JP2001029097A - Measurement of lipoxygenase activity in grain - Google Patents

Abstract

PROBLEM TO BE SOLVED: To conveniently and quickly measure lipoxygenase activity in grains e.g. for beer-brewing raw material which participates in oxidation reaction of lipid by reacting a grain extract with a substrate solution and then adding FOX(Ferrous oxidation/xylenol orange method) reagent to the reaction product, followed by measuring absorbance. SOLUTION: When reacting a grain extract e.g. comprising an extract of barley or malt with a substrate solution containing a free fatty acid (or its ester) such as linoleic acid or phosphatidylcholine dilinoleyl, dilinolein or trilinolein as a substrate, by terminating the reaction through the addition of an organic solvent such as methanol dissolving the free fatty acid or an organic solvent such as 1-butanol extracting neutral fat, or the like, recovering a fraction containing the reaction product, adding FOX reagent to the fraction and by measuring absorbance, lipoxygenase activity in grain which participates in oxidation reaction of lipid is measured conveniently in a short time.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring lipoxygenase activity in cereals, and more particularly to a method for measuring lipoxygenase activity present in cereals used as a raw material for beer brewing and involved in lipid oxidation reactions. .

[0002]

2. Description of the Related Art Lipoxygenase is well known as an oxidoreductase which generates peroxide by adding oxygen to a double bond of an unsaturated fatty acid, and is an effective method for measuring the degree of its activity in many fields. Has been studied. For example, in the field of plants, lipoxygenase is abundantly contained in cereals such as soybeans and barley. Regarding beer barley, which is a raw material of beer, lipid oxidation that significantly affects the quality of beer occurs in a malt production process and a brewing preparation process, and this is caused by lipoxygenase (hereinafter referred to as LOX). It is known that By the action of LOX, lipids are converted to lipid hydroperoxides,
These are said to be precursors of trihydroxyoctadecenoic acid (Sezo Yabuuchi, Journal of the Brewing Association, 75 , 273, 1980) having a foam retention inhibitory activity and trans-2-nonenal, which is considered to be a causative substance of aging odor. Have been done.

[0003] The proportion of various lipids in the lipids in germinated barley and malt is several percent of free fatty acids, about 70% of neutral fats, and about 30% of phospholipids. Despite being important to influence,
Most LOX studies so far have used free fatty acids as substrates, and few studies have used fatty acid esters as substrates. The reason is that a simple method for measuring LOX activity using lipid as a substrate has not been developed. For example, a method of measuring the production of linoleic acid hydroperoxide using linoleic acid as a substrate by ultraviolet absorption (Baxter; j. Inst. Brew., 88 , 390-396, 19
In 82), the substrate is limited to linoleic acid. Also, the method of monitoring enzyme consumption using an enzyme sensor requires a large amount of lipid as a substrate because of low sensitivity,
Fatty acid esters are so expensive that they cannot be used in practice.

[0004] Further, it is difficult to simultaneously process a large number of specimens by the conventional method, and it is difficult to simultaneously treat a variety of plants having low LOX, heat stability, p.
It was not possible to screen for plant varieties with low H stability and the like. In soybean, a lipoxygenase detection method using an enzyme-labeled antibody has been proposed (Evans et al .;
Crop. Sci., 34, 1529-1537, 1994). However, this method is a method for detecting an enzyme, and cannot measure the activity of the enzyme. In addition, this method has a disadvantage that LOX having different antigen specificities cannot be detected.

[0005] On the other hand, Piazza et al. Used purified soybean lipoxygenase as a material and prepared FOX (Ferrous oxidation / xyle).
Nol orange method) has been reported to measure the oxidation activity of fatty acid esters (JAOCS, 72 (4), 463-466, 1995).
The FOX method is based on the fact that lipid hydroperoxide oxidizes iron (II) to iron (III) and quantifies the complex of iron (III) and xylenol orange using absorbance (visible light at a wavelength of 540 nm). This is a method for indirectly quantifying lipid hydroxyperoxide in a sample. After reacting the enzyme with the substrate for a certain period of time, they extracted with chloroform / methanol, separated the generated hydroperoxide, dried the chloroform layer by spraying with nitrogen, dissolved again in methanol, and used in the FOX method. LOX activity is measured by reacting with a reagent (hereinafter, referred to as FOX reagent). For this reason, this method has many problems such that it takes time to dry and dissolve, and is not suitable for processing multiple samples.

