JP2006197842A - Composition having phospholipase a1 activity, 2-acyl type lysophospholipid obtained by using the same and method for producing those - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition having phospholipase A1 activity which can efficiently, readily and inexpensively be produced in large amounts from industrial viewpoints. <P>SOLUTION: The composition having phospholipase A1 activity is extracted from ovary of fishes. The 2-acyl type lysophospholipid is obtained by selectively hydrolyzing the 1-position acyl group of a phospholipid by using the composition. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a composition having phospholipase A1 activity, a 2-acyl lysophospholipid containing an unsaturated fatty acid such as DHA obtained by using this composition, and a process for producing them.

  Generally, docosahexaenoic acid (hereinafter referred to as DHA) is known to have excellent physiological activities such as improved memory learning ability, antiallergy, and antitumor, and many health foods containing this DHA are on the market. It is circulating.

  DHA is a kind of highly unsaturated fatty acid having 22 carbon atoms and 6 unsaturated bonds. Since this DHA cannot be mass-produced by chemical synthesis, it is currently isolated and commercialized in large quantities from fish oil bound to triacylglycerol (neutral lipid). However, the presence of DHA in a living body, particularly in the brain, is a form bound to phospholipids, and this DHA-bound phospholipid is more than triacylglycerol-type DHA in lipid improvement, brain function improvement, antitumor action, etc. Furthermore, it has become clear that physiological activity is high.

  On the other hand, lysophospholipid, which is a kind of phospholipid, is known to have higher surface activity than ordinary phospholipid. In addition, lysophospholipids are widely used in food and cosmetic emulsifiers because they have excellent properties such as strong emulsification and emulsification does not decrease even when metal ions are present in high concentrations. Can be done.

  This lysophospholipid refers to a phospholipid in which one fatty acid is bonded to an acyl group. Among them, 2-DHA-lysophosphatidylcholine (hereinafter referred to as 2-DHA-LPC) in which DHA is bonded to the 2-position of the glycerin skeleton is It has been reported that DHA transport ability to the brain and erythrocyte deformability are high. In order to produce 2-DHA-LPC, an enzyme having phospholipase A1 activity must be allowed to act on phosphatidylcholine having DHA bound at the 2-position as a substrate.

Phospholipase is reclassified into A1, A2, B, C, and D according to the action site of phospholipid, and among them, it is assumed that it hydrolyzes fatty acid bound to sn-1 position or 2 position of phospholipid. Are known, phospholipase A1 that hydrolyzes sn-1 position, A2 that hydrolyzes sn-2 position, and phospholipase B that hydrolyzes sn-1 or 2 position randomly. Among these, for phospholipase A1, for example, the following Patent Documents 1 to 3 are helpful.
JP-A-6-62850 JP-A-7-31472 JP-A-7-222592

  However, the currently commercially available phospholipase is phospholipase A2 derived from porcine pancreas and snake venom, but this does not give a 2-acyl lysophospholipid containing 2-DHA-LPC.

  Recently, methods for producing 2-acyl lysophospholipids utilizing phospholipase A1 activity found in microorganisms of the genus Aspergillus or Cyclobacter or in culture broth have been developed, but the culture method and the enzyme extraction method are complicated. Furthermore, phospholipase A1 is known to exist in mammalian brain, liver, pancreas, platelets, and the like, but at present, industrial production using these tissues as raw materials has never been performed.

  This invention is made | formed in view of such a subject, and it aims at providing the composition which has the phospholipase A1 activity which can be manufactured efficiently, in large quantities, easily and cheaply from an industrial viewpoint. Is. Furthermore, it is also an object of the present invention to provide 2-DHA-LPC or a lysophospholipid having a highly unsaturated fatty acid bonded to the 2-position using the composition having phospholipase A1 activity obtained in the present invention.

