JP2009240171A - Method for producing high 4-hydroxy isoleucine-containing fenugreek sprout using isoleucine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing fenugreek sprouts enriched in 4-OH-Ile to an unconventional extent. <P>SOLUTION: The method for producing the Fenugreek sprouts enriched in 4-hydroxy isoleucine (4-OH-Ile) includes growing fenugreek seeds in hydroponics, and supplies isoleucine in the hydroponics. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention provides fenugreek sprout with a high content of 4-hydroxyisoleucine and a method for producing the same using isoleucine.

  It has been conventionally known that branched chain amino acids such as 4-hydroxyisoleucine (hereinafter referred to as 4-OH-Ile) promote protein synthesis in skeletal muscle and liver and suppress degradation. In recent years, it has also been elucidated that branched chain amino acids have an action of regulating sugar metabolism. (Non-patent document 1) Therefore, 4-OH-Ile is expected to have a therapeutic effect on diseases involving sugar metabolism such as diabetes.

  On the other hand, Non-Patent Document 2 shows that when hydroponically cultivated fenugreek seeds, the 4-OH-Ile content per individual and per dry weight increased over time (cultivation period 1 week). . This indicates that 4-OH-Ile is biosynthesized in fenugreek sprout during cultivation. Further, in Non-Patent Document 2, a high molecular fraction (molecular weight 1000 or more) is obtained from the fenugreek sprout grinding liquid on the 5th day of hydroponics, and by adding isoleucine and coenzyme from the outside, 4-OH-Ile It is confirmed that it was synthesized. This indicates that the precursor of 4-OH-Ile is isoleucine, and 4-OH-Ile is enzymatically synthesized from isoleucine.

Chemistry and Biology, Vol. 45, no. 3, 203-210 (2007) Phytochemistry 44, 563-566 (1997)

  In Non-Patent Document 2, after a fenugreek sprout is pasted, a polymer fraction containing 4-OH-Ile synthase is separated, and isoleucine is added to the fraction to synthesize 4-OH-Ile. Is described. However, this method is difficult to apply industrially and is inefficient.

  In the conventional method, a plant having a high 4-OH-Ile content could not be produced.

  Accordingly, an object of the present invention is to provide a method for producing fenugreek sprout enriched with 4-OH-Ile.

The present invention includes the following inventions.
(1) A method for producing fenugreek sprout enriched with 4-hydroxyisoleucine (4-OH-Ile) by hydroponics from fenugreek seeds, wherein isoleucine is supplied in the hydroponics.
(2) The supply of isoleucine is a period of 72 hours or more, including a period of 48 hours centering on the point at which the rate of increase of 4-OH-Ile amount per unit dry weight of the growing sprout in the hydroponics is maximized. The method according to (1), wherein the method is performed for a period of time.
(3) The supply of isoleucine is performed at least for a period from the time when the total length of the hypocotyl part and the root part of the sprout is 1 cm to 6 cm in the hydroponics. Method.
(4) The method according to (1), wherein the supply of isoleucine is performed for a period from a time point 24 hours after the start of hydroponics to a time point 96 hours later.
(5) The method according to any one of (1) to (4), wherein isoleucine is supplied in the form of an aqueous solution.
(6) The method according to (5), wherein the concentration of the isoleucine aqueous solution is 10 to 100 mM.
(7) A fenugreek sprout produced by the method according to any one of (1) to (6), wherein the 4-OH-Ile content is 40 to 70 mg / g (dry weight basis).

  By the method of the present invention, it is possible to provide a fenugreek sprout containing a large amount of 4-OH-Ile, for example, 1.8 times or more the amount of ordinary fenugreek sprout (FIG. 4).

  By limiting the supply timing of isoleucine, it is possible to reduce the amount of isoleucine to be used, reduce the number of work steps to be supplied, and provide the fenugreek sprout at a low cost.

