JPH07222592A - Production of lysophospholipid by phospholipase a1 - Google Patents

Abstract

PURPOSE:To obtain the subject lysophospholipid, capable of inactivating the residual enzyme after reaction by mild heat treatment without regulating the pH, having a high lyso formation ratio and useful for foods, cosmetics, medicines, etc., by treating a phospholipid with an acidic phospholipase A1. CONSTITUTION:This method for producing a lysophospholipid is to culture a mold Aspergillus oryzae SANK-11870 strain of the genus Aspergillus in a culture medium composed of a mixture of bran with water in equal amounts at 30 deg.C for 6 days, then inoculate the cultured strain into a culture medium similar thereto, culture the inoculum at 30 deg.C for 15hr and further at 19 deg.C for 5 days, subsequently extract the resultant bran KOJI with water, add acetic acid to the extracted solution, regulate the pH to 4.0, add acetone thereto, cool the regulated extracted solution, separate the formed precipitate, provide acidic phospholipase A1, then add the prepared acidic phospholipase A1 to an aqueous suspension of a phospholipid and carry out the reaction at 37 deg.C for 3hr while stirring the suspension. The resultant lysophospholipid is good in quality and capable of inactivating the residual enzyme by mild heat treatment without requiring pH regulation in reacting the enzyme and has a high lyso formation ratio.

Description

Detailed Description of the Invention

[0001]

[Object of the Invention]

[0002]

TECHNICAL FIELD The present invention relates to a method for producing a lysophospholipid by treating a phospholipid with acid phospholipase A1.

[0003]

2. Description of the Related Art Lysophospholipids are partially decomposed phospholipids obtained by hydrolyzing a part of fatty acids bound to glycerin residues of phospholipids. Such lysophospholipids are
Compared to the original phospholipid, the water solubility increases and the O / W type emulsifying property becomes stronger, forming a more stable emulsion with respect to changes in pH and temperature, and also high in ions such as calcium and magnesium. It has been reported that excellent properties are imparted such that the emulsifying power does not decrease even when coexisting with the concentration. Therefore, lysophospholipid has a wider application range than phospholipid in the fields of food, cosmetics, pharmaceuticals, and the like. Also,
The lyso-formation of phospholipids in the phospholipid-containing substance may lead to improvement of the physical properties of food by the phenomenon alone or by a synergistic effect with other coexisting substances.

Phospholipase is known as an enzyme that decomposes phospholipids, and is further subdivided according to its site of action. Of these, phospholipase A1 or phospholipase A2 is known as a substance that decomposes phospholipids into lysophospholipids and fatty acids, and any of them can be used for the purpose of lysifying phospholipids.

At present, porcine pancreatic phospholipase A2 is exclusively used in the industrial method for producing lysophospholipid from phospholipid, but it has the following problems. The first point is that, generally, when phospholipase A acts, pH adjustment is required. When phospholipase A acts, the pH of the reaction system decreases due to free fatty acids,
The optimum pH of porcine pancreatic phospholipase A2 is neutral to weakly alkaline. Therefore, it is necessary to adjust the pH during the enzyme action, and a step for removing the substance added for pH adjustment after the enzyme reaction is required.

In order to solve this problem, solvent fractionation (US Pat. No. 3,652,397, etc.), enzymatic reaction in a nonionic nonpolar organic solvent (JP-A-3-98590) or free fatty acid and metal soap A method of adding a compound which makes the compound into the reaction (JP-A-62-14790, JP-B-4-814).
No. 31) is proposed. However, these methods are complicated in operation, and the safety of the product may be a problem in some cases.

The second point is that after the enzymatic reaction, porcine pancreatic phospholipase A2, which has extremely high stability to heat and organic solvents, remains in the obtained product. The presence of unhydrolyzed phospholipids in the final product
Phospholipase A2 may liberate new fatty acids from phospholipids, significantly impairing their commercial value. Therefore, a method of inactivating the residual enzyme by heat treatment is conceivable. However, the porcine pancreatic phospholipase A2 is not sufficiently inactivated only by heat-treating the enzyme reaction product at 95 ° C. for about 30 minutes, and When inactivated by heat treatment at about 120 ° C., problems such as deterioration of phospholipids or free fatty acids occur.