[0006]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to solve the above-mentioned problems and to reduce the LOX activity of a crude cereal extract, particularly an extract extracted from barley or malt, by a simpler method and in a short time. An object of the present invention is to provide a method for measuring LOX activity which can be measured in time. The present inventors have been studying to solve such a problem, and have found that an LO using the FOX method is used.
A method for measuring the activity of X was developed, and the present invention was completed.

[0007]

The present invention according to claim 1 is characterized in that after reacting a cereal extract with a substrate solution, a FOX reagent is added to the reaction product and the absorbance is measured. This is a method for measuring lipoxygenase activity in lipoxygenase. According to the present invention, the cereal extract is reacted with the substrate solution, and the reaction is performed by adding an organic solvent such as methanol for dissolving free fatty acids or an organic solvent such as 1-butanol for extracting neutral fats and the like. And then stopping the reaction, collecting a fraction containing the reaction product, adding a FOX reagent to the fraction, and measuring the absorbance, thereby measuring the lipoxygenase activity in the cereal. According to a third aspect of the present invention, in the method according to the first or second aspect, the substrate solution is a solution containing a free fatty acid or a fatty acid ester as a substrate. The present invention according to claim 4 is the measurement method according to claim 1 or 2, wherein the substrate solution is a solution containing any of linoleic acid, phosphatidylcholine dilinoleyl, dilinolein, and trilinolein as a substrate. The present invention according to claim 5 is the measurement method according to claim 1 or 2, wherein the cereal extract is an extract of barley or malt.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a method for measuring LOX activity in cereals, which method comprises extracting cereal seeds, germinated seeds or processed cereals using an appropriate buffer. After adding a solution containing a fatty acid or a fatty acid ester as a substrate and reacting,
The OX reagent is added and the absorbance is measured. Specifically, after reacting the cereal extract and the substrate solution for a predetermined time,
The reaction is stopped by adding methanol or 1-butanol, a fraction containing a lipid hydroperoxide, which is a reaction product, is separated, mixed with a FOX reagent, and the absorbance is measured.

In the present invention, barley is suitable as the cereal, and its seeds, germinated seeds or processed products thereof are specifically targeted, and specific examples thereof include barley malt and green malt. As the substrate, a fatty acid ester such as a free fatty acid, a neutral lipid or a polar lipid can be used. Among them, linoleic acid, phosphatidylcholine dilinoleyl, dilinolein and trilinolein are preferable. Next, F
The OX reagent is prepared by dissolving xylenol orange, ammonium iron (II) sulfate hexahydrate, sulfuric acid, and 2,6-di-t-butyl p-cresol in methanol. For example, 7.2 mg of xylenol orange, ammonium sulfate 9.8 mg of iron (II) hexahydrate, 69 μL of sulfuric acid and 88 mg of 2,6-di-t-butyl p-cresol were added to 9
It is a reagent prepared by dissolving in 100 mL of 0% methanol.

Hereinafter, the method for measuring LOX activity of the present invention will be described. First, the raw material grains are pulverized with a pulverizer such as a malt mill, and a buffer is added to the obtained pulverized product.
Extraction is performed by stirring at a temperature of 0 ° C., preferably at room temperature (20 ° C.) for 5 to 60 minutes, preferably 10 minutes. Next, the stirred product is rapidly cooled and filtered by a suitable means to obtain an extract. If necessary, the extract is subjected to solid-liquid separation such as centrifugation, and the obtained supernatant is filtered again.

A substrate solution is added to the cereal extract thus obtained and reacted. At this time, the substrate is used by being mixed with a surfactant, an emulsifier, a buffer, and the like. After reacting for a certain period of time, the reaction is stopped. When a free fatty acid such as linoleic acid or phosphatidylcholine dilinoleyl or a polar fatty acid ester is used as a substrate, the reaction is stopped using methanol, and a neutral lipid such as dilinolein or trilinolein is used as a substrate. Stops the reaction by adding 1-butanol. Thereafter, a lipid hydroperoxide-containing fraction, which is a reaction product, is separated from the reaction product. Specifically, a solid and a liquid are separated by a centrifugation method or another solid-liquid separation method, and when a free fatty acid or a polar fatty acid ester is used as a substrate, a supernatant fraction is collected. On the other hand, when a neutral lipid is used as a substrate, a 1-butanol fraction as a reaction terminator is recovered.