  As a result of intensive studies to achieve the above object, the present inventors have found that phospholipase A1 activity exists in the fish ovary and succeeded in extracting a composition containing it. Furthermore, as a result of carrying out an enzymatic reaction using several types of DHA-bound phospholipid as a substrate, it was revealed that 2-DHA-LPC can be concentrated, and the present invention has been completed.

  That is, according to the 1st main viewpoint of this invention, the composition which has the phospholipase A1 activity characterized by extracting from the ovary of fish is provided.

  Here, the ovary of the fish includes, but is not limited to, ovaries such as skipjack tuna, mackerel, salmon and trout, and cod. It is desirable to use these fish ovaries as fresh as possible.

  The ovary of this fish is mostly unused except for some fish species such as salmon and cod, so the present invention is also valuable from the viewpoint of effective utilization of unused marine products.

  Moreover, according to the second main aspect of the present invention, it is obtained by hydrolyzing phospholipids in a phospholipid or a phospholipid-containing material using a composition having phospholipase A1 activity extracted from the ovary of fish. 2-acyl lysophospholipids are provided.

  Furthermore, the phospholipid may be naturally derived or synthesized, but according to one embodiment of the present invention, the phospholipid is not limited thereto, but is derived from a marine product. Alternatively, it is a phospholipid in a phospholipid-containing material.

  According to one embodiment of the present invention, the 2-acyl lysophospholipid obtained by the composition having phospholipase A1 activity extracted from the ovary of the fish is not limited to this, but is in the second position. It is a lysophospholipid bound to highly unsaturated fatty acids. Furthermore, the polyunsaturated fatty acid is not limited to this, but is preferably docosahexaenoic acid.

  Soy lecithin and egg yolk lecithin, which are common phospholipids, have a low lysophospholipid content of about 0.5 to 4% in the phospholipid, and the bound fatty acids are unsaturated such as palmitic acid, oleic acid, linoleic acid, etc. It is a low-grade fatty acid. In addition, since soybean lysolecithin produced industrially as an emulsifier is prepared by phospholipase A2, it is a lysophospholipid having a saturated fatty acid bonded to the 1-position.

  Therefore, the present inventors have further studied to obtain a phospholipid having a higher lysophospholipid content. As a result of focusing on the fact that the composition ratio of lysophosphatidylcholine (LPC) in phospholipids is originally high in the ovary of fish such as skipjack and mackerel, the present inventors also have a high ratio of DHA composition in LPC. It was found that 2-acyl-lysophospholipids containing 2-DHA-LPC can be extracted directly from water using water or an organic solvent.

  That is, according to the third main aspect of the present invention, 2-acyl lysophospholipid having a highly unsaturated fatty acid bonded to the 2-position, characterized in that it is extracted from the ovary of fish. % Or more of the lipid composition is provided.

  Also, according to one embodiment of the present invention, there is provided a lipid composition wherein the polyunsaturated fatty acid is docosahexaenoic acid.

  Moreover, according to the 4th viewpoint of this invention, the manufacturing method of the composition which has the said phospholipase A1 activity, and 2-acyl type | mold lysophospholipid is provided.

  According to such a configuration, lysophospholipids in which DHA or highly unsaturated fatty acid is bonded to the sn-2 position can be easily and inexpensively concentrated at a high concentration.

  Further features and remarkable effects of the present invention will become apparent to those skilled in the art from the description of the embodiments of the present invention described below.

  Hereinafter, embodiments of the present invention will be described in detail.

  The present invention provides an enzyme having phospholipase A1 activity extracted from the ovary of fish.

  Here, the ovary of the fish includes, but is not limited to, ovaries such as skipjack tuna, mackerel, salmon and trout, and cod. It is desirable to use these fish ovaries as fresh as possible. Since most of the ovary of this fish is unused except for some fish species such as salmon and cod, the present invention has a remarkable effect of effectively utilizing unused marine products.

  Next, a method for extracting an enzyme having phospholipase A1 activity in this embodiment will be described.

