The Post-Harvest Application of UV-C Rays: Effects on the Shelf Life and Antioxidants of Fresh Green Asparagus (Asparagus officinalis L.)

Abstract

UV-C irradiation is an innovative postharvest technique for increasing the safety of fruits and vegetables. This study investigated the effect of UV-C rays (UV-C1 = 0.26 KJ/m²; UV-C2 = 0.40 KJ/m²; UV-C3 = 0.67 KJ/m²; and UV-C4 = 1.34 KJ/m²) on the preservation of the antioxidants, hardness, and color of fresh green asparagus during storage. UV-C1 and UV-C2 significantly maintained higher total phenolic content (10.6%), total flavonoid content (36%), rutin (14.3%), quercetin (27.03%), kaempferol-3-O-rutinoside (21.25%), and antioxidant activity (DPPH 7.5%). Over three weeks of storage, quercetin, ferulic acid, and kaempferol 3-O-rutinoside increased, while rutin and caffeic acid decreased. Storage caused a significant change in the color and hardness of the control sample, but UV-C4 counteracted hardening for up to three weeks, and UV-C3 was the best dose for stabilizing color during storage. This study indicates that the choice of UV-C dose can be modulated based on the characteristics that are intended to be preserved in green asparagus, maintaining a balance between nutraceutical and hedonic characteristics. To maintain the maximum level of nutraceutical compounds over time, UV-C2 can be adopted, while to preserve texture and color, UV-C3 and UV-C4 are a better choice.

1. Introduction

Asparagus (Asparagus officinalis L.) is recognized as a nutritional food due to its low caloric content and high concentrations of fiber and antioxidants such as flavonoids, phenolic acids, and vitamins [1,2], and it is solely eaten in the form of very young, thickened shoots called spears. The two main types are commercially recognized green (and green purple) and white. The green type traditionally has had a much bigger global market. Agronomically, the key difference is whether the shoots are harvested above ground (green) or underground before they reach the surface (white), but botanically, both types are a single species [3]. Global asparagus production is expected to reach 10.7 million metric tons by 2026, with an average annual growth rate of 1.4%. China is the world’s largest producer of asparagus, with 8.6 million metric tons in 2021. Peru, Mexico, and Germany rank second, third, and fourth, respectively [4].

The main flavonoid of green asparagus is represented by rutin, which has the highest antioxidant action [5,6], representing 70% to 90% of the total flavonoid concentration [7]. Rutin has been associated with reducing cholesterol levels and the risk of heart disease and may also have anti-cancer properties [8]. Besides rutin, asparagus contains quercetin and the hydroxycinnamic acids, ferulic and caffeic. Many factors affect the concentration of polyphenols in asparagus, including the cultivar, season, climate, light exposure, and portion of asparagus (top, middle, basal, and root) [6,9]. Polyphenols, particularly rutin, are present in high concentrations in green asparagus exposed to a high intensity and longer duration of sunlight [9,10], being produced in plants as a defense molecule against the UV irradiation in sunlight [11].

Asparagus is a highly perishable vegetable, and during storage, the quality of the spear is reduced due to hardening and a series of biochemical reactions [12]. Physiological changes during storage, which cause water and nutritional losses and infection by microorganisms, increase discarding and economic losses. About 30% of postharvest losses are due to spoilage caused mainly by Escherichia coli, yeasts, and molds [13]. Lignification is the main cause of hardening, which is a variation in texture, one of the main factors that determines the sensorial and cooking quality of fresh asparagus. Recent investigations have established that hardening is caused by the modification of cell wall composition and structure linkages by ferulic acid [14]. Moreover, the amount of ferulic acid increases during postharvest storage, and in white asparagus, the direct involvement was established of two dimers of ferulic acid, i.e., 8-0-4′diferulic acid (8-0-4′-di FA) and 8,5′-diferulic acid benzofuran (8,5′-BA di FA), in the increase in hardness during storage [15].

During senescence, phenylalanine ammonia lyase (PAL) is involved, and peroxidase (POD) acts as an antioxidant response in plant tissues. These enzymatic activities are involved in lignin accumulation and in the defense system, triggering a response from the antioxidant enzymes and antioxidant substances naturally present in vegetables [16]. A loss of quality is also noticeable from loss of the bright green color, which, in addition to bending, decreases postharvest quality [17]. Consequently, optimization of postharvest handling and processing, as well as storage conditions, is essential to reduce economic losses and improve asparagus supply chain management. Low temperatures and high humidity are commonly used conditions for many horticultural products to control senescence and increase shelf life [18].

In recent years, other methods for the preservation of fresh products have been developed, including cold plasma [19], ultraviolet (UV) irradiation [20], and gamma irradiation [21]. UV irradiation is widely used as an alternative to chemical sterilization and for the reduction in microorganisms in food products. The application of UV-C radiation to plant products brings significant benefits, such as reducing pesticide use and extending shelf life, which in turn reduce losses. Furthermore, modern LED lamps have very low electricity consumption and can be disposed of without harming the environment. There are four categories based on wavelength range: UV-A (315–400 nm), UV-B (280–315 nm), UV-C (200–280 nm), and vacuum UV (100–200 nm) [22]. The UV-C range has a high antimicrobial efficacy. UV-C can cause, according to some authors [23,24], biological stress in plants and trigger the defense mechanisms of plant tissues, with consequent increases in antioxidant compounds. The main documented effects of UV-C as a postharvest treatment are to reduce respiration rates [25], deterioration in fruit tissues [26], and delaying senescence and ripening in several fruit and vegetable species, such as apples, citrus fruits, tomatoes, peaches, grapes, and guavas [27]. As a result of the high economic value of green asparagus and its high perishability, there has been increase in the demand for new and milder technologies to enhance its shelf life. The short shelf life of asparagus is due to a high respiration rate, which continues after harvesting. Moreover, the domestic storage period is very short and not longer than 3–5 days [12].

