Foliar Selenium Biofortification in Temperate Fruit Crops: Impact on Selenium Accumulation and Nutritional Quality of Fruits and Juices

Abstract

As an essential mineral element, selenium (Se) must be consumed by organisms through food and beverages. A method used to raise the amount of Se in food made from plants is biofortification, which is the process that increases the bioactivity and content of Se in the edible parts of plants. Foliar fertilization is the most feasible method of introducing selenium into the food chain. The objective of this work was to determine the effect of foliar biofortification with Selenium on various quality attributes of fruit species suitable for fruit-based beverage production, with the main goal of verifying the incorporation of Se into plant tissues. During the growing season in 2023, sodium selenate was applied in an equivalent of 150 g/ha Se in professional raspberry, blueberry, redcurrant, honeysuckle and apple plantings, from which fruit-based juice was later produced and analyzed. There was significant increase (p ≤ 0.05) in the Se content in the fruit’s juice, which was the main goal. Furthermore, after the application of Se under the mentioned conditions, there was a significant (p ≤ 0.05) increase in nutritionally valuable parameters, such as antioxidant activity, ferulic acid and resveratrol, but also the content of glucose, fructose, malic acid, total acids, Mn, Ba, Ca, Li, myricetin and chlorogenic acid content. On the other hand, a decrease in some valuable indicators, but also heavy metals (Al, Cu, Cr), were noticed in some fruit juices.

1. Introduction

Mammals require selenium (Se), and dietary deficiencies in this element are a global issue. The goal of agronomic biofortification is to add selenium (Se) to crops so that humans can consume sufficient quantities of it [1]. Dietary selenium has a crucial function in regulating the mineral bioavailability required to produce selenoproteins, which are proteins containing selenocysteine (Sec) and necessary to key physiological pathways [2]. A multitude of chronic illnesses, including cancer, Alzheimer’s disease and thyroid dysfunction, have been linked to selenium deficiency [3]. Food generally has a low Se content. Nearly all the countries in Europe and Asia consume less Se than the daily recommended amount, which is insufficient to support human needs [4]. The World Health Organization states that an adult’s daily intake of selenium should be 55 μg (or 0.055 mg) [5]. The most common way that the human body obtains selenium (Se) is via eating foods like meat and fish, which provide a significant portion of the daily necessary amount of Se [6].

As primary sources of Se biofortification, plants and microbes are regarded as the main functional foods or food components of daily Se supplementation. The current recommendation is for Se-enriched vegetable and arable crops, as well as Se-yeast and Se-algae, to be the main sources of dietary Se for humans [7]. Although this element is not thought to be necessary for plants, it may be advantageous since, at low concentrations, selenium (Se) boosts production, antioxidant content and its concentration in the edible portion of the plant [8].

Enhancing the bioactivity and content of Se in the edible sections of plants is known as biofortification, and it is one method of increasing the content of Se in meals originating from plants [9]. The most practical technique to move selenium into the food chain is by foliar fertilization [10]. Enhancing the production of bioactive compounds and augmenting the amount of vital micronutrients in crops is possible by the application of micronutrients through biofortification [9].

The objective of this work was to determine the effect of foliar biofortification with Selenium on various nutraceutical quality attributes in selected fruit species suitable for beverage production, with the main goal of verifying the incorporation of Se into plant tissues.

2. Materials and Methods

2.1. Experimental Plot

The study was carried out during 2023 in the Dunajská Lužná area, in the southwest part of the Slovak Republic, 130 m a.s.l. The mentioned parcel is in a very warm and very dry agro-climatic area in a temperate climate zone, with an average January air temperature of −2 to 1 °C, an average July air temperature of over 20 °C and an average annual rainfall of 550–600 mm. The characteristics of the experimental plot are shown in

Table 1. Selected characteristics of the conducted study.

Fruit Species	Varieties	Number of Plants	Replications	Spacing	Planting Year
Raspberries	Kwanza, Kweli, Imara, Himbotop, Polka, Sugana	10	3	2.5 × 0.25 m	2021
Blueberries	Duke, Draper	10	3	3.5 × 1 m	2019
Redcurrants	Junifer, Jonkheer van Teets, Rovada	10	3	3.5 × 0.4 m	2019
Honeysuckle	Polaris	10	3	3.5 × 1 m	2019
Apples	Evelina	10	3	4 × 1 m	2018

. The plantings were highly intensive with computer-controlled irrigation and fertigation managed in integrated fruit production systems.