  First, ovaries such as bonito / tuna, mackerel, salmon / trout, cod, etc. are prepared, degreased from the tissue with an organic solvent such as acetone, ether, ethanol, etc., and then dried to obtain ovarian delipidated powder. . Next, enzyme extraction is performed with a buffer solution such as phosphoric acid or Tris-hydrochloric acid. The temperature during defatting, drying and extraction from the ovary is 30 ° C. or lower, preferably 5 ° C. or lower.

  Next, the obtained enzyme is reacted with the substrate in a buffer solution. As a substrate to be used, 2-DHA-LPC can be obtained at a higher concentration as the phospholipid has a higher DHA composition ratio at the sn-2 position. The reaction conditions may be any as long as the fatty acid is hydrolyzed by the enzyme, but the reaction temperature is preferably 0 to 50 ° C., the reaction time is 10 minutes to 48 hours, and the pH of the reaction solution is in the range of 3 to 8. In addition to the buffer solution, the reaction solution may be water, an organic solvent, or the like.

  After completion of the reaction, the enzyme can be deactivated by heating or adding an organic solvent, and the lipid can be separated by a usual production method. For example, chloroform / methanol is added to the enzyme reaction solution, and after stirring and centrifuging, the chloroform phase is recovered and dried under reduced pressure. Further, when recovering phospholipids, a chromatographic method using a silica gel column or the like can be used.

  Among the above materials, ovary such as skipjack and mackerel originally has a high LPC composition ratio in phospholipids and a high DHA composition ratio in LPC. Therefore, 2-DHA-LPC can be obtained by extraction directly from water or an organic solvent from fish ovaries.

  Next, the present invention will be described in detail with reference to the following examples.

  Example 1. Phospholipase A1 active enzyme was extracted from seven fish ovaries. Specifically, the ovary of skipjack, bluefin tuna, yellowfin, chub mackerel, sesame salmon, white salmon, and walleye pollock were each pulverized at low temperature, 4 volumes of acetone was added, and the mixture was stirred at 4 ° C. for 20 minutes. After the stirring, acetone was exchanged and this operation was performed three times. Next, the solvent was changed to ether and the mixture was further stirred for 20 minutes, and then the centrifugal residue was transferred to a filter paper and dried overnight in a refrigerator.

  Next, 4 times (w / v) Tris-HCl buffer (pH 7.4) was added to the dry powder, and the mixture was stirred at 4 ° C. for 1 hour 30 minutes. Thereafter, centrifugation (9000 rpm, 30 minutes, 4 ° C.) was performed, and the supernatant was used as a crude enzyme solution for the enzyme reaction.

  As a substrate, 10 mg of 1-palmitoyl-2-oleoylphosphatidylcholine, 0.5 ml of a crude enzyme solution, 35 mM of calcium and 0.5 ml of sodium cholate were reacted in 3 ml of Tris-HCl buffer for 2 hours at 20 ° C. Thereafter, lipids were collected from the reaction solution using a mixed solvent of chloroform and methanol, and phospholipids and free fatty acids (FFA) were fractionated using a silica gel column. The phospholipid fraction was divided into phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) by silica gel TLC, and then each band was scraped and the amount of phosphorus was quantified by the Bartlett method. Further, FFA was methyl esterified using hydrochloric acid-methanol and then quantified by gas chromatography.

  FIG. 1 shows the analysis results of fatty acid hydrolyzing ability (FIG. 1 a) and LPC production ability (20 ° C., 2 hours reaction: FIG. 1 b, reaction end: FIG. 1 c) of the crude enzyme solution extracted from seven fish ovaries. It was. In addition, FIG. 2 shows the analysis results of fatty acid hydrolyzing ability (FIG. 2a) and LPC production ability (20 ° C., 2 hours reaction: FIG. 2b, at the end of reaction: FIG. 2c) of the crude enzyme solution extracted from each part of skipjack. It was. 1b and 2b (% of PL / mg protein) are the amounts of fatty acids and LPC produced per 1 mg of protein in the crude enzyme, and 1c and 2c (% of PL) are obtained when the reaction time is finally finished. LPC amount. From these results, it was confirmed that the composition extracted from each part of fish ovary and skipjack had phospholipase A1 activity. It is particularly high in the bonito ovary, and the bonito ovary seems to be the most suitable raw material for obtaining a composition.