To our knowledge, there is little information about the effect of different doses of UV-C on the maintenance of freshness attributes and antioxidant compounds of green asparagus during storage. Therefore, the present study aims to investigate the effects of different doses of UV-C on green asparagus, to identify the best dose to reduce the effect of lignification on hardness and color, and, in the meantime, to save the antioxidant compounds for up to 3 weeks.

2. Materials and Methods

2.1. Plant Materials

All samples of the green asparagus (Asparagus officinalis L. cv. Grande) were harvested from a crop grown in compliance with the local agronomic practices (Foggia, Italy) on the same day (8 May 2023), and immediately transported to the laboratory. Spears were sorted by a mean diameter of 1.1 ± 0.2 cm, cut to a length of 15 cm, which represents the edible portion, and the remaining portion was discarded. The UV-C lamps and treatment area were enclosed in a box covered with aluminum foil. In Figure 1, the scheme of the treatment chamber is shown. Before use, the UV lamps were allowed to stabilize by turning them on for at least 15 min. Durations were 2, 3, 5, and 10 s; distance from the lamp to sample was 5 cm. Thereafter, 5 batches of 40 spears were subjected to the treatments with the following UV-C doses: 0.26 KJ/m² (UV-C1), 0.40 KJ/m² (UV-C2), 0.67 KJ/m² (UV-C3), and 1.34 KJ/m² (UV-C4), using 3 UV-C lamps (T8 30 W G13 UV-C light source, 12.6 W—254 nm, LEDVANCE SpA, Milan, Italy), continuously rotating the sample, to improve uniformity by exposing all surfaces to the UV-C light, in addition to the control sample. The doses used were chosen to fit them to industrial postharvest handling lines. The doses applied were determined by adapting them to the speed of the conveyor belt via which the asparagus was selected. The samples were treated with different doses of UV-C at T0 immediately upon harvest, stored in a cold room (G. Dentamaro, Bari Italy) (4 °C and 70% humidity) together with the untreated control for three weeks: (T0), 7th (T1), 14th (T2), and 21st (T3) days after the treatment [24].

2.2. Proximate Composition

Protein content was determined using the Dumas combustion nitrogen method, according to the AACC Approved Method 46–30.01 [28], and using FP528 (Leco Corp., Saint Joseph, MO, USA). A factor of 6.25 was used to convert the nitrogen to protein. All analyses were conducted in triplicate, and data were given as g/100 g dry matter (dm). Fiber content was determined through the Total Dietary Fiber assay kit (Megazyme International, Bray, Ireland), as proposed by Prosky et al. [29], and expressed as grams per 100 g of dry matter (g/100 g dm).

2.3. Hardness Determination

Asparagus hardness was measured by a TA. XT plus texture analyzer, equipped with a 25 kg load cell (Stable Micro System, Godalming, UK), according to Menga et al. [30], with a Warner-Bratzler blade. Hardness was quantified along the 15 cm of the edible spear selected. It represented the maximum force to cut the tips and the middle portion of the spears; the values were the mean of five measurements for each portion (tip and middle) and were expressed as Newton (N). The instrument was set up with a test speed of 1 mm/s and a travel distance of 30 mm.

2.4. Color Measurement

The color was registered using a Minolta CR200 colorimeter (Minolta Corp., Ramsey, NY, USA). Before use, the instrument was calibrated with a ceramic reference, CIE-L*a*b* coordinates, and D65 illuminant. Registered parameters were L* (lightness: L* = 0 means black, L* = 100 means maximum light intensity), a* (−a* = greenness, +a* = redness), and b* (−b* = blueness, +b* = yellowness). Three measurements were made on the tip and middle portion of each sample, and the mean values for the tip and middle are reported in the table separately. The difference between the color of the control and the color of the UV-C sample is calculated by Equation (1) according to Mokrzycki and Tatol [31]:

$$∆\mathrm{ELab} = \sqrt{{{(∆\mathrm{L}}^{*})}^2 + {{(∆\mathrm{a}}^{*})}^2 + {{(∆\mathrm{b}}^{*})}^2}$$

(1)

where ∆L* = L* sample (UV-C sample during each storage time) − L* control (at T = 0); ∆a* = a* sample (UV-C sample during each storage time) − a* control (at T = 0); and ∆b* = b* sample (UV-C sample during each storage time) − b* control (at T = 0).

2.5. Extraction and Analysis of Phenolic Compounds

Phenolic compounds were extracted according to Menga et al. [30], with minor modifications. Asparagus samples were freeze-dried (LIO 2000P, 5Pascal s.r.l., Trezzano Sul Naviglio, MI, Italy) and milled using a planetary mill with an agate jar and balls (Pulverisette 7 Planetary Micro 200 Mill, Classic Line; Fritsch GmbH Milling and Sizing, Idar-Oberstein, Germany) to obtain a fine powder. The samples, comprising 240 mg of ground powder, were extracted using 8 mL methanol acidified with 1% HCl (80:20), for 30 min in an ultrasonic bath at room temperature. The mixtures were centrifuged at 2000× g for 15 min at room temperature, and the supernatants were stored at −20 °C until further analysis, as reported below.

