2024 年 93 巻 1 号 p. 15-22
The effects of UV-C treatment on physicochemical quality, bioactive compounds and antioxidant activity of okra (Abelmoschus esculentus L.) during storage were investigated. Okra pods were exposed to UV-C irradiation dose at 1.5, 3.0 or 6.0 kJ·m−2 before storage at 10 ± 2°C for 12 days. The results showed that UV-C treatment had a significant effect on okra bioactive compounds, such as ascorbic acid, total phenolic, flavonoid contents and antioxidant activity. The highest values of these compounds were observed in okra treated with 6.0 kJ·m−2 UV-C irradiation. A statistical analysis of the data demonstrated that mucilage had a favorable association with antioxidant capabilities and bioactive substances in response to UV-C treatment. Moreover, UV-C treatment also effectively maintained the quality of okra during storage, as evidenced by lower weight loss and higher firmness without incidence of decay. Therefore, postharvest UV-C treatment can be a potential approach to enhance bioactive compounds and maintain the quality of okra during storage.
Ultraviolet (UV) radiation is a particular kind of solar radiation that can induce various physiological and biochemical changes in plants during development and after harvest (Khorrami et al., 2014). It is divided into three distinct categories depending on its waveband. The UV-C region, with a wavelength of around 260 nm, is considered the most effective germicidal region in the UV spectrum. It has been employed as an alternative to conventional disinfectants because of its capacity to damage microorganism DNA and RNA, as reported by Prajapati et al. (2021). The use of UV-C treatment has been documented to inhibit microbial growth and induce disease resistance in a variety of fresh produce, including mangosteen (Sripong et al., 2019), nectarine (Zhang et al., 2021), satsuma mandarin (Yamaga et al., 2021), and fresh-cut dragon fruit (Nimitkeatkai and Kulthip, 2016). In recent years, there has been a surge of interest in the use of UV-C irradiation to improve the quality and nutritional content of fruits and vegetables. For instance, irradiation with a UV-C dose of 10 kJ·m−2 was found to retain the highest chlorophyll content and delay postharvest senescence of broccoli florets (Costa et al., 2006). Exposure to UV-C was also shown to delay strawberry fruit softening (Pombo et al., 2009) and have an impact on cell wall-degrading enzymes in tomato fruit (Barka et al., 2000). One of the most widely recognized plant adaptations to increased UV radiation is the synthesis of flavonoids and other phenolic compounds, which are generated in postharvest fruit by triggering the phenylpropane pathway (Zhang et al., 2021). Phytochemicals and antioxidants are related to bioactive compounds that are abundant in plants and have been shown to have numerous health benefits, such as anti-inflammatory, anti-carcinogenic, and anti-diabetic properties. The use of UV-C irradiation has been found to be effective in increasing the levels of phenolic compounds and stimulating antioxidant system activity in okra seedlings (Khorrami et al., 2014), apple leaves (Kondo et al., 2011), and nectarine fruit (Zhang et al., 2021). Hence, UV-C treatment is regarded as a promising eco-friendly technology for maintaining the quality of fruit and vegetables during storage, while also enhancing their antioxidant characteristics. Furthermore, this process is free of chemicals and has no negative impacts on human health, making it a sustainable and safe alternative to traditional procedures.
Okra (Abelmoschus esculentus L.) is an important vegetable crop belonging to the Malvaceae family, with origins in Africa. It is abundantly cultivated and consumed in tropical and warm temperature regions worldwide. Okra is widely recognized for its health benefits due to its significant amounts of nutrients such as lipids, amino acids, dietary fiber, carbohydrates, vitamins (A and C), flavonoids, and phenolic compounds (Petopoulos et al., 2018; Raj et al., 2020; Zhang et al., 2018). In addition, mucilage is naturally found in okra pods and contains polysaccharides including D-galactose, L-galacturonic acid, and L-rhamnose units (Raj et al., 2020). Okra mucilage is rapidly becoming deployed in a variety of areas, including the pharmaceutical industry, culinary technology, and material development (Dantas et al., 2021). However, okra is highly perishable and prone to postharvest losses. Consequently, the application of appropriate postharvest treatments to maintain the quality of okra during distribution and storage is of great significance.