Various methods have been tried in order to obtain a lysophospholipid-containing substance having substantially no residual activity of phospholipase A2. For example, phospholipase A, which is a method combining solvent fractionation using various solvents and column treatment using silica gel in the manufacturing process.
After the phospholipid treated with 2 is dried, it is fractionated using a polar solvent (Japanese Patent Laid-Open No. 62-262998) or treated with an enzyme, and then phospholipase A2 in the reaction product is treated with a protease, and then, A method for inactivating the protease by heating (Japanese Patent Laid-Open No. 63-233750) and the like. But,
These operations are time-consuming, costly, and have various problems such as a decrease in yield.

[0009]

DISCLOSURE OF THE INVENTION The present inventors have, for many years, conducted extensive studies on a method for producing lysophospholipid from phospholipid using phospholipase, and confirmed that the phospholipid was treated with acid phospholipase A1 producing bacteria. By treating with acidic phospholipase A1 obtained by adding an aqueous organic solvent to the enzyme extract, it is possible to deactivate the residual enzyme by mild heat treatment without the need to adjust the pH during enzyme action, The present invention has been completed by finding a method for producing a lysophospholipid having a high lysolysis rate and high quality.

[0010]

[Constitution of the invention]

[0011]

The production method of the present invention is achieved by treating a phospholipid with acid phospholipase A1.

The acidic phospholipase A1 used is preferably pH 2.5 to 6 (preferably pH 3 to 5.
5) phospholipase A1 having an active region, more preferably phospholipase A1 having a stable upper limit temperature of 45 ° C to 90 ° C (preferably 50 ° C to 80 ° C), and even more preferably, Phospholipase A1 having an optimal pH of action of pH 3.2 to 5, pH stability of pH 3 to 8.5, and temperature stability of 55 ° C. or lower, and particularly preferably, having the above-mentioned properties, is Aspergillu
s ) Phospholipase A1 produced by filamentous fungi of the genus.

Examples of the producing bacterium that produces acidic phospholipase A1 include Aspergillus oryzae ( Aspergillus oryzae).
us oryzae : For example, SANK-11870, IFO-30102, etc.), Aspergillus niger : For example, IF
O-4407, ATCC-9642, etc.), Aspergillus usamii : For example, IFO-6082, IAM-2414
Etc.), Aspergillus awamor
i : For example, IFO-4033, IAM-2112, etc., Aspergillus fumigatus : For example, IA
M-2034, etc.), Aspergillus Soja (Aspergillus
sojae : For example, IAM-2666, etc., Aspergillus phoenisis : For example, IAM-2215
Etc.), Aspergillus went
ii : For example, filamentous fungi belonging to the genus Aspergillus such as IAM-2133 etc., preferably Aspergillus
It is Orize. The above-mentioned filamentous fungi are all known fungi (for example, Japanese Patent Publication No. 46-32792, J. Gen. Appl. Microbio
l., 17 , 281 (1971), Biochem. Z., 261 275 (1933) etc.)
And the Institute for Fermentat
ion Osaka: IFO), American TypeCulture Collection (ATCC),
Institute of Applied Microbiolog
y: IAM) etc. are stored under the respective numbers in storage organizations and can be freely distributed. In addition, SANK-11870,
It has been deposited at the Institute of Microbial Science and Technology, the Agency of Industrial Science and Technology, as Microorganism Research Institute No. 3887 (FERM BP-3887). Further, these strains include natural original strains and artificial mutants thereof.

Acid phospholipase A1 is obtained by the koji culture method,
It can be produced by any of the liquid culture methods, and in any case, by appropriately selecting the medium composition and culture conditions, it contains only phospholipase A1 as an enzyme having an activity of releasing fatty acids from phospholipids,
Moreover, phospholipase A1 without lipase activity can be selectively produced.

For example, the phospholipase A1 of the present invention is produced by the following method. That is, the strain is cultivated by a koji-type culturing method, in which 0.5 to 2 times by weight (preferably an equal amount) of water is added to bran, and a solid medium obtained by boiling is inoculated with an inoculum. . As the medium raw material, in addition to bran, cereals (for example, rice, wheat, etc.), various minor cereals (for example, corn, soybean, sesame, etc.), cottonseed meal and the like can be used alone or in combination.

