: 1-1: 10, the content of DHA and EPA in the sardine oil is respectively increased from 15.16% and 14.23% to more than 43% and more than 38%.
TABLE 4 enrichment Effect of immobilized TL Lipase at different substrate molar ratios
Example 4 enrichment purification of PUFAs in cod liver oil
(1) Base-catalyzed hydrolysis: firstly, 26.3g of NaOH is dissolved in 70.4mL of water and 422.4mL of 95% ethanol to prepare KOH alcohol-water solution, 150g of cod liver oil is added under the protection of nitrogen, and the mixture is refluxed for 1.5 hours under the condition of magnetic stirring at 65 ℃ to hydrolyze the grease. After the reaction, 381mL of water was added, and unsaponifiable matter was extracted with 500mL of 2N-hexane, waterThe layer was acidified with 3N HCL to pH =1, free fatty acids were extracted with 300mL x 2N-hexane, and then anhydrous CuSO was used4After drying, the solvent was removed by rotary evaporation to obtain free fatty acids.
(2) 9.70g of 7 parts of the free fatty acids prepared in step (1) (prepared by base-catalyzed hydrolysis of cod liver oil) and 3g of glycerol (molar ratio of free acid to glycerol is about 1: 1) were weighed into a 50mL reactor, and immobilized TL lipase (based on the mass of the reaction substrate) was added in the amount shown in Table 5, and the reaction was carried out at an agitation rate of 250rpm under vacuum conditions (gauge pressure reading of 0.3 kPa) at 60 ℃ for 12 hours. After completion of the reaction, the reaction mixture was separated by TLC (developer: n-hexane: ether: formic acid =80:20:2), and then a free fatty acid band was scraped off, and methyl esterification treatment was carried out, and the change in the content of PUFAs in the free acid was measured by GC analysis, and the results are shown in Table 5. At an enzyme dosage of 10%, the DHA and EPA contents of the cod liver oil are respectively increased from 9.13% and 9.56% to 47.22% and 43.15%.
TABLE 5 enrichment Effect of PUFAs with different enzyme dosages
Example 5 enrichment purification of PUFAs from herring oil
(1) Base-catalyzed hydrolysis: firstly, 36.8g of KOH is dissolved in 70.4mL of water and 422.4mL of 95% ethanol to prepare KOH alcohol-water solution, 150g of herring oil is added under the protection of nitrogen, and the mixture is refluxed for 2 hours under the condition of magnetic stirring at 70 ℃ so as to hydrolyze the grease. After the reaction was complete, 381mL of water was added, unsaponifiable material was extracted with 500mL of 2N-hexane, the hydrated layer was acidified to pH =1 with 3N HCl, free fatty acids were extracted with 300mL of 2N-hexane, and anhydrous P was used2O5After drying, the solvent was removed by rotary evaporation to obtain free fatty acids.
(2) 10.12g of 5 parts of the free fatty acid prepared in step (1) and 3g of glycerol (the molar ratio of free fatty acid to glycerol is about 1: 1) are weighed into a 50mL reactor, 10% of immobilized TL lipase (based on the mass of the reaction substrate) is added, and the reaction is carried out at a stirring rate of 250rpm under vacuum conditions (gauge reading of 0.3 kPa) of 25 ℃ to 80 ℃ for 12 hours. After completion of the reaction, the reaction mixture was separated by TLC, and then a band of free fatty acids was scraped off, subjected to methyl esterification, and the change in the content of PUFAs in the free acids was measured by GC analysis, and the results are shown in Table 6. Through gas chromatography analysis, the DHA and EPA contents in the original herring oil are respectively 8.24% and 6.18%, the enrichment effect of the immobilized TL lipase is greatly different at different reaction temperatures, and the oxidation of PUFAs can be caused by too high temperature.
TABLE 6 PUFAs enrichment Effect of immobilized TL Lipase at different reaction temperatures
The above examples are intended to illustrate the invention in further detail. It should be noted that the present invention is not limited to these specific embodiments. Equivalent alterations and modifications may be effected by those skilled in the art without departing from the background and spirit of the invention, and the content thereof is also intended to be covered by the appended claims.
