Abstract Postharvest browning in lotus stolon (Nelumbo nucifera) leads to significant reductions in quality and marketability. The objective of this study was to explore the potential of ascorbic acid (ASA) treatment in mitigating browning in lotus stolon, focusing on enzymatic browning and ASA-quinones redox cycling. Lotus stolons were immersed in ASA at concentrations of 0.2%, 0.5%, 1%, and 2% for 5 min, compared to distilled water, and stored at 29 ± 2 °C and 65 ± 5% relative humidity for 48 h. The results indicated that lotus stolon exhibited rapid browning within 12 h postharvest, with the severity increasing thereafter. Application of 0.2%, 0.5%, 1%, and 2% ASA can reduce browning index by 12%, 21%, 29%, and 31%, respectively, compared to the control throughout storage. The 1% and 2% ASA treatments increased endogenous ASA level, maintained total phenolics content, reduced quinones and H₂O₂ accumulations, inhibited polyphenol oxidase and peroxidase activities, and enhanced ascorbate peroxidase activity. Additionally, the 0.5% ASA treatment enhanced ASA/dehydroascorbate redox state with increased activities of monodehydroascorbate reductase and dehydroascorbate reductase, while the 0.2% ASA treatment had only a minimal impact on physiological processes. These findings suggest that ASA treatments reduce browning in lotus stolon through concentration-dependent mechanisms.

Keywords: Postharvest browning, Lotus stolon, Oxidation of phenolics, Ascorbic acid, Redox cycling

Citation: Vichaiya, T., Laohapornchaiphan, J., and Intarasit, S. 2026. Postharvest treatment of ascorbic acid in reducing browning development of lotus stolon: Insight into ascorbic acid-quinones redox cycling. Natural and Life Sciences Communications. 25(3): e2026063.

Graphical Abstract:

INTRODUCTION

The lotus stolon is the underground young stem of Nelumbo nucifera. It is long and cylindrical, with pale yellow-white skin, crisp, juicy flesh, and a neutral taste. It is commonly used in Southeast Asian cuisine, such as in sour curries and stir-fries, or as an ingredient to enhance nutritional value and medicinal properties. However, fresh lotus stolon has a limited shelf life of only a few days post-harvest, as the epidermis rapidly discolors and turns brown, which negatively impacts their visual quality and marketability (Min et al., 2017). Consequently, it is essential to develop techniques to prevent browning in lotus stolon after harvest.

Browning in fresh produce after harvest is a biological process involving several mechanisms associated with the oxidation of phenolic compounds. This process is primarily driven by enzymatic activities, particularly those of polyphenol oxidase (PPO) and guaiacol peroxidase (POD), which play pivotal roles (Furumo and Furutani, 2008). These enzymes catalyze the oxidation of phenolic substrates to form reactive quinones, which upon further biochemical transformations, lead to the polymerization and subsequent formation of complex brown polyphenolic compounds (Roginsky et al., 1999). One of the key factors contributing to this process is hydrogen peroxide (H₂O₂), a major molecule of reactive oxygen species (ROS) that not only plays a crucial role in plant responses to postharvest stress but also contributes to the initiation of enzymatic browning by facilitating the oxidation of phenolic substrates (Chomkitichai and Intarasit, 2024; Guan et al., 2024).

Ascorbic acid (ASA), widely known as vitamin C, is a pivotal antioxidant in plant cells. It plays a vital role in physiological defense by mitigating oxidative damage through the enzymatic pathways, such as those mediated by ascorbate peroxidase (APX) (Smirnoff, 2000). Additionally, ASA facilitates the conversion of quinones back to phenols, which leads to the formation of dehydroascorbate (DHA), helping to retard the browning process (Roginsky et al., 1999). This aligns with previous research indicating that plants rich in ASA exhibit a lower incidence of browning, as observed in lettuce leaves (Degl’Innocenti et al., 2005) and fresh cut potato (Tang et al., 2023). Moreover, plant cells can restore DHA to ASA through the activities of dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR). Previous study indicates that upregulation of DHAR and MDHAR activities to enhance the redox potential of DHA to ASA, which is positively correlated with the prevention of browning, has been reported in postharvest longan fruit (Chumyama et al., 2017).

However, the concentration of ASA in several crops is limited and may be insufficient to effectively reduce quinones, which are responsible for the browning characteristics (Degl’Innocenti et al., 2005; Tang et al., 2023). Therefore, the application of exogenous ASA treatment has been shown to effectively reduce postharvest browning in various crops. For example, immersion in 1% ASA for 5 min in fresh-cut apples (Özdemir and Gökmen, 2019), washing with 0.35% ASA for 2 h in mung bean sprouts (Sikora and Świeca, 2020), and dipping in 2% ASA for 2 min in fresh-cut eggplant slices (Sarengaowa et al., 2022) resulted in significant reductions in browning. Therefore, determining the optimal concentration of ASA for each species is crucial for effectively reducing browning. Although treatment with 3% ASA for 2 min has been reported to effectively inhibit postharvest browning in fresh-cut lotus roots (Gao et al., 2017), its efficacy in mitigating browning in lotus stolon remains unexplored. This knowledge gap motivated the present study.

This study aims to (1) investigate the optimal conditions for applying ASA to reduce browning in postharvest lotus stolon under ambient conditions, and (2) elucidate the physiological mechanisms through which exogenous ASA treatment affects browning development, with a particular focus on the modulation of enzymatic activity related to ASA-quinones redox cycling.