2.6. Total Phenolic Content (TPC)

TPC was determined using the Folin–Ciocalteu assay according to Suriano et al. [32] Briefly, 200 µL of extract was added to 1.5 mL of 10-fold diluted Folin–Ciocalteu reagent, then the solution was equilibrated for 5 min and mixed with 1.5 mL of 6% (w/v) sodium carbonate solution. After incubation at room temperature for 90 min, the absorbance was measured at 725 nm with a UV/Vis spectrometer (Lambda Bio20, PerkinElmer, Inc., Springfield, IL, USA). All assays were performed in triplicate, and the results were reported as mg/g of ferulic acid equivalents (mg FA/g dm).

2.7. Total Flavonoid Content (TFC)

TFC was determined according to Kim et al. [33] with minor modifications. Briefly, 250 µL of each extract was added to 1.25 mL of distilled water and 75 µL of 5% (w/v) NaNO₂. Six minutes later, 150 µL of 10% AlCl₃ (w/v) was added to the mixture. Then, 500 µL of 1M NaOH was added after five minutes, and the total volume was made up to 3 mL with distilled water. The solution was mixed, and its absorbance at 510 nm was measured with a UV/Vis spectrometer (Lambda Bio20, PerkinElmer, Inc., Springfield, IL, USA). All assays were performed in triplicate, and the results were reported as mg of catechin equivalents/g (mg CE/g dm).

2.8. Antioxidant Activity Measured by ABTS and DPPH Assays

Trolox equivalent antioxidant capacity (TEAC) was determined according to the procedure described by Fares et al. [34] with minor modifications. ABTS (2,2-azino-bis-[3-ethylbenzothiazoline 6-sulphonic acid]) was dissolved in water to a 7 mM concentration. ABTS radical cation was produced, allowing the reaction of the ABTS stock solution with potassium persulfate (2.45 mM, final concentration) for 12–16 h in the dark and at room temperature before use. ABTS radical cation solution was diluted with ethanol to an absorbance of 0.70 ± 0.02 at 734 nm (UV/Vis spectrometer Lambda Bio20, PerkinElmer, Inc., USA). TEAC values of the extracts were calculated, using a Trolox standard curve (0–15 µM), based on the percentage inhibition of absorbance at 734 nm and expressed in µmol of Trolox equivalents (µmol TE)/g sample on dry matter (dm).

DPPH assay (2,2′-diphenyl-1-picrylhydrazyl radical scavenging capacity assay) was determined according to the procedure of Suriano et al. [32] with some modifications. DPPH radical solution, having an absorbance value of 0.80 at 525 nm, was prepared daily by dissolving 5 mg of DPPH in 100 mL of a methanol/water mixture (50:50, v/v). For the determination of the DPPH scavenging activity, 20 µL of each sample was added to 4.98 mL of DPPH solution. The reaction mixture was incubated at 30° C in the dark for 30 min, and then the absorbance at 517 nm was measured. DPPH values of the extracts were calculated, using a Trolox standard curve, based on the percentage inhibition of absorbance at 525 nm and expressed in µmol of Trolox equivalents (µmol TE)/g sample on dry matter (dm).

2.9. Phenolic Acids and Flavonoids Content Quantification

The phenolic acids and flavonoids were determined according to Menga et al. [30]. The extracts were analyzed on a 1200 HPLC instrument (Agilent Technologies, Waldbronn, Germany) equipped with a diode array detector, and metabolite separation was achieved using a reverse phase C18 column (InfinityLAB Poroshell 120 RC-C18, 100 mm; 2.1 mm; particle size = 2.7 m) (Agilent Technologies, Santa Clara, CA, USA). The column temperature was maintained at 35 °C. The mobile phase consisted of (A) water with phosphoric acid 10-3 M and (B) acetonitrile at a flow rate of 0.5 mL/min, using the following linear gradient program: 5% B for 2.0 min, from 5% to 30% B for 10 min, from 30% B to 55% B for 1.0 min, from 55% to 70% for 2 min, isocratic at 70% for 1.0 min, linear gradient from 70% to 5% B for 6 min. Five microliters of the sample were injected using an autosampler. The wavelengths used for quantification of the phenolic acids were 280 and 320 nm. Phenolic compounds were identified by their characteristic UV/vis spectra and comparison of the retention times with those of pure standards and were quantified by the corresponding calibration curves (0–50 mg/L) and expressed as µg/g dm. The solvents were HPLC grade; solvents and standard compounds were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

2.10. Statistical Analysis

A two-way analysis of variance was performed on three replicates for each trait; means comparisons were carried out by the “Least Squares” method and separated by a post hoc Tukey’s test, and statistically significant differences were determined at the significance level of α = 0.05 (p < 0.05). To obtain a general, comprehensive characterization of the UV-C treatments and storage time, the traits were subjected to principal component analysis (PCA) based on correlations. The statistical treatments of the data and PCA were performed using the JMP software (SAS Institute Inc., Cary, NC, USA, version 8).