During the growing season, on 12 May 2023, sodium selenate (23.7–24.9% sodium, 39.7–44.0% selenium; Merck Life Science spol. s r.o., Bratislava, Slovakia) was applied at a concentration of 8.55 g per 10 L of water. This corresponds to a dose of 3 g of pure selenium, equivalent to 150 g Se per hectare. The application was carried out with a Stihl SR 200 backpack motor sprayer, Waiblingen, Germany) in windless and clear weather at an air temperature of 18 °C. The application was carried out on the same day for all plant species. Ten plants were Se-sprayed in three replications using a randomized block design in the treated variant. A control group of 10 plants was included, which did not receive any selenium application In addition to this application, the plants were treated as standard in terms of pruning and training, irrigation, nutrition, fertilization, and protection against pests, diseases and weeds.

2.2. Harvest, Storage and Juice Production

Depending on the variety, the fruits were collected at different times according to ripening in plastic tubs with a volume of 250 g, 500 g and 25 kg (apples). The collected fruits were transferred after harvest to the Laboratory of Beverages of Research Center AgroBioTech SAU in Nitra, Slovakia. Samples of small fruits (blueberries, honeysuckle, currants and raspberries) were placed in a refrigerator with a volume of 400 L and frozen at a temperature of −35 °C until their processing. Samples of apples were placed in refrigerators with a volume of 2000 L and stored at 2 °C until they were processed. Fruit processing and juice production were carried out on the day of analysis, depending on the specific analysis, between 6th and 10th of November 2023, by initial thawing 24 h before processing, rinsing with clean tap water and subsequent careful juicing using a Sana Juicer EUJ 707 (Sana, Prague, Czech Republic) horizontal shaft juicer. From berry fruit, 250 g fruit was used for one replication and there were three replications undertaken. From apples, 1000 g was used for one replication, with three replications in total.

2.3. Estimation of TSS, Glucose, Fructose, Total Sugar, Malic Acid, Total Acid Content and Ph

After obtaining the juice, we filtered it on a fine sieve with a mesh diameter of 0.2 mm. Then we collected the filtrate with a 15 mL syringe and applied a few drops into the FT-IR analyzer (AlphaWine Analyzer—module for juices, Bruker Optics, Billerica, MA, USA), where 120 scans were performed, resulting in a display of the analyzed parameters.

The parameters were determined in three replicates for each sample of extracted juice.

2.4. Determination of Total Polyphenols, Total Anthocyanins and Antioxidant Activity

All three parameters were analyzed using a Shimadzu UV/VIS 1240 spectrophotometer (Shimadzu, Japan). TPC was determined using a modified Folin–Ciocalteu method, based on the oxidation of phenolic compounds by a reagent mixture of H₃PW₁₂O₄₀ and H₃PMo₁₂O₄₀ in an alkaline environment, forming a blue-colored complex. The reaction mixture contained 0.2 mL of extract, 1.8 mL of distilled water and 2.5 mL of Folin–Ciocalteu reagent (Merck KGaA, Darmstadt, Germany). After a reaction time of 3 min, 4 mL of 7.5% aqueous sodium carbonate (Na₂CO₃) (Fisher Scientific, Vantaa, Finland) was added. The absorbance was measured at 765 nm after 1.5 h. A calibration curve was constructed using gallic acid in the range of 0.1 to 1.2 mg/L.

TAC was measured using a modified pH differential method based on the transformation of anthocyanins to the red-colored flavylium cation at low pH. One milliliter of extract was mixed with 1 mL of 0.01% HCl (Merck KGaA, Darmstadt, Germany) in 95% ethanol (PGChem, Nové Zámky, Slovakia) in two tubes. The first tube received 10 mL of 2% aqueous HCl (A1), while the second received 10 mL of a pH 3.5 buffer (A2) prepared from 0.2 M Na₂HPO₄ (Merck KGaA, Darmstadt, Germany) and 0.1 M citric acid. Absorbance was recorded at 520 nm, and the difference in absorbance was used to quantify TAC.

Antioxidant activity was assessed using the DPPH radical scavenging method, according to the procedure of [11]. This method measures the ability of antioxidant compounds in the extract to reduce the purple-colored DPPH radical to a colorless form, monitored spectrophotometrically by the decrease in absorbance [12].