The culturing temperature is 10 ° C. to 40 ° C. (preferably 15 ° C. to 35 ° C.), and the culturing time varies depending on the medium composition, the culturing temperature, etc., but is usually 3 days to 20 days (preferably 4 to 8 days). After culturing, add 1 to 20 times weight of water or an appropriate buffer solution (eg, acetate buffer solution, phosphate buffer solution, etc.) to the koji, mix well, and press-filter to obtain an enzyme solution. You can

For collecting the enzyme from the enzyme solution, a conventional method, for example, a salting-out method, an organic solvent precipitation method, an adsorption method using an ion exchanger or the like as an adsorbent, an ultrafiltration method, a vacuum drying method or the like is used. An enzyme having a desired quality can be prepared by using an appropriate combination. In the present invention, the organic solvent precipitation method, which is the most general method for collecting enzymes, is suitable.

Acid phospholipase A1 in the enzymatic reaction
The amount to be added varies depending on the reaction temperature, the reaction time, the pH during the reaction, the properties and quality of the substrate, the contaminating substance, the degree of the required effect, etc., but it is preferable to use 1000 units / g of enzyme. , For phospholipids (eg soybean phospholipids),
0.05 wt% to 10 wt% (particularly preferably 0.2
% To 2% by weight).

The substrate used is phospholipid itself or a phospholipid-containing substance, regardless of its source, content or property. They are, for example, plant phospholipids such as soybean, wheat, barley, corn, rapeseed, safflower, sunflower, peanut, cottonseed, egg yolk, animal brain (cow, sheep, pig, chicken, etc.) and the like. Phospholipids, chlorella cells,
It may be a microbial cell phospholipid such as a filamentous fungal cell, and is preferably soybean, wheat or egg yolk phospholipid.

The enzyme reaction for treating this enzyme and phospholipid is
It is carried out by bringing the enzyme and the substrate into contact with each other in an aqueous medium or in a wet state, and in some cases, a nonionic nonpolar organic solvent (eg, diethyl ether, ethers such as dioxane, hexane, benzene, toluene, etc.). It is also possible to carry out the enzyme reaction in the coexistence of hydrocarbons, esters such as ethyl acetate, butyl acetate, etc.).

The aqueous or wetting medium can be, for example, ion-exchanged water, distilled water, well water, tap water, etc., and if necessary,
Add an acid (such as acetic acid), an alkali (such as sodium hydroxide) or a buffer (such as acetate buffer),
The pH can be adjusted to 2.5 to 6.5 (preferably 3.5 to 5.5). When soft water such as ion-exchanged water or distilled water is used, it may be preferable to add calcium chloride or the like.

The enzyme reaction temperature is 10 ° C to 70 ° C (preferably 20 ° C to 65 ° C, particularly preferably 30 ° C to 6 ° C).
0 ° C), and the time required for the reaction varies depending on the reaction temperature, pH, etc., but is usually 10 minutes to 10 days (preferably,
1 hour to 2 days). In addition, it is usually not preferable that the phospholipase A1 activity remains in the obtained lysophospholipid, and after the enzymatic reaction is performed, if necessary, a simple operation, that is, a mild heat treatment, in the enzymatic reaction product, The residual phospholipase A1 activity of can be easily inactivated. This heat treatment is 45 ° C to 90 ° C
(Preferably 50 ° C. to 80 ° C.) and heating for 5 minutes to 5 hours (preferably 10 minutes to 2 hours).

In addition to the heat treatment, a conventional method for inactivating an enzyme, for example, pressure treatment, pH treatment, etc., may be used alone or in combination, and the residual phospholipase A1 may be used.
The activity can be deactivated.

According to the present enzymatic reaction, the reaction product can be used as it is, but if necessary, it is further concentrated or purified by a concentrator such as thin layer concentration, further spray-dried or freeze-dried. It is also possible to obtain a concentrated product, a purified product, or a dried product by subjecting it to a conventional drying method such as

The enzyme of the present invention can also be used when it is added directly to the dough for the purpose of improving the physical properties of flour products such as bread and noodles. At this time, phospholipase D and an emulsifier (for example, monoglyceride, calcium stearyl lactylate, etc.) can be added, if necessary. In addition, the effect of using the present enzyme appears in the physical properties of the dough and the product, and the dough becomes appropriate elasticity and extensibility by the addition of the present enzyme, and the adhesiveness is also suppressed, so that each work step Is easy to operate.

