US20130123525A1 - Method for producing oil containing polyunsaturated fatty acid using lipase - Google Patents

Method for producing oil containing polyunsaturated fatty acid using lipase Download PDF

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US20130123525A1
US20130123525A1 US13/700,647 US201113700647A US2013123525A1 US 20130123525 A1 US20130123525 A1 US 20130123525A1 US 201113700647 A US201113700647 A US 201113700647A US 2013123525 A1 US2013123525 A1 US 2013123525A1
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fatty acid
glyceride
lipase
reaction
area percent
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Hideo Ikemoto
Nobushige Doisaki
Yasuo Umehara
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Nissui Corp
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Nippon Suisan Kaisha Ltd
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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
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • A23D9/007—Other edible oils or fats, e.g. shortenings, cooking oils characterised by ingredients other than fatty acid triglycerides
    • A23D9/013—Other fatty acid esters, e.g. phosphatides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • A23D9/02—Other edible oils or fats, e.g. shortenings, cooking oils characterised by the production or working-up
    • A23D9/04—Working-up
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/001—Refining fats or fatty oils by a combination of two or more of the means hereafter
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/003—Refining fats or fatty oils by enzymes or microorganisms, living or dead
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/16—Refining fats or fatty oils by mechanical means
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C1/00—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids
    • C11C1/02—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils
    • C11C1/04—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils by hydrolysis
    • C11C1/045—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils by hydrolysis using enzymes or microorganisms, living or dead
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
    • C11C3/04—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fats or fatty oils
    • C11C3/10—Ester interchange
    • 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
    • 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
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to a method for producing oil containing polyunsaturated fatty acid using lipase reactions.
  • n-3 type polyunsaturated fatty acids such as docosahexaenoic acid and eicosapentaenoic acid
  • the ratio of intake amount of n-3 type polyunsaturated fatty acid to intake amount of n-6 type polyunsaturated fatty acid is important.
  • the industrialized world is characterized by a trend of decreased intake of n-3 type polyunsaturated fatty acids and a trend of increased intake of calories, saturated fatty acids, and n-6 type polyunsaturated fatty acids. This trend is considered to be related to various types of lifestyle-related diseases.
  • Fish oil is an oil that is rich in n-3 type polyunsaturated fatty acids.
  • the intake of such fish oil is widely recommended, and methods are being devised for the concentration of the n-3 type polyunsaturated fatty acids in fish oil for more efficient intake of n-3 type polyunsaturated fatty acids.
  • the concentration of polyunsaturated fatty acids using lipase reactions is one such method.
  • Lipases are enzymes that catalyze hydrolysis reactions to decompose oils into free fatty acids and glycerin.
  • Various types of animals and plants and microorganisms are known to have lipases.
  • a given type of lipase does not act similarly for all fatty acids, and the reactivity of a given lipase will vary according to bond position in the glyceride, carbon chain length of the fatty acid, number of double bonds, or the like. It is thus possible to selectively hydrolyze fatty acids using such lipases, and as a result, it becomes possible to concentrate a specific fatty acid within the glyceride fraction.
  • Patent Document 1 when a lipase produced by a kind of the genus Candida is used, it is known that hydrolysis reaction of fish oil results in concentration of polyunsaturated fatty acids, such as docosahexaenoic acid or the like, in the undecomposed glyceride fraction (Patent Document 1).
  • Hydrolysis reaction by lipase in this manner is a method that is effective for the concentration of polyunsaturated fatty acids.
  • concentration of polyunsaturated fatty acid in the glyceride fraction increases with progress of hydrolysis with respect to fatty acids other than those of the target polyunsaturated fatty acid.
  • the hydrolysis reaction slows down in practice, and excess enzyme must be added in order that the hydrolysis reaction proceed further.
  • excess addition results in a lowering of yield due to hydrolysis reaction of the target polyunsaturated fatty acid and a lowering of the concentration effect as hydrolysis increases.
  • lipase gradually loses activity with the passage of hydrolysis reaction time. It is thus possible to further promote hydrolysis by removing the deactivated enzyme and repeating the reaction using fresh enzyme. However, even in this case, there is a marked decline of yield when the degree of hydrolysis is excessive, and the concentration effect for the target polyunsaturated fatty acid is lost.