2.5. Determination of Elemental Content by ICP-OES

The analysis of the elemental composition of the fruit samples was carried out according to the procedure described in [13] with minor modifications. For microwave decomposition, we used 0.10–0.20 g of a dry sample, which we mineralized in an environment of 8 mL HNO₃ (69%) and 2 mL H₂O₂ (30%) ((Merck KGaA, Darmstadt, Germany); trace purity). For the separation, a gradient flow method of acetonitrile and H₃PO₄ (0.1% v/v) in an initial ratio of 95.5% was used. The mobile phase flow rate was set to 0.6 mL/min during the entire separation, which lasted 25 min. The separation temperature was set to 30 °C. The sample volume injected onto the column was 5 µL. All analyses were repeated 3×.

2.6. Determination of Phenolic Compounds by HPLC-DAD

The section describing the HPLC analysis of individual phenolic compounds and anthocyanins has been significantly expanded to ensure clarity, reproducibility and methodological transparency. The analysis was performed using an Agilent Infinity II HPLC system (Agilent Technologies GmbH, Waldbronn, Germany) equipped with a diode array detector (DAD) [14]. We used methanolic extracts for the analysis, which were filtered prior to injection using a Q-Max syringe filter (0.22 µm, 25 mm, PVDF; Frisenette ApS, Knebel, Denmark).

The separation was carried out on a reversed-phase column SUPERSPHER 100 RP 18.5 μm, 250 × 4.6 mm (Merck, Darmstadt, Germany) maintained at a constant temperature of 30 °C. The mobile phase consisted of two solvents: solvent A—water with 10% formic acid (v/v), and solvent B—a mixture of methanol/water/formic acid (45:45:10, v/v/v). The gradient elution was as follows: from 35% to 95% solvent B over 20 min, then from 95% to 100% in 5 min, followed by isocratic elution with 100% solvent B for 5 min. The flow rate was 0.8 mL/min and the injection volume was 10 μL.

Detection wavelengths were optimized for each group of compounds: 265 nm for 4-hydroxybenzoic acid, 320 nm for ferulic acid, chlorogenic acid, caffeic acid, cinnamic acid and coumaric acid, 372 nm for resveratrol, myricetin and quercetin, and 530 nm for individual anthocyanins.

2.7. Statistical Analyses

Standard methods were used for statistical evaluation using the statistical software Statgraphics Centurion XVII, version 17.2.07, Statgraphics Technologies, Inc., The Plains, VA, USA—multifactor analysis of variance (MANOVA), LSD test.

3. Results and Discussion

Interpreting the results of Se application requires great caution, as the boundary between its positive and negative effects, including (phyto)toxicity for both plants and humans, is very narrow. It is also essential to determine whether a reduction in a particular element following Se application is necessarily negative. In our study, it was confirmed that after the application of a specific dose of Se under the stated conditions, a demonstrable reduction in the content of aluminum (Al) in blueberry, currant, honeysuckle and apple juice was recorded, as well as a demonstrable reduction in Ni and Cr in raspberry, currant and apple juice, which is very positive. However, on the other hand, a demonstrable increase in Ni and Cr was observed in blueberry and honeysuckle juice.

The main objective of the study was to increase the organic selenium content in the fruits or juices by biofortifying it under the specified conditions avoiding a reduction in other valuable nutrients. This was confirmed by the comprehensive evaluation, which showed a demonstrable positive increase in the Se content (

Table 2. Parametric evaluation of significant changes (p ≤ 0.05) in the content of analyzed indicators in raspberries and blueberries after foliar application of sodium selenate.

