Biofortification and antioxidant improvement of onion bulbs using calcareous algae and storage

ABSTRACT Along the line that health-beneficial foods must also be accompanied by sustainable agricultural practices, the red alga Lithothamnium sp. (Rhodophyta), frequently used for animal and human nutrition, it was shown that it could be a biofertilizer, regarding their bioactive humic acid content, released by micronization. Also, onions present well-known benefits to health and are among the main vegetable crops grown worldwide. Thus, the objective this work was to evaluate the effects of foliar sprays with micronized Lithothamniun sp. on yield, mineral nutrients, flavonoids, phenolic content, and antioxidant activity before and after storage of bulbs of two organically grown onion cultivars. The yield, mineral content, antioxidant activity, and phenolic and flavonoid content in onion bulbs were improved through sprays, highlighting the dose of 1.5 g L-1 of Lithothamnium sp. in solution. Genotype interactions and storage effects were observed. The benefits with the use of Lithothamnium sp. as biofertilizer were towards the biofortification of organically grown onions by improving mineral nutrient acquisition as it was followed by upgrading antioxidant capacity.


INTRODUCTION
Onions (Allium cepa) are among the main vegetable crops grown worldwide.It isa bulb with a typical flavor sold and consumed in different ways, such as seasoning, cooked, fried, and dehydrated, with many beneficial effects on health (Marrelli et al., 2019).It has been present in human life since 3500 BC (Arshad et al., 2017).In addition to its unmistakable aroma, the onion is also well known for its nutritional attributes, as a source of calcium, phosphorus, and other essential nutrients.(Corzomartinez et al., 2007).
Onion have been used for their medicinal effects since ancient times.In addition, its antibacterial, anti-inflammatory, and antioxidant activities have been widely investigat-ed (Zamri & Hamid, 2019).Therefore, onions are part of our diet, not only for their flavor but for their functionality in the human body (Marrelli et al., 2019).
In this sense, healthy food production gains relevance health-beneficial foods should be accompanied by sustainable agricultural practices.Following this nature-friendly approach, the effect of algae extracts on the improvement of onion growth and yield has already been reported, regarding their biostimulant/ biofertilizer action (Sharma et al., 2014;Szczepanek et al., 2017;Mógor et al., 2021).
Recently, the red alga Lithothamnium sp.(Rhodophyta), frequently used in animal and human diets (Zenk et Aline Novaski et al. al., 2018), was reported as a natural plant biostimulant, promoting better growth related to the release of humic acid through a micronization process (Amatussi et al., 2020;Mógor et al., 2021).
The Lithothamnium sp. is a calcium-based alga, composed mainly of calcium and magnesium carbonate.It can absorb minerals from the aquatic environment and transform them into compounds that can be absorbed by plants (Melo & Moura, 2009).The depositions of calcareous algae at the bottom of the ocean could follow organic matter transformation, similar to that in the soil, where the algae organic fraction is being transformed into bioactive humic substances, promoting plant growth and yield (Amatussi et al., 2020).
Considering the importance of onion in the human diet, combined with the need to present natural alternatives to grow onions, the objective of this work was to evaluate the effect of micronized Lithothamniun sp. on yield, mineral nutrients, flavonoid, phenolic content, and antioxidant activity of organically grown onions.Also, to compare antioxidant profile in bulbs, before and after storage, considering that the onion storage by family farmers is customary to achieve better market prices despite causingbiochemical changes in the bulbs (Marrelli et al., 2019).; N = 30.3g kg −1 ; P = 8.5 g kg −1 ; K = 6.6 g kg −1 ; Ca = 8.1 g kg −1 ; Mg = 4.1 g kg −1 .The moisture was kept at 80% through irrigation and tensiometer.

Growing conditions and plant material
After the dispersion of the compost, beds were made with a dimension of 1.20 x 24 m.Four rows were spaced by 25 cm between them and 10 cm between plants, equivalent to a plant population of 250.000 per hectare.Two onion cultivars often used by organic growers in southern Brazil were chosen: i) Br-29, an open pollination cultivar, producing white, rounded, with dark yellow skin and ii) a hybrid cultivar "Perfecta F1", a white, rounded, with golden yellow skin, both short-day type from TopSeed ® , Agristar ® . .The sample humic acid concentration was determined through UV-VIS spectrophotometry and the ratio E4/E6 was calculated through readings at 465 nm and 665 nm in triplicates (Javanshah & Saidi, 2016), achieving a humic acid concentration of 31.36 μg.g −1 and E4/E6 of 1.35, indicating high humification degree (Saab & Martin-Neto, 2007).The sample (micronized powder) was suspended in deionized water at two concentrations: 1.5 g L -1 and 3.0 g L -1 (maximum soluble concentration) just before they were sprayed on the leaves of the onion plants.