  • Candida cylindracea -derived lipase When Candida cylindracea -derived lipase is used for hydrolysis of fish oil, it is possible to increase the acid value of the hydrolyzed oil by increasing the utilized amount of lipase, by prolonging the reaction time, or by repeatedly performing hydrolysis using lipase.
  • concentration of the target polyunsaturated fatty acid proceeds, when the acid value exceeds roughly 160, the degree of concentration of polyunsaturated fatty acid conversely decreases (Patent Document 1).
  • the target polyunsaturated fatty acid concentration factor stops increasing, and there is decline in the target polyunsaturated fatty acid concentration.
  • promotion of hydrolysis may cause a lowering of yield of glycerides.
  • a limit point occurs in the concentration of a polyunsaturated fatty acid utilizing the hydrolysis by lipase.
  • the added amount of enzyme, reaction time, or the like must be set to obtain a balance between the obtained oil product yield and efficiency of concentration of the target fatty acid.
  • Optimum temperature of the enzyme reaction is known to depend on the enzyme, and reactions are performed within the temperature range. Although lipase reacts within the temperature range thereof, viscosity of the target oil of the lipase reaction increases at low temperature, and the effectiveness of stirring the oil and enzyme-containing water worsens. Thus, the reaction is normally performed at from 30 to 40° C.
  • Patent Document 1 when Candida cylindracea -derived lipase is used for concentration of polyunsaturated fatty acid, the reaction temperature used in the working examples of Patent Document 1 (filed in 1982) was room temperature, and thereafter reaction temperatures were set in Patent Documents 2 to 7 (filed in 1988, 1993, 1994, 1995, 1996, and 1999, respectively) to 37, 37, 37, 30, 35, and 35° C., respectively.
  • the present invention focuses on the saturated fatty acids of the oil containing the polyunsaturated fatty acids.
  • Oils having a high concentration of polyunsaturated fatty acid are used for intake of useful components such as docosahexaenoic acid (abbreviated hereinafter as “DHA”), eicosapentaenoic acid (abbreviated hereinafter as “EPA”), or the like.
  • DHA docosahexaenoic acid
  • EPA eicosapentaenoic acid
  • the problem of the present invention is to provide an oil containing polyunsaturated fatty acids that has a further decreased content of saturated fatty acids.
  • the inventors of the present invention achieved the present invention by discovery of the ability to obtain unexpected results by adjustment of reaction temperature, which is a factor that had heretofore not been considered by anyone and for which thinking had become entrenched (i.e. thinking that from 30 to 40° C. was the optimum temperature).
  • the gist of the present invention is the method for lowering of the saturated fatty acid content of the below listed (1) and (2), and the glyceride having a low saturated fatty acid content of (3) to (14).
  • a method for lowering saturated fatty acid content include the step of concentrating polyunsaturated fatty acid using a lipase having low reactivity for the polyunsaturated fatty acid to react with a glyceride containing a polyunsaturated fatty acid; where the lipase reaction is performed at a temperature of not more than 25° C.
  • lipase is derived from a microorganism selected from among a group including microorganisms belonging to the genera Candida, Alcaligenes, Burkholderia, Pseudomonas, Thermomyces , and Rhizomucor.
  • area percent indicates the content fraction of a peak of a component as the ratio relative to the total peak area of all peak areas of the various components in an analytical graph using gas chromatography or thin-layer chromatography/flame ionization detector (TLC/FID) of a mixture of glyceride ingredients composed of various types of fatty acids.
  • the fatty acid composition was determined by gas chromatography by the method indicated in the working examples.
  • the lipid composition was determined using TLC/FID.
  • the polyunsaturated fatty acid such as EPA, DHA, or the like may be concentrated, and a glyceride may be produced that has a low saturated fatty acid content. It is possible to decrease the amount of intake of excess saturated fatty acid during the ingestion of polyunsaturated fatty acid desirable for health.