Next, the FOX reagent is added to the lipid hydroperoxide-containing fraction and mixed, and the mixture is stirred at room temperature for 30 to 90 minutes.
Preferably, it is left for 30 minutes. Thereafter, the absorbance at 540 nm is measured. The LOX activity is determined by comparing the obtained measured value with a previously prepared calibration curve. To prepare a calibration curve, a standard cumene hydroperoxide solution prepared by diluting cumene hydroperoxide with an appropriate buffer was used as a FOX.
This is performed by mixing with a reagent and measuring the absorbance of the mixture in the same manner as described above.

[0013]

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples. Example 1 Barley (mature seeds), green malt (germination day 1, 6), and roasted malt (roasted 1, 2, 3,
12 hours) and 20 g of freeze-dried malt,
The mixture was pulverized with an EBC malt mill, and 10 g of an acetate buffer (pH 5.0) was added to 1 g of the obtained pulverized product, followed by stirring at room temperature for 10 minutes. After stirring, quench to 0 ° C on an ice bath,
It filtered using the filter cloth. The cereal extract thus obtained was centrifuged (3000 rpm) at 4 ° C. for 15 minutes, and the obtained supernatant was filtered with a 0.45 μm filter.

Subsequently, 500 μL of the extract was placed in a 0.8 mm glass test tube on an ice bath, and 67 μL of 0.1 M sodium deoxycholate and 1 M Tris buffer (pH 8.0) were added thereto. Next, after keeping the temperature in an incubator set at 25 ° C. for 10 seconds, a 40 mM substrate solution was added to a final concentration of 2 mM to start an oxidation reaction. In addition, four types of linoleic acid (LH), phosphatidylcholine dilinoleyl (PC), gilenolein, and trilinolein were used as substrates. When linoleic acid was used, 1 g of linoleic acid was weighed, 1% Tween 20 was added thereto to a concentration of 40 mM, and the mixture was stirred to form an emulsion, which was used as the substrate solution. When phosphatidylcholine dilinoleyl, dilinolein, or trilinolein was used as a substrate, 40 mg of each was weighed, and 1% sodium deoxycholate and 1 M Tris buffer (pH 8.0) were adjusted to 40 mM. In addition, the mixture was stirred to form an emulsion, which was used as a substrate solution.

After a lapse of 30 minutes from the start of the reaction,
The reaction was stopped. When linoleic acid or phosphatidylcholine was used as a substrate, the reaction was stopped by adding 668 μL of 100% methanol and allowed to stand on an ice bath for 20 minutes. The reaction was then placed at 4 ° C. for 15 minutes.
The supernatant was collected by centrifugation (3000 rpm) and used as a sample. When dilinolein or trilinolein was used as the substrate, 668 μL of 1
-After stopping the reaction by adding butanol, 668
Add μL of chloroform, stir vigorously, then 4 ° C
And centrifugation (3000 rpm) for 15 minutes.
The butanol fraction was collected and used as a sample.

20 μL of each sample thus obtained
Was transferred to a microtiter plate, 200 μL of FOX reagent was added, and the mixture was allowed to stand at room temperature for 30 minutes. Thereafter, the absorbance at 540 nm was measured with a plate reader (Bio-Rad). The FOX reagent was prepared by adding 7.2 mg of xylenol orange, 9.8 mg of iron (II) sulfate hexahydrate, 69 μL of sulfuric acid, and 88 mg of 2,6-di-t-butyl p-cresol to 90 mg
It was prepared at any time during measurement by dissolving in 100 mL of% methanol. In addition, a standard cumene hydroperoxide solution was prepared by dissolving 50 mg of cumene hydroperoxide in methanol to a concentration of 10 mM, and diluting the solution with the enzyme extract to a concentration of 0.1 mM to 1 mM. Using this in advance, 540 nm in the same manner as above
Was measured and a calibration curve was prepared. The amount of lipid hydroperoxide in each sample was determined by comparing the measured values with a calibration curve, and the LOX activity was calculated.

The results when linoleic acid (LH) or phosphatidylcholine dilinoleyl (PC) was used as a substrate are shown in the figure. FIG. 1 shows the change over time in the amount of lipid hydroperoxide produced during the malting process of barley A and FIG. 2 shows that of barley B. The same tendency was observed when dilinolein or trilinolein was used as a substrate.

As a result, it was revealed that barley before germination had only the LH oxidation activity. Furthermore, after germination,
It was found that in addition to LH oxidation activity, PC oxidation activity was also induced. In addition, the measurement time of the LOX activity according to the present invention was significantly reduced as compared with the conventional method. According to the method of Piazza, separation of a chloroform layer (1-2 minutes), drying by blowing nitrogen (3-6 minutes), and re-dissolving in methanol (1) are performed to extract the reaction product hydroperoxide. ~ 2 minutes), which requires a minimum of 5-10 minutes per sample. According to the present invention, this operation can be omitted, and for example, processing about 100 samples can be reduced by about 8 to 16 hours.

