Influence of visible light wavelengths on bioactive compounds and GABA contents in barley sprouts

Barley seeds (H. vulgare L.) were purchased from a certified local agricultural distributor in Chiang Mai, Thailand. Analytical grade ethanol (70 %), γ-aminobutyric acid (GABA) standard, 9-fluorenylmethyl chloroformate (FMOC-Cl), acetonitrile (HPLC grade), trifluoroacetic acid (TFA), sodium acetate, ferric chloride (FeCl₃), Folin–Ciocalteu reagent, DPPH (2,2-diphenyl-1-picrylhydrazyl), and gallic acid were all obtained from Merck (Darmstadt, Germany). All solutions were freshly prepared.

A custom-designed LED germination chamber was constructed using aluminum panels (80 × 60 cm, thickness 5 mm) fitted with 30 LED bulbs (3 W each; total 90 W per panel). Two panels were installed per treatment condition, providing a total light output of 180 W. Light sources included red light (620 nm), blue light (450 nm), white light (400–700 nm spectrum) and combined red and blue light (equal number of red and blue LEDs).

Barley seeds were placed in trays lined with moistened filter paper and maintained at 25 ± 1 °C with relative humidity 70–80 %. Germination occurred in complete darkness for the first 3 days (steeping), followed by light exposure for 5 days under the respective LED treatments. This dark–light sequencing strategy has been described in LED-based seedling studies, where an initial dark phase is commonly applied to synchronize germination prior to light-induced metabolic activation [14]. The distance between the LEDs and seed surface was fixed at 20 cm. The experimental setup is shown in Fig. S1.

The Photosynthetic Photon Flux Density (PPFD) at seedling surface was directly measured using a calibrated PAR meter (model PM-01, Apogee Instruments Inc., USA) and found to be at 270 ± 18 μmol m⁻² s⁻¹. This value falls within the commonly accepted range for stimulating photomorphogenesis and metabolite accumulation in sprouting plants [15].

After light treatment began (Day 0 = start of light exposure), root elongation was monitored daily for 5 days using a vernier caliper (accuracy ±0.01 mm). Ten randomly selected seedlings per treatment were measured each day, and the average length was recorded.

Reducing sugars were quantified by the 3,5-dinitrosalicylic acid (DNS) method, modified from Wang et al. (2021) [10]. Germinated barley samples (1 g) were homogenized in 10 mL distilled water and centrifuged at 4,000 rpm for 10 min. The supernatant (1 mL) was mixed with 1 mL DNS reagent and incubated at 100 °C for 5 min. Absorbance was measured at 540 nm, and glucose was used to construct the calibration curve.

Total phenolic content (TPC) was determined using the Folin–Ciocalteu colorimetric method [12]. Barley powder (0.5 g) was extracted with 10 mL of 70 % ethanol for 1 h at room temperature. After centrifugation, 200 µL of the supernatant was mixed with 1.0 mL of Folin–Ciocalteu reagent (diluted 1:10) and incubated for 5 min. Then, 0.8 mL of 7.5 % sodium carbonate was added, and the mixture was kept in the dark for 30 min. Absorbance was measured at 765 nm. Results were expressed as milligrams of gallic acid equivalents per gram of sample dry weight (mg GAE/g DW).

The phenolic acid profile of germinated barley was determined using high-performance liquid chromatography (Agilent 1,260 Infinity, Agilent Technologies, USA), following Chen et al. (2021) [16] with minor modifications. The sample (0.5 g) was extracted with 10 mL of 70 % ethanol using sonication for 30 min at room temperature. The extract was centrifuged at 4,000 rpm for 10 min, and the supernatant was filtered through a 0.45 µm membrane before injection.

The analysis was performed using an HPLC system equipped with a Poroshell 120 EC-C18 column (4.6 × 150 mm, 4 µm) and a diode array detector (DAD). The mobile phases consisted of (A) water with 0.1 % formic acid (v/v) and (B) acetonitrile with 0.1 % formic acid (v/v), delivered at a flow rate of 0.8 mL/min. Gradient elution was applied as follows: 0 min, 5 % B; 20 min, 20 % B; 35 min, 30 % B; 37 min, 70 % B; 42–45 min, re-equilibration at 5 % B.

Detection wavelengths were set at 280 nm and 320 nm, targeting p-coumaric and ferulic acid, respectively [17], 18]. Quantification was performed using external calibration curves constructed with known concentrations of ferulic acid and p-coumaric acid standards. The results were expressed as mg per gram of dry weight (mg/g DW).