  • FIG. 1 is a graph showing proportions of saturated fatty acid and EPA+DHA contained in the glyceride fraction treated with lipase at each reaction temperature in Working Example 1, normalized such that results for the reaction at 40° C. are 100.
  • FIG. 2 is a graph showing proportions of saturated fatty acid and EPA+DHA contained in the glyceride fraction treated with lipase at each reaction temperature in Working Example 2, normalized such that results for the reaction at 40° C. are 100.
  • FIG. 3 is a graph showing change over time of saturated fatty acid, EPA, and DHA amounts during the lipase reaction of Working Example 3 (20° C., 600 units/mL oil).
  • FIG. 4 is a graph showing change over time of saturated fatty acid, EPA, and DHA amounts during the lipase reaction of Working Example 3 (20° C., 300 units/mL oil).
  • FIG. 5 is a graph showing change over time of saturated fatty acid, EPA, and DHA amounts during the lipase reaction of Comparative Example 1 (40° C., 600 units/mL oil).
  • FIG. 6 is a graph showing a comparison of saturated fatty acid, EPA, and DHA amounts in the lipase reaction of Working Example 4 (20° C.) and Comparative Example 2 (40° C.).
  • FIG. 7 is a graph showing results of the large-scale reaction of Working Example 5.
  • FIG. 8 is a graph showing change over time of saturated fatty acid, EPA, and DHA amounts in the lipase reaction of Working Example 7.
  • the polyunsaturated fatty acid of the present invention is taken to mean a fatty acid having at least 18 carbon atoms and having at least 3 double bonds, and more preferably a fatty acid having at least 20 carbon atoms and having at least 3 double bonds.
  • Such fatty acids are exemplified by ⁇ -linolenic acid (18:3, n-3), ⁇ -linolenic acid (18:3, n-6), arachidonic acid (20:4, n-6), dihomo- ⁇ -linolenic acid (20:3, n-6), eicosapentaenoic acid (20:5, n-3), docosapentaenoic acid (22:5, n-6), docosahexaenoic acid (22:6, n-3), or the like.
  • These polyunsaturated fatty acids are known to be contained in large amounts in certain microorganism oils, plant oils, marine animal oils, or the like.
  • Such polyunsaturated fatty acids are exemplified by: marine animal oils such as those of fishes including sardine, tuna, bonito, and the like and crustaceans including krill and the like; plant oils such as perilla oil, flaxseed oil, soybean oil, rapeseed oil; and oils produced by microorganisms belonging to the genera Mortierella, Penicillium, Aspergillus, Rhodotorula, Fusarium ; or the like.
  • marine animal oils such as those of fishes including sardine, tuna, bonito, and the like and crustaceans including krill and the like
  • plant oils such as perilla oil, flaxseed oil, soybean oil, rapeseed oil
  • saturated fatty acid for the present invention is a saturated fatty acid having 14, 16, or 18 carbon atoms. Although such saturated fatty acids are important nutrients as sources of calories, the intake of these saturated fatty acids from normal foods of the modern diet is often in excess of the required amount, and these saturated fatty acids are considered to be fatty acids that should not be ingested in excess.
  • the glycerides containing polyunsaturated fatty acids of the present invention are triglycerides, diglycerides, and monoglycerides containing as constituent fatty acids the aforementioned polyunsaturated fatty acids.
  • the aforementioned microorganism oils, plant oils, and marine animal oils are triglycerides that include polyunsaturated fatty acids.
  • any lipase may be used for the present invention as long as the lipase has low activity with respect to polyunsaturated fatty acid and has the property of concentrating polyunsaturated fatty acid in the undecomposed glyceride fraction by the hydrolysis reaction.
  • lipases derived from Candida cylindracea and Candida rugosa concentrate DHA, arachidonic acid, and ⁇ -linolenic acid.
  • Lipase derived from Rhizomucor miehei has the ability to concentrate DHA.
  • Lipases derived from Alcaligenes sp. and Pseudomonas sp. have the ability to concentrate EPA. All of these lipases are commercially marketed and may be readily obtained.
  • these lipases may be fixed prior to use.
  • the utilized lipase may be derived from Candida cylindracea , i.e. a lipase derived from the genus Candida .