3. Results

3.1. Asparagus Antioxidants Variation: Effect of UV-C Treatment, Storage Time, and Their Interaction

All phenolic acid, flavonoid, and antioxidant activity levels were affected by the UV-C treatment at nearly every evaluated intensity (

Table 1. Main effects of UV-C on antioxidants.

Traits	UV-C Treatments
	Control	UV-C1	UV-C2	UV-C3	UV-C4
TPC (mg FAE/g)	17.01 ± 1.53 b	16.19 ± 1.97 b	18.81 ± 1.52 a	19.28 ± 1.14 a	19.28 ± 1.53 a
TFC (mg CE/g)	3.64 ± 0.74 c	4.96 ± 0.57 a	4.60 ± 0.86 ab	4.30 ± 0.51 ac	4.16 ± 0.54 bc
ABTS (μM TE/g)	20.86 ± 1.26 b	19.01 ± 1.02 c	19.58 ± 1.00 bc	23.18 ± 1.36 a	23.89 ± 3.26 a
DPPH (μM TE/g)	32.54 ± 1.64 c	34.81 ± 1.28 ab	34.99 ± 1.78 a	33.27 ± 1.15 bc	33.83 ± 3.22 ac
Chlorogenic acid (μg/g)	200.76 ±147.33 b	221.76 ± 80.02 b	310.43 ± 195.81 a	143.43 ± 69.73 c	137.04 ± 177.48 c
Ferulic acid (μg/g)	101.51 ±15.96 bc	84.86 ± 24.22 c	86.27 ± 30.22 c	109.31 ± 31.86 ab	125.06 ± 48.55 a
Caffeic acid (μg/g)	326.75 ± 51.90 bc	377.47 ± 55.49 a	298.82 ± 136.94 c	347.59 ± 72.58 ab	357.49 ± 108.81 ab
trans-Cinnamic acid (μg/g)	37.77 ± 23.00 b	39.07 ± 20.70 b	50.50 ± 29.26 a	32.13 ± 13.24 b	31.25 ± 17.01 b
Rutin (μg/g)	5138.52 ± 1307.42 c	5497.53 ± 410.77 b	5874.36 ± 1103.94 a	5504.32 ± 928.05 b	5156.94 ± 1226.05 c
Quercetin (μg/g)	279.60 ± 49.74 c	300.77 ± 71.14 bc	355.18 ± 49.21 a	307.18 ±68.38 b	314.94 ±75.21 b
Kaempferol-3-O-rutinoside (μg/g)	167.05 ± 28.64 b	170.55 ± 29.86 b	203.10 ± 37.63 a	169.81 ±12.46 b	158.21 ± 16.08 b

Data represents the average UV-C ± SD values calculated over all storage times. Data are expressed as follows: TPC = total phenolic content; TFC = total flavonoid content; ABTS = 2,2-azino-bis-[3-ethylbenzothiazoline 6-sulphonic acid]; DPPH = 2,2′-diphenyl-1-picrylhydrazyl. Values in the same row followed by different letters are significantly different (Tukey test p < 0.05).

and Supplementary Table S1). TPC content significantly increased after UV-C2 by about 10.6% and the content did not vary up to UV-C4. TFC at UV-C1 increased significantly by about 36% and reached the highest value; the further application of UV-C2 and UV-C3 did not show a significant variation, but a significant drop was observed at UV-C4 with respect to UV-C1. ABTS at UV-C1 was significantly affected with a decrease of 8.85%; at the subsequent UV-C levels, significant increases were observed, and the highest value was measured at UV-C4 with an increase of about 14.5%. When compared to the control, DPPH showed a significant increase up to UV-C2 of about 7.5%, but the UV-C3 and UV-C4 applications had no effect, and the antioxidant activity (AA) measured at these intensities was the same as the control. Chlorogenic acid also showed a similar increasing trend up to UV-C2, but a significant drop in UV-C3 and UV-C4 of about 28.6% was observed. In respect of the control, ferulic acid increased significantly at UV-C3 and UV-C4 (7.7% and 23.2%, respectively) while caffeic acid increased significantly only in UV-C2 samples; rutin, quercetin, and kaempferol-3-O-rutinoside significantly increased up to UV-C2 of 14.3%, 27.03%, and 21.25%, respectively.

Storage time (

Table 2. Main effects of storage time on antioxidants.