GABA was extracted following a modified method of Khwanchai et al. (2014) [12]. Germinated barley (0.5 g) was homogenized in 5 mL of 70 % ethanol, then filtered through a 0.45 µm membrane. The filtrate was derivatized with FMOC-Cl and analyzed using an HPLC-MS system equipped with a C18 reversed-phase column. The mobile phase consisted of acetonitrile and 0.1 % TFA in water (gradient elution). Detection was performed in positive ESI mode with multiple reaction monitoring (MRM). GABA content was expressed as mg/g dry weight. Chromatographic profiles are shown in Fig. S2.

All experiments were conducted in triplicate (n = 3), and data were expressed as mean ± standard deviation (SD). A full-factorial design was employed to assess the effect of light treatment and germination day. One-way analysis of variance (ANOVA) was performed using SPSS version 26.0 (IBM, USA), followed by Duncan’s multiple range test for pairwise comparison. Statistical significance was considered at p < 0.05.

Root development was significantly influenced by light wavelength during the germination period (Figure 1). Red light (620 nm) significantly promoted early root elongation compared to other treatments (p < 0.05), particularly compared to blue light, with maximum growth observed by Day 2. This observation aligns with current understanding that light quality, particularly red or far-red wavelengths, plays a crucial role in elevating endogenous gibberellin (GA) levels, which drive cell elongation in early seedling growth. For instance, Li et al. (2019) demonstrated that far-red light promotes shoot elongation via GA signaling pathways in conifer seedlings, suggesting a conserved hormonal response to specific light cues across plant species [21].

Figure 1:

Effects of different light wavelengths on root length accumulation (cm) of germinated barley after treated by different light wavelength for 5 days. Different letter denoted by small letter indicate significant difference of root length among light treatment in the same irradiation day (p < 0.05).

In contrast, blue light (450 nm) significantly suppressed root growth, with root lengths remaining under 1.0 cm throughout the experiment. This inhibition may be attributed to enhanced abscisic acid (ABA) signaling under blue light, a hormone known to delay or inhibit root elongation by reducing cell wall extensibility [22]. The suppressive effect of blue light suggests its role in conserving energy resources during unfavorable environmental signaling.

Interestingly, the combined red-blue treatment resulted in intermediate root growth (∼1.6 cm), indicating a regulatory balance between GA promotion and ABA inhibition. White light (400–700 nm), which contains both red and blue components, produced slightly greater elongation (∼1.8 cm), likely due to its broader spectrum activating multiple photoreceptors that coordinate hormonal cross-talk during seedling development [5], 8].

These findings demonstrate that spectral composition of light can be a precise tool to modulate early root growth in barley. Red light may be preferred in applications aiming to accelerate germination and uniform sprouting, while blue light could be employed to delay elongation under specific cultivation conditions.

GABA content in germinated barley was significantly influenced by light wavelength (Figure 2). Red light treatment led to the highest accumulation and significantly different from blue and white light (p < 0.05), reaching 2.75 mg/g by day 5, substantially exceeding values reported for conventional malting methods (0.12–2.18 mg/g) [23], [24], [25], [26]. The temporal profile under red light revealed a biphasic pattern: an initial spike by day 2, followed by a brief decline and a secondary increase by day 5. This pattern reflects dynamic shifts in metabolic demands during germination.

Figure 2:

GABA content in germinated barley during the germination period under different light wavelengths, expressed in mg/kg. Different letter denoted by small letter indicate significant difference of GABA content among germinated day (p < 0.05); the uppercase letters indicate significant difference of GABA content among different light treatment (p < 0.05).

GABA is synthesized primarily via glutamate decarboxylation, catalyzed by the enzyme glutamate decarboxylase (GAD) [27]. Red light is known to activate phytochrome-mediated signaling, which can stimulate GAD gene expression either directly or via associated oxidative stress signals. Eprintsev et al. (2024) demonstrated that red light can induce ROS accumulation, which subsequently enhances GABA biosynthesis via activation of glutamate decarboxylase (GAD), suggesting a ROS-mediated regulatory pathway for GABA accumulation under red light exposure [28].

During the early germination period (day 0–2), GABA accumulation is likely driven by rapid glutamate decarboxylase activity stemming from available glutamic acid pools. This pattern is observed in germinating bean sprouts by [29]. The subsequent decline may reflect GABA consumption as a carbon and nitrogen source or its incorporation into secondary metabolic pathways [25]. The secondary increase on day 5 suggests reactivation of GAD or increased glutamate availability, possibly in response to light-induced stress during later stages of seedling development.

Blue and white light treatments also stimulated GABA accumulation but to a lesser extent. This could be due to blue-light-enhanced ABA signaling, which may counteract GAD induction or reduce glutamate pool size [30]. Combined red-blue light yielded intermediate GABA levels, possibly due to partial antagonism between red light–induced biosynthesis and blue light–mediated inhibition.