  • Lipase derived from Candida cylindracea is exemplified by Lipase OF, produced by Meito Sangyo Co., Ltd.
  • Alternative lipases are exemplified by lipases obtained from microorganisms belonging to Alcaligenes sp.
  • Lipases obtained from microorganisms belonging to Burkholderia cepacia (Lipase PS, produced by Amano Enzyme Inc.), lipases obtained from microorganisms belonging to Pseudomonas fluorescens (Lipase AK, produced by Amano Enzyme Inc.), lipases obtained from microorganisms belonging to Thermomyces lanuginosa (Lipozyme TLIM, produced by Novozymes), or the like.
  • the utilized amount of lipase relative to 1 g of triglyceride is normally from 10 to 2,000 units, and preferably is from 200 to 700 units.
  • 1 unit is the amount of enzyme that releases 1 ⁇ mol of fatty acid in 1 minute.
  • the hydrolysis reaction using lipase requires that the reaction be performed in the presence of a sufficient amount of water for expression of the hydrolysis activity of lipase.
  • the amount of water present is from 10 to 200 percent by weight, and preferably is from 50 to 150 percent by weight.
  • the hydrolysis is preferably performed under an inert gas atmosphere, such as dry nitrogen or the like.
  • An antioxidant may preferably also be used, such as tocopherol, ascorbic acid, t-butyl hydroquinone, or the like.
  • the hydrolysis reaction is performed at a temperature of not more than 25° C., preferably from 10 to 25° C., and more preferably from 15 to 20° C. Although as low a temperature as possible is preferred for causing a lowering of the content of saturated fatty acids, at temperatures of not more than 10° C., the viscosity of the oil increases and the rate of the enzyme reaction itself declines excessively. Thus a temperature of roughly from 15 to 20° C. is most preferable. In the case of a large-scale reaction, the reaction phase temperature is preferably set to be from 15 to 20° C., and the reaction may be performed while maintaining temperature within a range of about ⁇ 5° C.
  • the hydrolysis reaction is performed in a flow or the like caused by stirring, injecting an inert gas or the like.
  • Hydrolysis is performed until the proportion of docosahexaenoic acid contained in the constituent fatty acids reaches the target value.
  • Reaction conditions differ according to the raw material oil.
  • the reaction time is preferably at least 7 hours, and normally hydrolysis is performed for 5 to 24 hours.
  • the proportion of docosahexaenoic acid then becomes not less than 46 area percent by a single lipase reaction.
  • the reaction may also be performed for a longer time. There were no adverse effects even when the reaction was performed for 65 hours as indicated in the working examples.
  • acid value may be used as an indicator showing the degree of hydrolysis.
  • the fraction of docosahexaenoic acid normally becomes at least 46 area percent when the acid value becomes not less than 140.
  • a mixture of hydrolysate and unreacted triglyceride is obtained as the reaction liquid.
  • the proportion of polyunsaturated fatty acids i.e. docosahexaenoic acid, eicosapentaenoic acid, or the like
  • the concentration continues until the docosahexaenoic acid concentration at the time of completion of hydrolysis becomes at least 40 area percent.
  • most of the free fatty acids are fatty acids other than the polyunsaturated fatty acids.
  • the oil layer of the reaction liquid is obtained by removing the aqueous layer containing lipase, glycerin, and the like via centrifugal separation or the like. Then, the free fatty acids are removed.
  • Methods that may be adopted for the separation and removal of the free fatty acids include known methods such as the method of removal as alkaline salts, method using a liquid chromatographic device, fractional distillation method, and crystal separation method. However, molecular distillation and steam distillation are preferred.
  • a glyceride mixture is obtained of partial glycerides and triglycerides containing docosahexaenoic acid at high concentration.
  • a glyceride where docosahexaenoic acid is concentrated to a concentration of at least 40 area percent, and further concentrated to at least 46 area percent.
  • a glyceride may be obtained where the total amounts of saturated fatty acids having 14, 16, and 18 carbon atoms is not more than 12 area percent, and preferably is not more than 10 area percent.