Example 2 To 3.53 g of malt crushed by an EBC disk mill, 50
10 mL of tap water heated to 0 ° C was added, and the mixture was stirred for 30 seconds in a 50 ° C incubator. After stirring, the mixture was rapidly cooled to 4 ° C. on an ice bath, and filtered using a filter cloth. The filtrate was then centrifuged (3000 rpm) at 4 ° C. for 15 minutes.
m), and the obtained supernatant was filtered with a 0.45 μm filter to obtain a cereal extract.

Subsequently, a reaction solution was prepared in the same manner as in Example 1. Using linoleic acid as a substrate, a reaction temperature of 0 ° C., 25
The production amount of linoleic acid hydroperoxide at 50 ° C. and 50 ° C. was determined in the same manner as in Example 1. Also, 50
Enzyme inactivation by heat treatment at 20 ° C. for 20 minutes was tested. After the extract was treated at 50 ° C. for 20 minutes, the amount of linoleic acid hydroperoxide produced at 25 ° C. was determined in the same manner as in Example 1.

The results are shown in FIGS. FIG. 3 shows the change over time in the amount of linoleic acid hydroperoxide produced when malt was used as a raw material and the temperature conditions of the enzyme reaction were changed. FIG. 4 shows the change over time in the amount of linoleic acid hydroperoxide produced when heat treatment was performed at 50 ° C. for 20 minutes.

As apparent from FIG. 3, when the enzyme reaction was carried out at 0 ° C. and 25 ° C., the amount of linoleic acid hydroperoxide produced increased in proportion to the reaction time. On the other hand, when the enzyme reaction was performed at 50 ° C., the amount of linoleic acid hydroperoxide produced tended to gradually decrease with the lapse of reaction time. It was also found that the initial enzyme activity (reaction rate) was higher in the order of 50 ° C., 25 ° C., and 0 ° C. Furthermore, when the enzyme reaction was carried out at 0 ° C. and 25 ° C., it became clear that linoleic acid hydroperoxide reached almost the same level 40 minutes after the start of the reaction.

As is clear from FIG. 4, even when the heat treatment is performed at 50 ° C. for 20 minutes, the amount of linoleic acid hydroperoxide increases in proportion to the reaction time, similarly to the case without heat treatment. And malt L
It was found that OX hardly deactivated by heat treatment at 50 ° C. for 20 minutes.

[0024]

According to the measuring method of the present invention, the activity of the enzyme lipoxygenase, which has an important effect on the quality of raw cereals, malt quality and beer quality, can be measured more easily and in a shorter time than the conventional method. it can. Further, the present invention can be applied to screening of varieties lacking the enzyme lipoxygenase.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing the change over time in the amount of hydroperoxide produced during the malting process of barley A when linoleic acid or phosphatidylcholine is used as a substrate.

FIG. 2 is a graph showing the change over time in the amount of hydroperoxide produced during the malting process of barley B when linoleic acid or phosphatidylcholine is used as a substrate.

FIG. 3 is a graph showing the change over time in the amount of linoleic acid hydroperoxide produced when malt is used as a raw material and the temperature conditions of the enzyme reaction are changed.

FIG. 4 is a diagram showing a change with time in the amount of linoleic acid hydroperoxide produced when a heat treatment is performed at 50 ° C. for 20 minutes.

Claims (5)

[Claims] 1. A method for measuring lipoxygenase activity in cereals, comprising reacting a cereal extract with a substrate solution, adding a FOX reagent to the reaction product, and measuring the absorbance. 2. A cereal extract is reacted with a substrate solution, and the reaction is stopped by adding an organic solvent such as methanol for dissolving free fatty acids or an organic solvent such as 1-butanol for extracting neutral fats and the like. A method for measuring lipoxygenase activity in cereals, comprising collecting a fraction containing a reaction product, adding a FOX reagent thereto, and measuring the absorbance. 3. The method according to claim 1, wherein the substrate solution is a solution containing a free fatty acid or a fatty acid ester as a substrate. 4. The method according to claim 1, wherein the substrate solution is a solution containing any one of linoleic acid, phosphatidylcholine dilinoleyl, dilinolein and trilinolein as a substrate. 5. The method according to claim 1, wherein the cereal extract is an extract of barley or malt.