Analyzed Parameter	Unit	Raspberries		Blueberries
		Control	Se Application	Control	Se Application
Se	mg/kg	˂LOD	2.570 ± 0.306	0.715 ± 0.019 a	1.791 ± 0.066 b
pH		2.92 ± 0.01 a	2.843 ± 0.093 a	3.413 ± 0.015 b	3.06 ± 0.046 a
Mn	mg/kg	16.722 ± 0.036 b	6.501 ± 0.009 a	8.877 ± 0.044 a	11.616 ± 0.096 b
ferulic acid	mg/kg	1.13 ± 0.03 a	8.23 ± 0.07 b	16.783 ± 0.040 a	16.703 ± 0.032 a
AA DDPH	%	91.433 ± 0.850 a	91.8 ± 0.529 a	89.433 ± 0.635 b	79.633 ± 0.404 a
AA TEAC	μmolTE/g	12.717 ± 0.117 a	12.767 ± 0.757 a	12.437 ± 0.092 b	11.06 ± 0.056 a
AA FRAP	μmolTE/g	8.823 ± 0.118 a	8.853 ± 0.092 a	9.01 ± 0.04 b	8.893 ± 0.012 a
resveratrol	mg/kg	9.33 ± 0.04 b	8.763 ± 0.035 a	4.103 ± 0.350 a	3.830 ± 0.170 a
fructose	g/L	49.953 ± 0.263 a	57.77 ± 5.499 a	50.32 ± 1.121 a	49.733 ± 0.651 a
glucose	g/L	24.497 ± 1.192 a	28.193 ± 0.193 b	31.677 ± 0.719 a	32.483 ± 0.335 a
malic acid	g/L	13.207 ± 0.162 a	15.323 ± 0.289 b	8.387 ± 0.265 a	7.28 ± 0.14 b
total acid	g/L	14.06 ± 0.233 a	16.54 ± 0.299 b	8.597 ± 0.015 b	7.607 ± 0.101 a
Ba	mg/kg	3.226 ± 0.008 b	2.143 ± 0.006 a	2.103 ± 0.008 a	2.545 ± 0.022 b
Ca	mg/kg	3852.37 ± 9.625 b	1574.057 ± 3.215 a	1697.437 ± 10.451 a	3897.36 ± 46.455 b
Li	mg/kg	0.020 ± 0.001 a	0.017 ± 0.002 a	0.040 ± 0.000 b	0.024 ± 0.004 a
myricetin	mg/kg	˂LOD	˂LOD	5.103 ± 0.100 a	6.3 ± 0.750 a
chlorogenic acid	mg/kg	˂LOD	˂LOD	323.713 ± 2.067 a	410.43 ± 0.491 b
quercetin	mg/kg	˂LOD	˂LOD	7.017 ± 0.226 a	7.04 ± 0.061 a
TSS	Brix	7.645 ± 0.095 a	8.52 ± 0.108 b	8.87 ± 0.044 b	8.74 ± 0.061 a
total sugar	g/L	76.3 ± 2.151 a	82.567 ± 0.484 b	93.393 ± 1.082 b	90.2 ± 0.585 a
Cr	mg/kg	0.122 ± 0.017	˂LOD	0.370 ± 0.041 a	0.405 ± 0.024 a
Cu	mg/kg	7.024 ± 0.013 a	8.591 ± 0.029 b	8.293 ± 0.032 b	7.158 ± 0.020 a
Fe	mg/kg	25.379 ± 0.198 a	28.343 ± 0.084 b	58.010 ± 0.543 b	29.530 ± 0.364 a
Mg	mg/kg	984.823 ± 2.447 a	1025.78 ± 0.719 b	618.318 ± 2.047 b	449.990 ± 2.434 a
Na	mg/kg	150.444 ± 0.466 a	171.543 ± 0.076 b	219.412 ± 1.059 b	152.059 ± 0.348 a
Ni	mg/kg	4.184 ± 0.158 b	3.335 ± 0.006 a	2.554 ± 0.219 a	3.778 ± 0.316 b
Sr	mg/kg	29.188 ± 0.133 b	14.320 ± 0.013 a	15.927 ± 0.059 b	11.268 ± 0.053 a
caffeic acid	mg/kg	1.05 ± 0.03 a	1.15 ± 0.721 a	7.443 ± 0.031 b	7.133 ± 0.021 a
cinnamic acid	mg/kg	3.47 ± 0.01 b	3.097 ± 0.006 a	˂LOD	˂LOD
coumaric acid	mg/kg	6.873 ± 0.475 b	5.32 ± 0 a	6.927 ± 0.006 b	4.833 ± 0.023 a
4-OH benzoic acid	mg/kg	12.845 ± 0.205 b	10.497 ± 0.165 a	35.05 ± 0.411 b	22.413 ± 0.071 a
TPC	mg/kg	1176.053 ± 25.302 a	1449.08 ± 20.787 b	1842.053 ± 35.600 b	1595.627 ± 35.696 a
TAC	mg/kg	0.803 ± 0.006 a	0.61 ± 0.01 b	8.497 ± 0.083 b	6.337 ± 0.031 a
K	mg/kg	5354.833 ± 2.046 b	5038.893 ± 3.878 a	4226.373 ± 11.327 b	3833.81 ± 13.981 a
Zn	mg/kg	29.046 ± 0.299 b	7.936 ± 0.625 a	8.371 ± 0.254 a	13.790 ± 1.073 b
Al	mg/kg	19.759 ± 0.070 a	29.527 ± 1.074 b	50.383 ± 1.119 b	40.482 ± 0.927 a
rutin	mg/kg	7.193 ± 0.235 b	4.743 ± 0.035 a	9.14 ± 0.221 b	6.56 ± 0.305 a