Treatments
Ten foliar sprays of Lithothamnium sp.suspensions (Lit) were performed, starting 15 days after seedlings were transplanted and repeated weekly, using a pressurized sprayer at constant pressure (40 psi).The control plants were sprayed with deionized water.The plots with 1.20 × 1.0-m were distributed in a completely randomized design with four replications (n = 4).The plants were managed according to Brazilian regulations for organic agriculture.

Harvest and storage
Bulbs were harvested at 120 days after sowing, clas-Biofortification and antioxidant improvement of onion bulbs using calcareous algae and storage sified according to the mass and diameter following the Brazilian market classification.The yield was determined with bulbs in commercially viable classes: IV (70 mm to 90 mm) and class III (50 mm to 70 mm).Bulbs were randomly selected and used for biochemical and nutritional analysis.
The samples were separated, and the bulbs were randomly characterized as "at harvest" (Har) and "after storage" (Sto).The Har samples were immediately submitted to determinations of minerals, antioxidants, total phenolic, and total flavonoid contents.The Sto samples went on to the storage shed, where they remained for 60 days at room temperature (25 °C +/-2 °C).After, they were submitted to antioxidant, total phenolic content, and total flavonoid content analyses.

Mineral analysis
For nitrogen content analysis (N-total), bulbs were weighed into 15 mg samples.They were packed in tin capsules for posterior dry combustion in a CHONS analyzer.For determination of phosphorus (P), potassium (K), magnesium (Mg), calcium (Ca), manganese (Mn), iron (Fe), zinc (Zn) and copper (Cu) contents, samples with 0,3 g of dry bulb mass were used.These samples were diluted in HNO 3 and dissolved in H 2 O 2 .Then, the nutrient contents were determined using induction induced plasma optical emission spectroscopy Perkin Elmer Optima 4300 (ICP-OES) (Perkin Elmer, USA), in triplicate.

Antioxidant activity
The measurement of the sequential activity of the DPPH radical (1,1-diphenyl-2-picrilidrazil) was performed according to the methodology applied by Brand-Williams et al. (1995).To assess the antioxidant activity, measures of 0.05 g were used from the bulb samples (Cur and Har) diluted in 2 mL of distilled water and centrifuged for 5 minutes, then the supernatant was collected.After collecting 0.1 mL of each sample, and transferring to test tubes, 4.9 mL of the DPPH solution (0.1 mL) were added, then they sat in a dark room for 40 minutes for the reaction.The same procedure was carried out in a test tube

Total phenolic compounds
The content of total phenolic compounds was determined by the Folin-Ciocalteu spectrophotometric method using gallic acid as a reference standard (Folin & Ciocalteu, 1927).Folin Ciocalteu's reagent is a solution of complex polymeric ions formed from phosphomolybdic and phosphotungstic heteropoly acids.This reagent oxidizes the phenolates, reducing the acids to a blue Mo-W complex, in an alkaline medium.A measure of 0.5 g of each sample (no-cure and post-cure separately) was taken for extraction in 20 mL of methanol: water (40:60) solution, processed in a centrifuge for 20 minutes in two 10-minutes steps.The supernatant was transferred to a 25 mL volumetric flask and filled with distilled water.This extraction was stored in a refrigerator for later analysis.From this extract, a 0.1 mL aliquot was taken and transferred to a test tube, where 1.5 mL of distilled water were added, and then 0.1 mL of the reagent Folin Ciocalteu.This procedure was repeated in all 24 samples tested at a time, and by the time the last test tube was reached with the Folin-Ciocalteu reagent, enough time had passed to start adding 0.3 mL of sodium carbonate at 20% in the first tubes.The tubes were shaken and placed in a grid to take the water bath at 40 °C for 30 minutes.
Then, the absorbance reading was performed at 740 nm in a spectrophotometer.

Flavonoid content
The determination of flavonoid content was done according to the methodology proposed by Woisky & Salatino (1998).The technique is based on the absorbance measurement, at 420 nm, of the complex formed between the flavonoid and the aluminum of the color reagent, forming yellowish compounds.A measurement of approximately 0.05 g was taken from each sample (no-cure and post-cure separately), placed in test tubes and diluted in 2 mL of distilled water.A blank tube was made with only water and reagent.The tubes with samples were centrifuged and 0.5 mL of each were selected, transferred to other clean tubes, in duplicate, where 0.5 mL of 2% aluminum chloride were added to each tube.
All samples were incubated for one hour.Then the samples were read at 420 nm on a spectrophotometer.

Statistical calculations
The experiment was conducted in a completely randomized design, and the data collected were subjected to a factorial scheme being: i) cultivars (2) x treatments with Lit (2 plus control) for yield and mineral content, and ii) cultivars (2) x treatments with Lit (2 plus control) x harvest/storage (2) for antioxidant activity, total phenolic compounds, flavonoid content.The analysis was performed using Assistat statistical assistance software.