MATERIALS AND METHODS

Preparation of plant materials

Lotus stolon (Nelumbo nucifera) was harvested from a pond in Ayutthaya, Thailand and transported to the laboratory within one hour. The stolons were selected based on uniformity in length (65 ± 4 cm), diameter (1.0–1.2 cm), weight (43 ± 4 g), ground color (L* value 69.2 ± 0.3, chroma value 4.8 ± 0.2, and hue angle 79.2 ± 0.7), and freedom from defects and mechanical damage. The lotus stolons were washed with tap water and then cut into 10 cm (a total of 750 pieces) for further experimentation.

Experimental design

The experiment was conducted using a completely randomized design. The lotus stolons were divided into 5 groups (150 pieces per group) and immersed in ASA solution (Loba Chemie™, India) at concentrations of 0.2%, 0.5%, 1%, and 2% for 5 min, compared to distilled water as a control group. After treatment, 50 lotus stolons from each group were placed on a foam tray without any covering. Each tray represented an experimental unit, and each treatment included 3 replications (n = 3). The trays were stored at 29 ± 2°C with 65 ± 5% relative humidity (RH). During the storage period, 10 lotus stolons were randomly selected from each tray at 0, 12, 24, 36, and 48 h for analysis of: (1) browning surface color, (2) metabolite contents, including total phenolics, total quinones, ASA, DHA, and H₂O₂, as well as measurement of DPPH scavenging activity and pH, and (3) enzyme activities related to browning (PPO and POD), and ascorbate metabolism (APX, DHA and MDHAR).

Determination of surface color and browning index

Ten stolons in each tray were measured for color values at the middle position using a colorimeter (LS171, Linshang Technology) to record L*, a*, b*, chroma, and hue angle in the CIE color space (Pathare et al., 2013). Measurements were averaged per tray with three replications.

Determination of enzymatic browning activity

The enzymatic browning activity was determined based on PPO (EC 1.10.3.2) and POD (EC 1.11.1.7) activities, following the method of Furumo and Furutani (2008) with slightly modifications. Five grams of lotus stolon were homogenized at 4°C in 20 mL of 50 mM potassium phosphate (KP) buffer (pH 7) containing 1% polyvinylpyrrolidone (PVP), 1 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM phenylmethanesulfonyl fluoride (PMSF). The crude extract was centrifuged at 5,000 × g for 15 min at 4°C. The supernatant was collected for the determination of enzymatic activity and protein content.

The enzymatic activities were assayed by mixing 0.02 mL of crude extract with 0.2 mL of 50 mM KP buffer (pH 7), including specific substrates, such as 5 mM catechol for PPO and 4 mM guaiacol with 1 mM H₂O₂ for POD. The mixtures were incubated at 29°C, and their absorbance was monitored at one-minute intervals over a 5 min period. Specific wavelengths were measured using a SpectraMax ABS Plus spectrophotometer (Molecular Devices, USA) at 412 nm for PPO and 470 nm for POD. One unit of PPO and POD activity was defined as a 0.001 absorbance increase/min, with the blank containing the mixture without the enzyme extract.

The protein content in the crude extract was quantified using the Bradford method (1976), with bovine serum albumin (BSA) as a standard, and the enzyme activities were expressed as U mg⁻¹ protein.

Determination of total phenolics and total quinones contents

Five grams of lotus stolon were homogenized in 20 mL of ethanol at 4°C, and then centrifuged at 5,000 × g for 15 min.  The supernatant was collected to determine total phenolics and total quinones contents.

The total phenolics content was determined using the method proposed by Ketsa and Atantee (1998) with singly modification. The 0.03 mL of supernatant was mixed with 0.2 mL of 10% Folin-Ciocalteu reagent and 0.1 mL of 8% Na₂CO₃. The mixture was incubated at 29°C for 30 min before measuring the absorbance at 765 nm using the reaction mixture without the crude extract as the blank. The total phenolics content was determined by comparing to the gallic acid equivalent (GAE) at concentrations ranging from 0.05 to 0.50 mg/mL, and expressed as mg GAE g^-1 fresh weight (FW). Notably, the exogenous ASA from the treatment interfered with the Folin-Ciocalteu reagent, leading to an overestimation of the total phenolics content (Queval et al., 2008b). To correct for this interference, the estimated total phenolics in the ASA-treated group was calculated as: (measured total phenolics in the ASA-treated group at different storage times) – (disrupted value from ASA), where the disrupted value from ASA represents the difference between the measured total phenolics in the ASA-treated group and the measured total phenolics in the untreated group at the beginning of the experiment.

The total quinones content was determined using the method proposed by Homaida et al. (2017) with some modifications. The supernatant was diluted 10 times with ethanol, and the absorbance was measured at 420 nm, using ethanol as the blank.

Total quinones were quantified by comparison with quinone derivatives generated from catechol oxidation, following the procedure described by Ali et al. (2014).

Briefly, reaction mixtures containing 10–100 mM catechol were prepared, and the initial absorbance at 420 nm (A₁) was recorded. A portion of the reaction mixture was used to determine total phenolic content (C₁) using the Folin–Ciocalteu reagent, following the method described above. Subsequently, the reaction mixtures were incubated at 29°C for 24 h to allow oxidation, after which the absorbance at 420 nm (A₂) and total phenolic content (C₂) were measured. A standard curve was generated by plotting the change in absorbance (A₂ - A₁) against the reduction in catechol level (C₁ - C₂). The total quinone content was then calculated using the standard curve and expressed as mg of catechol oxidation per gram of fresh weight.