Traits	Storage Time
	T0	T1	T2	T3
TPC (mg FAE/g)	17.53 ± 1.77 ab	17.40 ± 1.76 b	18.74 ± 2.59 ab	18.79 ± 2.03 a
TFC (mg CE/g)	4.66 ± 0.75 a	4.52 ± 0.73 ab	4.21 ± 0.86 ab	3.93 ± 0.59 b
ABTS (μM TE/g)	20.41 ± 1.69 b	21.19 ± 2.46 b	22.80 ± 3.46 a	20.81 ± 2.05 b
DPPH (μM TE/g)	34.16 ± 1.31 a	34.13 ± 1.51 a	35.10 ± 2.32 a	32.15 ± 2.09 b
Chlorogenic acid (μg/g)	99.76 ± 39.20 c	109.38 ± 23.90 c	256.09 ± 128.775 b	345.51 ± 115.95 a
Ferulic acid (μg/g)	103.67 ± 17.93 b	97.27 ± 20.50 bc	123.28 ± 53.14 a	81.38 ± 21.48 c
Caffeic acid (μg/g)	396.93 ± 50.91 a	379.55 ± 72.06 a	328.62 ± 72.23 b	261.40 ± 105.13 c
trans-Cinnamic acid (μg/g)	19.66 ± 4.22 c	20.67 ± 5.03 c	49.21 ± 8.57 b	63.04 ± 20.35 a
Rutin (μg/g)	6477.31 ± 524.87 a	5878.37 ± 375.26 b	5030.87 ± 1000.21 c	4350.79 ± 616.53 d
Quercetin (μg/g)	233.50 ± 30.13 c	287.37 ± 52.09 b	362.71 ± 25.70 a	362.56 ± 39.32 a
Kaempferol-3-O-rutinoside (μg/g)	170.38 ± 14.56 b	153.15 ± 18.26 c	201.05 ± 35.51 a	170.39 ± 26.53 b

Data represents the average storage time ± SD values calculated over all UV-C. TPC = total phenolic content; TFC = total flavonoid content; ABTS = 2,2-azino-bis-[3-ethylbenzothiazoline 6-sulphonic acid]; DPPH = 2,2′-diphenyl-1-picrylhydrazyl. Values in the same row followed by different letters are significantly different (Tukey test p < 0.05). T0 = the first day of each UV-C treatment; T1 = 7th day of storage; T2 = 14th day of storage, and T3 = 21st day of storage.

and Supplementary Table S1) affected the content of all phenolic acids, flavonoids, and antioxidant activities. The TPC, after a slight decline at T1, showed a significant increase at T3 (7.19% compared to T1). The TFC showed a constant decrease throughout the storage times, with a loss of 15.66% going from T0 to T3. The antioxidant activity measured by ABTS showed no significant difference during the storage time, except for T2, where it reached the highest and significant value. On the contrary, no variation over time was observed for the AA measured by DPPH, which remained constant until T2. Only at T3 was a significant decrease of 5.88% measured. These behaviors were associated with variations in phenolic acids and flavonoids. Chlorogenic acid and trans-cinnamic acid showed a notable variation over time, with a minimum increase going from T0 to T1 (9.64% and 5.14%, respectively) but almost three-fold and four-fold at T2 and T3, respectively. Quercetin also showed an increasing trend over time, reaching 55% more at time T2 and remaining constant at T3. Ferulic acid and kaempferol 3-O-rutinoside showed the highest concentration at T2 with respect to T0, with an increase of about 18% for both metabolites. Rutin and caffeic acid showed a similar decreasing trend with a percentage of 9.24%, 22%, and 32% for T1, T2, and T3 in the case of rutin, and with a percentage decrease of 4.38%, 17.21%, and 34.14% at T1, T2, and T3 for caffeic acid.

As the main flavonoids in asparagus are quercetin and rutin, and the main hydroxycinnamic acids are ferulic and caffeic acids, the interaction of UV-C and storage for these compounds was evaluated (Figure 2 and Supplementary Table S1). A decreasing trend over time for rutin was observed, more significant in the control, where a loss of 48% from T0 to T3 was measured. In the samples treated with UV-C, rutin losses ranged from about 10% to 39% from T0 to T3, and only the UV-C2 treatment allowed the maintenance of high levels of rutin up to the second week. The opposite behavior was observed for quercetin, where the mean increases over time from 32% to 86%. Furthermore, all of the UV-C treatments enhanced the quercetin content up to the second week and maintained it until the third week. Ferulic acid showed significant increases in the second week, both with UV-C3 and UV-C4. Caffeic acid decreased during storage in all the UV-C-treated samples and the control.

3.2. Asparagus Shelf Life and Nutritional Composition: Effect of UV-C Treatment, Storage Time, and Their Interaction

The UV-C main effects are shown in

Table 3. Main effects of UV-C on hardness, color, and nutritional compounds.

Traits	UV-C Treatments
	Control	UV-C1	UV-C2	UV-C3	UV-C4
Hardness (N)	33.12 ± 6.89 bc	36.57 ± 8.62 a	36.11 ± 8.97 a	35.78 ± 9.23 ab	32.54 ± 8.59 c
Fiber content (%)	32.19 ± 3.14 a	31.83 ± 3.26 a	30.52 ±1.90 a	30.51 ± 1.98 a	30.14 ± 2.49 a
Protein (%)	38.02 ± 2.51 b	37.68 ± 2.05 c	39.71 ± 2.45 a	35.59 ± 1.32 d	35.58 ± 1.25 d
L* Tips	40.80 ± 1.65 a	40.56 ± 2.35 a	40.77 ± 3.12 a	41.49 ± 3.80 a	42.33 ± 3.78 a
a* Tips	−4.17 ± 1.49 bc	−2.33 ± 1.78 a	−2.63 ± 1.72 ab	−4.16 ± 2.08 bc	−5.04 ± 2.48 c
b* Tips	14.87 ± 1.58 a	11.43 ± 1.82 b	12.21 ± 1.87 b	14.37 ± 3.14 a	15.13 ± 2.83 a
L* Middle	46.45 ± 2.16 a	43.78 ± 2.11 bc	43.09 ± 1.28 c	45.37 ± 2.46 ab	43.82 ± 1.82 bc
a* Middle	−11.06 ± 2.04 b	−7.50 ± 3.73 a	−9.63 ± 2.80 ab	−11.00 ± 3.19 b	−9.73 ± 3.34 ab
b* Middle	24.24 ± 2.98 a	18.96 ± 2.47 c	21.50 ± 3.44 b	22.22 ± 4.00 ab	20.75 ± 3.73 bc