Values with different letters are significantly different at p < 0.05 by LSD test in ANOVA (Statgraphic XVII).

and

Table 3. Parametric evaluation of significant changes (p ≤ 0.05) in the content of analyzed indicators in redcurrants, honeysuckle and apples after foliar application of sodium selenate.

		Redcurrants		Honeysuckle		Apples
Analyzed Parameter	Unit	Control	Se Application	Control	Se Application	Control	Se Application
Se	mg/kg	˂LOD	8.975 ± 0.31 b	˂LOD	3.781 ± 0.592	˂LOD	1.227 ± 0.021
pH		2.943 ± 0.015 a	2.92 ± 0.026 a	3.167 ± 0.058 a	3.247 ± 0.115 a	3.59 ± 0.01 a	3.6 ± 0.1 a
Mn	mg/kg	7.758 ± 0.041 a	8.175 ± 0.044 b	9.048 ± 0.037 a	9.560 ± 0.064 b	5.018 ± 0.042 a	4.988 ± 0.033 a
ferulic acid	mg/kg	1.17 ± 0.01 a	1.107 ± 0.115 b	1.107 ± 0.006 a	8.307 ± 0.596 b	2.86 ± 0.017 a	5.633 ± 0.035 b
AA DDPH	%	75.233 ± 0.060 a	79.033 ± 0.503 b	89 ± 0.7 a	89.733 ± 0.321 a	60.9 ± 0.361 a	65.067 ± 0.252 b
AA TEAC	μmolTE/g	10.44 ± 0.085 a	10.973 ± 0.070 b	12.373 ± 0.102 a	12.477 ± 0.047 a	8.423 ± 0.047 a	9.007 ± 0.035 b
AA FRAP	μmolTE/g	7.07 ± 0.078 a	6.96 ± 0.02 a	9.227 ± 0.057 a	9.177 ± 0.057 a	4.7 ± 0.07 a	4.927 ± 0.098 b
resveratrol	mg/kg	3.797 ± 0.030 a	8 ± 0.044 b	1.183 ± 0.006 a	1.213 ± 0.907 a	3.793 ± 0.006 a	3.53 ± 0.236 a
fructose	g/L	42.953 ± 0.620 a	45.313 ± 0.277 b	53.153 ± 0.409 b	49.233 ± 0.503 a	90.31 ± 0.737 b	80.457 ± 0.405 a
glucose	g/L	21.313 ± 0.407 a	24.923 ± 0.076 b	38.383 ± 0.333 b	35.64 ± 0.472 a	10.04 ± 0.183 b	9.557 ± 0.083 a
malic acid	g/L	17.297 ± 0.115 b	15.483 ± 0.344 a	14.113 ± 0.215 a	15.99 ± 0.221 b	2.76 ± 0.145 a	3.553 ± 0.051 b
total acid	g/L	17.973 ± 0.081 b	16.27 ± 0.236 a	15.323 ± 0.582 a	15.033 ± 0.153 a	3.427 ± 0.215 a	4.333 ± 0.497 b
Ba	mg/kg	2.008 ± 0.007 b	1.406 ± 0.012 a	4.993 ± 0.020 a	5.108 ± 0.024 b	1.516 ± 0.013 a	1.780 ± 0.018 b
Ca	mg/kg	2047.043 ± 12.821 b	1574.05 ± 20.669 a	1946.153 ± 8.646 a	1990.273 ± 28.373 a	1143.68 ± 0.654 a	1198.76 ± 4.606 b
Li	mg/kg	0.078 ± 0.007 a	0.044 ± 0.004 a	0.012 ± 0.002 a	0.017 ± 0.003 a	0.045 ± 0.004 b	0.032 ± 0.002 a
myricetin	mg/kg	˂LOD	4.297 ± 0.115	˂LOD	˂LOD	˂LOD	˂LOD
chlorogenic acid	mg/kg	˂LOD	˂LOD	˂LOD	˂LOD	43.45 ± 0.308 a	48.793 ± 0.051 b