Although okra plants have been used as a model to study the mechanisms and kinetics of rapid UV protective responses (Neugart et al., 2021), no research has investigated the use of UV-C treatment as a non-thermal post-harvest treatment to maintain the quality, phytochemicals, antioxidants, and polysaccharides of okra during storage. Therefore, the objective of this study was to determine the parameter settings for the intensity of UV-C exposure in order to maintain the quality and associated health implications of okra, including its phytochemicals, antioxidants and mucilage during cold storage, which would be useful information for further studies and practical applications.
Fresh okra (A. esculentus L.) pods at the commercial maturity stage were purchased from a local grower in Phayao province, Thailand. The okra samples were transported to our laboratory within one hour in a temperature-controlled vehicle. Upon arrival, okra pods of uniform color and size (approximately 10 ± 1 cm in length) and without any defects were collected. The okra pods were then randomly divided into four groups of 90 fruits per group with each group represented by three replicates.
A germicidal UV-C radiator containing two UV-C emitting bulbs was used in this experiment. The UV-C radiation intensity was determined using a Solarmeter model 8.0 UV meter (Solar Light Company, Glenside, PA, USA). Samples were placed in a single layer at a distance of 30 cm from the UV-C lamps and rotated to the opposite side midway through the treatment. The applied UV-C intensity was calculated from a mean of 10 readings, and the applied dose was varied by altering the exposure time at a fixed distance and light intensity. In this experiment, the samples were exposed to UV-C doses of 0 (control), 1.5, 3.0, and 6.0 kJ·m−2, which were obtained after 0, 5, 10, and 20 min of exposure, respectively.
After the UV-C treatment, 10 okra pods from each treatment were placed in polystyrene foam trays (dimension 14 cm × 14 cm × 1.5 cm) and overwrapped with polyvinylchloride film before being transferred to storage at 10 ± 2°C for 12 days. Samples for the analysis of bioactive compounds and enzymatic browning activity were lyophilized and stored at −20°C until use.
Physical quality measurementThe physical quality evaluation of okra included measurements of weight loss, firmness, and color. Weight loss was measured periodically using an electronic weighing balance and was calculated as a percentage based on the initial weight on day 0 and at the end of each storage interval.
The firmness of okra pods was evaluated by manually pressing each pod with a fruit pressure tester model FT-3 (T.R. Turoni srl, Ascoli Piceno, Italy), and the results were expressed in Newtons (N).
The color of the okra pods was measured on the surface at the middle point of different sides and recorded in terms of lightness (L*) and greenness (a*) using a Minolta Chroma Meter (Konica Minolta Co., Tokyo, Japan) based on the CIE L*a*b* color system.
Biochemical analysisThe carotenoid concentration was determined using a modified method of Nagata and Yamashita (1992). Lyophilized samples were extracted with 10 mL of acetone-hexane mixture (4:6) for 15 min. The absorbance of the extract solution was measured at 453, 505, 645, and 663 nm. The carotenoid content was calculated using the following equation:
The ascorbic acid content was determined using the indophenol titrimetric method (Nielsen, 2017). Five grams of okra sample were ground in a solution of 3% metaphosphoric acid-acetic acid (5 mL) using a mortar and pestle, before being titrated with a dye solution of dichlorophenolindophenol.
The ascorbic acid content was calculated using the following equation:
Where X is the volume of the sample titration; B is the volume of the blank titration; F is the solution titer of dye use to titrate the ascorbic acid; E is the sample volume; V is the volume of the initial assay solution; and Y is the volume of the sample aliquot titrated. The results were expressed in mg of ascorbic acid per kg of fresh weight (mg·kg−1 FW).
Total phenolic and flavonoid contents were analyzed following the procedure described in our previous study (Jarerat et al., 2022) with slight modifications. One gram of lyophilized tissue was homogenized in 80% methanol and centrifuged at 10,000 × g for 10 min at 4°C. The supernatant was used to analyse the bioactive compounds and antioxidant activity. The reaction was initiated by mixing the supernatant with Folin-Ciocalteu phenol solution. After that, 1 mL of saturated Na2CO3 was added to the mixture and it was incubated at room temperature for 90 min. Total phenolic content was determined using an Evolution 201 UV-Vis spectrophotometer at 760 nm (Thermo Scientific; Madison, WI, USA) and expressed as milligrams of gallic acid equivalent per gram dry weight (mg GAE·g−1 DW).
To determine total flavonoid content, 0.5 mL of the supernatant was mixed with 0.5% NaNO2, 10% AlCl3·H2O, and 1 M NaOH. The absorbance was measured at 510 nm using a UV-Vis spectrophotometer. Total flavonoid content was expressed as milligrams of catechin equivalent per kilogram of dry weight (mg CE·kg−1 DW).
The extraction of okra mucilage was carried out using a modified method based on Wu et al. (1995). Briefly, okra was blended with hot water (80°C) for one minute using a low-speed blender. The resulting mucilage was mixed with ethanol at a ratio of 85:15 (mucilage:ethanol), separated by squeezing through a thin white cloth, and then centrifuged at 10,000 rpm for 30 min at 4°C. The resulting precipitate was dried at 55°C for 12 h and weighed to determine the yield.
Antioxidant propertiesThe antioxidant activity was determined using a DPPH (2,2-diphenyl-2-picrylhydrazyl) inhibition assay according to the method of Brand-Williams et al. (1995) with slight modification. Okra extract was obtained using the same method as for total phenolic measurement. The sample was mixed with DPPH solution in methanol. The mixture was kept in the dark for 30 min and the absorbance of the sample was measured against the reagent blank at 515 nm using a UV-Vis spectrophotometer (Thermo Scientific). The percent DPPH scavenging activity was determined using the formula: [(ADPPH − AS)/ADPPH × 100], where ADPPH is the absorbance of the DPPH solution and AS is the absorbance of the sample solution.
A Ferric Reducing Antioxidant Power (FRAP) assay was performed as described in our previous study (Jarerat et al., 2022). The results were expressed as μmol Fe (II)·kg−1 DW.
Statistical analysisThe experiments were conducted using a completely randomized design (CRD). Analysis of variance (ANOVA) was performed using SPSS software program version 18 to evaluate treatment effects. Significant differences between treatments were analyzed using Duncan’s New Multiple Range Test (DMRT) at the 95% and 99% confidence levels. The data were presented as mean values of at least three replications ± standard deviation (SD). Furthermore, Pearson correlation coefficients were used to examine the relationships among the measurements during okra storage at a significance level of 1% and 5%. Calculations were carried out using the SPSS software program.
Okra pod weight loss increased gradually as storage time progressed (Fig. 1A). However, exposure to UV-C irradiation at different doses (1.5–6.0 kJ·m−2) significantly maintained the water content of okra during storage. Okra treated with UV-C at 6.0 kJ·m−2 exhibited the lowest weight loss (1.9%), while the control okra showed the highest weight loss (3.6%) on day 12 of storage. A study by Prajapati et al. (2021) reported that untreated bitter gourd fruit had a weight loss rate approximately two-fold greater than that of UV-C treated samples at the end of storage. The authors suggested that UV-C radiation may have contributed to the formation of more compact wax structures, leading to a better protective layer, and altered the permeability of the pod surface to water vapor, thereby reducing water loss. Another possible impact of UV-C irradiation on weight reduction could be related to the respiration rate. Previous studies have reported that UV-C treatment reduced the respiration rate of broccoli (Costa et al., 2006) and mangosteen (Sripong et al., 2019).
Weight loss and firmness of untreated okra (control; CK) and treated okra with UV-C at different doses during storage at 10°C for 12 days. Results are the means of three replicates ± standard deviation. The different letters denote significant differences among treatments at P < 0.05.
The firmness of okra pods decreased after storage for 6 and 12 days (Fig. 1B). However, the application of UV-C treatment maintained the firmness of the okra pods significantly better than the control. The highest firmness at the end of the storage period was observed in okra pods treated with UV-C at 6.0 kJ·m−2 (21.62 N), followed by 3.0 kJ·m−2 (19.74 N), 1.5 kJ·m−2 (17.48 N) and the control (15.45 N). It was reported that softening of postharvest okra was linked to a lack of intercellular adhesion and cell wall detachment during storage (Dong et al., 2023). Several studies have demonstrated the effectiveness of UV-C treatment in maintaining the postharvest firmness of fruits and vegetables (Barka et al., 2000; Pombo et al., 2009). The delay in softening observed in UV-C treated tomato and strawberry fruit may be attributable to the downregulation of a group of genes, as well as the suppression of enzymes and proteins involved in cell wall deconstruction, such as endoglucanase, polygalacturonase (PG) and pectin-methylesterase (PME) (Barka et al., 2000; Pombo et al., 2009).
The color of the okra pods, as represented by the lightness (L*) and greenness (a*) values, are presented in Figure 2A and 2B. The lightness of the okra pods slightly increased during storage, with no significant difference observed among the different UV-C treatment doses and the control (Fig. 2A). During the first six days of storage, the greenness of the okra pods tended to be fairly consistent. The UV-C treatment at 1.5 kJ·m−2 significantly reduced the greenness of the okra pods, while UV-C treatments at higher doses of 3.0 and 6.0 kJ·m−2 showed no significant differences after 12 days of storage.
Color (L* and a* value) and total carotenoid content of untreated okra (control; CK) and treated okra with UV-C at different doses during storage at 10°C for 12 days. Results are the means of three replicates ± standard deviation. The different letters denote significant differences among treatments at P < 0.05.
Total carotenoid content of okra pods increased slightly during the first six days and there were no significant differences between the UV-C treatment and control (Fig. 2C). After 12 days, UV-C at 1.5 kJ·m−2 significantly increased the carotenoid content to the highest level among the UV-C dosage treatments (3.0 and 6.0 kJ·m−2) and control.
It has been reported that UV-C irradiation can delay color changes in several crops including broccoli (Costa et al., 2006) and the calyx of mangosteen (Sripong et al., 2019). Our study also showed that UV-C treatment can help maintain the green color of okra. The current results are in accordance with Costa et al. (2006), who found that UV-C irradiation delayed color changes in broccoli and resulted in a superficial color similar to a control. The authors suggested that a high dose of UV-C (14 kJ·m−2) reduced chlorophyll degradation, but did not delay the increase in pheophytin content. In addition, an increase in carotenoid content due to UV-C treatment was also observed in yellow bell pepper (Promyou and Supapvanich, 2012) and bitter gourd (Prajapati et al., 2021). Hermetic doses of UV-C induce plant defence systems and can also trigger the accumulation of carotenoids, which play an important role in protecting the fruit tissue by scavenging reactive oxygen species (ROS) (Gonzalez-Aguilar et al., 2010). This suggests that the increase in carotenoid content may be a response of the antioxidant system to UV-C stress. However, the effects of UV-C irradiation on carotenoid content in okra may depend on dosage and storage time, and these factors requires further investigation.
Effects of UV-C treatment on the biochemical attributes of okra podsUV-C Treatment with different doses (1.5–6.0 kJ·m−2) significantly induced the accumulation of ascorbic acid content in a dose-dependent manner after treatment throughout the storage period (Fig. 3A). On day 12 of storage, okra treated with UV-C at 1.5, 3.0, and 6.0 kJ·m−2 demonstrated 9.1, 17.5, and 39.4% higher ascorbic acid content, respectively, compared to the control fruit.