Data represents the average UV-C ± SD values calculated over all storage times. Values in the same row followed by different letters are significantly different (Tukey test p < 0.05).

and Supplementary Table S1. Regarding hardness, an increase up to UV-C2 was observed; thereafter, there were no statistical differences from the control at UV-C3 and UV-C4. The proteins reached the highest content with UV-C2 and showed significant decreases at UV-C4 with respect to the control. No significant effects were observed for the fiber content. The value of L* in the Tips did not show significant differences with increasing doses of UV-C, while in the Middle section, it showed a decreasing tendency. The Tip color parameters a* and b* had similar behavior, and it is observed that with UV-C3 and UV-C4, both the a* and b* values remained unchanged when compared to the control; vice versa, the lower doses (UV-C1 and UV-C2) determined a significant variation. The a* value increased by 44% and 37% at UV-C1 and UV-C2, respectively. Regarding the b* value, it decreased by about 23% and 17% at UV-C1 and UV-C2, respectively. Also, in the Middle section, the values of a*, b*, and L* varied due to the UV-C treatment. However, at UV-C3, it was observed that all the color parameters approached those of the control.

The main effects of storage time are reported in

Table 4. Main effects of storage time on hardness, color, and nutritional compounds.

Traits	Storage Time
	T0	T1	T2	T3
Hardness (N)	30.59 ± 6.66 b	35.20 ± 8.84 a	36.07 ± 8.86 a	37.43 ± 8.94 a
Fiber content (%)	31.45 ± 2.33 ab	28.37 ± 3.32 b	30.97 ± 3.10 ab	33.36 ± 2.86 a
Protein (%)	36.17 ± 1.65 b	35.46 ± 1.06 c	38.84 ± 2.16 a	38.79 ± 3.00 a
L* Tips	39.88 ± 2.28 b	40.54 ± 1.70 b	41.10 ± 2.82 ab	43.24 ± 3.81 a
a* Tips	−4.31 ± 1.82 a	−3.42 ± 2.13 a	−2.88 ± 1.75 a	−4.06 ± 2.53 a
b* Tips	13.78 ± 2.40 ab	13.15 ± 2.46 b	12.44 ± 2.47 b	15.05 ± 2.88 a
L* Middle	45.36 ± 2.62 a	44.70 ± 2.25 ab	44.22 ± 2.05 ab	43.73 ± 2.10 b
a* Middle	−10.91 ± 2.36 b	−9.49 ± 2.93 ab	−9.99 ± 2.67 ab	−8.75 ± 2.68 a
b* Middle	23.32 ± 2.96 a	20.72 ± 2.78 bc	22.34 ± 3.15 ab	19.76 ± 2.70 c

Data represents the average storage time ± SD values calculated over all UV-C. Values in the same row followed by different letters are significantly different (Tukey test p < 0.05). T0 = the first day of each UV-C treatment; T1 = 7th day of storage; T2 = 14th day of storage; T3 = 21st day of storage.

and Supplementary Table S1. The hardness was significantly influenced by storage, and an increase of 22% from T0 to T3 was observed. Fiber content did not show significant variations over time, unlike protein content, which increased significantly by 7% from T0 to T2 and T3. As regards the color of the spears during storage, in the Tips, an increase in L* can be seen up to T3, while a* and b* did not differ significantly from T0; in the Middle, on the contrary, a decrease in lightness was observed, compared to T0. During the storage time, variations in a* Middle and b* Middle were also observed for T0. The increase observed over time for a* was from −10.91 to −8.75, while regarding b*, it decreased from 23.32 to 19.76.

Figure 3 shows the trend in the samples over time concerning the hardness, and it is highlighted that only the maximum dose of UV-C showed a positive effect on hardening, which tended to reduce up to T3.

To verify whether during the three weeks of storage, the UV-C treatments kept the color of the asparagus samples unchanged, i.e., like the color of the freshly harvested product (measured at T0), we calculated the ∆E. As reported by Mokrzycki and Tatol [31], a clear color difference is noticed between 3.5 < ∆E < 5, identifying a color change perceivable by the eye.

Table 5. Difference between the color of the control and the color of the UV-C samples (ΔE) in the Tips and the Middle of the asparagus spears during storage.

