Extended Post-Harvest Eﬀect of Melatonin in Fresh-Cut Broccolini Plants (Bimi ® )

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Introduction
The Broccolini plant, also called baby broccoli, Chinese broccoli, Asparation, Broccole i, Tender Steam, or Bimi, was originally developed in 1999 by the Sakata Seed Company of Yokohama (Japan) as a hybrid of broccoli and Chinese kale plants (Brassica oleracea italica × alboglabra).They were patented and registered as Bimi ® [1].Broccolini belongs to the Brassicaceae family, and it is a green vegetable that resembles broccoli but has smaller florets and longer, thinner stalks.This family has numerous uses, such as in human and animal feed, condiments and seed oils, biofuel, biofumigant, and applications in medical issues, among many others.It should be mentioned that the most frequently utilized plant in research, Arabidopsis thaliana L., is a member of this family.The European production of Brassicaceae (Cruciferae) plants is estimated at approximately 70 million tons per year.Broccoli, broccolini, cauliflower, cabbage, collard greens, turnips, and radishes are just a few of the vegetables that have been singled out for their high consumption; all these vegetables are cultivars that come from Brassica rapa L. and Brassica oleracea L., which have a wide range of varieties and/or subspecies [2][3][4].
The consumption of fruits and vegetables is a key point in diminishing the risk of cardiovascular disease, stroke, inflammatory diseases, and some types of cancers [5,6].These vegetal-rich diets have been associated with the intake of numerous secondary metabolites and their great varieties in natural phytochemicals.Brassicaceae family plants, such as cabbage (Capitata group), cauliflower (Botrytis group), broccoli (Botrytis group), broccolini (Botrytis and Alboglabra groups), and kale (Acephala group), constitute very well-known crops with globally relevant nutritional and economic significance.In general, Brassica species contain a high content of carotenoids, phenolics (flavonoids and glucosinolates mainly), and dietary fiber and vitamins (A, C, and K).Most studies have focused on the presence of secondary metabolites, especially glucosinolates, even though horticultural brassicaceous plants are good suppliers of fiber, vitamins, and minerals.After a cell injury, such as mechanical damage, infection (fungi, bacteria, or parasites), or insect a acks, the cell degrades and the glucosinolates that were retained are now accessible to degradative enzymes, principally myrosinases and/or thioglucosidases, leading to various biochemical reactions that generate glucosinolate by-products (isothiocyanates, thiocyanates, etc.) [7][8][9].The biosynthesis of glucosinolates has been extensively explored and characterized, with three steps: (i) recruitment of precursor amino acids and sidechain elongation, (ii) creation of the core glucosinolate structure, and (iii) certain sidegroup alterations [10].Because of their high concentrations of glucosinolates and their derivative by-products, cruciferous vegetables have a strong negative relationship between their consumption and carcinogenic and cardiovascular illnesses.The mechanisms of action of glucosinolates remain a focus of significant research [11][12][13][14].However, epidemiological evidence shows that consuming isothiocyanates may reduce the incidence of cancer and other human health disorders [15].
In addition to glucosinolates and their by-products, the Brassicaceae are also a rich source of carotenoids such as lutein, zeaxanthin, β-carotene [16], and phenolic components such as flavonoids (apigenin, kaempferol, luteolin, quercetin), glycosides, hydroxylcinnamates, phenolic acids (caffeic and gallic), and anthocyanins [17][18][19].However, the perishability of broccoli and broccolini plants is a very important point during the postharvest period; above all, their visual and sensory quality is very diminished.Furthermore, due to their low durability their content in secondary metabolites, such as glucosinolates, can be rapidly reduced and, consequently, their nutritional, biochemical, and health properties are diminished.Respiration and transpiration are the most important processes that affect the storage life and quality of vegetables and regulating these processes, as well as the post-harvest life and quality of the product, can be prolonged and supported.Generally, cold maintenance below 5 °C is usually a common strategy during storage and transportation, being used to reduce the physiological and biochemical events associated with decay.However, because not all markets can sustain a comprehensive cold chain, alternative solutions such as modified-atmosphere packaging, light quality control, and chemical regulation are recommended [20][21][22].The control of the relative humidity is also a post-harvest strategy used with Brassica plants, which should be higher than 75% to avoid transpiration and weight loss [23].The blue-green color of the product is usually the main index of commercial quality for Brassica plants, since the appearance of yellowish tones due to the degradation of chlorophylls is the first visible symptom of the senescence of the floral tissue or florets.For that reason, pigment maintenance, especially chlorophylls, in Brassica plants is a very important step during the postharvest conservation, and many strategies are applied [24,25].
Melatonin (N-acetyl-5-methoxytryptamine) is an indolic molecule derived from tryptophan identified in cows that functions as a hormone in vertebrates, intervening in the regulation of processes such as locomotion, circadian rhythms, appetite, sexual behavior, and processes related to the immune system, among others [26,27].Melatonin (also known as phytomelatonin) was discovered in plants in 1995 by several researchers at the same time [28][29][30].Since then, several studies have been conducted to be er understand the role of melatonin in plant physiology [31][32][33].Melatonin has been demonstrated to influence nearly all plant responses, which enhances processes such as germination, growth, flowering, etc. [21,22,34,35]; however, the most essential melatonin activity is as a stress mediator, where it intervenes in biotic stress situations caused by herbivores, fungi, bacteria, parasites, and other plants, and abiotic stress processes related to salinity, drought, chemicals, UV-radiation, and physical and mechanical damage, among others [36][37][38][39][40][41][42][43][44][45].
Melatonin plays a significant function in regulating the biochemical and physiological aspects of the post-harvest physiology of fruits and vegetables [20][21][22].Some of the interesting results that have been obtained by studying the role of melatonin in post-harvest are delaying senescence and improving quality in plants [37], inhibition of browning of fresh-cut pear fruit [46] and litchi fruit [47], delaying post-harvest senescence and physiological deterioration of cassava [48] and Chinese cabbage [49], and improving the nutritional quality of tomatoes [50].Melatonin treatments in broccoli might be a useful method for preserving the quality of fresh-cut florets throughout cold storage and transportation [20,23,[51][52][53][54][55].
Considering the known data on the use of melatonin as a post-harvest conservation agent, the objective of this work is to extend the useful life of broccolini through treatments with natural substances, and that these are adaptable to current handling and marketing protocols for this vegetable.
We present original research on the efficacy of melatonin to promote broccolini preservation under cold commercial conditions.Changes in weight loss, color, chlorophyll, and carotenoid contents, as well as their potential impact on antioxidative enzymes and related parameters of broccolini, were studied for the first time in the presence of exogenous melatonin as a possible post-harvest expansive agent.