quercetin	mg/kg	˂LOD	˂LOD	˂LOD	˂LOD	˂LOD	˂LOD
TSS	Brix	6.593 ± 0.095 a	7.217 ± 0.075 b	10.773 ± 0.125 b	10.193 ± 0.021 a	11.737 ± 0.152 b	10.643 ± 0.049 a
total sugar	g/L	74.71 ± 0.159 a	80.477 ± 0.341 b	115.44 ± 1.002 b	110.473 ± 1.131 a	110.193 ± 1.042 b	100.453 ± 1.279 a
Cr	mg/kg	0.653 ± 0.022 b	0.252 ± 0.034 a	0.404 ± 0.532 b	0.197 ± 0.499 a	3.300 ± 0.009 b	0.944 ± 0.031 a
Cu	mg/kg	11.767 ± 0.044 b	7.437 ± 0.047 a	11.235 ± 0.421 a	12.228 ± 0.025 b	8.834 ± 0.032 b	8.024 ± 0.023 a
Fe	mg/kg	37.361 ± 0.080 b	28.060 ± 0.138 a	33.417 ± 0.211 a	34.652 ± 0.424 b	35.414 ± 0.253 b	26.002 ± 0.198 a
Mg	mg/kg	746.321 ± 0.493 b	667.480 ± 1.414 a	700.420 ± 0.887 b	671.668 ± 3.336 a	581.595 ± 0.900 a	625.427 ± 2.520 b
Na	mg/kg	258.507 ± 1.244 b	117.004 ± 0.654 a	211.202 ± 0.288 b	178.99 ± 0.418 a	155.083 ± 0.440 a	179.016 ± 0.573 b
Ni	mg/kg	11.938 ± 0.495 b	2.827 ± 0.118 a	1.724 ± 0.912 a	2.122 ± 0.079 b	3.748 ± 0.115 b	1.453 ± 0.162 a
Sr	mg/kg	15.627 ± 0.0156 b	9.603 ± 0.021 a	13.315 ± 0.050 a	15.489 ± 0.083 b	10.778 ± 0.027 a	11.644 ± 0.088 b
caffeic acid	mg/kg	1.157 ± 0.006 b	1.033 ± 0.006 a	7.017 ± 0.201 a	6.687 ± 0.085 a	4.667 ± 0.057	˂LOD
cinnamic acid	mg/kg	˂LOD	˂LOD	9.883 ± 0.015 b	8.19 ± 0.066 a	˂LOD	˂LOD
coumaric acid	mg/kg	˂LOD	˂LOD	6.993 ± 0.655 a	6.063 ± 0.071 a	˂LOD	˂LOD
4-OH benzoic acid	mg/kg	37.707 ± 0.503 b	4.14 ± 0.075 a	˂LOD	˂LOD	5.163 ± 0.061 a	6.597 ± 0.180 b
TPC	mg/kg	875.907 ± 19.031 b	711.053 ± 27.368 a	3245.587 ± 10.881 b	3163.733 ± 29.595 a	˂LOD	˂LOD
TAC	mg/kg	1.237 ± 0.031 a	1.387 ± 0.015 b	11.85 ± 0.062 b	11.457 ± 0.060 a	˂LOD	˂LOD
K	mg/kg	9949.86 ± 16.973 b	8134.497 ± 26.975 a	9727.08 ± 1.652 b	9436.3 ± 12.636 a	4545.91 ± 3.557 a	5673.103 ± 8.661 b
Zn	mg/kg	11.958 ± 0.570 b	8.084 ± 0.594 a	10.322 ± 0.229 b	8.999 ± 0.255 a	7.408 ± 0.756 b	4.377 ± 0.192 a
Al	mg/kg	38.522 ± 0.253 b	34.876 ± 0.554 a	36.030 ± 0.641 b	34.468 ± 0.556 a	50.441 ± 0.138 b	43.551 ± 1.116 a
rutin	mg/kg	8.807 ± 0.006 a	7.447 ± 0.306 b	130.29 ± 0.215 b	115.65 ± 0.478 a	˂LOD	1.17 ± 0.026

Values with different letters are significantly different at p < 0.05 by LSD test in ANOVA (Statgraphic XVII).

