JPH0534956B2 - - Google Patents

Description

[Detailed description of the invention]

[Object of the Invention] Industrial Application Field The present invention relates to a method for decomposing fats and oils containing highly unsaturated fatty acids using an enzyme. More specifically, the present invention relates to a method for decomposing fats and oils containing highly unsaturated fatty acids, such as aquatic animal fats and oils, using lipase produced by a strain belonging to the genus Aspergillus. BACKGROUND ART Methods for producing fatty acids and glycerol by hydrolyzing fats and oils have been practiced for a long time. The fatty acids produced are used as raw materials for detergents, surfactants, synthetic resins, rubber, paints, fibers, greases, medicines, cosmetics, etc., and glycerol is used for cosmetics, toothpaste,
Used as a raw material for medicines, etc. Methods for hydrolyzing fats and oils include the Teutschell method,
There are high-pressure decomposition methods, saponification decomposition methods, enzymatic decomposition methods, etc., and continuous high-pressure decomposition methods have mainly been used industrially. The high-pressure decomposition method involves the consumption of a large amount of energy, side reactions such as polymerization occur, and in the case of fatty acids containing many double bonds, double bond transfer occurs to produce isomers, and the produced fatty acids are colored. There are drawbacks such as: In contrast, the enzymatic decomposition method proceeds at room temperature and near normal pressure, and does not produce any by-products, so it has attracted particular attention in recent years and has been applied to the decomposition of beef tallow (among other things). Kosho 57-38239, same 57
-39638, 59-20357, JP-A-57-170193, 58
-146284, 59-175894, 59-179019, 60-
126090 etc.). Conventionally, the oil and fat raw materials for hydrolysis are:
Beef tallow, lard, and coconut oil are mainly used, and these fats alone account for about 80% or more. The fatty acids constituting the above-mentioned fats and oils such as beef tallow, lard, and coconut oil are mainly saturated fatty acids with 8 to 18 carbon atoms, or 12 to 18 carbon atoms, and saturated fatty acids with 1 to 1 double bond.
Consists of two unsaturated fatty acids. In contrast,
Aquatic animal fats and oils such as sardine oil and herring oil contain relatively large amounts of long-chain highly unsaturated fatty acids having 20 to 24 carbon atoms and 4 to 6 double bonds. Eicosapentaenoic acid, docosahexaenoic acid, and the like, which are types of these highly unsaturated fatty acids, are substances that are attracting attention for their effect on improving cardiovascular disorders as people become more health conscious. Fats and oils containing long-chain unsaturated fatty acids with carbon atoms of 20 or more are said to be less susceptible to hydrolysis by lipases than fats and oils containing fatty acids with carbon atoms of 18 or less. Utilizing these properties of lipase, a method of increasing the decomposition rate of fats and oils by combining a lipase that has positional specificity for triglycerides and a lipase that does not have such specificity has been proposed
130396), a method for leaving abundant polyunsaturated fatty acids in undecomposed glycerides produced by applying lipase to fats and oils (JP-A-60-234589), hydration of fats and oils containing long-chain polyunsaturated fatty acids using lipase A method of increasing the decomposition rate by adding alcohol during decomposition has been reported (Japanese Patent Laid-Open No. 60-234590). Problems to be Solved by the Invention The present invention provides a method for hydrolyzing fats and oils containing highly unsaturated fatty acids using an enzyme. In order to hydrolyze fats and oils industrially, a decomposition rate of at least 90% is required. However, the existence of an enzyme that decomposes fats and oils containing highly unsaturated fatty acids at a high decomposition rate has not yet been reported. The present inventors attempted to hydrolyze aquatic animal fats and oils containing highly unsaturated fatty acids using a number of commercially available lipases, but could only achieve a decomposition rate of about 80% at maximum. Therefore, an object of the present invention is to provide a method for hydrolyzing fats and oils containing highly unsaturated fatty acids, which increases the decomposition rate. [Structure of the Invention] Means for Solving the Problems According to the present invention, a strain belonging to the genus Aspergillus is produced in fats and oils containing highly unsaturated fatty acids such as aquatic animal fats and oils, and the specificity for the constituent fatty acids of the substrate triglyceride is improved. Provided is a method for decomposing fats and oils by using a lipase that has at least the property of being low in fat and oil. In the method of the present invention, low specificity for the constituent fatty acids of the substrate triglyceride means that the selectivity for the bonding position, chain length, and degree of saturation of the fatty acids constituting the triglyceride is low, and the ester bond is nonspecifically hydrolyzed. means quality. It has been known that Aspergillus bacteria produce lipase. However, for example, Aspergillus niger lipase acts well