1. Hydroponics Procedure Fenugreek sprout is a germinated vegetable obtained by hydroponically cultivating fenugreek seeds. The above hydroponics is also called hydroponics, and is a method of cultivating water alone or using the necessary nutrients (nitrogen, phosphate, potassium, calcium, magnesium, etc.) with liquid fertilizer (hybonica fertilizer, etc.) without using soil. Hydroponic cultivation of rice has been carried out for thousands of years.

  Typical cultivation methods for fenugreek sprout are as follows.

  First, fenugreek seeds are sterilized with a disinfectant such as hypochlorous acid aqueous solution or ethanol aqueous solution.

  The sterilized seeds were immersed in water in a dark place (eg, 20 to 30 ° C.) where the temperature was kept constant, and then sown in an environment (eg, 20 to 30 ° C.) where the temperature was kept constant. Later, water is supplied. Thereafter, the seeding time is set as the starting point (0 hour) (start of hydroponics), and the plant is grown in the dark or under light irradiation from 48 hours immediately after sowing to 96 to 168 hours immediately after sowing, and fenugreek sprout is harvested.

For the light irradiation, artificial light having a light intensity (20 μmol / m ² / sec) normally used in a conventional sprout is used.

  The temperature during cultivation is preferably 20 to 30 ° C., which is generally suitable for plant germination and growth.

  A container for production of sprout is a pot or a tray, and a chamber is a sprout production apparatus.

  For example, water is put into a container together with vermiculite or soil for cultivation.

  The sprout cultivation apparatus is an apparatus for easily cultivating sprout regardless of the place, and water and air can be automatically sprayed on the sprout simultaneously by a timer. If necessary, the container and the chamber are cultivated in a thermostatic bath capable of controlling temperature, light irradiation time and light intensity.

  The method for supplying isoleucine is not particularly limited as long as it can supply isoleucine to a plant body. However, considering the efficient absorption by sprout, a method of supplying isoleucine in the form of an aqueous solution of isoleucine is desirable.

  The method for supplying the isoleucine aqueous solution is not particularly limited. For example, there are (i) a method for supplying the aqueous solution to the whole plant, and (ii) a method for absorbing the aqueous solution from the root. Since isoleucine is expensive, it is desirable to circulate the isoleucine aqueous solution when supplying the isoleucine aqueous solution.

  It is preferable to supply the aqueous isoleucine solution in hydroponics continuously or intermittently. When performing intermittently, it can supply by methods, such as spraying, at the time interval of 6 hours-12 hours, for example.

  The concentration of isoleucine in the aqueous solution is 10 mM or more, preferably 25 mM to 100 mM, more preferably 25 mM to 50 mM. In 96 hours of cultivation, by using an isoleucine solution having a concentration of 10 mM or more, the amount of 4-OH-Ile per dry weight of the sprout individual is significantly increased with respect to the control group (water supply group). In the concentration range of 25 to 100 mM, the 4-OH-Ile content per dry weight of the sprout individual can be increased up to 1.6 times compared to the case of the water supply group (FIG. 2).

  When the isoleucine concentration is 25 to 100 mM, there is a difference in the amount of isoleucine observed in the sprout, but there is no difference in the amount of 4-OH-Ile accumulated in the sprout. -Ile synthesis amount does not increase. Therefore, a suitable isoleucine concentration for increasing 4-OH-Ile without wasting expensive isoleucine is 25 mM to 50 mM (FIG. 2).

2. When to supply isoleucine Since isoleucine is expensive, it is even more desirable if it can be supplied efficiently during hydroponics.

  The inventors surprisingly included isoleucine in a 48 hour period centered around the point at which the rate of increase of 4-OH-Ile per unit dry weight of growing sprout in hydroponics is maximized. It was found that if a period of 72 hours or longer is supplied, 4-OH-Ile equivalent to that obtained when isoleucine is supplied over the entire period of hydroponics is found. It is considered that the presence of a large amount of isoleucine at the time when the synthesis of 4-OH-Ile is prosperous induces a synthase and increases the subsequent 4-OH-Ile synthesis. Therefore, in the present invention, a sufficient amount of isoleucine is present in the sprout at a time when the synthesis rate of 4-OH-Ile becomes active. Here, “a 48-hour period centering on the point at which the rate of increase in the 4-OH-Ile amount per unit dry weight of the growing sprout is maximum” means 24 hours from the point 24 hours before the same point. It means a 48 hour period leading to a later time. The present invention is characterized in that isoleucine is supplied over a continuous period of 72 hours or more including this period. Further, “the point at which the increase rate of the 4-OH-Ile amount per unit dry weight of the growing sprout becomes maximum” can be determined as follows. First, hydroculture of fenugreek sprout is carried out under the same conditions as the hydroculture conditions of 4-OH-Ile-enriched fenugreek sprout except that isoleucine is not supplied. And the plant body after a hydroponics start is sampled with time, and the amount of 4-OH-Ile per unit dry weight is measured. From the relationship between the elapsed time after the start of hydroponics and the 4-OH-Ile amount, the time point (T) at which the increase rate of the 4-OH-Ile amount is the highest can be known.

  More preferably, isoleucine is used in hydroponics for a period of 72 hours from 24 hours before the maximum rate of increase in the amount of 4-OH-Ile per unit dry weight of growing sprout to 48 hours after that point. Supply. For example, in hydroponics for 168 hours, the rate of increase of 4-OH-Ile is maximized 48 hours after the start of cultivation, and the synthesis of 4-OH-Ile is almost completed 96 hours after the start of cultivation. Therefore, isoleucine is supplied for a period from 24 hours before the point at which the increase rate of 4-OH-Ile per unit dry weight of the sprout is maximized to 96 hours after the start of hydroponics (ie, 48 hours after that point). It is preferable to do. At this time, even if isoleucine is supplied from 96 hours after the start of cultivation, the increase amount of 4-OH-Ile is not different from the condition without addition of isoleucine.

  The “time point after 96 hours of hydroponics” and the “time point after 24 hours of hydroponics” described later refer to the sterilization seeds in the dark where the temperature is adjusted to a constant as described in “1. Starting from sowing after water immersion, the time point after 96 hours and the time point after 24 hours.

  The present inventors also found that in hydroponics of fenugreek sprout, the 4-OH-Ile amount increase curve correlates with the sprout growth (hypocotyl part + root length) curve, and the 4-OH-Ile amount It was found that the addition time of isoleucine can be defined from the growth of fenugreek without measuring the rate of increase in the growth rate. That is, if isoleucine is supplied for a period from the time when the total length of the sprout hypocotyl part and root part is 1 cm to 6 cm in hydroponics, it is equivalent to the case where isoleucine is supplied over the entire period of hydroponics. We found that 4-OH-Ile was accumulated.

  First, hydroponically cultivate fenugreek sprout under the same conditions except that it does not supply isoleucine with the desired cultivation conditions of 4-OH-Ile-enriched fenugreek sprout, and it is possible to confirm in advance the change in sprout length over time desirable. As shown in the Examples, the growth rate of fenugreek sprout does not vary depending on whether or not isoleucine is added.

  The present inventors also provided isoleucine for a period from the time point 24 hours after the start of hydroponics to the time point 96 hours later, when 4-OH-Ile equivalent to the case of supplying isoleucine over the entire hydroponics period is obtained. I found that it was accumulated.

  In any of the above forms, it is most efficient to supply isoleucine only for a predetermined period. However, it is also included in the scope of the present invention to supply isoleucine for a period including before and after the predetermined period. The

3. 4-OH-Ile -enriched fenugreek sprout The fenugreek sprout obtained by the method of the present invention contains 40-70 mg / g (dry weight basis) of 4-OH-Ile. Moreover, the fenugreek sprout obtained by the method of the present invention usually contains isoleucine supplied in the hydroponics process.

(experimental method)
The seed used was Indian fenugreek. Cultivation was carried out using the “special equipment for sprout cultivation” (Fig. 1). This is a device that can intermittently sprinkle air and water on seeds in a tray with a drain. Seed 25 g of 3 conditions aqueous solution of 5 conditions shown below: (1) water (control), (2) 10 mM isoleucine aqueous solution, (3) 25 mM isoleucine aqueous solution, (4) 50 mM isoleucine aqueous solution, (5) After soaking in 100 mM isoleucine aqueous solution for 16 hours, seeds are seeded at a high density in a tray (inner dimensions 33 cm x 6 cm) (0.28 g / cm ² as seed weight before water absorption) and sprayed with isoleucine aqueous solution periodically While growing at 25 ℃. Each spraying was about 4.5 ml / min / 1 tray, spraying and aeration time (performed simultaneously with spraying) was 15 minutes, and every 6 hours. Light irradiation was performed for 16 hours a day from 48 hours after 0 hours immediately after sowing. Fluorescent lamps (light intensity approx. 25 μmol / m ² / sec, illuminance approx. 2500 lx) and red LEDs (660 nm, light intensity approx. 25 μmol / m ² / sec, illuminance approx. 350 lx), 4-OH-Ile Since there was no difference in the effect on the amount, they were used without distinction. Harvested 96 hours after sowing, the amount of 4-OH-Ile per dry weight was measured.

  The amount of 4-OH-Ile per dry weight of fenugreek sprout was measured by the following method.

  50 g of sprout was weighed, 20 g of distilled water was added, and homogenized (10,000 rpm) for 8 minutes. About 1 g of the milled product was weighed (precisely weighed), made up to 20 ml with 70% ethanol, and left for 4 hours as a test solution for HPLC analysis. The amount of 4-OH-Ile was measured by a precolumn method using an amino acid derivatization reagent OPA using HPLC (1100 series manufactured by Agilent). The water content of the milled product was measured by separately weighing about 1 g of the milled product (precisely weighed) and drying at 105 ° C. for 16 hours. From the concentration of the 4-OH-Ile standard product, the 4-OH-Ile concentration in the 70% ethanol extract was calculated, and then the amount of 4-OH-Ile in the precisely weighed sample was calculated. Then, the amount of 4-OH-Ile per dry weight of fenugreek sprout was calculated from the amount of water in the ground product. Also, the isoleucine amount per dry weight is similar to the structure of isoleucine and 4-OH-Ile, so the 4-OH-Ile calibration curve was used, and the HPLC analysis area value of isoleucine was 4-OH. Substituted into the -Ile calibration curve, and calculated as 4-OH-Ile amount.

(result)
The amount of 4-OH-Ile synthesized with an aqueous isoleucine solution of 10 mM or more significantly increased compared to the control water supply section. Moreover, there was no change in the amount of 4-OH-Ile synthesized between 25 mM, 50 mM and 100 mM isoleucine aqueous solutions (FIG. 2). The amount of isoleucine present in the sprout was not seen at all in the water supply area, whereas it was as shown in FIG. 2 in the isoleucine supply area. From this, it was confirmed that the supplied isoleucine was absorbed into the sprout and converted to 4-OH-Ile, so that 4-OH-Ile increased compared to the water supply zone. In addition, it was confirmed that isoleucine in the sprout increased depending on the concentration of the aqueous isoleucine solution, but there was no change in the amount of 4-OH-Ile synthesized when the isoleucine concentration was between 25 and 100 mM ( Figure 2).

(experimental method)
The same seeds and cultivation apparatus as in Example 1 were used. Immersion and cultivation were performed under the following nine conditions.
Condition 1):
After 25 g of seeds were soaked in 3 times the amount of 50 mM isoleucine aqueous solution for 16 hours, seeds were sown in the same manner as in Example 1 and cultivated for 96 hours. The application amount and application time were the same as in Example 1.
Conditions (2) to (8):
After immersing 25 g of seeds in 3 times the amount of water for 16 hours, the seeds were sown on the tray at the same temperature and method as in Example 1 and cultivated for 96 hours. The application period of 50 mM isoleucine aqueous solution is 0 hours immediately after sowing, (2) 0 to 96 hours after sowing, (3) 0 to 72 hours after sowing, (4) 24 to 96 hours after sowing, (5 ) 24 to 72 hours after sowing, (6) 48 to 96 hours after sowing, (7) 24 to 48 hours after sowing, (8) 72 to 96 hours after sowing, and water on other days Scattered. The application amount and application time were the same as in Example 1.
Condition (9):
After immersing 25 g of seeds in 3 times the amount of water for 16 hours, seeds were sown in the same manner as in Example 1 and cultivated for 96 hours. The application amount and application time were the same as in Example 1.

  Light irradiation, harvesting, and measurement of 4-OH-Ile amount per dry weight of fenugreek sprout were performed in the same manner as in Example 1.

(result)
There was no significant difference in the amount of 4-OH-Ile, even if it was supplied from 0 to 96 hours after sowing, even if it was supplied from the whole cultivation period or from 24 hours after sowing, immediately after sowing as 0 hours ( (Figure 3). That is, if 50 mM isoleucine was supplied from 0 hours to 72 hours or 24 hours to 96 hours after seeding, the same amount of 4-OH-Ile as that supplied for 0 to 96 hours was synthesized. From this, it was confirmed that the use period of isoleucine can be limited.

(experimental method)
The same seeds and cultivation apparatus as in Example 1 were used. After soaking 25 g of seeds in 3 times the amount of water for 16 hours, seeding the trays in the same manner as in Example 1, and immediately after sowing under the following four conditions (1) to (4) as 0 hours, Cultivated for 168 hours. The application amount and application time were the same as in Example 1. The plants were harvested every 24 hours from 0 to 168 hours after sowing, and the amount of 4-OH-Ile per dry weight was measured. Light irradiation was performed at the same temperature and method as in Example 1.
Condition (1): Spraying from 0 to 168 hours after seeding with water Condition (2): Spraying with 50 mM isoleucine aqueous solution 24 to 168 hours after seeding (3): 96 hours after seeding with 50 mM isoleucine aqueous solution 168 hours spraying condition (4): 50 mM isoleucine aqueous solution sprayed 24 to 96 hours after seeding
In (2) to (4), water was sprayed during a period when the isoleucine aqueous solution was not sprayed.

  The amount of 4-OH-Ile per dry weight of fenugreek sprout was measured by the following method. 20 g of sprout was weighed out, 40 g of distilled water was added, and homogenized (10,000 rpm) for 8 minutes. About 2 g of the milled product was weighed (precisely weighed), made up to 20 ml with 70% ethanol, and left for 4 hours as a test solution for HPLC analysis. The amount of 4-OH-Ile was measured by a precolumn method using an amino acid derivatization reagent OPA using HPLC (1100 series manufactured by Agilent). The water content of the milled product was measured by separately weighing out about 2 g of the milled product (precise weighing) and drying at 105 ° C. for 16 hours. From the concentration of the 4-OH-Ile standard product, the 4-OH-Ile concentration in the 70% ethanol extract was calculated, and then the amount of 4-OH-Ile in the precisely weighed sample was calculated. Then, the amount of 4-OH-Ile per dry weight of fenugreek sprout was calculated from the amount of water in the ground product. Also, the isoleucine amount per dry weight is similar to the structure of isoleucine and 4-OH-Ile, so the 4-OH-Ile calibration curve was used, and the HPLC analysis area value of isoleucine was 4-OH. Substituted into the -Ile calibration curve, and calculated as 4-OH-Ile amount.

  The length of the sprout is the average value of the lengths of (hypocotyl + root) of the 20 sprout, and the raw weight is shown as an average value per 10 (N = 5).

(result)
The result of 4-OH-Ile amount is shown in FIG. In experimental condition (1), the amount of 4-OH-Ile per 168 hours after dry weight is 36 mg / g, whereas in experimental condition (2), 4-OH per 96 hours after dry weight. The amount of -Ile was 45 mg / g, and the amount after 168 hours was 63 m / g. In the hydroponics section of (1), the increase rate of 4-OH-Ile decreased after 96 hours. On the other hand, in the experimental groups under the conditions (2) and (4) using a 50 mM isoleucine aqueous solution, the amount of 4-OH-Ile greatly increased after 96 hours. Under conditions (2) and (4), there was almost no difference in the amount of 4-OH-Ile after 96 hours. Even after 96 hours after the synthesis of 4-OH-Ile stopped, even if isoleucine was supplied, the amount of 4-OH-Ile increased in the case of hydroponics in (1) was not changed (FIG. 5). From this, it was confirmed that the same amount of 4-OH-Ile was synthesized as when isoleucine was supplied from 24 hours to 96 hours after seeding and when it was supplied from 24 hours to 168 hours. After 120 hours after sowing, there is sufficient isoleucine under condition (3), but the amount of 4-OH-Ile was almost the same as the water supply area of (1). It was confirmed that even when isoleucine was supplied when the increase in the amount stopped, 4-OH-Ile did not increase (Fig. 5). FIG. 6 shows the sprout length (hypocotyl + root), and FIG. From the results of FIG. 6, it was found that there was a correlation between the 4-OH-Ile increase and the length of the sprout, and it was confirmed that the addition time of isoleucine could be defined by the length of the sprout. That is, 50 mM isoleucine may be supplied when the length of the sprout is 1 cm to 6 cm. On the other hand, there was no correlation between fresh weight and 4-OH-Ile.

It is a photograph of an apparatus dedicated to sprout cultivation. It is a figure which shows the relationship between isoleucine aqueous solution density | concentration, the amount of 4-OH-Ile in a sprout, and the amount of isoleucine (Ile). It is a figure which shows the relationship between the addition time of isoleucine aqueous solution, and the amount of 4-OH-Ile in a sprout. It is a figure which shows the relationship between the addition time of isoleucine aqueous solution, and the time-dependent change of the 4-OH-Ile amount in a sprout. It is a figure which shows the relationship between the addition time of isoleucine aqueous solution, and the time-dependent change of the amount of 4-OH-Ile in a sprout. It is a figure which shows the relationship between the addition time of isoleucine aqueous solution, and the time-dependent change of the 4-OH-Ile amount in a sprout. It is a figure which shows the time-dependent change of the length of a sprout. It is a figure which shows the time-dependent change of the raw weight of a sprout.

Claims (7)

  A method of producing fenugreek sprout enriched with 4-hydroxyisoleucine (4-OH-Ile) by hydroponics from fenugreek seed, wherein isoleucine is supplied in the hydroponics.   The supply of isoleucine is performed for a period of 72 hours or more, including a period of 48 hours centering on the point at which the rate of increase of 4-OH-Ile amount per unit dry weight of the growing sprout is maximized in the hydroponics. The method according to claim 1, wherein:   The method according to claim 1, wherein the supply of isoleucine is performed at least during a period from the time when the total length of the hypocotyl part and the root part of the sprout is 1 cm to 6 cm in the hydroponics.   The method according to claim 1, wherein the supply of isoleucine is performed for a period from a time point 24 hours after the start of hydroponics to a time point 96 hours later.   The method according to claim 1, wherein isoleucine is supplied in the form of an aqueous solution.   6. The method according to claim 5, wherein the concentration of the isoleucine aqueous solution is 10 to 100 mM.   A fenugreek sprout produced by the method according to any one of claims 1 to 6, wherein the 4-OH-Ile content is 40 to 70 mg / g (dry weight basis).

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