) in all fruit species. Since five species of fruit (juices) and 35 parameters were evaluated, it was necessary to make the evaluation clear. It was assumed that if the analyzed parameter significantly (p ≤ 0.05) increased or remained unchanged after Se application, it was considered a positive influence, whereas a decrease was regarded as a negative influence. The exception is heavy metals, where an increase in content was negative and a decrease was, on the contrary, positive.

Table 4. Parametric evaluation of significant changes (p ≤ 0.05) in the content of analyzed indicators in fruit juice after foliar application of sodium selenate.

Parameter	Raspberries	Blueberries	Redcurrants	Honeysuckle	Apples
Se	↑	↑	↑	↑	↑
pH	↔	↑	↔	↔	↔
Mn	↓	↑	↑	↑	↔
Ferulic acid	↑	↔	↑	↑	↑
AA (DPPH)	↔	↓	↑	↔	↑
AA (TEAC)	↔	↓	↑	↔	↑
AA (FRAP)	↔	↓	↔	↔	↑
Resveratrol	↓	↔	↑	↔	↔
Fructose	↔	↔	↑	↓	↓
Glucose	↑	↔	↑	↓	↓
Malic Acid	↑	↓	↓	↑	↑
Total Acid	↑	↓	↓	↔	↑
Ba	↓	↑	↓	↑	↑
Ca	↓	↑	↓	↔	↑
Li	↔	↓	↔	↔	↓
Myricetin	<LOD	↔	↑	<LOD	<LOD
Chlorogenic Acid	<LOD	↑	<LOD	<LOD	↑
Quercetin	<LOD	↔	<LOD	<LOD	<LOD
TSS	↑	↓	↑	↓	↓
Total Sugar	↑	↓	↑	↓	↓
Cr	↑	↔	↓	↓	↓
Cu	↑	↓	↓	↑	↓
Fe	↑	↓	↓	↑	↓
Mg	↑	↓	↓	↓	↑
Na	↑	↓	↓	↓	↑
Ni	↓	↑	↓	↑	↓
Sr	↓	↓	↓	↑	↑
Caffeic Acid	↔	↓	↓	↔	↓
Cinnamic Acid	↓	<LOD	<LOD	↓	<LOD
Coumaric Acid	↓	↓	<LOD	↔	<LOD
4-OH Benzoic Acid	↓	↓	↓	<LOD	↑
TPC	↑	↓	↓	↓	<LOD
TAC	↓	↓	↑	↓	<LOD
K	↓	↓	↓	↓	↑
Zn	↓	↑	↓	↓	↓
Al	↑	↓	↓	↓	↓
Rutin	↓	↓	↓	↓	↑

Values indicate changes compared with control; ↑ = Increase, ↓ = Decrease, ↔ = No Change, <LOD = Below Limit of Detection.

provides a thorough analysis of specific factors that changed, decreased or remained unchanged based on the fruit species.

It can be stated that the following parameters were somewhat increased or remained unchanged after Se application, which is considered a positive influence: content of Se, pH, Mn, ferulic acid, antioxidant activity (DDPH, TEAC and FRAP), resveratrol, fructose, glucose, malic acid, total acid, Ba, Ca and Li. A decrease in values was observed for the following parameters, leading to the conclusion that Se application had a negative impact on the evaluated parameters: caffeic acid, cinnamic acid, coumaric acid, 4-OH benzoic acid, TPC, TAC, K, Zn, Al and rutin. For detailed information, see Table 4. When looking at the results from the perspective of fruit species, raspberries, honeysuckle and apples can be grouped together, as the ratio of positive to negative effects of Se application on the measured parameters was higher in favor of positive effects (21–23:12–14). In contrast, for blueberries and redcurrants, a decrease in the measured parameters was observed after the application of Se at the given concentration (15–16:19–21). Foliar application, which is undertaken to raise the Se content in many crops, is more effective than soil fertilization because it avoids interference from problems with soil chemistry and microbiology. This is because even small amounts of Se solution can produce greater efficacy [15]. In our study, we also used foliar application of Se because of this phenomenon and we believe that in production practice it is easier to apply foliar Se than to apply it by watering into the soil after the necessary previous soil analysis. Moreover, after application by watering, the incorporation of Se into the tissues will take longer than direct application of Se to the leaves, from which the active substance is delivered very quickly and in a targeted manner.