Claims (11)
1. A method for enriching long chain polyunsaturated fatty acids (PUFAs) using immobilized lipase, the method comprising:
(1) reacting free fatty acid with alcohol in the presence of immobilized thermomyces lanuginosus lipase under vacuum pumping or protective atmosphere;
(2) separating free fatty acid enriched with free PUFAs from the reacted product;
the immobilized thermomyces lanuginosus lipase is the thermomyces lanuginosus lipase immobilized on ion exchange resin;
wherein the ion exchange resin is a weakly basic ion exchange resin.
2. A method for purifying long chain polyunsaturated fatty acids using immobilized lipase, the method comprising:
(1) reacting free fatty acid with alcohol in the presence of immobilized thermomyces lanuginosus lipase under vacuum pumping or protective atmosphere;
(2) separating free fatty acid containing free PUFAs from the reacted product;
the immobilized thermomyces lanuginosus lipase is the thermomyces lanuginosus lipase immobilized on ion exchange resin;
wherein the ion exchange resin is a weakly basic ion exchange resin.
3. A method of reducing the content of short-chain fatty acids in free fatty acids using immobilized lipase, the method comprising:
reacting free fatty acid with alcohol in the presence of immobilized thermomyces lanuginosus lipase under vacuum pumping or protective atmosphere;
the immobilized thermomyces lanuginosus lipase is the thermomyces lanuginosus lipase immobilized on ion exchange resin;
wherein the ion exchange resin is a weakly basic ion exchange resin.
4. A method of increasing the content of long chain polyunsaturated fatty acids in free fatty acids using immobilized lipase, the method comprising:
reacting free fatty acid with alcohol in the presence of immobilized thermomyces lanuginosus lipase under vacuum pumping or protective atmosphere;
the immobilized thermomyces lanuginosus lipase is the thermomyces lanuginosus lipase immobilized on ion exchange resin;
wherein the ion exchange resin is a weakly basic ion exchange resin.
5. The method according to any one of claims 1 to 4, wherein the free fatty acids are obtained by base-catalyzed hydrolysis, enzymatic hydrolysis or high-pressure high-temperature hydrolysis of fats and oils containing PUFAs.
6. The method of claim 5, wherein the free fatty acid is prepared by:
(a) carrying out base catalytic hydrolysis on grease containing PUFAs under the condition of vacuum pumping or protective atmosphere;
(b) adding an extracting agent for extraction, acidifying the obtained water layer, and extracting again to obtain free fatty acid.
7. The method according to any one of claims 1 to 4, wherein the Thermomyces lanuginosus lipase immobilized on a weakly basic ion exchange resin is prepared by: (i) contacting the ion exchange resin with Thermomyces lanuginosus lipase to obtain immobilized Thermomyces lanuginosus lipase.
8. The method of claim 7, wherein the method further comprises the steps of: (ii) drying the immobilized thermomyces lanuginosus lipase.
9. The method as claimed in claim 8, wherein in step (i), the weakly basic ion exchange resin is contacted with Thermomyces lanuginosus lipase at 25-35 ℃ at 150-.
10. The method of claim 9, wherein 0.1 to 5g of weakly basic ion exchange resin is added per ml of Thermomyces lanuginosus lipase solution.