The pronounced GABA accumulation under red light highlights its utility in modulating non-protein amino acid content for functional food development. Given GABA’s established health benefits – such as anxiolytic effects, blood pressure regulation, and neurological support – this light-based approach presents a sustainable, non-chemical strategy for enhancing the nutraceutical profile of cereal sprouts.

Reducing sugar content increased progressively from day 0 to day 5 under all light treatments (Figure 3), indicating active carbohydrate metabolism during germination. However, light wavelength significantly modulated the extent of sugar accumulation (p < 0.05).

Figure 3:

Effects of different light wavelengths on reducing sugar content in germinated barley (mg/kg). Different letter denoted by small letter indicate significant difference of sugar content among germinated day in same light treatment (p < 0.05); the uppercase letters indicate significant difference of sugar content among different light treatment in same day (p < 0.05).

The initial increase in reducing sugars during Days 1–3 likely results from amylase-driven starch hydrolysis to support early germination. The decline from Day 3 to Day 4 may reflect the utilization of reducing sugars for biosynthesis of GABA and phenolic compounds [31]. The subsequent rise in reducing sugars between Days 4 and 5 could be due to renewed enzymatic degradation of starch reserves by the embryo as seedling development progresses [32].

Red light consistently induced the highest levels of reducing sugars, reaching 170.0 ± 10.0 mg/g by day 5, were significantly higher than blue (110 ± 8 mg/g) and white (125 ± 7 mg/g) light (p < 0.05). This enhancement can be attributed to red light-induced activation of phytochrome receptors, which are known to stimulate gibberellic acid (GA) synthesis [33]. It was found that activation of phytochrome B (PhyB) by red light leads to the degradation of transcription factor PIF1, which normally represses GA biosynthetic genes. This early red-light signaling thus promotes endogenous GA accumulation [14]. GA in turn upregulates hydrolytic enzymes such as α-amylase and β-amylase, promoting starch degradation into simple sugars like maltose and glucose [3], 6]. The early and sustained sugar accumulation under red light reflects this hormonal and enzymatic synergy.

In contrast, blue light resulted in significantly lower sugar levels. The suppressive effect may stem from blue-light activation of cryptochromes, which are involved in abscisic acid (ABA) signaling pathways [22]. ABA negatively regulates amylase expression and promotes dormancy-related gene expression, thus slowing starch-to-sugar conversion. As such, the blue light group exhibited a delayed metabolic activation pattern.

White and combined red-blue light produced intermediate sugar levels, reflecting the balancing effects of red-light-enhanced starch degradation and blue-light-suppressed carbohydrate mobilization. Interestingly, the red-blue combination resulted in a less pronounced sugar peak than red light alone, highlighting that blue light may partially antagonize GA-driven sugar metabolism.

These findings suggest that targeted red LED exposure during barley germination can significantly improve sugar content, which is crucial for malt quality and fermentable sugar availability in brewing and functional food formulations.

Total phenolic content (TPC) in germinated barley significantly increased during the five-day germination period, with a strong dependence on light wavelength (Figure 4). Red light (620 nm) induced the highest accumulation of phenolics, reaching 1.35 ± 0.15 mg GAE/g DW on day 5 (p < 0.05). This elevated response is consistent with the stimulatory effect of red light on the phenylpropanoid biosynthesis pathway. In particular, red light upregulates phenylalanine ammonia-lyase (PAL), a key enzyme that catalyzes the deamination of phenylalanine to cinnamic acid, an early precursor of many phenolic compounds [34], 35].

Figure 4:

Total phenolic content (TPC) of germinated barley under different light treatments, expressed as mg gallic acid equivalent per gram of dry weight (mg GAE/g DW). Different letter denoted by small letter indicate significant difference of total phenolic content among germinated day in same light treatment (p < 0.05); the uppercase letters indicate significant difference of total phenolic content among different light treatment in same day (p < 0.05).

Red light has been previously reported to increase PAL gene expression in sprouts and medicinal plants, correlating with elevated flavonoid and phenolic acid content [34]. In barley sprouts, the observed phenolic accumulation may also be linked to photomorphogenic signaling via phytochrome activation, which modulates defense-related metabolic routes during early seedling development.

Blue light may partially enhance PAL expression through cryptochrome-mediated signaling, although its concurrent stimulation of ABA-related genes could counteract full metabolic activation [30]. Consequently, blue light alone elicited only a moderate phenolic response.

