We conducted experiments to establish the growing conditions of hydroponic young green barley plants in which functional ingredients were applied and absorbed during the cultivation period. No studies have conducted experiments on functional ingredients applied during the cultivation of young green barley. In this study, glucosamine (GlcN) and collagen (Cgn) were used, both of which are widely known as functional ingredients and are in high market demand. In the GlcN application experiment, young green barley plants were cultivated with only liquid fertilizer during the early growing period for 9 days, and then the plants were cultivated in 0.25% GlcN-water solution for the following 3 days. The plants used in this experiment absorbed 0.60% of GlcN. Furthermore, an experiment was conducted to test plant absorption of collagen. Collagen absorption was evaluated using hydroxyproline (Hyp) as an indicator ingredient. Under control conditions, the Hyp content was 0.04% after 14 days of cultivation. Meanwhile, in the application experiment of Cgn derived from pigs (average molecular weight is 3000), plants were cultivated in 1% Cgn-water solution for 14 days. As a result, the Hyp content increased to 0.28%. Thus, this study clarifies, for the first time, that barley plants can absorb exogenous functional ingredients applied from the outside. The nutrient component contents in young green barley plants were measured. The amino acid and vitamin C content in hydroponic young green barley significantly increased, as compared to those grown in organic soil. Furthermore, in 0.1% Cgn-water solution cultivation, glutamic acid and aspartic acid, both of which are a functional amino acids, increased approximately three folds as compared to the plant grown in organic soil under natural sunlight. Furthermore, K and Na, which are inorganic ingredients in young green barley, were measured. The K content in the plants grown in 1% Cgn-water solution cultivation for 14 days decreased by 80% as compared with the control hydroponic plant grown with liquid fertilizer. It was inferred that the plant might be used as food for dialysis patients.
In recent years, there has been a growing demand for highly functional and enriched vegetables from general consumers, which are used to prevent lifestyle-related diseases that are associated with an aging society. The majority of studies on the production of useful materials that originate in organisms, but are later incorporated into plants via genetic modification, have focused on the research and development of medicines. For instance, studies have targeted medicines linked to vaccine generation and antibody production, but it has been quite difficult to produce such pharmaceutical products [
Meanwhile, regarding the cultivation of general vegetables and flowers, cost reduction strategies have been developed through the innovation of light-emitting diode (LED) light sources, the establishment of cultivation systems using information technology (IT), and an increase in established plant factories. With such developments in cultivation technology, the research and development of vegetables with functional ingredients is advancing, and an increasing number of studies have reported the enhancement of functional ingredients possessed by plants as well as the addition of new functional ingredients to plants.
Furthermore, in cereal grains, it was reported that sprouting leads to an increase in the content of plant ingredients and enhances the associated functions [
Meanwhile, research suggests a method of enriching the content of functional ingredients in vegetables through the application of exogenous materials, not by enhancing the content that the vegetables initially pos- sessed. For example, there are reports about the application of ascorbic acid to butterhead lettuce [
The health food industry in Japan, reflecting its aging society, has mainly introduced products with added ingredients such as collagen, glucosamine, and hyaluronic acid, which are all said to help improve age-related diseases. However, it is necessary to be mindful of the allergic responses to added ingredients, as many are high in animal protein. Therefore, this study attempted to produce hydroponic young green barley plants with high functional ingredient content, in which allergenic substances were avoided via the application of glucosamine and collagen during the growth of the plants.
In 2002, research and development of the cultivation of vegetables with low potassium (K) content, designed for dialysis patients, was initiated, and some studies have reported cultivation methods for low K spinach [
Two-rowed barley (Hordeum vulugare f. distichon) Nishinohoshi was harvested in 2014. Seeds of barley were surface-disinfected for 1 min with hypochlorous acid [(HClO), approximately 0.5% of active chlorine] and then rinsed several times with sterilized water.
Surface-disinfected seeds were sown in mesh tray that is set into the hydroponics container (L 25. 5 cm × W 12. 5 cm × H 7 cm, YAMATO PLASTIC, Japan) containing 900 mL of nutrient solution (
Seed were sown in planters (41 cm × 20 cm, soil area 820 cm2) containing organic fertilizers (SHOEI, Miyazaki, Japan), grown in two different biotron at temperature of 15˚C and 20˚C, 70% relative humidity.
Zero point onegram of sample was homogenized in 10 mL of 6 mol/L hydrochloric acid, and the mixture was allowed to stand for 16 h at 100˚C. Evaporation of liquid mixture, obtain dry products, it dissolves in water and neutralization, and transferred into a 20 mL volumetric flask, adding water to volume than filtered. The supernatants were analyzed using the DX 500 HPLC System (Nippon Dionex K. K) equipped with a Dionex CarboPac PA1 (4.0 mm × 250 mm) maintained at 32˚C. The mobile phase consisted of 0.05 mol/L sodium hydroxide solution at a flow rate of 1.0 mL/min.
As an oxidative reaction, one gram of sample was mixed with 20 mL of 20% hydrochloric acid (content in 0.04% 2-mercaptoethanol), and the mixture was allowed to stand for 24 h at 100˚C. The mixture solution, bring the final volume up to 100 mL by adding water. Than take 10 mL of solution (pH 2.2) dilute to 10 mL with sodium citrate buffer (pH 2.2) as a test sample. The supernatants were analyzed using JLC-500/V AminoTac™ Amino Acid Analyzer (JEOL Ltd., Japan) equipped with a LCR-6 (4.0 mm × 120 mm) column. The mobile phase was sodium citrate buffer (H-01-H-04, JEOL Ltd., Japan) at a flow rate of 0. 42 mL/min and the reaction solution (JEOL for ninhydrin coloring solution kit-II, Wako Pure Chemical Industries, Ltd., Japan) at a flow rate of 0.22 mL/min.
Amino acid (except for tryptophan) in the young green barley were determined by automatic amino acid analyzer JLC-500/V (JEOL Ltd., Japan), and tryptophan in the young green barley was measured by LC-20AD liquid chromatography unit (Shimadzu Ltd., Japan).
Determination of vitamin C: Two to six grams of young green barley was homogenized in 50 mL of 5% metaphosphoric acid and centrifuged. Onemillilitre of extract was mixed with 1 mL of 5% metaphosphoric acid, 200 μL of 0.2% 2,6-dichlorophenol-indophenol and 2 mL of 2% thiourea-5% metaphosphoric solution. As an oxidative reaction 0.5 mL of 2% 2,4-dinitrophenyl-hydrazine 4.5 mol/L sulfuric acid solution was added, and the mixture was allowed to stand for 16 hours at 38˚C - 42˚C. Then, 3 mL of ethyl acetate was added, and the mixture was shaken for about 60 min. The supernatants were analyzed using the LC-20AT (Shimadzu Co., Ltd.) system equipped with a Senshupak Silica-1100-N column (Senshu Scientific Co., Ltd.) maintained at 35˚C. Extract was eluted in the mobile phase (ethyl acetate/hexane/acetic acid/water = 60/40/5/0.05) at a flow rate of 1.5 mL/min. The absorbance of the eluent was measured at 495 nm.
Determination of vitamin E: Young green barley was extracted using 0.3 g pyrogallol, 2 mL of 10 g/L sodium chloride solution, and 10 mL of ethanol (approximately 1 - 2.5 g of sample/12 mL of solvent) and 2 mL of 600 g/L potassium hydroxide. The mixture was boiled and then refluxed for 30 min at 70˚C. Next, the mixture was extracted in 20 mL of 10 g/L sodium chloride solution, then 14 mL of hexane/ isopropanol/ethyl acetate (9/1.5/1) was added, and the mixture was centrifuged (5 min, 1500 rpm) three times and filtrated through a 0.45-μM PTFE membrane syringe filter. The liquid chromatography LC-20AT system, equipped with an RF-10A fluorescence detector (Shimadzu Co., Ltd., Japan) was used for analysis. The peak was detected using an excitation wavelength of 298 nm and an emission wavelength of 325 nm. The mobile phase was hexane/isopropanol/acetic acid (1000/6/5 with butylated hydroxytoluene), and the flow rate was 1.5 mL/min.
In a hydroponic experiment using water and liquid fertilizer, we conducted a growth test in GlcN-water solution with either 0.25% or 0.50% GlcN content, and the plants grown in 0.50% GlcN-water solution withered and died. Plant growth in 0.25% GlcN-water solution was largely inhibited as compared to that of the control plant that was exposed to liquid fertilizer, and the height and weight of the plants was inhibited to 72% and 57% on day 10 following germination, respectively (
GlcN was not detected in the control plants grown only in liquid fertilizer for 12 days. The GlcN content was 0.60% in the young green barley plants grown only in liquid fertilizer for 9 days and then in 0.25% GlcN-water solution for the following 3 days. However, the content was 0.20% in the plants grown in liquid fertilizer for 9 days and then in 0.25% GlcN liquid fertilizer solution for the following 3 days. It became clear that GlcN accumulated in the stems and leaves of young green barley without inhibiting plant growth under conditions where
Culture solution | 9 days after germination | 10 days after germination | 12 days after germination | 14 days after germination | ||||
---|---|---|---|---|---|---|---|---|
Height (cm) | Fresh weight (g) | Height (cm) | Fresh weight (g) | Height (cm) | Fresh weight (g) | Height (cm) | Fresh weight (g) | |
S1 | 49.4 ± 3.0 | 0.44 ± 0.05 | ||||||
S2 | 35.5 ± 1.6 | 0.25 ± 0.02 | ||||||
S3 | 18.9 ± 1.7 | 0.13 ± 0.02 | ||||||
S1 | 43.6 ± 1.6 | 0.35 ± 0.03 | ||||||
6th-S2 | 38.1 ± 1.9 | 0.26 ± 0.03 | ||||||
6th-S4 | 38.4 ± 2.1 | 0.28 ± 0.02 | ||||||
S1 | 51.8 ± 4.8 | 0.41 ± 0.04 | ||||||
9th-S2 | 51.5 ± 5.0 | 0.40 ± 0.04 | ||||||
9th-S4 | 52.9 ± 5.2 | 0.40 ± 0.04 | ||||||
S1 | 32.7 ± 1.4 | 0.29 ± 0.03 | ||||||
S5 | 23.8 ± 1.4 | 0.15 ± 0.02 | ||||||
S6 | 27.6 ± 2.0 | 0.17 ± 0.02 | ||||||
4th-S6 | 30.3 ± 2.0 | 0.23 ± 0.03 | ||||||
4th-S7 | 30.8 ± 1.5 | 0.25 ± 0.02 | ||||||
S1 | 50.7 ± 3.9 | 0.39 ± 0.05 | ||||||
S5 | 29.9 ± 1.2 | 0.19 ± 0.02 | ||||||
S6 | 35.7 ± 1.2 | 0.22 ± 0.01 | ||||||
9th-S6 | 49.1 ± 4.3 | 0.36 ± 0.03 | ||||||
9th-S7 | 49.2 ± 3.2 | 0.36 ± 0.02 |
S1: Liquid fertilizer; S2: 0.25% GlcN-liquid fertilizer; S3: 0.50% GlcN-liquid fertilizer; S4: 0.25% GlcN-water solution; S5: Tap water; S6: 1% Cgn-water solution; S7: 1% Cgn-liquid fertilizer.
plants were cultivated only in liquid fertilizer for 9 days, and then for 3 days in the GlcN added solution.
In hydroponic cultivation with water and liquid fertilizer, a 9-day cultivation test was conducted in 1% Cgn content solution. We cultivated young green barley plants under the following five conditions: (1) control with only liquid fertilizer, (2) control with only water, (3) 1% Cgn content water solution, (4) only liquid fertilizer for 4 days and then 1% Cgn content water solution for the following 5 days, (5) only liquid fertilizer for 4 days and then in 1% Cgn content liquid fertilizer solution for the following 5 days (
The plant height and weight increase of young green barley in the 9-day cultivation test are indicated as follows: (1) > (5) > (4) > (3) > (2). Furthermore, collagen addition during the latter 5 days induced slight growth inhibition as compared to the plant growth in (1) control with only liquid fertilizer, with 86% and 79% in the fresh weight of (5) and (4), respectively (
As an index of the Cgn content, the hydroxyproline (Hyp) amount was measured [
We discovered that exogenous functional ingredients could be added during the cultivation period of young
Culture solution | Hydroxyproline contents (mg/100g dry weight) | |
---|---|---|
9 days after germination | 14 days after germination | |
S1 | 47 | 39 |
S5 | 53 | - |
S6 | 165 | - |
S6 | - | 284 |
4th-S6 | 120 | - |
4th-S6 | - | 202 |
9th-S6 | - | 98 |
4th-S7 | 94 | - |
9th-S7 | - | 100 |
S1: Liquid fertilizer; S5: Tap water; S6: 1% Cgn-water solution; S7: 1% Cgn-liquid fertilizer.
green barley; however, it is not meaningful if that addition is accompanied by a large decrease in the basic nutrient components of the plant. Therefore, an organic soil cultivation test was conducted in planters set inside a biotron, and the results were compared to the analyzed amino acid content, vitamin C, and vitamin E data from the previous hydroponic cultivation tests. In hydroponic culture, we analyzed the nutrient components of young green barley plants grown only in liquid fertilizer for 14 days and those of plants cultivated only in liquid fertilizer for 4 days and then in 1% Cgn-water solution for more 10 days. The total amino acid content of plants grown under these conditions was 1.46 times larger than those grown under control conditions (
Amino acid contents (g/100g dry weight) | ||||
---|---|---|---|---|
Organic soil culture | Hydroponic culture | |||
Natural sun light | Artificial sun light | 1% collagen water solution | Liquid fertilizer | |
Arginine | 1.40 | 0.89 | 1.61 | 1.35 |
Lysine | 1.54 | 1.04 | 1.71 | 1.53 |
Histidine | 0.54 | 0.39 | 0.61 | 0.55 |
Phenylalanine | 1.28 | 0.90 | 1.29 | 1.24 |
Tyrosine | 0.91 | 0.61 | 0.91 | 0.83 |
Leucine | 1.98 | 1.38 | 2.02 | 1.93 |
Isoleucine | 1.01 | 0.69 | 1.06 | 1.00 |
Methionine | 0.49 | 0.31 | 0.52 | 0.47 |
Valine | 1.39 | 0.95 | 1.57 | 1.37 |
Alanine | 1.64 | 1.10 | 2.01 | 1.71 |
Glycine | 1.32 | 0.93 | 1.50 | 1.39 |
Proline | 1.17 | 0.81 | 1.18 | 1.12 |
Glutamic acid | 2.62 | 1.66 | 6.99 | 2.93 |
Serine | 1.10 | 0.73 | 1.39 | 1.05 |
Threonine | 1.16 | 0.79 | 1.27 | 1.12 |
Aspartic acid | 2.27 | 1.55 | 7.30 | 2.80 |
Tryptophan | 0.58 | 0.36 | 0.51 | 0.48 |
Cystine | 0.32 | 0.20 | 0.40 | 0.32 |
Total amino acid | 22.72 | 15.29 | 33.85 | 23.19 |
Vitamin contents (mg/100g dry weight) | ||||
---|---|---|---|---|
Organic soil culture | Hydroponic culture | |||
Natural sun light | Artificial sun light | 1% collagen water solution | Liquid fertilizer | |
Total ascorbic acid | 367.5 | 262.5 | 450.0 | 434.0 |
α-tocopherols | 12.8 | 27.5 | 20.2 | 12.1 |
γ-tocopherols | 3.4 | 5.0 | 4.6 | 3.0 |
β and δ-tocopherols were not detedted.
The K and Na content in young green barley plants grown for 9 days in only liquid fertilizer or only in water are shown in
The component profiling of young green barley has already been developed, and various studies have been conducted on the ingredients [
In the present study, we examined the application and plant absorption of glucosamine (GlcN) and collagen (Cgn), both of which are functional ingredients contained in young green barley plants in small amounts. An experiment was initially conducted on the plant absorption of glucosamine (GlcN), which is a low-molecular- weight functional ingredient. In the 13-day cultivation experiment with a liquid fertilizer control, no peak of the GlcNcontent was observed; thus, it was understood that GlcN was not synthesized in vivo. Next, when young green barley plants were grown only in liquid fertilizer for 9 days and then in 0.25% GlcN-liquid fertilizer medium for the following 3 days, it was observed that 0.2 g/100g of GlcN dry weight was absorbed into plants. Meanwhile, when grown in 0.25% GlcN-water medium for the latter 3 days, it was found that 0.6 g/100g of GlcNdry weight was absorbed into plants. The present study is the first research reporting the absorption of exogenous functional ingredients from young green barley roots.
Next, we examined the plant absorption of Cgn, a functional ingredient that is much higher in molecular weight than GlcN. Inoue et al. reported in their study on lettuce that only those exogenous functional ingredients that were less than 1000 in molecular weight could be absorbed through the roots [
Culture solution | K contents (mg/100g dry weight) | Na contents (mg/100g dry weight) | |
---|---|---|---|
9 days after germination | 14 days after germination | ||
S1 | 5940 | - | 111 |
S5 | 1690 | - | 945 |
S6 | 1230 | 1220 | - |
4th-S6 | 3820 | 2400 | - |
S1: Liquid fertilizer; S5: Tap water; S6: 1% Cgn-water solution.
to be close to the composition of human Cgn. Recent studies have shown that not all mammalian Cgn is broken down and absorbed as amino acids, but are instead absorbed and exist in the blood as dimers and oligomers [
The Hyp content was 109 mg/100g on average in the 5-day cultivation in 1% Cgn-water solution, 165 mg/100g in the 9-day cultivation, and 284 mg/100g in the 14-day cultivation (
It is understood that Cgn in mammals is absorbed and exists in the form of dimers such as Pro-Hyp and Hyp- Gly, or in the form of oligopeptides such as Pro-Hyp-Glyn and Pro-Hyp-Hyp-Gly [
As young green barley is utilized widely as a functional ingredient material, it is not meaningful if nutrition components that originally exist in the plants decrease, even though the exogenous functional ingredients GlcN and Cgn are absorbed by plants. Therefore, we conducted an experiment to grow young green barley in organic soil planters set inside a biotron, as an original growing model of young green barley, and we subsequently compared the amino acid, vitamin C, and vitamin E contents of the plants grown in the biotron to those grown in 1% Cgn nutrient medium conditions (
Furthermore, the total amino acid content in plants grown in the 1% Cgn solution was approximately 45% higher than that of plants grown in LF and NSL. Glutamic acid (Glu) and aspartic acid (Asp), in particular, weighed approximately 2.5 times more in comparison with controls grown in liquid fertilizer. This particular increase in the Glu and Asp contents did not reflect the amino acid composition of pig Cgnthat was originally applied. Thus, it was suggested that this unique increase in the two amino acids (Glu and Asp) might be due to an effect on the tricarboxylic acid (TCA) cycle made by C and N sources derived from Cgn. Glu and Asp have been used as functional amino acids, and this research suggests that young green barley with a new function might be produced.
As a food material for dialysis patients, there is a demand for the production of low K content vegetables. Research suggests that the K content in spinach decreased 80% by decreasing the K supply in water culture solution during the latter half of hydroponic cultivation [