11. The method of claim 9, wherein the weakly basic ion exchange resin is present in an amount of 0.4 to 2.5 g.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310161447.2A CN104130860B (en) | 2013-05-03 | 2013-05-03 | Method for enriching long-chain polyunsaturated fatty acid by utilizing immobilized thermomyces lanuginosus lipase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310161447.2A CN104130860B (en) | 2013-05-03 | 2013-05-03 | Method for enriching long-chain polyunsaturated fatty acid by utilizing immobilized thermomyces lanuginosus lipase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104130860A CN104130860A (en) | 2014-11-05 |
| CN104130860B true CN104130860B (en) | 2020-03-31 |
Family
ID=51803718
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310161447.2A Active CN104130860B (en) | 2013-05-03 | 2013-05-03 | Method for enriching long-chain polyunsaturated fatty acid by utilizing immobilized thermomyces lanuginosus lipase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104130860B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105385675A (en) * | 2015-12-31 | 2016-03-09 | 厦门大学 | Immobilization method of candida antarctica lipase B |
| CN109852643B (en) * | 2019-03-18 | 2022-05-06 | 威海深蓝奇迹生物科技有限公司 | Method for preparing sn-1-DHA-lyso-phosphatidylserine |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1726181A (en) * | 2002-11-14 | 2006-01-25 | 普罗诺瓦·比奥凯尔有限公司 | Lipase-catalysed esterification of marine oil |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9404483D0 (en) * | 1994-03-08 | 1994-04-20 | Norsk Hydro As | Refining marine oil compositions |
| DK2172558T3 (en) * | 2007-07-30 | 2017-09-25 | Nippon Suisan Kaisha Ltd | Process for producing an EPA-enriched oil and DHA-enriched oil |
| CN101348807B (en) * | 2008-08-26 | 2011-07-27 | 江南大学 | Method for enrichment of n-3 polyunsaturated fatty acid glyceride from fish oil |
| CN102839166B (en) * | 2011-06-23 | 2015-11-25 | 丰益(上海)生物技术研发中心有限公司 | Tl immobilized enzyme and application thereof |
| CN102604913B (en) * | 2012-04-05 | 2013-08-14 | 湖南尤特尔生化有限公司 | Preparation method and application of thermomyces lanuginosus lipase |
| CN102660593B (en) * | 2012-04-28 | 2014-04-02 | 北京化工大学 | Method for preparing glyceride rich in algal oil n-3 polyunsaturated fatty acid through enzyme process |
-
2013
- 2013-05-03 CN CN201310161447.2A patent/CN104130860B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1726181A (en) * | 2002-11-14 | 2006-01-25 | 普罗诺瓦·比奥凯尔有限公司 | Lipase-catalysed esterification of marine oil |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104130860A (en) | 2014-11-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2649337C (en) | Process for preparing concentrated polyunsaturated fatty acid oil | |
| JP5204776B2 (en) | Method for producing EPA concentrated oil and DHA concentrated oil | |
| Carvalho et al. | Eco-friendly production of trimethylolpropane triesters from refined and used soybean cooking oils using an immobilized low-cost lipase (Eversa>® Transform 2.0) as heterogeneous catalyst | |
| JP3720194B2 (en) | Method for producing partial glycerides | |
| SG140551A1 (en) | Production of biodiesel and glycerin from high free fatty acid feedstocks | |
| Vandamme et al. | Impact of harvesting method on total lipid content and extraction efficiency for Phaeodactylum tricornutum | |
| CN111088296A (en) | Method for enriching n-3 polyunsaturated fatty acid glyceride in grease | |
| DE502005006779D1 (en) | Process for the accelerated enzymatic synthesis of triglycerides of unsaturated fatty acids | |
| KR101828769B1 (en) | Method for manufacturing succinic acid esters | |
| CN104130860B (en) | Method for enriching long-chain polyunsaturated fatty acid by utilizing immobilized thermomyces lanuginosus lipase | |
| CN104046662A (en) | Transesterification preparation method for 1,3-dioleic acid-2-triglyceride palmitate | |
| CN106399406A (en) | Coupling process for preparing biodiesel and enriching polyunsaturated fatty acid esters | |
| CN112266939B (en) | Method for enriching polyunsaturated fatty glyceride in grease by enzyme method | |
| US10870869B2 (en) | Enzymatic method for preparing glyceryl butyrate | |
| CN106609286A (en) | Preparation method for phosphatide containing long-chain polyunsaturated fatty acid | |
| CN110029133B (en) | Method for separating saturated fatty acid and unsaturated fatty acid from DHA algae oil | |
| US11655489B2 (en) | Method for improving n-3 polyunsaturated fatty acids enrichment efficiency in glyceride | |
| dos Santos Corrêa et al. | Enzymatic synthesis of dioctyl sebacate | |
| JP6083431B2 (en) | Method for producing glycerin carbonate acrylate compound | |
| US20130192126A1 (en) | Method for producing biodiesel from algal lipid with dimethyl carbonate | |
| JP3880095B2 (en) | Method for purifying highly unsaturated fatty acids | |
| CN115058463A (en) | Enzymatic preparation method of n-3 polyunsaturated fatty glyceride | |
| JPH10176182A (en) | Refining method for omega-3 highly unsaturated fatty acid | |
| CN115572745A (en) | Method for preparing structured fat by two-step enzyme method | |
| Awang et al. | Thermomyces lanuginosus lipase-catalyzed esterification of 9, 10-dihydroxystearic acid and monohydric alcohol |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |