JP3715839B2 - Method for measuring lipoxygenase activity in cereals - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for measuring lipoxygenase activity in cereals, and more particularly to a method for measuring lipoxygenase activity that is present in cereals used as a raw material for beer brewing and is involved in lipid oxidation reaction.
[0002]
[Prior art]
Lipoxygenase is well known as a redox enzyme that generates peroxide by adding oxygen to the double bond of unsaturated fatty acid, and an effective method for measuring the degree of its activity has been studied in many fields. .
For example, in the field of plants, lipoxygenase is particularly abundant in cereals such as soybeans and barley. Beer barley, which is a raw material of beer, undergoes lipid oxidation that has an important influence on the quality of beer in the malt production process and the beer brewing process, which is caused by lipoxygenase (hereinafter referred to as LOX). It is known that
By the action of LOX, lipids are converted to lipid hydroperoxides, which are considered to be the causative substances of trihydroxyoctadecenoic acid (Seizo Tsujiuchi, Shuzokai, 75 , 273, 1980) and foam aging odor. It is said to be a precursor of trans-2-nonenal.
[0003]
The percentage of various lipids in germinated barley and malt lipids is as follows: free fatty acids are several percent, neutral fats are about 70%, and phospholipids are about 30%. The oxidation of these lipids affects the quality of beer. Despite the importance, most of the previous LOX studies have been based on free fatty acids as substrates, and few studies have been conducted on fatty acid esters as substrates.
This is because a simple LOX activity measurement method using lipid as a substrate has not been developed. For example, in the method in which the production of linoleic acid hydroperoxide is measured by ultraviolet absorption (Baxter; j. Inst. Brew., 88 , 390-396, 1982) using linoleic acid as a substrate, the substrate is limited to linoleic acid.
In addition, the method of monitoring enzyme consumption using an enzyme sensor requires a large amount of lipid as a substrate because of its low sensitivity. However, fatty acid esters are very expensive and cannot be used in practice.
[0004]
Furthermore, in the conventional method, it is difficult to simultaneously process multiple specimens, and plant varieties with low LOX and plant varieties with low thermal stability, pH stability, etc. could not be screened. 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 the activity of the enzyme cannot be measured. In addition, this method has a drawback that LOX having different antigen specificities cannot be detected.
[0005]
On the other hand, Piazza et al. Have reported a method for measuring the oxidation activity of fatty acid esters by the FOX method (Ferrous oxidation / xylenol orange method) using purified soybean lipoxygenase (JAOCS, 72 (4), 463-466, 1995). . The FOX method utilizes the fact that lipid hydroperoxide oxidizes divalent iron to trivalent iron, and quantifies trivalent iron and xylenol orange complex using absorbance (absorbance of visible light having a wavelength of 540 nm). This is a method for indirectly quantifying lipid hydroxyperoxides in data.
They react the enzyme with the substrate for a certain period of time, then extract with chloroform / methanol, separate the hydroperoxide formed, dry the chloroform layer by blowing nitrogen, dissolve it again in methanol, and use it 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 as that it takes time to dry and dissolve and is not suitable for processing a large number of samples.
[0006]
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to solve the above-mentioned problems and to measure the LOX activity of a crude cereal extract, particularly an extract extracted from barley or malt, in a simpler manner and in a short time. It is to provide a measurement method.
The present inventors have repeatedly studied to solve such problems, developed a method for measuring LOX activity using the FOX method, and completed the present invention.
[0007]
[Means for Solving the Problems]
In the first aspect of the present invention, after reacting a cereal extract with a solution containing a free fatty acid or a polar fatty acid ester as a substrate, methanol is added to stop the reaction product, and then the reaction product is separated by a solid-liquid separation method. A method for measuring lipoxygenase activity in cereals, comprising collecting a fraction containing the mixture and measuring the absorbance by adding a FOX reagent thereto.
The present invention according to claim 2 contains a reaction product by a solid-liquid separation method after reacting a cereal extract with a solution using a neutral lipid as a substrate and then adding 1-butanol to stop the reaction. A method for measuring lipoxygenase activity in cereals, comprising collecting fractions and measuring the absorbance by adding a FOX reagent thereto.
According to a third aspect of the invention, the substrate solution is a measurement method of claim 1, wherein a solution containing linoleic acid or phosphatidylcholines dilinoleyloxy as substrate.
The present invention according to claim 4 is the measuring method according to claim 2 , wherein the substrate solution is a solution containing dilinolein or trilinolein as a substrate.
According to a fifth aspect of the invention, cereal extract is a measuring method according to claim 1 or 2 which is extract of barley or malt.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method for measuring LOX activity in cereals, and this method comprises the extraction of cereal seeds, germinated seeds or processed cereal products using a suitable buffer, fatty acid or fatty acid ester as a substrate. After adding a solution containing, a FOX reagent is added to the reaction product 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, and the fraction containing the lipid hydroperoxide as a reaction product is separated, and the FOX reagent Mix and measure absorbance.
[0009]
In the present invention, barley is suitable as the cereal, and its seeds, germinated seeds or processed products thereof are targeted, and specific examples thereof include barley malt and green malt.
As the substrate, fatty acid esters such as free fatty acids, neutral lipids or polar lipids can be used, and among them, linoleic acid, phosphatidylcholine dilinoleyl, dilinolein and trilinolein are preferable.
Next, the FOX reagent is prepared by dissolving xylenol orange, ammonium iron (II) sulfate hexahydrate, sulfuric acid and 2,6-di-t-butyl p-cresol in methanol. It is a reagent prepared by dissolving 2 mg, 9.8 mg of iron (II) sulfate hexahydrate, 69 μL of sulfuric acid and 88 mg of 2,6-di-t-butyl p-cresol in 100 mL of 90% methanol.
[0010]
Below, the measuring method of the LOX activity of this invention is demonstrated.
First, the raw cereal is pulverized with a pulverizer such as a malt mill, and a buffer solution is added to the obtained pulverized product, and the temperature is 0 to 50 ° C., preferably room temperature (20 ° C.) for 5 to 60 minutes, preferably 10 minutes. Extract with stirring. Next, the stirred product is rapidly cooled and then filtered by an appropriate 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.
[0011]
The substrate solution is added to the cereal extract thus obtained and reacted. At this time, the substrate is used by mixing with a surfactant, an emulsifier, a buffer solution and the like.
After reacting for a certain time, the reaction is stopped. When using free fatty acids or polar fatty acid esters such as linoleic acid or phosphatidylcholine dilinoleyl as substrates, the reaction is stopped using methanol, and when neutral lipids such as dilinolein or trilinolein are used as the substrate. Stops the reaction by adding 1-butanol.
Thereafter, the fraction containing lipid hydroperoxide, which is a reaction product, is separated from the reaction product. Specifically, the solid and the liquid are separated by a centrifugal separation method or other solid-liquid separation methods, 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 neutral lipid is used as a substrate, the 1-butanol fraction which is a reaction terminator is collected.
[0012]
Next, the FOX reagent is added to the lipid hydroperoxide-containing fraction and mixed and left at room temperature for 30 to 90 minutes, preferably 30 minutes. Thereafter, the absorbance at 540 nm is measured.
The obtained measured value is collated with a calibration curve prepared in advance to determine LOX activity. A calibration curve is prepared by mixing a standard cumene hydroperoxide solution prepared by diluting cumene hydroperoxide with an appropriate buffer with FOX reagent, and measuring the absorbance of the mixture in the same manner as described above.
[0013]
【Example】
EXAMPLES Next, although an Example demonstrates this invention in detail, this invention is not limited to these.
Example 1
Barley (mature seeds), green malt (germination 1, 6th day), dried malt (roasted 1, 2, 3, 12 hours) and malt were lyophilized during the malting process of two types of barley A and B 20 g of each product was pulverized with an EBC malt mill, 10 mL of acetate buffer (pH 5.0) was added to 1 g of the pulverized product, and the mixture was stirred at room temperature for 10 minutes. After stirring, the mixture was rapidly cooled to 0 ° C. on an ice bath and filtered using a filter cloth.
The cereal extract thus obtained was centrifuged (3000 rpm) at 4 ° C. for 15 minutes, and the resulting supernatant was filtered with a 0.45 μm filter.
[0014]
Subsequently, 500 μL of the extract was placed in a 0.8 mm thick 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 incubating for 10 seconds in an incubator set to 25 ° C., a 40 mM substrate solution was added to a final concentration of 2 mM to initiate the oxidation reaction.
As substrates, four types of linoleic acid (LH), phosphatidylcholine dilinoleyl (PC), zirenolein, and trilinolein were used. Among these, when linoleic acid was used, 1 g of linoleic acid was weighed, and 1% Tween 20 was added to this to be 40 mM and stirred to form an emulsion, which was used as the substrate solution. When phosphatidylcholine dilinoleyl, dilinolein, and trilinolein were used as substrates, 40 mg was weighed, and 1% sodium deoxycholate and 1 M Tris buffer (pH 8.0) were added to give 40 mM. In addition, an emulsion formed by stirring was used as a substrate solution.
[0015]
After 30 minutes from the start of the reaction, the reaction was stopped. When linoleic acid or phosphatidylcholine dilinoleyl 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. Thereafter, the reaction product was centrifuged (3000 rpm) at 4 ° C. for 15 minutes to recover the supernatant, which was used as a sample.
When dilinolein or trilinolein was used as a substrate, the reaction was stopped by adding 668 μL of 1-butanol, and then 668 μL of chloroform was added and stirred vigorously, followed by centrifugation at 4 ° C. for 15 minutes ( 3000 rpm) to collect the 1-butanol fraction, which was used as a sample.
[0016]
After 20 μL of each sample thus obtained was transferred to a microtiter plate, 200 μL of FOX reagent was added and left at room temperature for 30 minutes. Thereafter, the absorbance at 540 nm was measured with a plate reader (Bio-Rad).
As for the FOX reagent, 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 are dissolved in 100 mL of 90% methanol. From time to time, it was prepared at the time of measurement.