Interestingly, the red-blue combined treatment and white light condition produced significantly lower phenolic levels. This outcome may reflect an antagonistic interaction between red-light-induced biosynthesis and blue-light-associated inhibition or photoreceptor competition. White light, which covers the full visible spectrum, simultaneously activates multiple photoreceptors namely phytochromes (responsive to red light) and cryptochromes (responsive to blue light).

These photoreceptors often trigger antagonistic signaling cascades, influencing the expression of phenylpropanoid pathway genes. Studies have shown that under combined spectral activation, signaling components like HY5 and PIFs can mediate opposing regulation of downstream pathways [36]. This complex photoreceptor cross-talk under white light likely dilutes the specific induction of PAL and phenolic synthesis, accounting for the attenuated response observed.

The enhanced TPC under red light suggests its application in producing barley sprouts with higher antioxidant potential, given that phenolic compounds are major contributors to free radical scavenging and oxidative stress mitigation [37].

The levels of individual phenolic acids, specifically ferulic acid and p-coumaric acid, were significantly influenced by the light treatments during barley germination (Figure 5). Ferulic acid and p-coumaric acid are the two predominant hydroxycinnamic acids typically found in barley [38]. These compounds are major representatives of the phenylpropanoid pathway and are closely associated with antioxidant activity and plant defense mechanisms. Red light (620 nm) resulted in the highest accumulation of both compounds, with ferulic acid reaching 0.83 mg/g DW and p-coumaric acid reaching 0.31 mg/g DW by day 5 (p < 0.05). The concentrations we observed (∼0.02–0.08 mg/g DW) may be approaching the detection limits of the HPLC-DAD method. Supporting this, Silva et al. (2021) reported LOD values of 0.08–0.83 mg/L and LOQ of 0.27–2.78 mg/L for phenolic compounds using a comparable HPLC-DAD setup [39]. These findings are consistent with the elevated total phenolic content (TPC) observed under red light (Figure 4), suggesting that red light not only increases phenolic quantity but also promotes the biosynthesis of specific hydroxycinnamic acids through the phenylpropanoid pathway. It is known that red light influences the phenylpropanoid biosynthetic pathway by activating specific photoreceptors (e.g., phytochromes), which in turn regulate the expression of key genes such as PAL (phenylalanine ammonia-lyase), C4H (cinnamate-4-hydroxylase), and 4-CL (4-coumarate-CoA ligase). These enzymes control the metabolic flux through the phenylpropanoid pathway, ultimately increasing the accumulation of hydroxycinnamic acids like ferulic acid and p-coumaric acid. This mechanism is detailed in recent studies [40].

Figure 5:

The amount of ferulic acid (A) and p-coumaric acid (B) of germinated barley under different light treatments, expressed as mg per gram of dry weight (mg/g DW). Different letter denoted by small letter indicate significant difference of total phenolic content among germinated day in same light treatment (p < 0.05); the uppercase letters indicate significant difference of total phenolic content among different light treatment in same day (p < 0.05).

Both ferulic and p-coumaric acids are synthesized downstream of phenylalanine ammonia-lyase (PAL) activity, with p-coumaric acid serving as a precursor to ferulic acid. Red light is known to stimulate PAL expression via phytochrome-mediated signaling, thus enhancing the metabolic flux toward these compounds [34]. In contrast, blue light and the red-blue combination yielded moderate levels of both phenolic acids, while white light produced the lowest concentrations. The relatively lower content under white light may reflect interference from competing photoreceptor signaling pathways, resulting in weaker PAL induction or metabolic diversion.

Notably, the proportion of these two phenolic acids relative to TPC was substantial, accounting for approximately 85 % of the total phenolic content under red light conditions. This high contribution supports the hypothesis that hydroxycinnamic acids are the major phenolics in germinated barley and play a key role in its antioxidant capacity. Ferulic acid, in particular, has strong electron-donating properties and contributes significantly to both DPPH and FRAP assay results [41]. While hydroxycinnamic acids (e.g., ferulic and p-coumaric acid) are predominant in germinated barley, it is standard practice to express total phenolic content (TPC) in gallic acid equivalents (GAE) when using the Folin–Ciocalteu assay. This approach ensures comparability across studies and is widely adopted [42], [43], [44].

The enhancement of ferulic and p-coumaric acid levels under red light provides further evidence that targeted spectral treatments can modulate specific branches of phenolic biosynthesis. These compounds are known for their antioxidant, anti-inflammatory, and neuroprotective activities, supporting their value as functional ingredients in health-oriented food products [45], [46], [47].

Antioxidant capacity of germinated barley, as determined by DPPH radical scavenging assay, was significantly influenced by the wavelength of light exposure during germination (Figure 6A). Red light treatment yielded the highest antioxidant activity on day 5, reaching 40.40 mg/g, followed by blue light, while red-blue and white light treatments showed significantly lower activity (p < 0.05).