Determination of ASA and DHA contents

The ASA and DHA were quantified using the method proposed by Gómez Ruiz et al. (2016) with some modifications. Five grams of lotus stolon were homogenized in 20 mL of 0.1% trichloroacetic acid (TCA) containing 1% PVP at 4°C and then centrifuged at 5,000 × g for 15 min. The supernatant was collected to determine ASA and DHA contents.

The ASA content was assayed by adding 0.03 mL of the supernatant to 0.3 mL of 0.08% 2,6-dichlorophenolindophenol (DCPIP). The reaction mixture was incubated at 29°C for 10 min, and absorbance was measured at 600 nm, using the reaction mixture without the crude extract as the blank. The concentration of ASA was determined by comparison to the standard (1–8 mM), and the ASA content was reported as μmol g⁻¹ FW.

The DHA content was also assayed by reducing DHA to ASA. A 0.03 mL aliquot of the supernatant was mixed with 0.02 mL of 5 mM dithiothreitol (DTT) and incubated at 29°C for 30 min. Then, 0.02 mL of 5 mM N-ethylmaleimide (NEM) was added to clear the excess DTT. Afterward, 0.3 mL of DCPIP was added to develop color for the determination of total ASA + DHA according to the method described above. The DHA content was calculated as (total ASA + DHA) – ASA.

The ASA redox potential was calculated using the Nernst equation:

where E₀ is the standard redox potential of the ASA/DHA redox couple (+90 mV, Noctor, 2006); R is the gas constant (8.314 J·K⁻¹·mol⁻¹); F is the Faraday constant (96,485 C.mol⁻¹); T is the experimental temperature (302 K); n is the number of electrons involved in the reaction (2 e-); and [DHA]/[ASA] is the concentrations of the oxidized per reduced forms. The positive ASA redox potential reflects its tendency to undergo oxidation (electron loss), as seen in the conversion of ASA to DHA. In contrast, the negative redox potential reflects its tendency for reduction (electron gain), as seen in the conversion of DHA to ASA.

Notably, our method reliably detects ASA concentrations as low as 1 mM. The values below this threshold are reported as not detected (N.D.), and consequently, the ASA/DHA ratio and redox potential cannot be calculated and are also reported as N.D.

Determination of H₂O₂ content

The H₂O₂ content was determined following Rahman et al. (2023). Lotus stolons (5 g) were homogenized in 20 mL of 0.1% TCA containing 1% PVP at 4°C, centrifuged at 5,000 × g for 15 min, and the supernatant was collected. A 0.05 mL aliquot was mixed with 0.3 mL of ferrous oxidation-xylenol orange (FOX) reagent and incubated at 29°C for 10 min. Absorbance was measured at 560 nm and compared to a 10-100 uM H₂O₂, with results expressed as μmol g⁻¹ FW. Notably, the exogenous ASA from the treatment interfered with the FOX reagent, leading to an overestimation of the H₂O₂ levels (Queval et al., 2008a). To correct for this interference, the estimated H₂O₂ in the ASA-treated group was calculated as: (measured H₂O₂ in the ASA-treated group at different storage times) - (disrupted value from ASA), where the disrupted value from ASA represents the difference between the measured H₂O₂ in the ASA-treated group and the measured H₂O₂ in the untreated group at the beginning of the experiment.

Determination of DPPH scavenging activity

The radical scavenging activity was measured using the DPPH assay (Mun’im, 2003) with minor modifications. Lotus stolons (5 g) were homogenized in 20 mL of ethanol at 4°C, centrifuged at 5,000 × g for 15 min, and the supernatant was collected. A 0.03 mL aliquot was mixed with 3 mL of 0.2 mM DPPH in ethanol and incubated in darkness at 29°C for 30 min. Absorbance was measured at 517 nm, compared with ethanol. The DPPH radical scavenging activity was calculated as % inhibition   where A blank represents the absorbance of DPPH solution without the sample.

Determination of enzymatic ascorbate metabolism

The APX (EC 1.11.1.11), DHAR (EC 1.8.5.1), and MDHAR (EC 1.6.5.4) activities were determined following the method of Hodges and Forney (2000) with some modifications. Five grams of lotus stolon were homogenized at 4°C in 20 mL of 50 mM KP buffer (pH 7) containing 1% PVP, 1 mM EDTA, and 1 mM PMSF. The crude extract was centrifuged at 5,000 × g for 15 min at 4°C. The supernatant was collected for the determination of enzymatic activity and protein content.

The enzymatic activities were assayed by mixing 0.02 mL of crude extract with 0.2 mL of 50 mM KP buffer (pH 7), including specific substrates, such as 1 mM ASA with 1 mM H₂O₂ for APX, 2 mM GSH with 0.2 mM DHA for DHAR and 0.2 mM NADH, 2 mM ASA with 0.2 U ascorbate oxidase for MDHAR. The mixtures were incubated at 29°C, and their absorbance was monitored at one-minute intervals over a 5 min period. Specific wavelengths used were 290 nm for APX, 265 nm for DHAR, and 340 nm for MDHAR.  One unit of APX activity was defined as the decomposition of 1 μmol of ASA per minute, based on a molar absorptivity of 2.8 mM⁻¹ cm⁻¹, while one unit of DHAR activity was defined as the formation of 1 μmol of ASA per minute, based on a molar absorptivity of 14.7 mM^-1 cm^-1 and one unit of MDHAR activity was defined as the oxidation of 1 μmol of NADH per minute, based on a molar absorptivity of 6.2 mM^-1 cm^-1. The blank contained the same mixture without the enzyme extract. 

The protein content in the crude enzyme extract was determined according to the method of Bradford (1976). The activities of APX, DHAR, and MDHAR were expressed as U mg⁻¹ protein.

Measurement of crude extract pH

Crude extract pH was determined by homogenizing 5 g of lotus stolon in 50 mL of distilled water (pH 7.0) at 4°C. The homogenate was then filtered through Watchman No.1 paper. The pH of the resulting extract was measured using a pH meter (EC500 EXTECH, Santa Technology).

Statistical analysis

Our study includes five treatments (0.2%, 0.5%, 1%, and 2% ASA and control), with data collected at five different time points (0, 12, 24, 36, and 48 h), resulting in 25 data points (5 treatments × 5 time points). All data were analyzed via one-way analysis of variance (ANOVA), and mean values were compared by Tukey's multiple range test (P ≤ 0.05). The statistical analyses were performed using SPSS26 statistical software package (IBM SPSS Statistics).

The data are expressed as the mean ± standard error (SE), based on three replicates (n = 3). The percentages of reductions or increases in the treated group compared to the control group were calculated and averaged across the five time points throughout the experimental period.

RESULTS

Effects of exogenous ASA on postharvest browning of lotus stolon

The results showed that the control group exhibited increased browning during storage, with the browning index rising from 6.77 initially to 32.76 at 48 h. In comparison, the 0.2%, 0.5%, 1%, and 2% ASA-treated groups exhibited slower browning development, with average reductions of 12%, 21%, 29%, and 31%, respectively, compared to the control group throughout storage. Notably, the 0.5%, 1%, and 2% ASA treatments effectively reduce browning, while 0.2% ASA has a minimal effect (Figures 1A and 1B).

Changes in the color of the lotus stolon surface can be described by the L*, hue angle, and chroma. In the control group, L* decreased steadily from 69.26 to 62.21 and hue angle from 79.22 to 61.61 over 48 h of storage, while chroma increased from 4.85 to 15.55 during the same period. In comparison, the 0.5-2% ASA-treated groups exhibited a slower decline in L*, which remained on average 4% higher than the control throughout storage. The chroma value in the 0.5-2% ASA-treated groups slowly increased during storage but remained on average 9-14% lower than that of the control. The hue angle in the 0.5-2% ASA-treated groups decreased during storage but remained on average 20-27% higher than that of the control. Notably, the 0.2% ASA treatment showed only a slight effect on L*, hue angle, and chroma when compared to the control throughout the experimental period (Figures 1C, 1D, and 1E).

Figure 1. Changes in visual appearance (A), browning index (B), L* value (C), chroma value (D) and hue angle (E) in untreated and ASA treated groups of postharvest lotus stolon during storage at 29 ± 2°C with 65 ± 5% RH for 48 h. The values are presented as mean ± SE (n = 3). Different letters above the bars indicate significant differences between treatments throughout the storage time, as determined by Tukey's multiple range test (P ≤ 0.05).

Effects of exogenous ASA on changes in enzymatic browning activity and level of total phenolics and quinones in postharvest lotus stolon

The PPO activity in the control group increased significantly during storage, rising from 33.5 U mg⁻¹ protein initially to 59.1 U mg⁻¹ protein at 48 h. In comparison, the PPO activity in 0.2%, 0.5%, 1%, and 2% ASA-treated groups were initially inhibited, with reductions of 24%, 39%, 41%, and 86%, respectively. Subsequently, PPO activity gradually increased during storage, but remained lower than in the control group throughout, with average reductions of 23%, 36%, 34%, and 50%, respectively (Figure 2A).

The POD activity in the control group increased significantly during storage, rising from 96.1 U mg⁻¹ protein initially to 357.8 U mg⁻¹ protein at 48 h. In comparison, the POD activity in 2% ASA-treated groups were initially inhibited, with reductions of 86%. Subsequently, POD activity in 0.5%, 1%, and 2% ASA-treated groups gradually increased during storage, but remained lower than in the control group throughout, with average reductions of 33%, 34%, and 39%, respectively. Notably, the 0.2% ASA treatment exerted only a minor effect on POD activity, resulting in an average reduction of 16% compared with the control throughout the experimental period (Figure 2B).

The total phenolics content in the control group steadily decreased during storage, from 6.47 mg GAE g⁻¹ FW at the initial time point to 4.56 mg GAE g⁻¹ FW at 48 h. In comparison, the total phenolics content in the 0.5%, 1%, and 2% ASA-treated groups significantly maintained higher level with average increases of 14%, 32%, and 48%, respectively, relative to the control throughout the storage period. No significant change in total phenolics content was observed in the 0.2% ASA-treated group (Figure 2C).

The total quinones content in the control group increased continuously during storage, rising from 0.36 mg of catechol oxidation g⁻¹ FW at the initial time point to 0.73 mg of catechol oxidation g⁻¹ FW at the end of the storage period (48 h). In comparison, the total quinones content in the 0.2%, 0.5%, 1%, and 2% ASA-treated groups was markedly inhibited at the early stage, showing reductions of 24%, 42%, 35%, and 37%, respectively. Although quinones level in ASA-treated groups gradually increased during storage, they remained consistently lower than those in the control group, showing average reductions of 20–23% across the 0.5–2% ASA treatments. Notably, the 0.2% ASA treatment exhibited only a minor effect on total quinones accumulation, showing an average reduction of 12% compared with the control throughout the experimental period (Figure 2D).

Figure 2. Changes in PPO activity (A), POD activity (B), total phenolics content (C) and total quinones content (D) in untreated and ASA treated groups of postharvest lotus stolon during storage at 29 ± 2°C with 65 ± 5% RH for 48 h. The values are presented as mean ± SE (n = 3). Different letters above the bars indicate significant differences between treatments throughout the storage time, as determined by Tukey's multiple range test (P ≤ 0.05).

Effect of exogenous ASA on changes in H₂O₂ content, DPPH scavenging activity and ascorbate peroxidase activity of postharvest lotus stolon

The H₂O₂ content in the control group continuously increased during storage, rising from 0.68 μmol g⁻¹ FW initially to 1.42 μmol g⁻¹ FW at 48 h. In comparison, the H₂O₂ content in the 0.5%, 1%, and 2% ASA-treated groups increased more slowly and remained lower than the control group from 36 to 48 h, with average reductions of 17%, 16%, and 23%, respectively. Notably, the H₂O₂ level in the 0.2% ASA-treated group was comparable to those of the control throughout the experimental period (Figure 3A).

The DPPH scavenging activity in the control group declined significantly during storage, decreasing from 16.56% at the initial time point to 7.85% after 48 h. In comparison, the DPPH scavenging activity in the 0.5%, 1%, and 2% ASA-treated groups increased significantly after treatment, reaching 1.4-, 2.8-, and 3.4-fold, respectively. Subsequently, the DPPH scavenging activity in ASA-treated groups gradually declined with prolonged storage, but remained higher than that of the control by approximately 32%, 96%, and 107%, respectively. Notably, the 0.2% ASA treatment had only a marginal effect on DPPH scavenging activity relative to the control (Figure 3B).

The APX activity in the control group exhibited a transient increase, rising from 30 U mg⁻¹ protein at day 0 to a maximum of 41 U mg⁻¹ protein within the first 24 h, before declining markedly to 11 U mg⁻¹ protein at 48 h. A similar pattern was observed in the 0.2%, 0.5%, 1%, and 2% ASA-treated groups, where APX activity increased sharply within the first 12 h by approximately 2.2-, 2.3-, 2.6-, and 3.1-fold, respectively, before gradually declining during subsequent storage. Nevertheless, the APX activity in ASA-treated groups remained consistently higher than that in the control throughout storage, with average increases of 76%, 100%, 106%, and 102%, respectively (Figure 3C).

Figure 3. Changes in H₂O₂ content (A), DPPH scavenging activity (B) and APX activity (C) in untreated and ASA treated groups of postharvest lotus stolon during storage at 29 ± 2°C with 65 ± 5% RH for 48 h. The values are presented as mean ± SE (n = 3). Different letters above the bars indicate significant differences between treatments throughout the storage time, as determined by Tukey's multiple range test (P ≤ 0.05).

Effects of exogenous ASA on changes in endogenous ASA form and redox status of postharvest lotus stolon

The endogenous ASA content in the control group declined rapidly from an initial level of 6.34 μmol g⁻¹ FW and became undetectable at 36 and 48 h. In comparison, the endogenous ASA content in the 0.5%, 1%, and 2% ASA-treated groups increased immediately after treatment by approximately 2.3-, 3.0-, and 4.6-fold, respectively. Although endogenous ASA content gradually decreased during subsequent storage, it remained significantly higher than that of the control by approximately 125%, 183%, and 277%, respectively, throughout the experimental period. Notably, the 0.2% ASA treatment exerted only a marginal effect on endogenous ASA content compared with the control (Figure 4A).

The DHA content in the control group increased slightly from 13.60 μmol g⁻¹ FW at the initial time point to 18.10 μmol g⁻¹ FW at 36 and 48 h. In comparison, treatments with 0.5%, 1%, and 2% ASA led to a continuous increase in DHA levels, which remained significantly higher than the control by approximately 20%, 33%, and 36%, respectively, throughout storage.  Notably, the 0.2% ASA treatment showed only a minimal effect on DHA content compared with the control (Figure 4B).

Changes in the ASA redox state, as indicated by the ASA/DHA ratio, were observed. The ASA/DHA ratio in the control group declined from an initial value of 0.47 and became undetectable at 36 and 48 h. In comparison, the ASA/DHA ratios in the 0.5%, 1%, and 2% ASA-treated groups increased immediately after treatment by approximately 2.1-, 2.8-, and 4.5-fold, respectively, before gradually decreasing during subsequent storage. Nevertheless, these ratios remained consistently higher than those of the control by approximately 100%, 129%, and 210%, respectively, throughout the experimental period. Notably, the 0.2% ASA treatment did not significantly alter the ASA/DHA ratio in comparison with the control (Figure 4C).

The redox potential of the control group increased from +99.6 mV at the initial time point to +117.8 mV at 24 h, and became undetectable at 36 and 48 h. In comparison, the 0.5%, 1%, and 2% ASA-treated groups exhibited an immediate decrease of 17%, 19%, and 28%, respectively, followed by a gradual increase over time. Despite this rise, the redox potential in ASA-treated groups remained lower than that of the control by 12%, 14%, and 19%, respectively. Notably, treatment with 0.2% ASA did not significantly affect the redox potential compared to the control
(Figure 4D).

Figure 4. Changes in ASA content (A), DHA content (B), ASA/DHA ratio (C) and ASA redox potential (D) in untreated and ASA treated groups of postharvest lotus stolon during storage at 29 ± 2°C with 65 ± 5% RH for 48 h. The values are presented as mean ± SE (n = 3). Different letters above the bars indicate significant differences between treatments throughout the storage time, as determined by Tukey's multiple range test (P ≤ 0.05).

Note: the N.D. indicates not detected or that the value could not be determined.

The DHAR activity in the control group increased slightly from 5.23 U mg⁻¹ protein initially to 7.58 U mg⁻¹ protein at 24 h, then gradually decreased to 4.37 U mg⁻¹ protein at 48 h. In comparison, the DHAR activity in 0.2% and 0.5% ASA-treated groups dramatically increased significantly by 2.0- and 2.3-fold, respectively, at 24 h, followed by a gradual decline. However, it remained higher than the control throughout the experimental period by 24% and 28%, respectively. On the other hand, DHAR activity in 2% ASA-treated group sharply decreased by 59% initially, before gradually increasing and remaining 30% lower than the control throughout storage, while the 1% ASA-treated group showed no significant change (Figure 5A).

The MDHAR activity in the control group remained stable at 0.47-0.52 U mg⁻¹ protein for the first 24 h, then rapidly decreased to 0.14 U mg⁻¹ protein at 48 h. In comparison, ASA-treated groups initially decreased by an average of 33%, and then the 0.2% and 0.5% ASA groups gradually increased, remaining 23% higher than the control between 24-36 h. However, the 1% and 2% ASA treatments gradually decreased during storage, staying 26% and 28% lower than the control, respectively (Figure 5B).

Figure 5. Changes in DHAR activity (A) and MDHAR activity (B) in untreated and ASA treated groups of postharvest lotus stolon during storage at 29 ± 2°C with 65 ± 5% RH for 48 h. The values are presented as mean ± SE (n = 3). Different letters above the bars indicate significant differences between treatments throughout the storage time, as determined by Tukey's multiple range test (P ≤ 0.05).

Effects of exogenous ASA on changes in pH of the lotus stolon crude extract

The ASA solution of experiment at concentrations of 0.2%, 0.5%, 1%, and 2% had pH values of 3.25, 3.05, 2.75, and 2.65, respectively, while distilled water (control) had pH value of 6.95 (Figure 6A). The pH of crude extract decreased promptly in proportion to the ASA concentration, reaching 5.86, 5.73, 5.58, and 5.17, respectively, compared to the initial pH of 6.08 in the untreated group. After 48 h, the pH of crude extract slightly decreased during storage, reaching 5.35, 5.26, 4.95, and 4.96, respectively, in the 0.2%, 0.5%, 1%, and 2% ASA-treated groups, and 5.44 in the untreated group (Figure 6B).

Figure 6. The pH of treatment solution (A) and changes in pH of crude extract (B) in untreated and ASA treated groups of postharvest lotus stolon during storage at 29 ± 2°C with 65 ± 5% RH for 48 h. The values are presented as mean ± SE (n = 3). Different letters above the bars indicate significant differences between treatments throughout the storage time, as determined by Tukey's multiple range test (P ≤ 0.05).

DISCUSSION

Mechanisms of postharvest browning in untreated lotus stolon: The interplay between enzymatic browning and ASA redox status

Upon harvesting, lotus stolon undergoes chemical changes that lead to browning, primarily driven by PPO and POD activities, which oxidize phenolic compounds into quinones (Homaida et al., 2017; Min et al., 2017). Our results show that PPO and POD activities increased during storage (Figures 2A and 2B), which is accompanied by a decrease in total phenolics content and an increase in quinones (Figures 2C and 2D). This correlates with a significant intensification of browning, as indicated by the decrease in L* value and hue angle, while chroma value and browning index increased (Figures 1A, 1B, 1C, 1D and 1E).

Notably, the activity of POD is 2.9-6.4 times higher than that of PPO during storage (Figures 2A and 2B), suggesting that POD is the key enzyme primarily driving the development of browning in lotus stolon. Additionally, other factors contribute to the accelerated oxidation of phenolic compounds into quinones. H₂O₂ is a major ROS in post-harvest crops, generated during exposure to environmental stresses such as extreme temperatures, mechanical damage, and dehydration (Chomkitichai and Intarasit, 2024; Guan et al., 2024). The increase in H₂O₂ level during storage
(Figure 3A) may promote the oxidation of phenolics, enhancing quinone formation and contributing to the browning process, as H₂O₂ acts as a cofactor for POD (Wang et al., 2024) (Figure 7A).