  • the concentration of palmitic acid (having 16 carbon atoms), which is contained at the highest concentration among saturated fatty acids is not more than 8 area percent, and preferably is not more than 6 area percent.
  • the proportion of triglyceride in the lipid composition of the glyceride in the obtained reaction oil became high.
  • the proportion of triglyceride at 40° C. was about the 70 area percent level.
  • the obtained proportion of triglyceride was not less than 80 area percent.
  • deacidification, decoloration, and deodorizing treatment of the lipase reaction oil of the present invention polyunsaturated fatty acids are concentrated, and a glyceride may be obtained that has a reduced content of saturated fatty acids.
  • the processing methods of deacidification, decoloration, and deodorizing may be any methods.
  • Deacidification treatment is performed by distillation.
  • Decolorization treatment is performed by treatment using activated earth, activated carbon, or the like.
  • Deodorization treatment is performed by steam distillation or the like.
  • Monoglycerides are simultaneously removed during deacidification treatment by distillation, and it is thus possible to further increase the proportion of triglycerides in the obtained oil. It is possible to obtain a proportion of triglyceride of not less than 85 area percent, and preferably not less than 90 area percent.
  • Fatty acid composition of the fish oil used as the raw material was measured by ethyl esterification of the fish oil and measurement by gas chromatography. That is to say, 1 mL of 1N sodium ethylate in ethanol solution was added to 40 ⁇ L of fish oil, and the mixture was agitated for about 30 seconds. Thereafter, 1 mL of 1N hydrochloric acid was added to neutralize the mixture, and 2 mL of hexane and 3 mL of saturated ammonium sulfate aqueous solution were added. After stirring and then allowing the mixture to sit, content of the supernatant was measured by gas chromatography.
  • the fatty acid composition of the glyceride fraction of the oil after the enzyme reaction was measured by ethyl esterification of the glyceride fraction, removal of free fatty acids (i.e. byproduct of the enzyme reaction), and measurement by gas chromatography. That is to say, 1 mL of 1N sodium ethylate in ethanol solution was added to 70 ⁇ L of reaction oil, and the mixture was agitated for about 30 seconds. Thereafter, 1 mL of 1N hydrochloric acid was added to neutralize the mixture, and 700 ⁇ L of hexane and 3 mL of saturated ammonium sulfate aqueous solution were added.
  • Carrier gas helium (2.9 mL/min, constant flow)
  • acid value was measured based on Standard Methods for the Analysis of Fats, Oils, and Related Materials (2003 edition, edited by the Japan Oil Chemists' Society).
  • the fatty acid composition (area percent) of the glyceride fraction of the obtained reaction oil, the acid value, and the fatty acid composition of the refined fish oil 1 (area percent) are shown in Table 1.
  • the total of the myristic acid (C14:0), palmitic acid (C16:0), and stearic acid (C18:0) content (area percent) in the glyceride fraction obtained during gas chromatography is indicated as the saturated fatty acid content (hereinafter, “saturated fatty acid content” described in the working examples is taken to mean this value).
  • the fatty acid composition (area percent) of the glyceride fraction of the obtained reaction oil, the acid value, and the fatty acid composition (area percent) of the refined fish oil 2 are shown in Table 2 (only typical fatty acids are shown in the fatty acid composition).
  • the glyceride content here was calculated by calculating the free fatty acid content equivalent to oleic acid from the acid value, and then subtracting the free fatty acid content from the total reaction oil.
  • the total of the myristic acid (C14:0), palmitic acid (C16:0), and stearic acid (C18:0) content (area percent) in the glyceride fraction obtained during gas chromatography is indicated as the saturated fatty acid content.
  • FIG. 2 shows the EPA and DHA content and the saturated fatty acid content of the glyceride fraction under each temperature condition, when the result for the 40° C. reaction oil is normalized to 100. From this figure, it is understood that, under 10 to 25° C. conditions, there was a great decrease in the saturated fatty acid content, while the EPA and DHA contents remained high.
  • the saturated fatty acid content (area percent) and the EPA and DHA content (area percent) at each time interval are shown in FIGS. 3 and 4 .
