CN111088296B - Method for enriching n-3 polyunsaturated fatty acid glyceride in grease - Google Patents
Method for enriching n-3 polyunsaturated fatty acid glyceride in grease Download PDFInfo
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- CN111088296B CN111088296B CN202010105081.7A CN202010105081A CN111088296B CN 111088296 B CN111088296 B CN 111088296B CN 202010105081 A CN202010105081 A CN 202010105081A CN 111088296 B CN111088296 B CN 111088296B
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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
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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
Abstract
The invention discloses a method for enriching n-3 polyunsaturated fatty acid glyceride in grease, which comprises the steps of putting the grease, lipase and buffer solution into a reactor, reacting for a certain time to obtain a mixture, and neutralizing and deacidifying the mixture by alkali liquor to obtain the glyceride rich in n-3 polyunsaturated fatty acid. The invention provides a method for enriching n-3 polyunsaturated fatty acid glyceride in grease, a lipase catalytic hydrolysis method directly takes fish oil as a raw material, no other organic solvent is added in the reaction process, the reaction raw materials are the fish oil and a buffer solution, and the catalyst is lipase; compared with the enzymatic transesterification and esterification, the method is safer, has lower cost, simpler process and mature method, and is more suitable for industrial production.
Description
Technical Field
The invention relates to the field of deep processing of grease, in particular to a method for enriching n-3 polyunsaturated fatty glyceride in grease.
Background
The n-3 type polyenoic acid (n-3PUFAs) has important biological significance to human bodies, the physiological functions of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the polyenoic acid (n-3PUFAs) are studied for a long time at home and abroad, and as essential fatty acid which cannot be synthesized by human bodies, the DHA and the EPA can obviously prevent and treat cardiovascular diseases, inhibit the growth of cancer cells, repair inflammation, promote the normal development of sense organs of infants and young children, and prevent and treat senile dementia to a certain extent, so the n-3 type polyenoic acid is widely applied to health foods at present.
The n-3PUFAs are mainly derived from raw materials such as deep sea fish oil, algae oil and the like, mainly exist in the form of glyceride, but the content of the glyceride is relatively low and is about 30 percent, so that the health care and medicinal requirements of people cannot be met. Therefore, the content of n-3PUFAs in the fish oil glyceride is increased, the health care value of the fish oil can be improved, and the method has important economic significance. The method for enriching n-3PUFAs mainly comprises physical methods such as supercritical fluid extraction and molecular distillation, chemical methods such as urea complexing low-temperature solvent crystallization, enzyme methods such as lipase catalytic hydrolysis and lipase catalytic transesterification, and currently, fish oil products mainly enriched by the physical and chemical methods account for most of the market.
The n-3PUFAs products on the market mainly have three forms of ethyl ester type, glyceride type and free fatty acid type, and most of ethyl ester type fish oil is easily obtained by physical and chemical methods such as urea embedding. However, researches report that the ethyl ester type fish oil is difficult to digest and absorb in human bodies, and in addition, reaction conditions required by a chemical method often have side reactions, so that the quality of products is reduced. Compared with the traditional chemical method, the method for enriching n-3PUFAs by the enzyme method has the advantages of greenness, safety, few byproducts and the like. However, in the prior art, the method for enriching n-3PUFAs by the enzyme method mainly synthesizes n-3PUFAs ethyl ester and glyceride type by the enzyme method esterification and ester exchange method, the lipase-catalyzed ester exchange method and the lipase-catalyzed esterification method have higher cost, and other organic solvents are added in the reaction process, so that the method does not conform to the green safety concept, and meanwhile, the process is complex and is not suitable for industrial production, so that the method for enriching n-3PUFAs by the enzyme method, which is suitable for industrial production and conforms to the green safety concept, is urgently needed in the field.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made in view of the above and/or the problems of the conventional methods for enriching n-3 polyunsaturated fatty acid glycerides in fats and oils.
Therefore, the invention aims to overcome the defects in the prior art and provide a method for enriching n-3 polyunsaturated fatty acid glyceride in grease.
In order to solve the technical problems, the invention provides the following technical scheme: a method for enriching n-3 polyunsaturated fatty acid glyceride in grease comprises the steps of putting grease, lipase and buffer solution into a reactor, reacting for a certain time to obtain a mixture, and removing free fatty acid in the mixture to obtain the glyceride rich in n-3 polyunsaturated fatty acid.
As a preferable scheme of the method for enriching n-3 polyunsaturated fatty acid glyceride in the grease, the method comprises the following steps: the oil is oil containing n-3 polyunsaturated fatty acid.
As a preferable scheme of the method for enriching n-3 polyunsaturated fatty acid glyceride in the grease, the method comprises the following steps: the oil comprises fish oil and algae oil.
As a preferable scheme of the method for enriching n-3 polyunsaturated fatty acid glyceride in the grease, the method comprises the following steps: the lipase is derived from one or more of Candida cylindracea (Candida cylindracea) and Rhizopus oryzae (Rhizopus oryzae).
As a preferable scheme of the method for enriching n-3 polyunsaturated fatty acid glyceride in the grease, the method comprises the following steps: the lipase derived from the candida cylindracea is one or more of AY 'Amano' 400SD and AY 'Amano' 30 SD.