ASA is a powerful reducing agent that inhibits postharvest browning via quinones recycling and H₂O₂ scavenging (Smirnoff, 2000). However, the content of ASA in lotus stolons is limited (~6 µmol/g FW or ~100 mg/100 g FW, Figure 4A), when compared with guava fruit (~220 mg/100 g FW) and chili paper (~240 mg/100 g FW) (Yang and Xu., 2016). Our results demonstrate that the reduction in ASA occurs concomitantly with an increase in DHA (Figures 4A and 4B) suggesting that ASA donates electrons to scavenge ROS and reduce quinones, suggesting that ASA donates electrons to scavenge ROS and reduce quinones. This process leads to the oxidation of ASA into DHA, which ultimately results in a lower ASA redox status (Figure 4C). Moreover, a reduction in ASA redox status leads to lower DPPH scavenging activity (Figure 3B) and decreased APX activity (Figure 3C). Since ASA acts as both a non-enzymatic antioxidant and a cofactor for APX, this reduction diminishes H₂O₂ scavenging, contributing to increased browning, as observed in fresh-cut lotus roots (Ali et al., 2020).

Although ASA is limited in plant tissue, DHA (oxidized form) can be recycled to ASA (reduced form) through the activities of MDHAR and DHAR. MDHAR is an enzyme responsible for recycling MDHA to ASA, coupling the conversion of nicotinamide adenine dinucleotide (phosphate) from its reduced form (NAD(P)H) to its oxidized form (NAD(P)⁺). In contrast, DHAR recycles DHA to ASA, coupling the conversion of glutathione from its reduced form (GSH) to its oxidized form (GSSG) (Chumyam et al., 2017). However, our results show that the activities of MDHAR and DHAR in lotus stolon decreased during storage (Figures 5A and 5B), resulting in a delayed conversion of DHA to ASA. This indicates a higher ASA redox potential (Figure 4D), reflecting that ASA was oxidized to DHA rather than DHA being recycled back to ASA. Notably, the activity of DHAR increased within 24 h (Figure 5A), which may be related to the response to postharvest stress. However, it was insufficient to prevent the oxidation of ASA to DHA, resulting in a lower ASA redox status, which contributes to increased browning that has been reported in postharvest longan fruit (Chumyama et al., 2017) and fresh-cut potato (Tang et al., 2023).

Mechanisms of ASA treatments in inhibiting browning of lotus stolon: Roles of PPO/POD inhibition and ASA-quinones redox cycling

ASA serves as a powerful antioxidant and reducing agent, playing a crucial role in mitigating browning across a wide range of harvested crops (Gao et al., 2017; Ali et al., 2020; Hou et al., 2022). In this study, the acceptable visual quality was defined by the initial fresh state of the lotus stolon at 0 h. Colorimetrically, a decrease in L* and hue angle reflects tissue darkening and a shift towards brown, whereas an increase in chroma indicates intense brown discoloration (Pathare et al., 2013). Therefore, an effective anti-browning treatment must mitigate these specific shifts to maintain the visual quality close to the initial fresh state. Application of 0.5–2.0% ASA effectively suppressed these changes, as indicated by maintaining significantly higher L* and hue angle values (with average increases of 4% and 20–27%, respectively) and lower chroma values and browning index (with decreases of 9–14% and 21–31%, respectively) compared to the control throughout the storage time (Figures 1B–1E). This significant delay in discoloration helped preserve an acceptable visual quality for up to 24 h, remaining visibly comparable to the initial fresh state at 0 h (Figure 1A). In contrast, 0.2% ASA had a minor effect on reducing browning. Our findings align with previous studies, such as Özdemir and Gökmen (2019) and Ali et al. (2020), which showed that 1–2% ASA reduced browning in fresh-cut apples and lotus root slices.

Although the role of ASA in browning inhibition is known, its specific mechanism in lotus stolon has yet to be fully elucidated. One possible explanation is that exogenous ASA suppresses PPO and POD activities by lowering the cellular pH below their optimal functional range of 5.5–7.5 (Li et al., 2023). In support of this, 0.5%–2% ASA treatments (pH 2.65–3.05) significantly increased the acidity of the lotus stolon crude extract, lowering its pH to 4.96–5.73 compared to 5.44–6.08 in the untreated group (Figures 6A and 6B). This significant drop in pH likely created an unsuitable environment that inhibited PPO and POD activities throughout the storage period (Figures 2A and 2B). Consequently, the inhibited enzyme activities led to a lower accumulation of quinones and a higher retention of total phenolics (Figures 2C and 2D). These findings suggest that ASA treatment effectively limits the precursors required for the formation of complex brown polymers, thereby mitigating postharvest browning (Figures 7B and 7C).

H₂O₂ is another important factor contributing to the browning process, as it serves as a cofactor for POD-mediated phenolic oxidation and has been widely reported in many postharvest crops (Chomkitichai and Intarasit, 2024; Guan et al., 2024; Wang et al., 2024). Our results demonstrate that 0.5-2% ASA treatments can reduce H₂O₂ level (Figure 3A), which correlated with an increase in APX activity and DPPH scavenging activity (Figures 3B and 3C). These findings indicate that ASA treatment promotes scavenging H₂O₂ through the enzymatic APX pathway (Figures 7B and 7C). In addition, previous research has shown that ASA can directly scavenge H₂O₂, with DHA being produced as a byproduct (Smirnoff, 2000). Our data showed that the increase in endogenous ASA and DHA levels by 1% and 2% ASA treatments (Figures 4A and 4B) was consistent with the reduction in H₂O₂ (Figure 3A), which suggests that this treatment may enhance H₂O₂ scavenging through the ASA/DHA redox cycle (Figure 7B). Our results are consistent with previous reports in postharvest longan (Intarasit and Saengnil, 2021) and pineapple fruit (Hou et al., 2022), exogenous ASA is suggested to activate APX, enhance antioxidant scavenging capacity, reduce H₂O₂ accumulation, and ultimately delay browning.