on triglycerides containing medium-chain fatty acids, and its positional specificity is at the 1st and 3rd positions, and fatty acids at the 2nd position are said to be difficult to decompose ( Science and Industry, Vol. 58, pp. 60-69, 1984). The lipase used in the present invention belongs to the genus Aspergillus and can be collected from the culture obtained by culturing in a medium a strain that produces a lipase that has at least the property of having low specificity for the constituent fatty acids of the substrate triglyceride. . Suitable examples of the lipase-producing strain used in the present invention include strains having the following properties, which were discovered by the present inventors by screening soil. (1) Growth status on each medium Malt extract agar medium: Growth is good, mycelium is white, conidia are well formed, and the color is dark brown to dark reddish brown to purplish black. The underside is smooth and the color ranges from colorless to yellowish white to yellowish brown. Tuapetsk agar medium: Growth is good, mycelium is white, conidia are well produced, and the color is dark brown to dark reddish brown. The underside is smooth and the color is colorless to dark yellowish brown. (2) Morphology on Czapetsk agar medium Conidial head: radial or lobed to form a cylindrical shape. The length reaches 500μ. Conidiophore: length 0.2~1.5mm, diameter 5~
11μ, smooth surface. Apical capsule: spherical to subglobular, elliptical in some cases. Diameter 10~55μ. Small infarcts usually occur throughout the apical capsule. Metre: Not formed. Small stalks: Size 6-9μ x approx. 3μ Conidia: Globular to subglobular, rarely oval to oval. The surface is thorn-like. Diameter 3.5~
4.5μ. Ascospores: Not formed. (3) Physiological properties The conditions and range for growth on malt extract agar medium or malt extract liquid medium are as follows. Optimal growth conditions: Temperature around 30℃, slightly acidic PH
It grows well in Growth range: Does not grow at 45°C (cultured for 5 days). It does not grow at 5°C (cultured for 14 days).
It grows well in the pH range of 3 to 9. The above mycological properties were evaluated by Barron, GL:
the Genera of Hyphomycetes from Soil,
Williams & Wilkins, Baltimore, 1968), it was found that the conidiophores are solitary without aggregation, that the conidia are chained, that the conidia have no septa, and that there are numerous small It is classified as a member of the genus Aspergillus because of its stalk formation. For species, see Raper and DIFennel (KBRaper and DIFennel: the Genus
Aspergillus, Williams & Wilkins, Baltimore,
1965) and Iizuka (Noka, Vol. 27, p. 801,
(1953), it was found that the color of the conidia was black rather than green, that the conidia formed well on Tuapetsk agar medium, and that the conidial heads were radial, indicating that Aspergillus.
It can be seen that it belongs to the Aspergillus niger group. Since there is no metre (that is, there is only one stage of sycamore), it is a close relative of A. japonicus or A. aculeatus. The conidia of Aspergillus aquiuleatus are oval, the apical capsule is 50 to 80 μm, and the conidiophore is 3 to 4 mm. In contrast, in this strain, most of the conidia are spherical to subglobular, and the apical capsule is 10 to 10.
55μ, and the conidiophores are relatively short and 0.2
Since it is ~1.5 mm, it is appropriate to identify it as Aspergillus japonicus. This strain was then transformed into Aspergillus japonicus No. 213F (Aspergillus japonicus No. 213F).
japonicus No.213F). This strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, No. 8608.
It has been deposited under the accession number FERM P-8608. The physicochemical properties of the lipase produced by Aspergillus japonicus No. 213F are as follows. (1) Action, substrate specificity: Hydrolyzes ester bonds at all positions of triglyceride to produce fatty acids and glycerol. It acts on a wide range of natural oils and fats, especially on carbon numbers.
It effectively breaks down triglycerides containing more than 20 highly unsaturated fatty acids. It also decomposes triglycerides containing saturated or unsaturated fatty acids having 4 to 18 carbon atoms well. (2) Optimum PH: 6 to 7 (shown in Figure 1) (3) Stable PH range: PH 4 to 8 (shown in Figure 2) (4) Optimum temperature: approximately 50℃ (shown in Figure 2) (5) Temperature stability: Stable below approximately 50°C at pH 7 (shown in Figure 4) (6) Activity measurement method: Commercially available lipase measurement kit (Lipase Kit S, Dainippon Pharmaceutical Co., Ltd.) PH7.0, 37℃
After incubation for 15 minutes at 412nm
Measure the increase in absorbance at . The unit of lipase was defined as the amount of enzyme that increases the OD by 1 under the above conditions. The lipase used in the present invention is particularly preferably obtained by culturing Aspergillus japonicus No. 213F strain in a nutrient medium and collecting it from the resulting culture. When used in the present invention, the obtained lipase may be in the culture itself or in a crude or purified state. In carrying out the present invention, a product with a high decomposition rate can be obtained by mixing an oil or fat containing a highly unsaturated fatty acid with the above-mentioned lipase and carrying out a hydrolysis reaction. In the present invention, the fats and oils targeted for decomposition are fats and oils containing unsaturated fatty acids having 20 or more carbon atoms and 4 to 6 double bonds as constituent fatty acids. Examples of fats and oils containing these fatty acids include aquatic animal fats and oils such as sardine oil, saury oil, herring oil, squid oil, tuna oil, and cod oil. In the method for decomposing fats and oils according to the present invention, the reaction conditions may be any conditions as long as they are within the range where hydrolysis is carried out. Particularly preferably, the temperature is 30-60