Chemicals
Sigma-Aldrich Co. (Madrid, Spain) provided the solvents, chemicals, and reagents needed.In this investigation, ultra-pure water from the Milli-Q system (Milli-Q Corp, Merck KGaA, Darmstadt, Germany) was employed.Spectrophotometric measurements were performed using a Perkin-Elmer Lambda-2S UV-visible spectrophotometer (Überlingen, Germany) with a controlled temperature at 25 ± 0.1 °C by use of Thermo-Haake D1G equipment (Karlsruhe, Germany).

Plant Material
The place where the broccolini (Brassica oleracea italica × alboglabra) plants were harvested at commercial maturity was in Lorca (37°40′16.28″N; 1°42′6.12″W) from the region of Murcia (Murcia, Spain) by a horticultural company.To reduce bruising and to preserve moisture, the gathered samples were stored in polystyrene boxes with ice.Broccolini plants were transferred to our laboratory, where sections of uniform size and color, free of visible illness or injury, were chosen at random for treatment.

Broccolini Treatments
Ten broccolini plants were submerged in distilled water (as control without melatonin for 30 min) or in different melatonin solutions (as treatments, 50 and 100 µM) prepared in water for 15 and 30 min, respectively.The broccolini sections were then air-dried for 30 min.All processes were carried out at room temperature.Following that, each broccolini treatment group was covered in clear plastic film and kept in the dark for 28 days at 5 °C and 75% relative humidity in a controlled cold room.Each treatment was carried out three times.To measure weight loss and color, samples were taken at 0, 7, 14, and 28 days.Broccolini were then quickly frozen in liquid nitrogen and kept at −80 °C for further measurement of pigment (chlorophyll and carotenoid) levels, total antioxidant activity, total phenolic and total flavonoid contents, lipid peroxidation by MDA content determination, and the antioxidant enzymes catalase and peroxidase [56].