As a preferable scheme of the method for enriching n-3 polyunsaturated fatty acid glyceride in the grease, the method comprises the following steps: the lipase derived from rhizopus oryzae is DF "Amano" 15.
As a preferable scheme of the method for enriching n-3 polyunsaturated fatty acid glyceride in the grease, the method comprises the following steps: the buffer solution is one of phosphoric acid buffer solution and citric acid buffer solution, wherein the concentration of the buffer solution is 0.05-0.5 mol/L, and the pH value range of the buffer solution is 5-8.
As a preferable scheme of the method for enriching n-3 polyunsaturated fatty acid glyceride in the grease, the method comprises the following steps: the mass ratio of the buffer solution to the grease is 0.2-3: 1.
As a preferable scheme of the method for enriching n-3 polyunsaturated fatty acid glyceride in the grease, the method comprises the following steps: the reaction time is 4-48 h, and the reaction temperature is 10-60 ℃.
As a preferable scheme of the method for enriching n-3 polyunsaturated fatty acid glyceride in the grease, the method comprises the following steps: the addition amount of the lipase accounts for 0.04-1% of the mass percentage of the grease.
The invention has the beneficial effects that:
(1) the invention provides a method for enriching n-3 polyunsaturated fatty acid glyceride in grease, a lipase catalytic hydrolysis method directly takes fish oil, algae oil and the like as raw materials, no other organic solvent is added in the reaction process, the reaction raw materials are the fish oil and buffer solution, the catalyst is lipase, and compared with the traditional chemical method, the method has the advantages of mild reaction conditions, less by-products and environmental protection; compared with the enzymatic transesterification and esterification, the method is safer and has lower cost.
(2) The AY 'Amano' 400SD and AY 'Amano' 30SD lipases selected by the invention have substrate 'discriminative property' in the aspect of hydrolysis speed, the hydrolysis rate of triglyceride containing saturated fatty acid and low unsaturated fatty acid is far higher than that of triglyceride containing polyunsaturated fatty acid, and the enrichment of EPA and DHA in glyceride is achieved by moderate hydrolysis by utilizing the characteristic.
(3) Compared with the existing process for enriching n-3PUFAs by an enzyme method, the process provided by the invention is simpler, the method is mature, and the method is more suitable for industrial production.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is a gas chromatographic chart of fatty acid composition before fish oil reaction in example 1 of the present invention. Putting 50mg fish oil into a 10mL graduated tube, adding 2mL of 0.5mol/L potassium hydroxide-methanol solution, saponifying at 65 ℃ for 30min, cooling, adding 2mL of 25% volume fraction boron trifluoride-methanol solution, and carrying out water bath at 70 ℃ for 5 min; adding 2mL of n-hexane, oscillating for 3-4min to extract fatty acid methyl ester, adding 4mL of saturated NaCl solution, taking the upper layer solution, adding anhydrous sodium sulfate, oscillating (centrifuging at 10000rpm for 5min), sucking by a syringe, passing through a membrane, detecting by gas chromatography, and calculating the content of n-3 PUFAs.
FIG. 2 is a compositional analysis liquid chromatography (differential detector) chart of the oil phase product obtained after hydrolysis of the fish oil in example 1 of the present invention. And (3) taking 20mg of the hydrolyzed mixed fish oil product, adding 1mL of mobile phase (normal hexane: isopropanol: formic acid: 15:1:0.003) to dissolve, passing through a membrane, detecting by liquid chromatography, and calculating the content of free fatty acid under hydrolysis.
Fig. 3 is a gas chromatographic chart of the fatty acid composition of the glyceride type fish oil product obtained after the fish oil is hydrolyzed and deacidified in example 1. Taking 50mg of the prepared glyceride rich in n-3PUFAs in a 10mL graduated tube, carrying out sample preparation steps in the same operation mode as the operation mode shown in the figure 1, and calculating the content of the n-3 PUFAs.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.
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.
The lipase AY 'Amano' 400SD, 400000U/g in the invention is purchased from Japan Tianye biological enzyme preparation Co.Ltd; AY "Amano"30SD, 400000U/g, from Japanese Tian-Ye
Enzyme preparations Co., Ltd; DF "Amano"15, 400000U/g, was purchased from Japan Nature Bio-enzyme preparations Ltd.
The oil used in the invention is commercially available, wherein the fish oil 1 (tuna oil) has EPA (in percentage of 6.06%) and DHA (in percentage of 24.23%); fish oil 2(1812 fish oil) EPA% 18.02%, DHA% 11.39%; algae oil EPA 3.32% and DHA 37.28%. Other reagents are not specifically indicated and are all commercially available.
Example 1
Accurately weighing 3.0g of fish oil 1 sample (fatty acid composition of the fish oil sample is shown in figure 1, wherein EPA% is 6.06%, DHA% is 24.23%), 1.5g of phosphate buffer solution (0.1mol/L, pH 7), and 2.4mg of hybrid lipase of AY "Amano"400SD and AY "Amano"30SD (mass ratio of the two is 2:3), adding into a reaction kettle, placing into a magnetic rotor, and sealing. The reaction kettle is placed on a magnetic stirrer, circulating water is introduced into the reaction kettle, the temperature of the water is constant at 37 ℃, and the reaction kettle is kept for 12 hours. The oil phase of the hydrolysate obtained had a mass fraction of glycerides of 58.21% and a mass fraction of free fatty acids of 41.79% (see fig. 2).
After the reaction was completed, the resulting mixture was transferred to a separatory funnel, and 30mL of n-hexane was added thereto to separate the fish oil from water. Adding phenolphthalein indicator, titrating with KOH-ethanol aqueous solution until the system turns red, standing for layering, removing the lower aqueous phase, washing with distilled water to remove possible residual fatty acid salt and KOH in the oil phase, taking the upper clear oil phase, and evaporating to remove the solvent to obtain the n-3 PUFAs-rich fish oil glyceride. The fatty acid composition of the glyceride product increased n-3PUFAs from 34.11% in the crude oil to 60.98% after hydrolysis, and the total content of EPA and DHA increased from 30.29% to 56.11% (see FIG. 3).
Fig. 1 and fig. 3 are gas chromatograms of fatty acid composition before and after fish oil hydrolysis, and it can be seen that the DHA mass fraction is significantly increased. Fig. 2 shows the mass ratio of triglyceride, diglyceride, monoglyceride, fatty acid in the oil phase system after hydrolysis, so that the degree of hydrolysis can be seen.
Example 2
Accurately weighing 3.0g of refined fish oil 1 sample, 1.5g of phosphate buffer solution (0.1mol/L, pH 7) and 2.4mg of AY 'Amano' 400SD lipase, adding into a reaction kettle, placing into a magnetic rotor, and sealing. The reaction kettle is placed on a magnetic stirrer, circulating water is introduced into the reaction kettle, the temperature of the water is constant at 37 ℃, and the reaction kettle is kept for 12 hours. The mass fraction of glyceride in the oil phase of the hydrolysate obtained was 59.46%, and the mass fraction of free fatty acid was 40.54%.
After the reaction was completed, the resulting mixture was transferred to a separatory funnel, and 30mL of n-hexane was added thereto to separate the fish oil from water. Adding phenolphthalein indicator, titrating with KOH-ethanol aqueous solution until the system turns red, standing for layering, removing the lower aqueous phase, washing with distilled water to remove possible residual fatty acid salt and KOH in the oil phase, taking the upper clear oil phase, and evaporating to remove the solvent to obtain the n-3 PUFAs-rich fish oil glyceride. The fatty acid composition of the glyceride product is increased from 34.11% to 62.36% after hydrolysis, and the content of n-3PUFAs is increased from 30.29% to 55.33%.
Example 3
Accurately weighing 3.0g of refined fish oil 1 sample, 3.0g of phosphoric acid buffer solution (0.1mol/L, pH 6) and 2.4mg of AY 'Amano' 30SD lipase, adding into a reaction kettle, placing into a magnetic rotor, and sealing. The reaction kettle is placed on a magnetic stirrer, circulating water is introduced into the reaction kettle, the temperature of the water is constant at 37 ℃, and the reaction kettle is kept for 12 hours. The mass fraction of glyceride in the oil phase of the hydrolysate obtained was 73.25%, and the mass fraction of free fatty acid was 26.75%.
After the reaction was completed, the resulting mixture was transferred to a separatory funnel, and 30mL of n-hexane was added thereto to separate the fish oil from water. Adding phenolphthalein indicator, titrating with KOH-ethanol aqueous solution until the system turns red, standing for layering, removing the lower aqueous phase, washing with distilled water to remove possible residual fatty acid salt and KOH in the oil phase, taking the upper clear oil phase, and evaporating to remove the solvent to obtain the n-3 PUFAs-rich fish oil glyceride. The fatty acid composition of the glyceride product is increased from 34.11% to 54.12% after hydrolysis, and the total content of EPA and DHA is increased from 30.29% to 46.89%.
Example 4
Accurately weighing 3.0g of refined fish oil 1 sample, 1.5g of citric acid buffer solution (0.1mol/L, pH is 6) and 3.6mg of mixed lipase of AY 'Amano' 400SD and AY 'Amano' 30SD (2:3), adding into a reaction kettle, placing into a magnetic rotor, and sealing. The reaction kettle is placed on a magnetic stirrer, circulating water is introduced into the reaction kettle, the temperature of the water is constant at 50 ℃, and the reaction kettle is kept for 6 hours. The mass fraction of glyceride in the oil phase of the hydrolysate obtained was 75.69%, and the mass fraction of free fatty acid was 24.31%.
After the reaction was completed, the resulting mixture was transferred to a separatory funnel, and 30mL of n-hexane was added thereto to separate the fish oil from water. Adding phenolphthalein indicator, titrating with KOH-ethanol aqueous solution until the system turns red, standing for layering, removing the lower aqueous phase, washing with distilled water to remove possible residual fatty acid salt and KOH in the oil phase, taking the upper clear oil phase, and evaporating to remove the solvent to obtain the n-3 PUFAs-rich fish oil glyceride. The fatty acid composition of the glyceride product is increased from 34.11% to 50.83% after hydrolysis, and the total content of EPA and DHA is increased from 30.29% to 44.23%.
Example 5
Accurately weighing 3.0g of refined fish oil 1 sample, 3.0g of citric acid buffer solution (0.1mol/L, pH 6) and 3.6mg of AY 'Amano' 400SD lipase, adding into a reaction kettle, placing into a magnetic rotor, and sealing. The reaction kettle is placed on a magnetic stirrer, circulating water is introduced into the reaction kettle, the temperature of the water is constant at 37 ℃, and the reaction kettle is kept for 12 hours. The mass fraction of glyceride in the oil phase of the hydrolysate obtained was 65.49%, and the mass fraction of free fatty acid was 34.51%.
After the reaction was completed, the resulting mixture was transferred to a separatory funnel, and 30mL of n-hexane was added thereto to separate the fish oil from water. Adding phenolphthalein indicator, titrating with KOH-ethanol aqueous solution until the system turns red, standing for layering, removing the lower aqueous phase, washing with distilled water to remove possible residual fatty acid salt and KOH in the oil phase, taking the upper clear oil phase, and evaporating to remove the solvent to obtain the n-3 PUFAs-rich fish oil glyceride. In the fatty acid composition of the glyceride product, n-3PUFAs accounts for an increase from 34.11% of the crude oil to 57.21% after hydrolysis, wherein the total content of EPA and DHA is increased from 30.29% to 51.29%.
Example 6
Accurately weighing 3.0g of refined fish oil 1 sample, 6.0g of phosphoric acid buffer solution (0.1mol/L, pH 5) and 1.2mg of mixed lipase of AY 'Amano' 400SD and AY 'Amano' 30SD (2:3), adding into a reaction kettle, placing into a magnetic rotor, and sealing. The reaction kettle is placed on a magnetic stirrer, circulating water is introduced into the reaction kettle, the temperature of the water is constant at 20 ℃, and the reaction kettle is kept for 24 hours. The mass fraction of glyceride in the oil phase of the hydrolysate obtained was 72.23%, and the mass fraction of free fatty acid was 27.77%.
After the reaction was completed, the resulting mixture was transferred to a separatory funnel, and 30mL of n-hexane was added thereto to separate the fish oil from water. Adding phenolphthalein indicator, titrating with KOH-ethanol aqueous solution until the system turns red, standing for layering, removing the lower aqueous phase, washing with distilled water to remove possible residual fatty acid salt and KOH in the oil phase, taking the upper clear oil phase, and evaporating to remove the solvent to obtain the n-3 PUFAs-rich fish oil glyceride. The fatty acid composition of the glyceride product is increased from 34.11% to 52.29% after hydrolysis, and the content of n-3PUFAs is increased from 30.29% to 46.87%.
Example 7
Accurately weighing 3.0g of refined fish oil 1 sample, 0.6g of citric acid buffer solution (0.1mol/L, pH 5) and 1.2mg of AY "Amano"30SD lipase, adding into a reaction kettle, placing into a magnetic rotor, and sealing. The reaction kettle is placed on a magnetic stirrer, circulating water is introduced into the reaction kettle, the temperature of the water is constant at 50 ℃, and the reaction kettle is kept for 6 hours. The mass fraction of glyceride in the oil phase of the hydrolysate obtained was 80.01%, and the mass fraction of free fatty acid was 19.99%.
After the reaction was completed, the resulting mixture was transferred to a separatory funnel, and 30mL of n-hexane was added thereto to separate the fish oil from water. Adding phenolphthalein indicator, titrating with KOH-ethanol aqueous solution until the system turns red, standing for layering, removing the lower aqueous phase, washing with distilled water to remove possible residual fatty acid salt and KOH in the oil phase, taking the upper clear oil phase, and evaporating to remove the solvent to obtain the n-3 PUFAs-rich fish oil glyceride. The fatty acid composition of the glyceride product is increased from 34.11% to 46.98% by n-3PUFAs, and the total content of EPA and DHA is increased from 30.29% to 40.19%.
Example 8
Accurately weighing 3.0g of refined fish oil 1 sample, 6.0g of phosphoric acid buffer solution (0.1mol/L, pH 8) and 2.4mg of AY 'Amano' 400SD lipase, adding into a reaction kettle, placing into a magnetic rotor, and sealing. The reaction kettle is placed on a magnetic stirrer, circulating water is introduced into the reaction kettle, the temperature of the water is constant at 37 ℃, and the reaction kettle is kept for 24 hours. The mass fraction of glyceride in the oil phase of the hydrolysate obtained was 71.55%, and the mass fraction of free fatty acid was 28.45%.
After the reaction was completed, the resulting mixture was transferred to a separatory funnel, and 30mL of n-hexane was added thereto to separate the fish oil from water. Adding phenolphthalein indicator, titrating with KOH-ethanol aqueous solution until the system turns red, standing for layering, removing the lower aqueous phase, washing with distilled water to remove possible residual fatty acid salt and KOH in the oil phase, taking the upper clear oil phase, and evaporating to remove the solvent to obtain the n-3 PUFAs-rich fish oil glyceride. The fatty acid composition of the glyceride product is increased from 34.11% to 62.86% after hydrolysis, and the content of n-3PUFAs is increased from 30.29% to 56.02%.
Example 9
Accurately weighing 3.0g of refined fish oil 1 sample, 0.6g of citric acid buffer solution (0.1mol/L, pH 7) and 3.6mg of AY 'Amano' 30SD lipase, adding into a reaction kettle, placing into a magnetic rotor, and sealing. The reaction kettle is placed on a magnetic stirrer, circulating water is introduced into the reaction kettle, the temperature of the water is constant at 20 ℃, and the reaction kettle is kept for 6 hours. The mass fraction of glyceride in the oil phase of the hydrolysate obtained was 78.93%, and the mass fraction of free fatty acid was 21.07%.
After the reaction was completed, the resulting mixture was transferred to a separatory funnel, and 30mL of n-hexane was added thereto to separate the fish oil from water. Adding phenolphthalein indicator, titrating with KOH-ethanol aqueous solution until the system turns red, standing for layering, removing the lower aqueous phase, washing with distilled water to remove possible residual fatty acid salt and KOH in the oil phase, taking the upper clear oil phase, and evaporating to remove the solvent to obtain the n-3 PUFAs-rich fish oil glyceride. The fatty acid composition of the glyceride product is increased from 34.11% to 49.96% by hydrolysis, and the total content of EPA and DHA is increased from 30.29% to 43.06%.
Example 10
Accurately weighing 3.0g of refined fish oil 1 sample, 1.5g of phosphoric acid buffer solution (0.1mol/L, pH 8) and 2.4mg of AY 'Amano' 400SD lipase, adding into a reaction kettle, placing into a magnetic rotor, and sealing. The reaction kettle is placed on a magnetic stirrer, circulating water is introduced into the reaction kettle, the temperature of the water is constant at 20 ℃, and the reaction kettle is kept for 24 hours. The mass fraction of glyceride in the oil phase of the hydrolysate obtained was 72.13%, and the mass fraction of free fatty acid was 27.87%.
After the reaction was completed, the resulting mixture was transferred to a separatory funnel, and 30mL of n-hexane was added thereto to separate the fish oil from water. Adding phenolphthalein indicator, titrating with KOH-ethanol aqueous solution until the system turns red, standing for layering, removing the lower aqueous phase, washing with distilled water to remove possible residual fatty acid salt and KOH in the oil phase, taking the upper clear oil phase, and evaporating to remove the solvent to obtain the n-3 PUFAs-rich fish oil glyceride. The fatty acid composition of the glyceride product is increased from 34.11% to 53.09% after hydrolysis, and the content of n-3PUFAs is increased from 30.29% to 46.59%.
Example 11
Accurately weighing 3.0g of fish oil 2 sample (wherein EPA% ═ 18.02%, DHA% ═ 11.39%), 1.5g of phosphate buffer solution (0.1mol/L, pH 7) and 2.4mg of hybrid lipase of AY "Amano"400SD and AY "Amano"30SD (the mass ratio of the two is 2:3), adding into a reaction kettle, putting into a magnetic rotor, and sealing. The reaction kettle is placed on a magnetic stirrer, circulating water is introduced into the reaction kettle, the temperature of the water is constant at 37 ℃, and the reaction kettle is kept for 12 hours. The mass fraction of glyceride in the oil phase of the hydrolysate obtained is 59.81%, and the mass fraction of free fatty acid is 40.19%.
After the reaction was completed, the resulting mixture was transferred to a separatory funnel, and 30mL of n-hexane was added thereto to separate the fish oil from water. Adding phenolphthalein indicator, titrating with KOH-ethanol aqueous solution until the system turns red, standing for layering, removing the lower aqueous phase, washing with distilled water to remove possible residual fatty acid salt and KOH in the oil phase, taking the upper clear oil phase, and evaporating to remove the solvent to obtain the n-3 PUFAs-rich fish oil glyceride. In the fatty acid composition of the glyceride product, the n-3PUFAs accounts for an increase from 33.75% of the crude oil to 58.27% after hydrolysis, wherein the total content of EPA and DHA is increased from 29.41% to 52.96%.
Example 12
Accurately weighing 3.0g of algae oil sample (wherein EPA% (-) 3.32%, DHA% (-) 37.28%), 1.5g of phosphate buffer solution (0.1mol/L, pH 7) and 2.4mg of mixed lipase of AY "Amano"400SD and AY "Amano"30SD (the mass ratio of the two is 2:3), adding into a reaction kettle, placing into a magnetic rotor, and sealing. The reaction kettle is placed on a magnetic stirrer, circulating water is introduced into the reaction kettle, the temperature of the water is constant at 37 ℃, and the reaction kettle is kept for 12 hours. The mass fraction of glyceride in the oil phase of the hydrolysate obtained is 63.12%, and the mass fraction of free fatty acid is 36.88%.
After the reaction was completed, the resulting mixture was transferred to a separatory funnel, and 30mL of n-hexane was added thereto to separate the fish oil from water. Adding phenolphthalein indicator, titrating with KOH-ethanol aqueous solution until the system turns red, standing for layering, removing the lower aqueous phase, washing with distilled water to remove possible residual fatty acid salt and KOH in the oil phase, collecting the upper clear oil phase, and evaporating to remove the solvent to obtain the n-3 PUFAs-rich algal oil glyceride. In the fatty acid composition of the glyceride product, n-3PUFAs accounts for an increase from 43.03% of crude oil to 67.69% after hydrolysis, wherein the total content of EPA and DHA is increased from 40.60% to 65.64%.
Example 13
Accurately weighing 3.0g of fish oil 1 sample (wherein EPA% ═ 6.06%, DHA% ═ 24.23%), 1.5g of phosphate buffer solution (0.1mol/L, pH 7) and 2.4mg of hybrid lipase of AY "Amano"400SD and AY "Amano"30SD (the mass ratio of the two is 2:3), adding into a reaction kettle, putting into a magnetic rotor, and sealing. The reaction kettle is placed on a magnetic stirrer, circulating water is introduced into the reaction kettle, the temperature of the water is constant at 37 ℃, and the reaction kettle is kept for 12 hours. The mass fraction of glyceride in the oil phase of the hydrolysate obtained is 58.21%, and the mass fraction of free fatty acid is 41.79%.
And after the reaction is finished, transferring the obtained mixture into a molecular distillation apparatus, and separating to obtain the heavy-phase fish oil glyceride rich in n-3 PUFAs. The fatty acid composition of the glyceride product is increased from 34.11% to 59.33% after hydrolysis, and the content of n-3PUFAs is increased from 30.29% to 55.78%.
Example 14
Accurately weighing 3.0g of fish oil 1 sample (wherein EPA% ═ 6.06%, DHA% ═ 24.23%), 1.5g of phosphate buffer solution (0.1mol/L, pH 7) and 2.4mg of hybrid lipase of AY "Amano"400SD and AY "Amano"30SD (the mass ratio of the two is 2:3), adding into a reaction kettle, putting into a magnetic rotor, and sealing. The reaction kettle is placed on a magnetic stirrer, circulating water is introduced into the reaction kettle, the temperature of the water is constant at 37 ℃, and the reaction kettle is kept for 12 hours. The mass fraction of glyceride in the oil phase of the hydrolysate obtained is 58.21%, and the mass fraction of free fatty acid is 41.79%.
After the reaction is finished, transferring the obtained mixture to a small deodorization tower, separating free fatty acid to obtain heavy-phase fish oil glyceride rich in n-3 PUFAs. The fatty acid composition of the glyceride product is increased from 34.11% to 57.12% after hydrolysis, and the content of n-3PUFAs is increased from 30.29% to 53.40%.
Example 15
Accurately weighing 3.0g of fish oil 2 sample (wherein EPA% ═ 18.02%, DHA% ═ 11.39%), 1.5g of phosphate buffer solution (0.1mol/L, pH 7) and 2.4mg of hybrid lipase of AY "Amano"400SD and AY "Amano"30SD (the mass ratio of the two is 2:3), adding into a reaction kettle, putting into a magnetic rotor, and sealing. The reaction kettle is placed on a magnetic stirrer, circulating water is introduced into the reaction kettle, the temperature of the water is constant at 37 ℃, and the reaction kettle is kept for 12 hours. The mass fraction of glyceride in the oil phase of the hydrolysate obtained was 60.33%, and the mass fraction of free fatty acid was 39.67%.
And after the reaction is finished, transferring the obtained mixture into a molecular distillation apparatus, and separating to obtain the heavy-phase fish oil glyceride rich in n-3 PUFAs. The fatty acid composition of the glyceride product is increased from 33.75% to 56.73% after hydrolysis, and the content of n-3PUFAs is increased from 29.41% to 52.39%.
Example 16
Accurately weighing 3.0g of algae oil sample (wherein EPA% (-) 3.32%, DHA% (-) 37.28%), 1.5g of phosphate buffer solution (0.1mol/L, pH 7) and 2.4mg of mixed lipase of AY "Amano"400SD and AY "Amano"30SD (the mass ratio of the two is 2:3), adding into a reaction kettle, placing into a magnetic rotor, and sealing. The reaction kettle is placed on a magnetic stirrer, circulating water is introduced into the reaction kettle, the temperature of the water is constant at 37 ℃, and the reaction kettle is kept for 12 hours. The mass fraction of glyceride in the oil phase of the hydrolysate obtained is 63.39%, and the mass fraction of free fatty acid is 36.61%.
After the reaction is finished, the obtained mixture is transferred to a small deodorization tower, free fatty acid is separated out, and the heavy-phase algal oil glyceride rich in n-3PUFAs is obtained. The fatty acid composition of the glyceride product is increased from 43.03% to 65.32% after hydrolysis, and the content of n-3PUFAs is increased from 40.60% to 63.07%.
The reaction conditions of examples 1 to 16 are shown in Table 1.
TABLE 1
Examples 1-16 reaction results for different reaction conditions, including the mass fraction of glycerides in the oil phase after hydrolysis, the deacidified n-3PUFAs content, and the deacidified EPA + DHA content, are shown in table 2.
TABLE 2
As can be seen from Table 2, the n-3PUFAs content after deacidification of the oil and fat under different reaction conditions is different from the EPA + DHA content after deacidification, and the EPA + DHA content after deacidification is optimal under the conditions of example 1, namely the reaction conditions are optimal. Under the same conditions, the raw material in example 11 is changed into another fish oil and algae oil, and a better enrichment effect is also obtained. Also, examples 13-16 show that several deacidification methods did not have a significant effect on the enrichment results.
Examples 17 to 29
Referring to the steps of examples 1 and 2, one of the reaction conditions was changed, and the remaining steps and parameters were not changed, and the changed conditions are shown in Table 3.
TABLE 3
Examples 17 to 29 reaction results corresponding to different reaction conditions, including the mass fraction of glycerides in the oil phase after hydrolysis, the deacidified n-3PUFAs content and the deacidified EPA + DHA content, are shown in table 4.
TABLE 4
Comparative example 1
Accurately weighing 3.0g of refined fish oil 1 sample, 1.5g of phosphate buffer solution (0.1mol/L, pH 7) and 2.4mg of Lipozyme435 lipase, adding into a reaction kettle, placing into a magnetic rotor, and sealing. The reaction kettle is placed on a magnetic stirrer, circulating water is introduced into the reaction kettle, the temperature of the water is constant at 37 ℃, and the reaction kettle is kept for 12 hours. The mass fraction of glyceride in the oil phase of the hydrolysate obtained is 71.76%, and the mass fraction of free fatty acid is 28.24%.
After the reaction was completed, the resulting mixture was transferred to a separatory funnel, and 30mL of n-hexane was added thereto to separate the fish oil from water. Adding phenolphthalein indicator, titrating with KOH-ethanol aqueous solution until the system turns red, standing for layering, removing the lower aqueous phase, washing with distilled water to remove possible residual fatty acid salt and KOH in the oil phase, taking the upper clear oil phase, and evaporating to remove the solvent to obtain the n-3 PUFAs-rich fish oil glyceride. The fatty acid composition of the glyceride product is increased from 34.11% to 40.09% after hydrolysis, and the total content of EPA and DHA is increased from 30.29% to 35.12%.
Comparative example 2
Accurately weighing 3.0G of refined fish oil 1 sample, 1.5G of phosphate buffer solution (0.1mol/L, pH 7) and 2.4mg of G 'Amano' 50 lipase, adding into a reaction kettle, placing into a magnetic rotor, and sealing. The reaction kettle is placed on a magnetic stirrer, circulating water is introduced into the reaction kettle, the temperature of the water is constant at 37 ℃, and the reaction kettle is kept for 12 hours. The mass fraction of glyceride in the oil phase of the hydrolysate obtained was 94.12%, and the mass fraction of free fatty acid was 5.88%.
After the reaction was completed, the resulting mixture was transferred to a separatory funnel, and 30mL of n-hexane was added thereto to separate the fish oil from water. Adding phenolphthalein indicator, titrating with KOH-ethanol aqueous solution until the system turns red, standing for layering, removing the lower aqueous phase, washing with distilled water to remove possible residual fatty acid salt and KOH in the oil phase, taking the upper clear oil phase, and evaporating to remove the solvent to obtain the n-3 PUFAs-rich fish oil glyceride. In the fatty acid composition of the glyceride product, n-3PUFAs accounts for an increase from 34.11% of the crude oil to 40.12% after hydrolysis, wherein the total content of EPA and DHA is increased from 30.29% to 35.65%.
Comparative example 3
Accurately weighing 3.0g of refined fish oil 1 sample and 2.4mg of AY 'Amano' 400SD lipase, adding into a reaction kettle, placing into a magnetic rotor, and sealing. The reaction kettle is placed on a magnetic stirrer, circulating water is introduced into the reaction kettle, the temperature of the water is constant at 37 ℃, and the reaction kettle is kept for 12 hours. The mass fraction of glyceride in the oil phase of the hydrolysate obtained is 90.89%, and the mass fraction of free fatty acid is 9.11%.
After the reaction was completed, the resulting mixture was transferred to a separatory funnel, and 30mL of n-hexane was added thereto to separate the fish oil from water. Adding phenolphthalein indicator, titrating with KOH-ethanol aqueous solution until the system turns red, standing for layering, removing the lower aqueous phase, washing with distilled water to remove possible residual fatty acid salt and KOH in the oil phase, taking the upper clear oil phase, and evaporating to remove the solvent to obtain the n-3 PUFAs-rich fish oil glyceride. The fatty acid composition of the glyceride product is increased from 34.11% to 37.79% after hydrolysis, and the content of n-3PUFAs is increased from 30.29% to 33.59%.
Comparative example 4
Accurately weighing 3.0g of refined fish oil 1 sample, 1.5g of phosphate buffer solution (0.1mol/L, pH 9) and 2.4mg of AY 'Amano' 400SD lipase, adding into a reaction kettle, placing into a magnetic rotor, and sealing. The reaction kettle is placed on a magnetic stirrer, circulating water is introduced into the reaction kettle, the temperature of the water is constant at 37 ℃, and the reaction kettle is kept for 12 hours. The mass fraction of glyceride in the oil phase of the hydrolysate obtained was 95.36%, and the mass fraction of free fatty acid was 4.64%.
After the reaction was completed, the resulting mixture was transferred to a separatory funnel, and 30mL of n-hexane was added thereto to separate the fish oil from water. Adding phenolphthalein indicator, titrating with KOH-ethanol aqueous solution until the system turns red, standing for layering, removing the lower aqueous phase, washing with distilled water to remove possible residual fatty acid salt and KOH in the oil phase, taking the upper clear oil phase, and evaporating to remove the solvent to obtain the n-3 PUFAs-rich fish oil glyceride. The fatty acid composition of the glyceride product is increased from 34.11% to 39.98% by n-3PUFAs, and the total content of EPA and DHA is increased from 30.29% to 35.43%.
The reaction conditions of comparative examples 1 to 4 are shown in Table 5.
TABLE 5
The reaction results corresponding to different reaction conditions of comparative examples 1 to 4, including the mass fraction of glycerides in the oil phase after hydrolysis, the n-3PUFAs content after deacidification, and the EPA + DHA content after deacidification, are shown in table 6.
TABLE 6
As can be seen from Table 6, different enzyme pairs have higher EPA + DHA content after deacidification, and the enzyme Lipozyme435 is selected, so that the EPA + DHA content after deacidification is lower. Meanwhile, different pH values have great influence on the enzymolysis effect.
Comparative example 5
The effect of different enzyme addition ratios (AY "Amano"400SD and AY "Amano"30SD) on the EPA + DHA content after deacidification was investigated without changing other conditions in example 1, and the experimental design is shown in Table 7.
TABLE 7
According to the experiments and data in the examples, it can be seen that AY "Amano"400SD has better hydrolysis effect (saturated and low unsaturated fatty acid under hydrolysis), but also has a certain hydrolysis effect on EPA, and the hydrolysis effect is greatly weakened by the reduction of the addition amount. AY "Amano"30SD has good hydrolysis effect, and has much weak hydrolysis to EPA. Two enzymes were mixed for hydrolysis, and as can also be seen in several of the experiments in comparative example 5, AY "Amano"400 SD: the AY Amano 30SD has better effect when the ratio is 2:3, the integral hydrolysis effect is influenced by the overhigh ratio of the AY Amano 30SD, and the EPA content is reduced by the overhigh ratio of the AY Amano 400 SD.
The invention does not need to disturb and recombine fatty acid hydrolysis, avoids the generation of oxidation and cis-trans isomerization and can obtain more natural glyceride products. Meanwhile, the inventors found that the enzyme has hydrolysis-preferential selectivity for saturated and unsaturated fatty acids, substrate discrimination for EPA and DHA, and particularly higher DHA content. Using AY "Amano"400 SD: AY 'Amano' 30SD is mixed hydrolase of 2:3, so that EPA loss is reduced, and the process is more complete.
In the prior art, triglyceride is hydrolyzed into free fatty acid by a chemical method, and then the free fatty acid type of n-3PUFAs is enriched by physicochemical methods such as urea complexation, molecular distillation, low-temperature crystallization and the like, or n-3PUFAs ethyl ester and glyceride type are synthesized by enzymatic esterification and ester exchange methods. The scheme directly carries out enzymatic hydrolysis on triglyceride, and the selected enzyme has specificity: has hydrolysis-preferential selectivity for saturated and unsaturated fatty acids and substrate discrimination for EPA and DHA, especially DHA. The selection range of saturated and low unsaturated fatty acid can be enlarged by selecting the mixed enzyme, thereby achieving better hydrolysis effect.
The chemical hydrolysis may partially destroy natural all-cis n-3PUFAs, the scheme does not need to disturb and recombine fatty acid hydrolysis, oxidation and cis-trans isomerization are avoided, and more natural n-3PUFAs glyceride can be obtained. In the aspect of process, the technical scheme has the advantages of simple operation flow, good effect and higher industrial feasibility degree. In addition, the technical scheme can obtain more than 60 percent of n-3PUFAs glyceride from common fish oil in the market, and can be realized only by the specific process conditions of the invention. Compared with the existing process for enriching n-3PUFAs by an enzyme method, the process provided by the invention is simpler, the method is mature, and the method is more suitable for industrial production.
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 (3)
1. A method for enriching n-3 polyunsaturated fatty acid glyceride in grease is characterized in that: comprises the steps of (a) preparing a mixture of a plurality of raw materials,
taking oil, lipase and buffer solution to react in a reactor for a certain time to obtain a mixture, and removing free fatty acid in the mixture to obtain glyceride rich in n-3 polyunsaturated fatty acid;
wherein the oil is fish oil or algae oil containing n-3 polyunsaturated fatty acid;
the lipase is derived from Candida cylindracea: (Candida cylindracea) The enzyme mixture consists of AY 'Amano' 400SD and AY 'Amano' 30SD according to the mass ratio of 2: 3;
the buffer solution is one of phosphoric acid buffer solution and citric acid buffer solution, wherein the concentration of the buffer solution is 0.05-0.5 mol/L, and the pH value range of the buffer solution is 5-8;
the addition amount of the lipase accounts for 0.08-0.5% of the mass percent of the grease;
the reaction temperature is 20-60 ℃.
2. The method for enriching n-3 polyunsaturated fatty acid glycerides in oils and fats according to claim 1, wherein: the mass ratio of the buffer solution to the grease is 0.2-3: 1.
3. The method for enriching n-3 polyunsaturated fatty acid glycerides in oils and fats according to claim 1, wherein: the reaction time is 4-48 h.
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