°C, the water content in the reaction mixture is 0.2 relative to fat.
~20 parts by weight, PH4-8, amount of enzyme used is 1g of fat or oil
10 to 1000 units per unit. To accelerate the hydrolysis reaction, emulsifiers such as polyvinyl alcohol and fatty acid esters, enzyme activators such as magnesium ions, manganese ions, and calcium ions,
Buffers such as phosphate buffers can also be added. Further, in order to prevent deterioration of highly unsaturated fatty acids which are decomposition products, the reaction can also be carried out in the presence of an inert gas and an antioxidant. During the reaction, it is preferable to maintain the emulsion by stirring or other means. The lipase used in the present invention can also be immobilized on a carrier to act on fats and oils. Immobilized lipase can be used repeatedly and is economical. Methods for immobilizing lipase are known, such as physical adsorption, carrier bonding using ionic or covalent bonds, crosslinking using a multifunctional reagent, and entrapment in a polymer lattice or microcapsule. prepared. Separation of fatty acids and glycerol from the hydrolysis reaction mixture is carried out according to conventional methods. for example,
The reaction mixture is allowed to stand and can be separated into an oil layer containing fatty acids and glycerol water (sweet water). As mentioned above, the lipase used in the present invention has characteristics not found in conventional lipases. By utilizing this property, for example, after decomposing fats and oils containing long-chain highly unsaturated fatty acids with a conventional first lipase having fatty acid selectivity or position specificity and separating the generated glyceride, the present invention It is also possible to concentrate long chain polyunsaturated fatty acids by decomposing glycerides with a second lipase used in The present invention will be explained in further detail by referring to Reference Examples and Examples below. Reference example Production of lipase Soy flour 2.5%, starch 1.0%, soybean oil 0.5%, ammonium phosphate 0.5%, and dipotassium phosphate 0.2
Aspergillus japonicus in a medium consisting of %
No. 213F was inoculated and cultured at 30°C for 3 days.
The resulting culture was filtered to remove bacterial cells, and the filtrate was concentrated by ultrafiltration. A three-fold amount of ethanol was added to the concentrated solution, and the resulting precipitate was collected and vacuum-dried to obtain a lipase sample of 5200 u/g. Example 1 Sardine oil 0.5g, 0.5M phosphate buffer (PH7.0)
1.5ml, 1.25ml of lipase obtained in the above reference example,
8.0 ml of a 2.5, 5 or 10 u/ml aqueous solution and 30 glass beads with a diameter of 5 mm were placed in a 100 ml flask and heated at 40°C with shaking (140 rpm,
amplitude 3 cm) and incubated for 24 hours. After extracting the oil layer in the reaction mixture with petroleum ether and removing the solvent, its acid value (AV) and saponification value (SV) were measured. The decomposition rate [AV/SV (x100%)] was calculated from the obtained measured values. Figure 5 shows the relationship between the amount of enzyme used and the decomposition rate of fats and oils. Comparative Example 1 In Example 1, commercially available lipase derived from Pseudomonas bacteria (product name: Lipase P, manufactured by Amano Pharmaceutical Co., Ltd.) and lipase derived from Candida bacteria (product name: Lipase OF, The same procedure was carried out except that the method (manufactured by Meito Sangyo Co., Ltd.) was used. Figure 5 shows the relationship between the amount of enzyme used and the decomposition rate of fats and oils. As can be seen from FIG. 5, according to the method of the present invention, the decomposition rate of fats and oils reached a maximum of 96%, but it was 82% with lipase of Pseudomonas genus and 69% with lipase of Candida genus. Example 2 3.0 g of sardine oil, 4.5 ml of the aqueous solution containing 120 units of lipase from the above reference example, and 30 glass beads with a diameter of 5 mm were placed in a 100 ml flask and heated at 37° C. while shaking (140 rpm, amplitude 3 cm). Incubated for 24 hours. The decomposition rate of fats and oils was 94%. (See Table 1.) Comparative Example 2 In Example 2, a commercially available lipase derived from a Rhizopus bacterium (trade name:
Lipase N, produced by Amano Pharmaceutical Co., Ltd.), lipase derived from Mucor bacteria (product name: Lipase M, produced by Amano Pharmaceutical Co., Ltd.), lipase derived from Aspergillus niger (product name:
Lipase A, Amano Pharmaceutical Co., Ltd.), Candida-derived lipase (product name: Sakemate, Meito Sangyo Co., Ltd.), or Pseudomonas-derived lipase (product name: Lipase P, Amano Pharmaceutical Co., Ltd.). was operated in the same way. The decomposition rate of fats and oils was as shown in Table 1.