Weight Loss
Weight loss was estimated by weighing broccolini before and after the storage period.The values were presented as the weight loss rate related to the storage time, expressed as grams per day (g/day).

Color
Hue Angle (color parameter) values were measured at one spot on the surface of four broccolini using an automated colorimeter PCE-XXM 20 (PCE Instruments, Tobarra, Spain), calibrated according to the manufacturer's specifications.The Hue angle values were determined following the equation H = 180° + tan −1 (b*/a*) when a* < 0 and b* > 0 [57].

Determination of Chlorophyll and Carotenoid Contents
The variations in the chlorophyll and carotenoid contents were measured as described by Lichtenthaler and Wellburn [58].To extract the pigments, a mortar was used to ground the frozen broccolini florets (0.3 g) with 5 mL acetone (80%) three times.The extraction obtained was centrifuged at 10,000× g for 10 min at 4 °C.Absorbances at 470, 645, 652, and 663 nm to estimate the concentrations of chlorophylls a and b, total chlorophylls, and carotenoids were recorded.The measures were conducted in triplicates.Results were expressed in mg of chlorophyll (a or b or total) or mg of carotenoid/g FW.

Determination of Total Antioxidant Activity
Antioxidant capacity was measured using our ABTS/H2O2/HRP method [59].Briefly, 0.2 g of plant material (broccolini florets) and 5 mL ice-cold buffer (50 mM potassium phosphate, pH 7.0) were ground to obtain the extract.Then, the extract was centrifuged at 10,000× g for 10 min at 4 ℃.An aliquot of extract was mixed with the medium reaction containing radical ABTS, and the diminution in absorbance at 730 nm, which is proportional to the ABTS radical quenched, was determined after 6 min.A standard curve of ascorbic acid (AA) was used to estimate the antioxidant capacity by evaluating the similarity between the values of our samples with the values from the standard curve of AA. Results are expressed as milligrams of ascorbic acid equivalents per gram of fresh weight (mg eq.ASC/g FW).The determinations were made in triplicates.

Total Phenolic Content
Some modifications were made in the process described by Singleton and Rossi to determine the total phenolic compounds by using Folin-Ciocalteu reagent in the samples [60].Briefly, to generate the extract, 0.2 g of plant material (broccolini florets) were ground in the presence of 5 mL ice-cold buffer (50 mM potassium phosphate, pH 7.0).The obtained extract was centrifuged at 10,000× g for 10 min at 4 °C.Then, 50 microliters of sample were transferred into a glass tube containing 50 µL of 1 M sodium carbonate, 50 µL of Folin-Ciocalteu reagent, and 950 µL of distilled water, and after it was mixed, it was left in a water bath at 30 °C for 15 min, and the absorbance at 715 nm was recorded.The standard used was gallic acid.The total phenolic content of the samples was expressed as milligrams of gallic acid equivalent per gram of fresh weight (mg GAE/g FW).The determinations were made in triplicates.

Total Flavonoid Content
The total flavonoid content was determined by applying the aluminium chloride colorimetric method, modified by Woisky and Salatino [61].A calibration curve was made from 25, 50, 100, and 150 µg•mL −1 of quercetin prepared in 80% ethanol and diluted to obtain the concentration of each standard.The broccolini extracts were obtained by grounding 0.2 g of broccolini florets and 5 mL ice-cold buffer (50 mM potassium phosphate, pH 7.0).The extracts were centrifuged at 4 ℃ for 10 min at 10,000× g.To determine the flavonoid content samples or standard solutions, 0.5 mL was transferred into a glass tube containing 2.8 mL of distilled water, 1.5 mL of 95% ethanol, 0.1 mL of 10% aluminium chloride, and 0.1 mL of 1 M potassium acetate and mixed.The final step consisted in an incubation for 30 min at room temperature, and finally the wavelength used to determine the absorbance of the reaction mixture was 415 nm.The units used to express the results were milligrams of quercetin equivalents per gram of fresh weight (mg QE/g FW).Experiments were conducted in triplicates.