Yield
The foliar sprays of Lithothamnium sp.suspensions (Lit) improved the average yield of the cultivar Perfecta-F1 (PF) (Table 1).Yield increments were 26.7% for Lit 1.5 g L -1 and 21.7% for 3.0 g L -1 over the control.

Minerals
The foliar sprays of Lit influenced the content of all nutrients in onion cultivars (Fig. 1).Interactions were found among cultivars and treatments for nitrogen (N), iron (Fe), zinc (Zn), and copper (Cu), and effect of treatments at both cultivars on average for phosphorous (P), potassium (K), calcium (Ca), magnesium (Mg) and manganese (Mn).
Regarding the interactions among cultivars and treatments, the N content (Fig. 2a) in BR-29 (BR) bulbs was incremented by Lit at both concentrations (1.5 g L -1 and 3.0 g L -1 ).Perfecta-F1 (PF) presented a higher content in the control-treatment, indicating genotype differences among cultivars.The Fe content (Fig. 2f) was incremented only in BR by Lit 1.5 g L -1 , while Cu (Fig. 2i) was incremented only in BR by both Lit concentrations.On the other hand, Zn (Fig. 2g) content was incremented in PF by Lit 1.5 g L -1 and in BR by both Lit concentrations, yet when comparing cultivars, PF had higher Zn content than BR.
Regarding to both cultivars on average, the treatments showed similar effects for the contents of P (Fig. 2b) and K (Fig. 2c), being incremented by Lit 1.5 g L -1 over the control.Whereas, both Litconcentrations did not differing each other for P and K, and also Lit 3.0 g L -1 not differing from control, indicating that doubling Lit concentration did not increment P and K uptake by onion.The Ca content (Fig. 2d) was incremented only by Lit 1.5 g L -1 , while Mg and Mn were incremented by both Lit concentrations, showing differences in each other, with Lit 1.5 g L -1 promoting higher Mg and Mn accumulation in the onion bulbs than 3.0 g L -1 .
In general terms, the foliar sprays of Lithothamnium sp.micronized suspensions at 1.5 g L -1 were efficient to improve the mineral content in onion bulbs, in which genotype interactions are taken into account.

Antioxidants
Data in Table 2 indicate that the total content of flavonoids (Flav) was affected by the three factors (Lit x 'Cult' x Har/Sto).At Har, both Lit treatments have increased Flav over the control in PF, not affecting BR.After Sto, both Lit increased Flav over the control in BR, while Lm 1.5 g L -1 improved Flav content in PF by 26.7% over the control.
The phenolic content (Phe) in bulbs was affected by Lit, 'Cult' and Sto; however, without interaction between them.Comparing Har/Sto, the Phe was higher at Har in comparison to after Sto.The comparison between 'Cult' Table 1: Yield of two organically grown onion cultivars (PF = Perfecta-F1, BR = BR-29) submitted to foliar sprays with solutions containing Lithothamnium sp.micronized (1.5 g L -1 and 3. 0 g L -1 ) Average yield (ton ha

C x T *
Onions cultivars: P = Perfecta F1; B = BR 29.Means followed by the same letter do not differ statistically at the 5% probability level by the Tukey test (n = 4) ± SD.Upper case letters at lines, lowercase letters at columns.ANOVA: ns = not significant, * and ** significant at p 0.05 and 0.01 respectively.C = cultivars.T = treatments and C x T = interactions.
Biofortification and antioxidant improvement of onion bulbs using calcareous algae and storage showed that Phe was higher in PF bulbs than BR and the comparison betweenLit, showed that Phe was improved at Lit 1.5 g L-¹ by 23.5 % over the control, but not differing from 3.0 g L -1 which in turn did not differ from control.
The antioxidant activity (Aox) was affected by Har/Sto and Lit treatments.Comparing Har/Sto, the 60-day storage at room temperature improved Aox by 17.4%.The Lit at 1.5 g L-¹ improved Aox by 23% over the control, but not differing from 3.0 g L -1 , which in turn, did not differ from control.
In general terms, Lit 1.5 g L -1 increased Flav, Phe and Aox in bulbs.The Sto also increased Flav and Aox but reduced Phe content in bulbs.It could be seen in 'Cult' that PF was richer in Phe than BR, although PF had responded mainly to Lit before.On the other hand, BR showed responses after Sto.