  • the saturated fatty acid content decreased greatly with reaction time, and the EPA and DHA content increased with reaction time.
  • the reaction was performed by the same procedure and under the same conditions as Working Example 3 using 600 units/mL of the lipase.
  • Saturated fatty acid content (area percent) and EPA and DHA content (area percent) at each time interval are shown in FIG. 5 .
  • the saturated fatty acid content decreased down to 15.0 percent at 2 hours of reaction time, further decrease was not found even when the reaction time was further prolonged, and there was a shift to a high saturated fatty acid content of about 15 percent.
  • the reaction was performed under the same conditions as those mentioned above for Working Example 4, except for change of temperature to 40° C. Except for temperature, all conditions and procedures were the same.
  • the saturated fatty acid content (area percent) and the EPA and DHA content (area percent) of Working Example 4 and Comparative Example 2 are shown in FIG. 6 . From this figure, it is understood that the saturated fatty acid content greatly decreased and the EPA and DHA content somewhat increased under the 20° C. temperature condition in comparison to 40° C. temperature condition.
  • Lipid composition (area percent) was measured in the glyceride fraction of the reaction oils produced under the 10, 20, 40, and 50° C. reaction conditions of Working Example 1. As shown in Table 3, it was possible to obtain a triglyceride fraction having a triglyceride proportion of at least 80 area percent when the reaction was performed at 10 and 20° C. On the other hand, the triglyceride proportion was about 72.8 area percent and 59.9 area percent when the reaction was performed at 40 and 50° C., respectively. By performing the lipase reaction at low temperature, in addition to being able to lower the saturated fatty acid content, it was shown possible to increase the triglyceride proportion.
  • Acid value of the obtained reaction oil, fatty acid composition (area percent) of the glyceride fraction, degree of hydrolysis (percent), and fatty acid composition (area percent) of the refined fish oil 5 are shown in Table 4.
  • the degree of hydrolysis was calculated by the following formula from the saponification value (206.04) of refined fish oil 5 and the acid value.
  • One object of intake of fatty acids having physiological activity is to prevent diseases of the heart and vascular system such as hypercholesterolemia or the like.
  • saturated fatty acids are important as a source of calories, the modern diet often results in excessive intake of saturated fatty acids, particularly during middle age, and aggressive intake of saturated fatty acids is undesirable.
  • the amount of co-ingested saturated fatty acid is preferably as low as possible.
  • the oil produced by the present invention concentrates polyunsaturated fatty acid and further reduces the amount of saturated fatty acid. The present invention is thus suitable for use as a health food or supplement for supplying n-3 type polyunsaturated fatty acids.

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EP3848466A4 (en) * 2018-09-04 2022-06-15 Nippon Suisan Kaisha, Ltd. METHOD FOR PRODUCTION OF GLYCERIDE CONTAINING POLYUNSATURATED FATTY ACID BY MEANS OF LIPASE HYDROLYSIS REACTION
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US10399922B2 (en) 2012-05-14 2019-09-03 Nippon Suisan Kaisha, Ltd. Highly unsaturated fatty acid or highly unsaturated fatty acid ethyl ester with reduced environmental pollutants, and method for producing same
US11034643B2 (en) 2012-05-14 2021-06-15 Nippon Suisan Kaisha, Ltd. Highly unsaturated fatty acid or highly unsaturated fatty acid ethyl ester with reduced environmental pollutants, and method for producing same
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WO2016152174A1 (en) * 2015-03-25 2016-09-29 Kewpie Corporation Method of producing lower alcohol ester of fatty acid -containing composition
US11771106B2 (en) * 2015-06-01 2023-10-03 Cargill, Incorporated Oil composition with mono-acylglycerides
EP3848466A4 (en) * 2018-09-04 2022-06-15 Nippon Suisan Kaisha, Ltd. METHOD FOR PRODUCTION OF GLYCERIDE CONTAINING POLYUNSATURATED FATTY ACID BY MEANS OF LIPASE HYDROLYSIS REACTION
US11840714B2 (en) 2018-09-04 2023-12-12 Nissui Corporation Enriching DHA in glyceride fractions

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