Determination of Malondialdehyde Content
The thiobarbituric acid (TBA) method was assayed to measure the malondialdehyde (MDA) content, a product of lipid peroxidation, which is used to determine cell membrane damage [62].Briefly, the homogenized solution constituting of 0.2 g of frozen plant material (broccolini florets) and 5 mL of 5% trichloroacetic acid (TCA) was obtained and then centrifuged at 10,000× g for 10 min at 4 ℃.Then, 1 mL of the supernatant obtained was mixed with 4 mL of TCA (20%) and TBA (0.5%) solution.The mixture was heated at 90 °C for 30 min in a water bath to stop the reaction and the solution was transferred to an icebath for rapid cooling and then centrifuged at 10,000× g for 10 min at 4 °C.Absorbances at 450, 532, and 600 nm were recorded.MDA was used as a standard by preparing a standard curve, and the expression of the results were milligrams of MDA per gram of fresh weight (mg MDA/g FW).The determinations were made in triplicates.

Measurement of the Antioxidant Activity of Enzymes
To determine the activities of the antioxidant enzymes catalase (CAT) and peroxidase (POD), a plant extract was obtained as follows: fresh plant tissue of broccolini florets (0.3 g) was homogenised in ice-cold 50 mM potassium phosphate buffer (pH 7.0).After centrifugation of the homogenate (10,000× g for 10 min at 4 °C), a supernatant was obtained that was used as the source to measure CAT and POD activities.The decomposition of H2O2 at 240 nm for 2 min was used to estimate the CAT activity.The reaction mixture was made of 50 mM phosphate buffer (pH 7.0), 15 mM H2O2, and 100 µL of enzyme extract.The activity was expressed as UI/g FW (µmol formed product/g FW) and an extinction coefficient of 43.6 mM −1 cm −1 was used [63,64].The monitorisation of ABTS oxidation, traduced in the increase in absorbance at 414 nm, was used to estimate the POD activity.The extinction coefficient used in this measurement was 31.1 mM − 1 cm −1.The reaction mixture was made of 50 mM glycine buffer (pH 4.5), 0.2 mM H2O2, ABTS 0.5 mM, and 10 µL of enzyme extract.The activity was expressed as UI/g FW (µmol formed product/g FW) [65].The determinations were conducted three different times.

Statistical Analysis
SigmaPlot graphics program version 14.0 (SYSTAT Software Inc., Palo Alto, CA, USA) was used to create the graphics.The statistical significance of the results was tested using a two-ways analysis of the variance (ANOVA) with the statistical analysis software SPSS 20 (IBM, Chicago, IL, USA) with a confidence level of 95.0% (p < 0.05).All the data were presented as means ± SE.

Results and Discussion
Based on the results of the preliminary study in melatonin-treated broccoli florets with different immersion times, and stored at 5 °C, it was decided to study the outcomes of storage at a low temperature (5 °C) on MEL-treated broccolini.Based on the results previously obtained with broccoli, the concentrations of 50 and 100 µM were selected because they maintained the initial color throughout the storage time compared to all the other treatments applied [55].The immersion time is also a relevant parameter; it must not be too short that it avoids the good absorption of melatonin by the tissues, and it must not be too long that it can hinder the production chains of broccolini in the vegetable packaging industry.Therefore, we selected two treatment times per immersion, at 15 and 30 min, according to the previous study on broccoli [53,55].

Effect of Melatonin on Weight Loss
One of the most important aspects during the storage at 5 °C of fresh-cut broccolini plants is weight loss (WL) because of the water loss.This process could cause stalk hardening and bud-cluster turgidity loss [66,67].Figure 1 shows that over all monitored days, the WL rate was consistently greater in the control samples than in the MEL-treated samples.Melatonin treatments show that melatonin acts by delaying water loss and, consequently, weight loss.This occurs because melatonin has a function related to the stomata in plants, intervening in the process of the closing and opening of these plant structures [22,68].Of all the treatments, the 15 min melatonin treatment was the most effective, reducing water loss around 28 and 21% compared to the control after 14 and 28 days of storage, respectively, and 100 µM melatonin at 30 min was the least effective in reducing water loss and weight loss.