  Example 2 The characteristics of the skipjack ovary crude enzyme solution were examined. The method is the same as in Example 1. FIG. 3 shows the results of examining the temperature dependence, and FIG. 4 shows the results of examining the pH dependence. Further, in FIG. 5, 1-C16: 0-2-C18: 1-PC (1-palmitoyl-2-oleoylphosphatidylcholine), 1-C18: 1-2-C16: 0-PC (1-oleoyl) as a substrate. Substrate dependence using 1-C16: 0-2-C18: 1-PE (1-palmitoyl-2-oleoylphosphatidylethanolamine) and (2-palmitoylphosphatidylcholine) In the case of studying with 5a, LysoPL production amount: FIG. LysoPL is meant to mean both in the figure because the substrates used in this experiment are PC and PE, and those obtained by reaction are LPC and LPE. From the above results, the optimum reaction conditions for the phospholipase A1 activity of the skipjack ovary extract were 20-30 ° C. and pH 6-7, but a relatively high activity was observed even in low temperature zones such as 0 ° C. and 10 ° C. . Since polyunsaturated fatty acids are generally easily oxidized, it was considered that higher quality lysophospholipids can be obtained by reacting the extracted enzyme at a low temperature of 10 ° C. or lower depending on the production conditions.

  Example 3 Total lipids were extracted from bonito ovary using a mixed solvent of chloroform and methanol, and fractionated with a silica gel column to obtain phospholipids. A crude oil was obtained from the lyophilized bonito ovary by ethanol extraction. Using these as substrates, an enzyme reaction was carried out with a skipjack ovary crude enzyme solution at 20 ° C. for 6 hours. First, FIG. 6 uses phospholipid as a substrate, and changes with time (mol%: diagram) of phospholipid composition (LPC: lysophosphatidylcholine, SPM: sphingomyelin, PC: phosphatidylcholine, PE: phosphatidylethanolamine, Others: other phospholipids). 6a), changes in the amount of C16: 0 (palmitic acid), C18: 0 (stearic acid), C18: 1n-9 (oleic acid), C22: 6n-3 (docosahexaenoic acid (DHA)) as a fatty acid produced over time ( FIG. 6 b) shows the results of examining the time course of DHA in phospholipid (FIG. 6 c). Next, FIG. 7 shows the results of examining changes in the LPC composition ratio over time (FIG. 7a) and changes in the DHA composition ratio over time (FIG. 7b) using total lipids as substrates. FIG. 8 shows the results of examining the time course of the LPC composition ratio using crude refined oil as a substrate. In any experiment, skipjack ovary crude enzyme hydrolyzed the contained phospholipid, and increased the LPC composition ratio and DHA composition ratio in the phospholipid.

  Example 4 A total of 21 ovaries discharged from the 6 plants were obtained from the bonito and namaribushi factories in Yaizu City, Shizuoka Prefecture. From these, lipids were extracted with a mixed solvent of chloroform and methanol, and fractionated with a silica gel column to obtain phospholipids. After fractionating this with TLC, phosphorus was measured by the Barlett method, and the amount of lysophospholipid in the phospholipid was measured. As a result, it was found that lysophospholipid was contained in the phospholipid in an average of 35.8%. In order to fractionate 1-acyl-lysophospholipid and 2-acyl-lysophospholipid, phospholipase C (from Bacillus cereus) was reacted with 5 mg of phospholipid fractionated on a silica gel column and bound to the sn-3 position. The phosphoric acid group is hydrolyzed. Next, 1-acyl-lysophospholipid-derived 1-monoglyceride and 2-acyl-lysophospholipid-derived 2-monoglyceride were obtained by TLC using a nonpolar developing solvent. After these were methyl esterified, the fatty acid composition according to the binding position of lysophospholipid was measured by gas chromatography (FIG. 9). As a result, lysophospholipids having a highly unsaturated fatty acid bonded to the 2-position accounted for 54.0% of the lysophospholipids, which corresponded to an average of 19.3% of the total phospholipids.