Ascorbic acid, total flavonoid and total phenolic contents of untreated okra (control; CK) and treated okra with UV-C at different doses during storage at 10°C for 12 days. Results are the means of three replicates ± standard deviation. The different letters denote significant differences among treatments at P < 0.05.
UV-C radiation is a well known abiotic physical elicitor of stress resistance mechanisms, which lead to stimulation of secondary metabolite synthesis that includes ascorbic acid, phenolics, antioxidants, and carotenoids during storage in a dose-dependent manner (Pinheiro et al., 2015; Prajapati et al., 2021). In ascorbic acid in okra, higher amounts were found immediately after UV-C treatment. Conversely, studies have also reported that similar UV-C treatment resulted in the suppression of ascorbic acid reduction in nectarine (Zhang et al., 2021) and apple (Kondo et al., 2011). It has been discovered that the ascorbic acid in plants is a potent source of antioxidants that can protect against abiotic stressors that cause oxidative stress (Fenech et al., 2019). It is also believed that this could be a protective mechanism of plant tissues against excessive radiation (Yoshikawa et al., 2007). Fenech et al. (2019) proposed that an increase in ascorbate biosynthesis occurs in order to detoxify the ROSs caused by UV radiation, resulting in an increase in ascorbate.
There were no significant differences in total flavonoid content among control and treated okra pods immediately after UV-C treatment (Fig. 3B). During storage, flavonoids tended to decrease more markedly in control okra, and after 12 days of storage 3.0 and 6.0 kJ·m−2 UV-C-treated okra maintained a higher level of flavonoids than 1.5 kJ·m−2 UV-C and control okra.
Total phenolic content also decreased after six days of storage in control okra and increased thereafter as senescence occurred (Fig. 3C). On day 12 of storage, higher levels of phenolics were found in UV-C treated okra at 1.5, 3.0, and 6.0 kJ·m−2 with a 13.4, 16.7, and 26.1% increase, respectively, compared to the control.
The increase in total flavonoid and phenolic content following UV-C treatments could be interpreted as an adaptive mechanism of okra to UV-C stress. Evidence suggests that UV radiation results in the accumulation of flavonoids and other phenolics that absorb UV light, particularly in the plant’s epidermal tissues (Pinheiro et al., 2015). UV-C irradiation has been linked to promotion of the phenylpropanoid pathway in several fruit species by inducing phenylalanine ammonia lyase (PAL), a crucial enzyme in the system (Pombo et al., 2011). The authors found an increase in the expression and activity of PAL at both early and late storage time points after irradiation. Our results showed that higher doses of UV-C resulted in greater increases in bioactive compounds such as ascorbic acid, phenolics, and flavonoids. Neugart et al. (2021) also noted a dose-dependent relationship between flavonoid content and UV-C irradiation. Thus, longer-term moderate UV-C exposure could have a positive impact on plant constituents.
As shown in Figure 4, the mucilage content in okra decreased as the storage time progressed. However, the application of UV-C at 1.5–6.0 kJ·m−2 effectively delayed the decreases in mucilage content after storage for 6 and 12 days. On day 12, the highest amount of mucilage was observed in samples treated with 6.0 kJ·m−2 UV-C, followed by 3.0 and 1.5 kJ·m−2 UV-C which exhibited a 57.7, 34.1, and 14.6% increase from the control, respectively.
Mucilage content of untreated okra (control; CK) and treated okra with UV-C at different doses during storage at 10°C for 12 days. Results are the means of three replicates ± standard deviation. The different letters denote significant differences among treatments at P < 0.05.
The increase in mucilage in UV-C treated okra can be attributed to the copolymerization effect of UV-C irradiation on bio-polysaccharide. Many natural polymers have been reported to be manipulated by crosslinking, which typically leads to improved products. UV radiation has also been used to initiate graft copolymerization of okra mucilage by crosslinking (Raj et al., 2020).