Effect of Melatonin on Color and Pigments
The Hue angle (H) parameter (Figure 2) was used to measure yellowing in broccolini plants, leading to a decrease in the visual quality of these vegetables [20,57].The initial H values in fresh-cut broccolini samples were around 133 degrees (day 0), remaining constant during the first 7 days of storage in all cases.At 28 days of storage, the H value of the control samples dropped to 128 degrees, however, 15 min MEL-treatments maintained the H values around 133 degrees for 28 days of storage.Moreover, the color of melatonintreated broccolini is conserved, indicating that the broccolini plants' shelf life could be increased by a MEL-treatment, reaching a storage period of 28 days with acceptable color.The determination of chlorophyll and carotenoid contents was carried out (Figure 2) as a way to establish their correlation with the Hue angle values obtained in all samples.The catabolism of chlorophylls results in the loss of the green/blue characteristic color of these plants and the apparition of the yellow color, a symptom related to a time-dependent process known as senescence.
The total chlorophyll content, as result of chlorophyll a and b contents (Figure 3A,B), showed a decline in the non-treated (control) samples during the cold storage time, indicating that the blue-green color of the samples has been lost due to postharvest senescence (Figure 3C); however, in the case of the MEL-treated plants, the loss of chlorophyll (blue/green color) is lower, showing similar or high chlorophyll content at 7 and 14 days than at initial state (day 0).On day 28, only 50 µM melatonin with the 15 min treatment showed total chlorophyll values similar to those of the control, showing that the other treatments had a negative response (Figure 3A-C).These differences in chlorophyll content support the values of the Hue angle obtained in the different treatments.
In a similar way, the total carotenoid contents did not have a significant variation up to 14 days in the melatonin-treated broccolini plants (Figure 3D), and with an important carotenoid decrease in control samples.After 28 days of storage, only 50 µM melatonin with the 15 min treatment showed total carotenoid content values similar to those of the control.

Effect of Melatonin on Antioxidative Status: Antioxidant Activity, Total Phenolic, Flavonoid, Malondialdehyde Contents and Enzymes
During cold post-harvest, fresh-cut broccolini suffers progressive deterioration, which is reflected in its antioxidative status, the levels of phenolic compounds, and damage to cell membranes.Figure 4A shows the values of total antioxidant activity (TAA).This parameter reflects the antioxidative status of plants through their free radical scavenger capacity [65].Melatonin is a good antioxidant molecule, as can be observed in practically all treatments, increasing the TAA during the cold storage time compared to non-treated samples (control samples) at 7, 14, and 28 days.At 28 days, the control samples' TAA decreased by up to 50% compared to day 0; in contrast, samples treated with melatonin for 28 days presented a high TAA, close to that of 7 and 14 days (Figure 4A).
Regarding the phenolic content, an increase in the total content of phenolics (Figure 4B) was observed, with generally higher values in the samples treated with melatonin compared to the control, throughout the storage time.However, 100 µM and the 30 min treatment does not seem to be effective in maintaining the total phenolic levels.The total flavonoid content is shown in Figure 4C, where higher values of total flavonoid content are observed in the samples treated with melatonin compared to the control during 7 and 14 days of storage.The content in total flavonoids seems to be similar among the control and melatonin treatments for 28 days of storage.Post-harvest cold damage is usually related to the malondialdehyde (MDA) content of tissues, a by-product of the lipid peroxidation of biological membranes [52].Broccolini control samples showed high values of MDA content at 7, 14, and 28 days due to cold storage (Figure 4D).Melatonin treatments (specially 50 µM) induced a minor MDA content due to the protective antioxidant action of melatonin on the membranes [56,69].
The enzymatic activities of catalases and peroxidases in broccolini samples during cold storage have been studied.Melatonin-treated samples showed a decrease in catalase activity compared to control samples at each storage time (Figure 5A), while peroxidase activity increased (Figure 5B).This dual effect can be explained by the different mode of action of melatonin in stress conditions [70][71][72].Thus, while melatonin co-participates with catalases as a scavenger of hydrogen peroxide, it is not necessary for the transcriptional induction of these enzymes.In the case of peroxidases, it seems that the up-regulation of these enzymes occurs with its effect on the increase in enzymatic activity at 7 and 14 days, and especially in the case of 100 µM for 30 min melatonin treatment, at 14 and 28 days.