Figure 6:

DPPH scavenging activity (A) and ferric reducing antioxidant power (FRAP) values (B) of germinated barley under different light wavelengths, expressed in mg/g. Different letter denoted by small letter indicate significant difference of DPPH scavenging activity among germinated day in same light treatment (p < 0.05); the uppercase letters indicate significant difference of DPPH scavenging activity among different light treatment in same day (p < 0.05).

The elevated DPPH scavenging capacity under red light closely correlates with the higher total phenolic content observed in the same group (Figure 4). Phenolic compounds are primary antioxidants that donate hydrogen atoms or electrons to neutralize DPPH free radicals, thereby reducing oxidative stress [34], 37]. As such, the DPPH assay is a direct reflection of the phenolic antioxidant potential stimulated by red light.

Blue light also showed a moderate increase in antioxidant activity, consistent with its partial enhancement of phenolic biosynthesis. However, the lower efficiency compared to red light suggests that the phenolic profile under blue light may be qualitatively different (e.g., lower levels of highly active hydroxylated flavonoids), or that blue light limits phenolic transport or localization within seedling tissues [41].

White light produced the lowest antioxidant response, while red-blue combined light showed moderate levels that lower than red light but higher than blue and white light treatments. This could stem from antagonistic photoreceptor activation leading to attenuated phenolic accumulation, as previously noted in TPC results. Alternatively, broader-spectrum white light may induce oxidative stress response pathways without sufficient induction of phenolic biosynthesis, leading to a mismatch between ROS generation and antioxidant defense.

The antioxidant capacity measured by the FRAP assay showed a similar trend to the DPPH results but with lower absolute values (Figure 6B). Red light treatment produced the highest FRAP activity, followed by blue light, while white and red-blue light treatments yielded lower reducing power. The differences among treatments were statistically significant (p < 0.05).

While both DPPH and FRAP assays assess antioxidant capacity, they operate through different mechanisms. The DPPH assay measures the ability of antioxidants to donate hydrogen atoms or electrons to neutralize free radicals, primarily reflecting radical scavenging activity. In contrast, the FRAP assay evaluates the capacity of antioxidants to reduce ferric ions (Fe³⁺ to Fe²⁺), a mechanism more sensitive to the presence of reducing agents such as polyphenols, flavonoids, and certain vitamins [35].

The lower FRAP values observed in this study may be due to the specific phenolic composition induced under each light treatment. Red light likely promoted the synthesis of phenolic compounds with strong redox potential, such as hydroxycinnamic acids, which are effective at reducing metal ions. Conversely, blue and broad-spectrum light may have induced phenolics with weaker electron-donating capacity or more complex structures less reactive toward ferric ions [41].

The alignment of FRAP and DPPH results under red light reinforces the role of red light in enhancing both the quantity and quality of antioxidant compounds during germination. However, the discrepancy in sensitivity between assays also suggests that DPPH may detect a broader range of antioxidants, while FRAP is more specific to strong reducing agents.

These findings underscore the importance of selecting appropriate light wavelengths to modulate not only total phenolic levels but also their functional redox behavior, which is crucial for oxidative stability in food applications and potential health benefits.

A comparative analysis between red LED light treatment and conventional malting methods is presented in Table 1. Notably, red light treated barley exhibited superior GABA accumulation (2.75 mg GAE/g), outperforming values reported in traditional germination processes (range: 0.12–2.18 mg GAE/g) [23], [24], [25], [26]. This substantial enhancement underscores the efficacy of spectral modulation in stimulating GABA biosynthesis through non-chemical, abiotic means.

While the total phenolic content (TPC) under red light (1.35 ± 0.15 mg GAE/g) fell within the lower to mid-range of conventional values (0.2–3.83 mg GAE/g), it was accompanied by a proportionally high antioxidant response, particularly in DPPH activity. This suggests that the quality of phenolics rather than quantity alone, may be optimized by red light, potentially yielding higher radical scavenging efficiency per unit of phenolic content.

In terms of reducing sugar content, red light treatment produced slightly higher values (170.0 ± 10.0 mg GAE/g) than conventional malting under normal light (158.75 mg GAE/g). This improvement is consistent with enhanced α-amylase activation and starch degradation under phytochrome stimulation [48].

Collectively, these results demonstrate that targeted red LED light can match or surpass conventional germination methods in improving key nutritional and functional parameters of barley sprouts. The simplicity, precision, and energy efficiency of LED setups further strengthen their applicability in scalable functional food production.