ΔE
		T0	T1	T2	T3
Tips	UV-C1	4.11 ± 0.16	6.50 ± 0.33	1.65 ± 0.52	5.25 ± 0.22
	UV-C2	4.53 ± 0.28	2.1 ± 0.14	1.86 ± 0.07	4.38 ± 0.03
	UV-C3	1.87 ± 0.07	0.85 ± 0.08	2.28 ± 0.11	1.33 ± 0.25
	UV-C4	1.74 ±0.35	0.92 ± 0.11	2.18 ± 0.04	5.14 ± 0.20
Middle	UV-C1	15.64 ± 0.06	1.74 ± 0.06	5.09 ± 0.01	5.76 ± 0.07
	UV-C2	8.44 ± 0.06	4.46 ± 0.08	1.38 ± 0.10	6.00 ± 0.11
	UV-C3	2.21 ± 0.13	1.77 ± 0.11	2.44 ± 0.06	6.85 ± 0.21
	UV-C4	3.95 ± 0.08	4.68 ± 0.28	6.15 ± 0.07	4.76 ± 0.08

T0 = the first day of each UV-C treatment; T1 = 7th day of storage; T2 = 14th day of storage; T3 = 21st day of storage.

shows the measured values. The color variation (∆E) on Tips, caused by the UV-C treatment during the storage time, was perceptible only in the UV-C1 and UV-C2 samples (except for T2). On the contrary, the color variation was not perceptible in the samples treated with the highest doses. During the storage time, the color variation registered in UV-C3 and UV-C4 samples was maintained at low; at time T3, only UV-C4 samples showed an increase in ∆E (5.4). The UV-C treatments caused a more evident color variation in the Middle, during the storage time. Only ∆E in UV-C3 samples showed the same evolution over time observed in the Tips; therefore, it was not perceptible to an observer up to T2. At T3, samples reached the same variation.

3.3. PCA

To evaluate the associations between the effect of UV-C, storage time, and the studied variables, the dataset was subjected to a principal component analysis (PCA), and only the components with eigenvalues ≥ 1.0 were considered as having a major contribution to the total variation [35]. Six principal components were chosen based on this criterion, and the relative percentage of variability explained, as well as the related eigenvectors, are shown in Supplementary Table S2. The first two components explain 30.1% and 19.6%, respectively, of the total variability, and Figure 4 shows the score plot (a) and the loading plot (b) of PC1 and PC2, while Supplementary Figure S1 shows the overview score plot of the six components. The traits of protein, hardness, chlorogenic acid, trans-cinnamic acid, and quercetin were positively correlated with PC1, while rutin and caffeic acid were negatively correlated with PC1 (Table S2). The a* Tips, TFC, and DPPH were positively correlated with the PC2, while b* Tips and ABTS were negatively correlated with PC2, respectively (Table S2). The difference between UV-C treatment and storage time was evident at times T0 and T1, which were separated from T2 and T3 along the PC1 component, while the UV-C1 and UV-C2 were separated from UV-C3 and UV-C4 along PC2. Independent of the storage time, the control was grouped with UV-C3 and UV-C4 (Figure 4a). UV-C1 and UV-C2 treatments were more closely related to the main antioxidant compounds over the storage period. Similarly, hardness and proteins were associated with milder UV-C treatments, although their effects were more evident during the last storage times (T2 and T3). The separation of the samples treated with the highest UV-C doses is more influenced by ferulic acid and the L* Middle, but only at T0 and T1. As highlighted by PCA in our study, the control was grouped with UV-C3 and UV-C4, indicating that high intensity has overall fewer effects than low intensity, especially evident for hardness and antioxidants.

4. Discussion

The quality attributes of whole spears and tips of green asparagus include appearance, color, and texture. According to the EU Commission [36], the color of green asparagus must be green for at least 80% of the length. The tip must be compact, and the spear slightly curved (for the best classification). For consumers, texture is important as well as aroma and flavor. The latter is determined by sugars, organic acids, and phenolic compounds such as rutin and hydroxycinnamic acids [37]. Among phenolic acids detected in the asparagus cell wall, the most representative are p-coumaric and ferulic acids. In our study, the most representative hydroxycinnamic acid was caffeic acid, followed by ferulic acid, but no trace of p-coumaric acid was detected. This difference could be attributed to differences in cultivars and agronomic practices [11]. Regarding flavonoids, quercetin exerts its antioxidative effects through multiple mechanisms. Firstly, it acts as a scavenger of free radicals, neutralizing them and preventing them from causing damage to cell membranes. Additionally, quercetin enhances the activity of superoxide dismutase, catalase, and glutathione peroxidase, which are endogenous antioxidant enzymes, and further contribute to the cellular defense against oxidative stress. The antioxidant effects of quercetin are due to its structural arrangement, which includes several hydroxyl groups attached to different positions on the benzene rings. Moreover, the conjugation of double bonds in the aromatic rings strengthens the antioxidant activity, creating a complex structure that plays a crucial role in quercetin’s bioactivity and physiological effects. Rutin is a glycoside of quercetin, which is a compound made up of quercetin linked to a sugar called rutinose and has a lower antioxidant activity in respect to quercetin [38]. The concentration of rutin in asparagus depends on the variety chosen; moreover, environmental conditions have a significant impact, and the positive effect of sunlight on accumulation, specifically on rutin, has been documented by several authors [11,39].