Conclusions
MEL-treatments via immersion induced positive effects in broccolini plants from a post-harvest focus, in a manner comparable to broccoli florets.Figure 6 shows a model of action for melatonin, summarizing all the effects exerted in the broccolini plant's postharvest process.The most relevant effects can be observed in the reduced weight loss of melatonin-treated broccolini during cold storage time.The application of melatonin treatments on broccolini plants may diminish the respiration and transpiration rate, resulting in a decrease in water and weight loss.Moreover, the melatonin treatments' effect on the chlorophyll and carotenoid contents of broccolini plants is reflected by an increase in the duration of conservation of these vegetables, showing a higher commercial shelf life.The application of melatonin as a post-harvest agent could maintain the green-blue color of broccolini plants, extending the shelf life of fresh-cut broccolini plants by almost 7 days with respect to melatonin-untreated plants.The data on the levels of antioxidant activity, total phenolics, and total flavonoids were higher in the melatonin-treated plants.These higher levels of antioxidants could be implicated in their minor degradation, improving their human functional properties such as nutritional, biochemical, and health properties.The levels of malondialdehyde, as a marker of lipid peroxidation of membranes, is lower in broccolini plants treated with melatonin, which revealed a minor level of damage in these plants such that the quality and visual characteristics are extended over time.This study demonstrates for the first time the effectiveness of melatonin treatments in extending the shelf life of broccolini during cold storage between 7 and 14 days with respect to melatonin-untreated broccolini, as well as its possible commercial use.These treatments by immersion with melatonin are presented as economically affordable for any company or agricultural cooperative specialized in the commercialization of fresh vegetables and does not require machinery or complex treatments.h ps://www.um.es/en/web/phytohormones/, accessed on 10 September 2023 (Phytohormones and Plant Development Lab).

Figure 1 .
Figure 1.Broccolini plants' weight loss (WL) rate by treatment (0, 50, and 100 µM MEL) for 15 and 30 min, and stored for 7, 14, and 28 days in the dark at 5 °C.Data represent mean value for each treatment, different le ers indicate statistical significative differences using Tukey HSD test with p value < 0.05.

Figure 2 .
Figure 2. Hue angle for broccolini plants treated for 15 with 0, 50, and 100 µM of melatonin (MEL), and stored for 7, 14, and 28 days in the dark at 5 °C.Data are the mean value ± standard error (n = 3) for each treatment, different le ers denote statistical significative differences at a p-value < 0.05 using a Tukey HSD test.Insert: graphical representation of H value in the CIE L*C*h Color Space.

Figure 3 .
Figure 3. Chlorophyll a (A), chlorophyll b (B), total Chlorophyll (C), and carotenoid (D) content in broccolini treated with 50 and 100 µM of MEL for 15 and 30 min and stored in the dark at 5 °C for 7, 14, and 28 days.Data are the mean value ± standard error (n = 3) for each treatment, different le ers denote statistical significative differences at a p-value < 0.05 using a Tukey HSD test.

Figure 4 .
Figure 4. Antioxidant activity (A), total phenolics compounds (B), total flavonoids (C), and malondialdehyde (D) content in broccolini treated with 50 and 100 µM of MEL for 15 and 30 min and stored in the dark at 5 °C for 7, 14, and 28 days.Data are the mean value ± standard error (n = 3) for each treatment, different le ers denote statistical significative differences at a p-value < 0.05 using a Tukey HSD test.

Figure 5 .
Figure 5. Catalase (A) and peroxidase (B) activity in broccolini treated with 50 and 100 µM of MEL during 15 and 30 min and stored in the dark at 5 °C for 7, 14, and 28 days.Data are the mean value ± standard error (n = 3) for each treatment, different le ers denote statistical significative differences at a p-value < 0.05 using a Tukey HSD test.

Figure 6 .
Figure 6.Schematic illustration of the exogenous melatonin treatments' effect on broccolini florets during the post-harvest period.Green and red arrows represent increase and decrease in the process, respectively.