The previous study has demonstrated that ASA can facilitate the conversion of quinones back to phenols (Roginsky et al., 1999). Our results showed that 1% and 2% ASA treatments effectively suppressed quinones accumulation (Figure 2D) consistent with a higher total phenolics content (Figure 2C) and a higher ASA redox potential (Figure 4D). This suggests that ASA serves as a vital electron donor, preventing quinone polymerization and subsequent browning (Figures 7B). Our findings are consistent with those of Irchad et al. (2022), who demonstrated that exogenous ASA application can inhibit the oxidation of phenolics, thereby reducing peel browning in dried figs. It is noteworthy that total phenolics content in the ASA-treated group significantly increased during storage, and this change could not be explained solely by reduction of quinones to phenols. This suggests that ASA treatment may stimulate phenolic synthesis in response to postharvest stress, as reported by Zhu et al. (2009).

The ASA status is critical for regulating browning by suppressing quinone accumulation and scavenging H₂O₂ (Roginsky et al., 1999; Smirnoff, 2000). Consequently, a high ASA/DHA ratio reflects a robust redox balance that is directly associated with reduced browning in postharvest crops (Chumyam et al., 2017). Our results demonstrate that while 0.5–2% ASA treatments significantly elevated the ASA/DHA ratio, the underlying mechanisms appear to differ. High doses (1–2%) primarily directly expand the internal ASA pool, whereas the 0.5% dose had a minimal impact on the absolute ASA content (Figure 4C). To further elucidate why the 0.5%-dose maintained a favorable redox state despite its minimal effect on total ASA levels, we investigated the ASA-recycling system. Our results showed that 0.5% ASA treatment efficiently upregulated the activities of both MDHAR and DHAR (Figures 5A and 5B). These results suggest that lower dose ASA optimizes the activities of these enzymes by altering an appropriate redox balance between substrates and products, whereas higher ASA concentrations may disrupt redox equilibrium, leading to feedback inhibition (Mehmood et al., 2024). Although 0.2% ASA promotes DHAR activity (Figure 5A), it fails to elevate the ASA/DHA ratio. This suggests that ASA at this low concentration neither expands the internal ASA pool nor provides a sufficient enzymatic boost to alter the redox status. Ultimately, the maintenance of a high ASA/DHA ratio through enzymatic regulation appears to be a key strategy for browning control. Our results align with Chumyam et al. (2017), who reported that ClO₂ treatment increased MDHAR and DHAR activities, resulting in a higher ASA redox status, along with reduced postharvest browning in longan fruit.

The collective results demonstrate that different concentrations of ASA treatment have varying inhibitory effects on the browning of lotus stolon. The 1% and 2% ASA treatments effectively reduced postharvest browning by boosting endogenous ASA levels and creating an acidic environment. This dual action directly suppresses enzymatic browning (PPO and POD), reduces quinones back to phenolics, and facilitates H₂O₂ detoxification through both enzymatic (APX) and non-enzymatic pathways (Figure 7B). In comparison, the 0.5% ASA treatment mitigates browning primarily by inhibiting enzymatic browning and H₂O₂ detoxification through the APX-mediated pathway, while uniquely modulating the ASA redox status via MDHAR and DHAR activities (Figure 7C). However, the 0.2% ASA treatment has a limited impact on reducing browning and minor effects on physiological processes.

Figure 7. The physiological mechanisms of browning development in lotus stolon during storage at 29 ± 2°C and 65 ± 5% RH for 48 h (A). The proposed mechanisms of 1% and 2% ASA (B) and 0.5% ASA (C) treatments in reducing browning during storage at 29 ± 2°C and 65 ± 5% RH for 48 h. The symbol ← represents activation of ASA treatment; the symbol ⊢ represents inhibition of ASA treatment. The solid black line ( ^___ ) indicates significant effects, while the dashed grey line ( ^___ ) indicates minor effects.

CONCLUSION

Application of 0.5%, 1%, and 2% ASA effectively reduces browning in lotus stolon, although physiological responses varied according to the concentration applied. High-dose treatments (1–2% ASA) provide a dual action by boosting endogenous ASA levels and creating an acidic environment, which directly suppresses PPO and POD activities and enhances H₂O₂ scavenging and quinone reduction via enzymatic (APX) and redox pathways. Conversely, the 0.5% dose mitigates browning by optimizing the ASA redox balance via enzymatic recycling (MDHAR and DHAR), thereby facilitating efficient H₂O₂ detoxification. In contrast, 0.2% ASA provides limited browning suppression and exerts only minor effects on the overall physiological processes.

AUTHOR CONTRIBUTIONS

Thanakorn Vichaiya: Conceptualization (Lead), Methodology (Lead), Investigation (Lead), Formal Analysis (Lead), Validation (Equal), Visualization (Lead), Supervision (Lead), Writing – Original Draft (Lead); Jutitorn Laohapornchaiphan: Methodology (Supporting), Resources (Supporting), Formal Analysis (Supporting), Validation (Supporting), Writing – Review & Editing (Supporting); Sitthisak Intarasit: Validation (Equal), Writing – Review & Editing (Lead), Formal Analysis (Supporting).

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

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