UV-C could cause an increase in the concentration of bioactive compounds, and as indicated in the present work, the positive effects depended on the dose applied. For TPC, TFC, ABTS, and DPPH, a general increase was observed, but not with all doses. Similarly, it was observed for phenolic acids and flavonoids, including rutin and quercetin, whose content increased at a low dose (UV-C2). There are conflicting opinions in the literature on this topic. Some authors have indicated that high doses of UV-C decrease antioxidants, presumably due to damage to the cell membrane [40]. This would lead to a reduction in antioxidants for their activity in contrast to the oxidative stress. However, other authors have found an increase in antioxidants in fresh-cut mangoes, spinach, tomato, and blueberries with moderate doses of UV-C, like our UV-C doses [41,42,43]. In our opinion, the question of why only some phenolics and flavonoids showed an increase after UV-C treatments is due to their specific involvement in quenching reactive oxygen species (ROS) [38]. The increase in quercetin observed after UV-C treatment can be explained by those mechanisms in which quercetin is involved, firstly as a scavenger of ROS and after as an enhancer of endogenous enzymes, as indicated by the increase in ABTS. Indeed, ROS are produced after UV-C application because they cause direct damage to the cell membrane. Consequently, the most reactive compounds are increased, as observed in the present study for quercetin, and consequently, the activation of endogenous antioxidant enzymes, which in turn determines the increase in ABTS. Unlike other studies, and as already mentioned, the most representative hydroxycinnamic acid was caffeic acid, followed by ferulic acid [11]. Regarding flavonoids, the concentration of rutin in asparagus is also dependent on the variety chosen. In our control sample, rutin content was 6990 µg/g of dry weight, a concentration in agreement with Fuentes-Alventosa et al. [7]. The authors found a concentration ranging between 259 and 763 mg/kg fresh weight of total flavonoids in 32 commercial green hybrids of asparagus; considering that approximately 90% of the weight of this vegetable is water, the concentration can be considered comparable.

Storage time affected the content of all phenolic acids, flavonoids, and antioxidant activities, and as indicated in Table 2, antioxidant activity measured by ABTS showed no significant difference during the storage time, except for T2, where it reached the highest and most significant value. On the contrary, no variation over time was observed for the antioxidant activity measured by DPPH, which remained constant until T2. The observed behaviors were associated with variations in phenolic acids and flavonoids. Ferulic acid and kaempferol-3-O-rutinoside showed the highest content at T2 with respect to T0, with an increase of about 18% for both metabolites. Our results agree with those of Jaramillo et al. [15], who found that during postharvest storage, the amount of cell wall phenolics (mainly ferulic acid) increased significantly. Choosing the right dose of UV-C to apply is important to induce the hormetic effect, that is, the dose that induces the desired effects [44,45]. Our study demonstrated that treatment with low doses of UV-C could be useful to limit the losses of phenolic compounds and flavonoids. The UV-C2 best prolonged the storability of rutin up to two weeks, while for quercetin, all the UV-C tested allowed the maintenance of the highest levels up to three weeks. In effect, our results indicated that the losses of rutin in the UV-C-treated samples were significantly lower than the control. This finding was in accord with Gonzales-Aguilar et al. [41], who studied the effect of UV-C on the storability of antioxidant compounds on mangoes and found an increasing content after UV-C treatments.

Postharvest treatment, with UV-C on asparagus and vegetables in general, could also have an effect in increasing the shelf life of the products, and decreasing ripening and senescence. Our findings indicated that hardness was significantly influenced by storage, while fiber content did not show significant variations over time, unlike protein content, which increased significantly by 7% from T0 to T2 and T3. Thus, the range of UV-C applied in this study did not affect the fiber content, which seemed not to be involved in hardening during storage. Therefore, this result indicated the prevalent involvement of phenolic cross-links in determining hardening during storage as previously found by Jaramillo et al. [15]. This study confirmed that protein content increased during storage [46], but also that the negative effect of the denaturation process caused by the highest UV-C doses can occur, as observed by Csapo et al. [47].

Regarding the interaction of UV-C and storage time on the hardness, only the maximum dose of UV-C showed a positive effect on hardening, which tended to reduce up to T3. Poubol et al. [48] have found that the lowest UV-C doses were the best in preventing hardening, while higher doses did not cause appreciable changes. Our findings showed that the UV-C4 prevented the hardening for a longer period of time than the other doses, suggesting that it may represent the hormetic dose for hardening. Indeed, in the control sample, after the first week (T1), the cultivar Grande tested in this study showed significant hardening, which remained unchanged up to the last week of storage (T3). On the contrary, the application of UV-C4 exerted a positive effect on the hardness, which always remained the lowest for two weeks. A similar effect with low doses of UV-C was observed in red tomatoes [49]. Regarding spear color, several studies [50,51] showed that low doses of UV-C produced no effects, and our results have also confirmed this finding.

Finally, as highlighted by PCA, the control was grouped with UV-C3 and UV-C4, indicating that high intensity has fewer overall effects than low intensity, especially evident for hardness and antioxidants.

5. Conclusions

The application of UV-C on green asparagus was tested in the present study, and the effect during three weeks of storage on antioxidant activity, phenolics, hardness, and color was unveiled. This study has indicated that the UV-C dose can be tailored to the characteristics desired, i.e., to maintain a balance between nutraceutical and hedonic characteristics. To maintain the maximum level of nutraceutical compounds over time, UV-C2 will be adopted, whereas to preserve the texture and color, UV-C3 and UV-C4 will be a better choice. The PCA results indicate that high-dose UV-C (UV-C-3 and 4) were grouped with the control, suggesting their effectiveness in preserving the same characteristics of the fresh product. Finally, the choice of the cultivar with a high content of phytochemicals could also be a good starting point for maintaining high nutritional value during storage. Given the importance of green asparagus cultivation (agronomic, nutritional, nutraceutical, and economic), it would be necessary to expand research on the response of a large panel of cultivars to UV-C. Our work has explored the effects of low doses of UV-C on a single cultivar.