Similar results were also observed by Phornvillay et al. (2020), who reported that chilling-injured okra pods displayed mucilage bursting as observed by scanning electron micrography. This is considered to be a plant tissue defense mechanism against extreme environmental stress. However, the penetrating ability of UV-C radiation is limited to the outer pericarp and an increase in phenolic compounds, mainly in the skin, is to be expected with a lower impact on more inner layers of tissue (Pombo et al., 2011). As a result, an increase in the mucilage response may require a higher dosage of UV-C irradiation.
Effects of UV-C treatment on the antioxidant activity of okra podsAs shown in Figure 5, antioxidant activity assessed by FRAP assay in control okra pods continuously decreased during storage. Nevertheless, UV-C treatment at 1.5–6.0 kJ·m−2 significantly suppressed the decrease in antioxidants at all storage times. In contrast to the FRAP analysis, there was no significant difference in DPPH inhibition between UV-C irradiation and the control during the first six days of storage. On day 12, DPPH inhibition of okra treated with 6.0 kJ·m−2 UV-C retained the highest value, while no significant difference was observed in untreated okra and with lower doses of UV-C irradiation (1.5 and 3.0 kJ·m−2).
FRAP assay and DPPH inhibition of untreated okra (control; CK) and treated okra with UV-C at different doses during storage at 10°C for 12 days. Results are the means of three replicates ± standard deviation. The different letters denote significant differences among treatments at P < 0.05.
UV-C treatment significantly increased the antioxidant capacity of okra pods and maintained greater antioxidant levels throughout storage than control okra. Several studies have reported that exposure to UV-C increases antioxidant levels. Previous research on okra seedlings found that UV-C treatment enhanced phenolic compounds and antioxidant system activity, which can be used as biomarkers of UV radiation stress intensity (Khorrami et al., 2014).
To identify the factors that contribute to the treatment responses, Pearson’s correlation analysis was performed among these measurements during okra storage. As shown in Table 1, mucilage was positively correlated with antioxidant properties such as FRAP and DPPH, as well as with bioactive compounds such as ascorbic acid, flavonoid, and phenolic compounds. Additionally, weight losses showed a negative relationship with antioxidant properties and bioactive compounds. However, no significant correlation between colors (L* and a*) and other parameters was detected.
Pearson’s correlation coefficients of weight loss (WL), L*-value, a*-value, texture (TT), ascorbic acid (AsA), flavonoid (Fla), phenolic compound (Phe), FRAP, DPPH, carotenoid (Car), and mucilage (Muc) in okra.
Our study found little effect of carotenoid as an antioxidant in irradiated okra, suggesting that there may be other compounds, such as ascorbic acid, that contribute to the antioxidant activity. Alternatively, concurrent oxidation processes may have interfered with the results. UV-C radiation has been shown to enhance flavonoids and phenolic compounds in many species, while also increasing antioxidant activity. Moreover, okra mucilage carbohydrates have antioxidant activity that can prevent cell damage caused by reactive oxygen species and increase the levels of superoxide dismutase (SOD), favoring the antioxidant mechanism (Dantas et al., 2021; Zhang et al., 2018).
In summary, our study suggests that UV-C treatment at 1.5–6.0 kJ·m−2 maintains okra firmness and prevents weight loss. The significant increase in antioxidant activity corresponds to the presence of bioactive compounds such as ascorbic acid, phenolics, and flavonoids and may protect the plants against the major deleterious effects of UV-C irradiation. The response of bioactive compounds and antioxidant activity to UV-C irradiation was dose-dependent. Mucilages are generally normal products of metabolism formed within cells (intracellular formation), and are produced without injury to the plant (Jani et al., 2009). However, the effect of UV-C on the induction of okra mucilage during storage and the pathway involved remain unclear and require further investigation. The obtained results lead us to conclude that applying postharvest UV-C treatment has the potential to increase the levels of bioactive compounds in okra and preserve its quality during storage.