[0026]

INDUSTRIAL APPLICABILITY The method for producing lysophospholipids using the acidic phospholipase A1 of the present invention does not require adjustment of pH at the time of enzyme action, and not only has the effect of producing highly pure lysophospholipids, but also has mild heating. Since the residual enzyme can be inactivated by the treatment, the quality of the produced lysophospholipid can be prevented from deteriorating and the energy can be saved by a simple operation.

[0027]

EXAMPLES Examples and production examples of the present invention will be shown below.
The present invention is not limited to these.

[Example 1] Production of lysophospholipid 10% (w / w) suspension of 5 g of SLP-white ( manufactured by Trurecitin) in 45 ml of water and 0.2 g of calcium chloride in a 100 ml beaker And was thoroughly dispersed with an ultrasonic homogenizer. After preheating at 37 ° C, add 865 units of phospholipase A1-a to -c each, and stir it with a stir bar at 37 ° C to prevent the water during the reaction from evaporating. (Manufactured by K.K.) to allow the enzymatic reaction to proceed. At the start of the reaction, 1 hour, 2 hours, and 3 hours later, the reaction solution was collected, and the free fatty acid in the sample was quantified using a free fatty acid quantification reagent Determiner NEFA. The apparent phospholipid lysolysis rate is 76% of the average molecular weight of phospholipids in SLP-white.
It was calculated using the formula A / B x 100 A: number of moles of lysophospholipid (number of moles of fatty acid released by enzymatic reaction) B: number of substrate phospholipids. The results are shown in Table 1.

[0029]

[Table 1] ──────────────────────────────────── Experiment Addition enzyme Apparent phospholipid lysolysis rate (%) Number (pH value) Reaction time 0 hours 1 hour 2 hours 3 hours ──────────────────────────────── ──── 1. Phospholipase A1-a 0 70 84 89 (6.4) (4.7) (4.6) (4.6) Comparative Example 2. Phospholipase A1-b 0 65 83 90 (6.4) (4.7) (4.6) (4.7) 3 Phospholipase A1-c 0 68 86 90 (6.4) (4.7) (4.6) (4.6) ───────── ──────────────────────────── The phospholipase A1-a of the present invention is a phospholipase A1-b (phospholipase A1-) shown as a comparative example. a
Is further purified) and phospholipase A1-c ( As
( from Pergillus niger ) showed almost the same excellent phospholipid lysolysis rate.

[Example 2] Inactivated substrate of enzyme remaining by heat treatment [10 (w / w)% SLP-white (manufactured by Trulecithin)]
50 ml and 0.2 g calcium chloride were placed in a 100 ml beaker and thoroughly dispersed with an ultrasonic homogenizer. After preheating at 37 ° C, phospholipase A1-
2160 units of each of a to -c were added, and the enzyme reaction was allowed to proceed at 37 ° C for 5 hours while stirring with a stirring bar. As the buffer, 5 mM acetate buffer (pH 4.5) was used. Further, the apparent phospholipid lysation rate after 5 hours was 90% or more. After the enzymatic reaction, about 7 g of each reaction solution was dispensed into several Somogyi test tubes, covered with a sealing film to prevent water from evaporating, and at 50 to 80 ° C.
Left for 30 minutes. The phospholipase A1 activity remaining in each reaction solution was measured by the method described below and expressed as a percentage with respect to the phospholipase A1 activity in the reaction solution without heat treatment. The results are shown in Table 2.

[0031]

[Table 2] ──────────────────────────────────── Experiment added enzyme Remaining enzyme activity (%) No. Treatment temperature Untreated 50 ℃ 60 ℃ 70 ℃ 80 ℃ ───────────────────────────────────── 1 Phospholipase A1-a 100 77 68 0 0 Comparative example 2 Phospholipase A1-b 100 80 70 0 0 3 Phospholipase A1-c 100 78 73 19 0 ───────────────────── ───────────────── The phospholipase A1-a of the present invention includes phospholipase A1-b (further purified phospholipase A1-a) and phospholipase A1- which are shown as comparative examples. c ( Aspe
( from rgillus niger ), it could be completely inactivated by mild heat treatment.