  From the above, by utilizing the fish ovary-derived phospholipase according to the present invention, lysophospholipids having DHA or highly unsaturated fatty acid bound to the sn-2 position can be concentrated at a high concentration. Moreover, the DHA concentrate obtained by this invention can be utilized as a health food and a food additive raw material besides medicine.

  Although specific preferred embodiments and examples of the present invention have been described and illustrated above, the present invention is not limited to these one embodiment and examples, and does not change the gist of the present invention. Various modifications are possible.

The graph which compared the phospholipase A1 (PLA1) activity of fish classification. The graph which compared the PLA1 activity of each bonito part. The graph which showed the temperature dependence of skipjack ovary phospholipase. The graph which showed the pH dependence of skipjack ovary phospholipase. The graph which showed the substrate dependence of skipjack ovary phospholipase. The graph which showed the result of the enzymatic decomposition of substrate phospholipid. The graph which showed the result of the enzyme decomposition | disassembly of a substrate total lipid. The graph which showed the result of the enzymatic decomposition of the substrate crude refined oil. The figure which showed the composition of the coupling | bonding fatty acid of a lysophospholipid.

Claims (15)

  A composition having phospholipase A1 activity, which is extracted from ovary of fish. The composition of claim 1, wherein
A composition having phospholipase A1 activity, wherein the ovary of the fish is an ovary of a skipjack, a tuna, or a mackerel.   A 2-acyl lysophospholipid obtained by hydrolyzing a phospholipid or a phospholipid in a phospholipid-containing material using the composition of claim 1. The 2-acyl lysophospholipid of claim 3,
A 2-acyl lysophospholipid characterized in that a highly unsaturated fatty acid is bonded to the 2-position. In the 2-acyl lysophospholipid of claim 4,
The 2-acyl lysophospholipid, wherein the polyunsaturated fatty acid is docosahexaenoic acid.   A 2-acyl type lysophospholipid having a highly unsaturated fatty acid bonded to the 2-position, wherein the composition of claim 1 is used to hydrolyze a phospholipid derived from a marine product or a phospholipid-containing phospholipid. . The 2-acyl lysophospholipid of claim 6,
The 2-acyl lysophospholipid, wherein the polyunsaturated fatty acid is docosahexaenoic acid.   A lipid composition containing 10% or more of 2-acyl lysophospholipid having a highly unsaturated fatty acid bonded to the 2-position, which is extracted from fish ovary. The lipid composition of claim 8, comprising
The lipid composition, wherein the polyunsaturated fatty acid is docosahexaenoic acid. The process of preparing fish ovaries;
Extracting the composition having phospholipase A1 activity from the ovary of the fish. A method for producing a composition having phospholipase A1 activity. The method of claim 10, wherein:
The fish ovary is a bonito, tuna or mackerel ovary. The process of preparing fish ovaries;
A step of extracting a composition having phospholipase A1 activity from the ovary of the fish, and a 2-acyl lysophospholipid by hydrolyzing phospholipids in the phospholipid or the phospholipid-containing material using the composition having phospholipase A1 activity A process for producing 2-acyl lysophospholipid, comprising the step of: The method of claim 12, wherein
The 2-acyl lysophospholipid is a method in which a highly unsaturated fatty acid is bonded to the 2-position. 14. The method of claim 13, wherein
The method according to claim 2, wherein the highly unsaturated fatty acid of the 2-acyl lysophospholipid is docosahexaenoic acid. The method of claim 12, wherein
The method according to claim 1, wherein the phospholipid or the phospholipid-containing material is derived from a marine product.

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