DISCUSSION
The effect of humic substances (HS) is widely discussed in literature in regards to their auxin-hormone like action on plant metabolism, promoting increases in the H + ATPase enzyme activity with consequences in the cell expansion and biomass accumulation (Canellas et al., 2015).
The effect on onion yield through the use of HS was previously reported, showing improvements on bulbs biomass and caliber (Bettoni et al., 2016), increasing marketable yield of bulbs, as well as enhancing the average weight of bulbs through foliar application (Kandil et al., 2013), which are effects also presented by Lit applications (Table 1).Aline Novaski et al.
The experiment with HS is, in general, was conducted using the mineral leonardite obtained from mining, a source used in biostimulants to promote plant growth (Du Jardin, 2015).The novelty in this work is that HS is released from calcareous red algae through micronization, showing improvements on onion yield by 26.7% for Lit 1.5 g L -1 and 21.7% for 3.0 g L -1 over the control (Table 1), an achievement that is in line with the sustainable agricultural practices.
The effect of HS on improving root growth can also improve the mineral uptake by onion plants (Gemin et al., 2019).Considering that the diets of over two-thirds of the world's population lack one or more essential mineral elements, agronomic practices to increase the concentration of minerals on food (biofortification) are strategic for an adequate human nutrition (White & Broadley, 2009).Thus, Lit treatments improved the nutritional quality of onion bulbs (Fig. 1).
The N content in food supply is determinant in terms of dietary protein adequacy (Shaheen et al., 2016), in this sense, Lit has contributed by improving N content in bulbs on average up to 23.7% through foliar sprays with 1.5 g L -1 .Just as well, Lit has showed effects over major mineral nutrients in bulbs, those that play key roles in our body Analysis done at harvest (Har) and after storage (Sto).Onion cultivars -'Cult' (PF = Perfecta-F1, BR = BR-29).Lithothamnium sp.micronized -Lm (1.5 g L -1 and 3.0 g L -1 ).For flavonoids, ANOVA found triple interaction (Har/Sto x 'Cult' x Lm) = upper case letters at lines, lowercase letters at columns.There were no interactions for phelonics, with significances (Har/Sto, 'Cult', Lm) on averages X ̄.There were no interactions for antioxidant activity, with significances (Har/Sto, Lm) on averages X ̄.Means followed by the same letter do not differ statistically at the Tukey test = * (p ≥ 0.05) and **(p ≥ 0.01).(n = 4) +/−SD.
The significant increases in mineral concentrations in bulbs promoted by Lit could be related to the achieved biochemical changes (Table 2) as the nutrients may participate in secondary metabolites pathways in plants, such as calcium, magnesium, iron and manganese, linked to the biosynthesis of Phe and Aox (Lattanzio et al., 2009).
Phenolics are known to be the largest group of secondary metabolites in plants, varying from single aromatic rings to more complex ones.Phenols are divided into several groups such as phenolic acids and Flav (Sharma et al., 2019).Onions are a recognized source of Flav, strongly linked to the Aox, with beneficial roles in human health as often discussed (Rodrigues et al., 2017).
As a consequence of Lit sprays, the Aox of bulbs were improved, as well the Phe and Flav content, effect which was more pronounced at 1.5 g L -1 .
The Aox and Phe often increase during storage (Rodrigues et al., 2017).However, a decrease was observed in to its other varied Phe (Kefeli et al., 2003).
The increase in Aox compounds in onion bulbs can bring many benefits for their consumers (Rodrigues et al., 2017), enforcing the advantages of Lit sprays to grow onions beyond the biofortification, with the improved mineral nutrient acquisition, which is followed by a better antioxidant capacity.
solution, under the evaluated conditions can improve yield and mineral content in onion bulbs, antioxidant activity in bulbs with their phenolic and flavonoid contents.

CONCLUSIONS
Foliar sprays with micronized Lithothamnium sp.
solution can improve yield and mineral content in onion bulbs also the antioxidant activity in bulbs improving their phenolic and flavonoid contents.

Figure 1 :
Figure1: Contents of nutrients in bulbs of two organically grown onion cultivars submitted to foliar sprays with solutions containing Lithothamnium sp.micronized (1.5 g L-1 and 3.0 g L-1).For N, Fe, Zn and Cu, upper case letters = cultivars, lowercase letters = treatments.For P, K, Ca, Mg and Mn, the ANOVA did not found interaction among cultivars and treatments, being lowercase letters = treatments.PF = Perfecta-F1 light gray column, BR = BR-29 dark gray column.Means followed by the same letter do not differ statistically at the 5% probability by the Tukey test.Bars represent standard error.

Phe
after Sto in comparison to 'Cult'.Such decrease could be related to metabolic changes to other phenolics in BR over the Sto period, such as phenolic acids, and other, not detected by the analytical method.These same acids may have been degraded or transformed during Sto, as increases in Aox and Flav concentration were observed after Sto.The greatest Aox potentials of onions are in its Flav, compared

Table 2 :
Values of antioxidants (flavonoid, phenolics and antioxidant activity) in onion bulbs of two organically grown cultivars submitted to foliar sprays with solutions containing Lithothamnium sp.micronized (1.5 g L -1 and 3.0 g L -1 )