[Production Example] Phospholipase A1-a Aspergillus oryzae SANK11
870 strains, 12g medium consisting of a mixture of bran and water
The cells were cultured at 30 ° C for 6 days. Then, 600 g of a mixture of bran and water was added to a metal dish (42 x 24 x 7 (depth) cm).
The above culture was inoculated into a medium that had been placed in a container and cooked under pressure at 120 ° C for 30 minutes, then at 30 ° C for 15 hours, and at 19 ° C for 5
Cultured for a day. To the bran koji thus formed, 3 liters of water was added, mixed well, allowed to stand at 37 ° C. for 2 hours, then filtered to give a phospholipase A1 titer of 5.9 units / m 2.
l of enzyme extract 2.87 l was obtained. Acetic acid was added to this enzyme extract to adjust the pH to 4.0, cooled acetone was added in an amount of 3 times, and the mixture was allowed to stand in a cool place overnight. Then discard the supernatant,
The precipitate was thoroughly washed with acetone and vacuum dried to obtain 11.1 g of phospholipase A1-a.

Main enzyme activities per 1 g of this sample are shown in Table 3.
Shown in. Each enzyme activity was measured by the method described below.

[0034]

[Table 3]
Unit ──────────────────────────────────── Phospholipase A1 1,170 units Lower limit of lipase measurement (10 units) Amylase 8,740 units Acidic protease 38,800 units Neutral / alkaline protease 230,000 units ────────────────────────────── [Comparative Example 1] Phospholipase A1-b 10 g of phospholipase A1-a obtained in Production Example was dissolved in about 100 ml of water, 1N acetic acid was added to adjust pH to 4.0, and water was added. After making up to 200 ml, 200 ml of cold acetone was added and mixed, and left for 1 hour. afterwards,
The mixed solution was centrifuged to obtain a first precipitate. 600 in the supernatant
ml of cold acetone was added and mixed and left for 1 hour. Then, the mixed solution was centrifuged to obtain 5 g of a second precipitate. 5 g of the precipitate was added to 50 mM acetate buffer (pH 5.5) 5
It was dissolved in 00 ml and 300 g of ammonium sulfate was added for salting out. The precipitate obtained by centrifugation was dissolved in 50 mM acetate buffer (pH 5.5) containing 1 M ammonium sulfate, and impurities were filtered off, followed by column chromatography [column: Butyl Toyopearl Pack 650S (Butyl Toy
opearl pak 650S), manufactured by Tosoh Corporation, effluent solvent: ammonium sulfate-containing 50 mM acetate buffer (pH 5.5), ammonium sulfate concentration, 1 to 0 M gradient elution]. Ammonium sulfate was added to the eluted phospholipase A1 active fraction for salting out, the mixture was allowed to stand overnight, and the precipitate obtained by centrifugation was subjected to column chromatography [column: Superose 12 manufactured by Pharmacia,
Elution solvent: deionized water] for desalting. The eluted phospholipase A1 active fraction was lyophilized to obtain 0.034 g of phospholipase A1-b. The molecular weights of the two types of phospholipase A1 contained in this sample are SDS-
It was 37,000 or 35,000 and the isoelectric point was 3.9 or 4.3, respectively, as measured by polyacrylamide gel electrophoresis.

The main enzyme activities per 1 g of this sample are shown in Table 4.
Shown in. Each enzyme activity was measured by the method described below.

[0036]