【table】

〔Effect of the invention〕

According to the method of the present invention, a method is provided for decomposing fats and oils containing highly unsaturated fatty acids at a high decomposition rate using lipase produced by Aspergillus bacteria. The method of the present invention consumes less energy than conventional high-pressure decomposition methods, and is free from problems such as the production of by-products due to polymerization of heat-labile fatty acids and the like, and coloring of the produced fatty acids.

[Brief explanation of the drawing]

Figure 1 shows Aspergillus japonicus No.
FIG. 2 is a diagram showing the optimal pH of lipase produced by 213F, and FIGS. 2, 3, and 4 are diagrams showing the stable PH range, optimal temperature, and temperature stability, respectively. Fifth
The figure shows the relationship between the amount of lipase used and the decomposition rate of fats and oils when lipase produced by Aspergillus japonicus No. 213F and commercially available lipases derived from Pseudomonas and Candida are applied to sardine oil. be.

Claims (1)

[Scope of Claims] 1. A lipase produced by a strain belonging to the genus Aspergillus, which has at least the property of having low specificity for fatty acids constituting the substrate triglyceride, is made to act on fats and oils containing polyunsaturated fatty acids. A method for decomposing fats and oils containing saturated fatty acids. 2. The method for decomposing fats and oils containing highly unsaturated fatty acids according to claim 1, wherein the strain belonging to the genus Aspergillus is Aspergillus japonicus No. 213F. 3. The method for decomposing fats and oils containing highly unsaturated fatty acids according to claim 1, wherein the fats and oils containing highly unsaturated fatty acids are aquatic animal fats and oils.