The standard cumene hydroperoxide solution was prepared by dissolving 50 mg of cumene hydroperoxide in methanol to 10 mM and diluting it with the enzyme extract to 0.1 mM to 1 mM. Using this, the absorbance at 540 nm was measured in the same manner as described above to prepare a calibration curve.
The amount of lipid hydroperoxide in each sample was determined by comparing the measured value with a calibration curve, and LOX activity was calculated.
[0017]
Results are shown when using linoleic acid (LH) or phosphatidylcholine dilinoleyl (PC) as substrate. FIG. 1 shows changes over time in the amount of lipid hydroperoxide produced during the barley making process of barley A and FIG. 2 shows that of barley B. The same tendency was observed when dilinolein and trilinolein were used as substrates.
[0018]
As a result, it became clear that only LH oxidation activity exists in the barley before germination. Furthermore, after germination, it was found that PC oxidation activity was also induced in addition to LH oxidation activity.
In addition, the measurement time of LOX activity according to the present invention was significantly shortened as compared with the conventional method. In the Piazza method, for extraction of the reaction product hydroperoxide, separation of the chloroform layer (1-2 minutes), drying by blowing nitrogen (3-6 minutes), and dissolution in methanol again (1 ~ 2 minutes) is required, but this requires a minimum of 5-10 minutes per specimen. According to the present invention, this operation can be omitted and, for example, about 8 to 16 hours can be shortened to process 100 samples.
[0019]
Example 2
10 mL of tap water heated to 50 ° C. was added to 3.53 g of malt crushed by an EBC disc mill, and the mixture was stirred for 30 seconds on an incubator at 50 ° C. After stirring, the mixture was quenched on an ice bath to 4 ° C. and filtered using a filter cloth. Next, the filtrate was centrifuged (3000 rpm) at 4 ° C. for 15 minutes, and the resulting supernatant was filtered with a 0.45 μm filter to obtain a cereal extract.
[0020]
Subsequently, a reaction solution was prepared in the same manner as in Example 1. Using linoleic acid as a substrate, the amount of linoleic acid hydroperoxide produced at reaction temperatures of 0 ° C., 25 ° C., and 50 ° C. was quantified in the same manner as in Example 1.
In addition, enzyme inactivation by heat treatment at 50 ° 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 quantified in the same manner as in Example 1.
[0021]
The results are shown in FIGS. FIG. 3 shows changes 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 shows the change over time in the amount of linoleic acid hydroperoxide produced when heat treatment is performed at 50 ° C. for 20 minutes.
[0022]
As apparent from FIG. 3, when the enzyme reaction was performed 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 production amount of linoleic acid hydroperoxide tended to gradually decrease with the lapse of the reaction time.
Moreover, it became clear that the initial enzyme activity (reaction rate) is high in the order of 50 ° C, 25 ° C, and 0 ° C. Furthermore, when the enzymatic reaction was performed at 0 ° C. and 25 ° C., it was revealed that linoleic acid hydroperoxide reached almost the same level 40 minutes after the start of the reaction.
[0023]
In addition, as is apparent from FIG. 4, even when heat treatment is performed at 50 ° C. for 20 minutes, the amount of linoleic acid hydroperoxide produced increases in proportion to the reaction time, as in the case of no heat treatment. It became clear that malt LOX was hardly inactivated by heat treatment at 50 ° C. for 20 minutes.
[0024]
【The invention's effect】
According to the measurement method of the present invention, the activity measurement of the enzyme lipoxygenase, which has an important influence on the quality of raw cereals, malt quality and beer quality, can be carried out more easily and in a shorter time than the conventional method.
The present invention can also be applied to screening for varieties lacking the enzyme lipoxygenase.
[Brief description of the drawings]
FIG. 1 is a graph showing changes in hydroperoxide production over time during the malting process of barley A when linoleic acid or phosphatidylcholine dilinoleyl 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 dilinoleyl is used as a substrate.
FIG. 3 is a graph showing changes 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 graph showing a change over time in the amount of linoleic acid hydroperoxide produced when heat treatment is performed at 50 ° C. for 20 minutes.

Claims (5)

  After reacting the cereal extract with a solution containing free fatty acid or polar fatty acid ester as a substrate, methanol is added to stop, and then the fraction containing the reaction product is recovered by solid-liquid separation method. A method for measuring lipoxygenase activity in cereals, wherein the absorbance is measured by adding a FOX reagent.   After reacting the cereal extract with a solution using a neutral lipid as a substrate, 1-butanol is added to terminate the reaction, and then a fraction containing the reaction product is recovered by solid-liquid separation, and FOX is collected therein. A method for measuring lipoxygenase activity in cereals, comprising adding a reagent and measuring the absorbance. Substrate solution, the measurement method of claim 1, wherein a solution containing linoleic acid or phosphatidylcholines dilinoleyloxy as substrate. The measuring method according to claim 2 , wherein the substrate solution is a solution containing dilinolein or trilinolein as a substrate. Cereal extract is measuring method according to claim 1 or 2 which is extract of barley or malt.

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