[Table 4] ─────────────────────────────────── Phospholipase A 72,100 units Lipase 100 units or less Amylase 3,990 units Acidic protease 50,500 units Neutral / alkaline protease 49,000 units ─────────────────────────────── Comparative Example 2 Phospholipase A1-c Aspergillus niger ATCC96
12 g of a medium consisting of 42 strains and an equal amount of bran and water
After culturing at 30 ° C. for 6 days and then preculturing all strains, 600 g of a mixture of bran and water was added to a metal dish (42 × 2).
4 x 7 (depth) cm), inoculate the medium that was cooked at 120 ° C for 30 minutes, incubate at 30 ° C for 15 hours, then 19 ° C
The cells were cultured for 5 days. Add 3 parts of water to the bran so prepared.
l, mixed well, left at 37 ° C. for 2 hours,
An enzyme extract was obtained by filtration. Acetic acid was added to this enzyme extract to adjust the pH to 4.0, cooled acetone was added in an amount of 3 times, and the mixture was allowed to stand in a cool place overnight. After that, the supernatant is discarded, the precipitate is thoroughly washed with acetone, and dried in vacuum to obtain an enzyme sample with an activity of 119 units / g that liberates fatty acids from phospholipids.
19.2 g was obtained. 10 g of this enzyme sample is used for about 100 m of water.
Dissolve in 1 and add 1N acetic acid to adjust to pH 4.0,
After adding water to 200 ml, 200 ml of cold acetone was added and mixed, and the mixture was left for 1 hour. Then, the mixed solution was centrifuged to obtain a first precipitate. To the supernatant, 600 ml of cold acetone was added and mixed, and the mixture was left for 1 hour. Then, the mixed solution was centrifuged to obtain a second precipitate. 5 of this precipitate
It was dissolved in 500 ml of 0 mM acetate buffer (pH 5.5) and 300 g of ammonium sulfate was added for salting out. The precipitate obtained by centrifugation was dissolved in 50 mM acetate buffer (pH 5.5) containing 1 M ammonium sulfate, the insoluble matter was filtered off, and then Butyl Toyopearl pak 650S (Butyl Toyopearl pak) was added.
650S, manufactured by Tosoh Corporation, Q-Sepharose
, Pharmacia), and Super Loose 12 (Sup
Erose 12, manufactured by Pharmacia), and purified by column chromatography using phospholipase A1 titer of 2,100 units / g phospholipase A1-c 0.0.
15 g was obtained. The molecular weight of phospholipase A1 contained in this sample was estimated to be 32,000 by the gel filtration method using Superose 12, and the isoelectric point was 3.0.

Main enzyme activities per 1 g of this sample are shown in Table 5.
Shown in. Each enzyme activity was measured by the method described below.

[0038]

[Table 5] ─────────────────────────────────── Phospholipase A 2, 100 units Lower limit of lipase measurement (10 Unit) or less Amylase 17 units Acid protease 280 units Neutral / alkaline protease 700 units ───────────────────────────────── Next, the phospholipases A1-a to -c obtained in Production Examples and Comparative Examples 1 to 2 each had ^14- position palmitoyl group.
C-labeled L-α-dipalmitoylphosphatidylcholine (NE)
N company NEC764) and L-α-dipalmitoylphosphatidylcholine (NEN company NEC 682) labeled with ¹⁴ C at the 1- and 2-position palmitoyl groups. As a result, 1 palmitic acid 2-position of the palmitoyl group was ¹⁴ C labeled only from ¹⁴ C-labeled phosphatidylcholine were liberated. From this result, it was confirmed that the fatty acid-releasing activity from the phospholipid contained in the phospholipase A1-a to -c is derived from the release of fatty acid from the 1-acyl group. In addition, it was confirmed that none of the phospholipases A1-a to -c was accompanied by lipase activity (about 0.1% or less of phospholipase A1 activity).

The characteristics of the phospholipase A1-a of the present invention are shown in Comparative Examples 1 and 2 below.
And shown in comparison with -c. (1) Optimum pH of action The optimum pH of action is pH 3.2 to 5, as shown in FIG. The phospholipase A1 activity was measured by the method described below, and the activity at pH 4 was defined as 100%.

(2) Optimum temperature of action The optimum temperature of action was 50 to 60 ° C. as shown in FIG. The phospholipase A1 activity was measured by the method described below, and the activity at 37 ° C was defined as 100%. (3) pH stability The pH stability is pH 3 to 8.5 as shown in FIG. The phospholipase A1 activity was about 10 units / phospholipase A1 using 0.2 M acetate buffer (pH 3.2 to 6.5) and 0.01 M Tris-HCl buffer (pH 3.2 to 9.0). Prepare ml of enzyme solution and keep at 37 ℃
Then, after heating for 60 minutes, measure by the method described below,
The maximum value of residual activity when heated at 37 ° C. for 60 minutes was defined as 100%.

(4) Temperature stability The temperature stability at pH 4 is 30 ° C. as shown in FIG.
To 55 ° C. The phospholipase A1 activity is
After the enzyme solution was heated at 30 to 70 ° C. for 30 minutes, it was measured by the method described below, and the activity when it was not heated was defined as 100%.

Each enzyme activity was measured by the following method.

(I) Phospholipase A1 activity and fatty acid release activity test from phospholipids 2.0 (w / v)% SLP-white (manufactured by Trurecitin Industry Co., Ltd.) and 4 (v / v)% Triton X -100
0.1 ml of 0.1 M calcium chloride in 0.5 ml of aqueous solution
0.25 ml of 0.2 M acetate buffer (pH 4.0) was added. Then, 0.1 ml of the enzyme solution was added, and the mixture was stirred to make it uniform, and allowed to stand at 37 ° C. for 10 minutes to carry out an enzyme reaction. Then, 0.1 ml of 1N hydrochloric acid was added to stop the enzymatic reaction. 0.02 ml of the reaction solution was taken, and the free fatty acid was quantified using a free fatty acid quantification reagent Determiner NEFA (manufactured by Kyowa Medex Co., Ltd.). The enzyme activity that produces 1 μmol of fatty acid per minute of the enzyme reaction was defined as 1 unit.

(Ii) Lipase activity test 2.0 (w / v)% olive oil (manufactured by Wako Pure Chemical Industries) emulsion [0.5 g (w / w)% in 0.2 g of olive oil]
10 ml of an aqueous solution of gum arabic was added and dispersed for 5 minutes with an ultrasonic homogenizer] 0.5 ml of 0.1 M calcium chloride 0.05 ml and 0.02 M acetate buffer (p
H6) was added. Then add 0.1 ml of enzyme solution,
The mixture was stirred to make it uniform, left to stand at 37 ° C., and subjected to an enzymatic reaction for 10 minutes. Then, 0.1 ml of 1N hydrochloric acid was added to stop the enzymatic reaction. The reaction solution (0.02 ml) was sampled and the free fatty acid was quantified using the free fatty acid quantification reagent Determiner NEFA. The enzyme activity that produces 1 μmol of fatty acid per minute of the enzyme reaction was defined as 1 unit.

(Iii) Amylase (saccharification type) activity test In a test tube, the substrate [2 g of soluble starch and 20 ml of water (appropriate amount) was added and dissolved by heating, and after cooling, 0.2 M acetate buffer (pH 4.5) ) Was added to 20 ml, and water was further added to adjust the volume to 50 ml] 0.5 ml was added, and after preheating in a 37 ° C. constant temperature water tank, 0.25 ml of the enzyme solution was added. After the enzymatic reaction for 30 minutes, the reaction was stopped by adding 0.25 ml of 0.5N sodium hydroxide. Using this as an enzyme reaction solution, the Somogyi-Nelson reducing sugar assay method (J. Biol. Chem., 160 , 61-68 (1945), J. B.
iol. Chem., 153 , 375-380 (1944)) was used to quantify the reducing sugar produced by the enzymatic reaction. The enzymatic activity for producing a reducing sugar equivalent to 1 μmol of glucose per minute of the enzymatic reaction was defined as 1 unit.

(Iv) Protease activity test Hagiwara method (enzyme research method 2 Asakura Shoten, 237-246 (1956)
) Was measured. The substrate pH was 3.0 (acidic protease) or 7.0 (neutral / alkaline protease). The unit of enzyme activity is Northrop.
p) and Anson were defined as 1 unit of the enzyme activity that produces a non-protein substance that absorbs 1 μg of tyrosine at 275 nm per minute under standard conditions.

[Brief description of drawings]

FIG. 1 shows the optimum pH of action of phospholipase A1-a of Production Example and phospholipases A1-b to -c of Comparative Examples 1 and 2.

FIG. 2 is the optimum action temperature of phospholipase A1-a of Production Example and phospholipases A1-b to -c of Comparative Examples 1 and 2.

FIG. 3 shows pH stability of phospholipase A1-a of Production Example and phospholipases A1-b to -c of Comparative Examples 1 and 2.

FIG. 4 is the temperature stability of phospholipase A1-a of Production Example and phospholipases A1-b to -c of Comparative Examples 1 and 2.

Claims (4)

[Claims] 1. A method for producing lysophospholipid, which comprises treating phospholipid with acid phospholipase A1. 2. The method for producing a lysophospholipid according to claim 1, wherein the acid phospholipase A1 is phospholipase A1 obtained by adding an aqueous organic solvent to an enzyme extract of an acid phospholipase A1 producing bacterium. 3. The method for producing a lysophospholipid according to claim 1, wherein the acid phospholipase A1 is phospholipase A1 produced by a filamentous fungus of the genus Aspergillus . 4. The method for producing a lysophospholipid according to claim 1, 2 or 3, wherein the acid phospholipase A1 is phospholipase A1 produced by Aspergillus oryzae .