On average, foliar spraying of fruit plants is more effective than soil application because it results in a higher uptake of selenium (Se), no residual effects and a lower consumption of Se salts. This makes it the most cost-effective and environmentally friendly method [15,16]. In pomegranates and apples, fruit quality was improved by foliar spray supplementation with Se [17]. Specifically, in apples, higher levels of soluble solid content, titrable acidity, hardness of the flesh and antioxidant enzyme activity were noted in addition to the increased Se content [18]. In contrast, Se fertilization significantly increased the amount of antioxidants, phenolic compounds and anthocyanins in pomegranates [17]. Although olives were foliarly enriched with selenium, the nutritional quality of the drupes was also altered. Compared with the untreated control group, the concentrations of B, Na, Mg, K, Cr, Mn, Fe and Cu were much higher [19]. Similar results were also obtained in our study, positively confirming that when Se is incorporated into plant tissues, no significant decrease in health-promoting substances in the produced juices is observed. It is difficult to find parallels within the discussion, because identical studies have not been practically conducted on the given types of fruit and analytical parameters, but the aforementioned studies partially confirm our conclusions.

Absorption of selenium is almost immediate and targeted; we bypass often unknown soil attributes, thereby preventing problems with incompatibility of individual elements in soil and selenium.

Wine grapes treated with Se amino-acid-chelated fertilizer produced increased levels of acid invertase activity, total soluble sugar and Se content in vitis grapes compared with the untreated control. Furthermore, soluble sugar, soluble protein, soluble solid and lower levels of organic acid were all enhanced by selenium fertilizer; however, it had no influence on the polyphenol antioxidants found in Eurasian species. Furthermore, Se fertilization can be utilized to lower the buildup of heavy metals Pb, Cr, Cd, As and Ni in addition to raising the Se content and nutritional quality of grapes [20].

We can confirm a similar reduction in some heavy metals in our study after the application of Se, namely a reduction in the content of Al, Cu and Cr in some types of juices. In a dose-dependent way, adding inorganic selenium may significantly raise the selenium content of lupine seeds [21]. The selenium content of soybeans that have germinated may be enhanced by the addition of sodium selenite, as stated by [22]. Selenium levels increased in other cereal seeds during seed germination, such as chickpeas [23] or brown rice [24]. These results show that Se application also affects other horticultural and field crops, including the Se content in seeds.

To produce Se-rich potatoes, foliar selenite spraying during the tuber bulking stage was effective [25]. In broccoli, Se fortification at developmental stages increased SeMeSeCys content [26]. The Se fortification of lettuce and other green vegetables, including spinach, basil, endive and chicory, has also been the subject of extensive research [7]. The results are also supported by our own multi-year studies on carrots and chili peppers [27,28].

These studies demonstrate that the beneficial benefits of foliar selenium administration extend to various field and horticultural crops with comparable outcomes.

4. Conclusions

The results of this study clearly confirm that the application of Se leads to an increase in Se in fruits and in juices. This biofortification process is particularly interesting in the context of the growing demand for functional foods enriched with essential trace elements. According to an extensive number of examined parameters, it can be stated that when a dose of pure selenium equivalent to 150 g/ha was applied to apple trees, gooseberries, currants, raspberries and blueberries, a significant increase in the amount of selenium in the fruit juices was always observed, indicating that the selenium was incorporated into the tissues. When juices treated in this manner are consumed, the recommended quantity of selenium is obtained directly and organically. Furthermore, it can be stated that after the application of Se under the mentioned conditions, there was a demonstrable increase in nutritionally valuable parameters, such as antioxidant activity measured by three methods (DPPH, TEAC, FRAP), ferulic acid and resveratrol, but also the content of glucose, fructose, malic acid, total acids, Ba, Ca, Li, Mn, myricetin and chlorogenic acid.

A decrease was observed in elements such as aluminum (Al), as well as a demonstrable reduction in Ni and Cr in raspberry, currant and apple juice, but on the other hand, there was a demonstrable increase in Ni and Cr in the case of blueberry and honeysuckle juice. A decrease must also be noted in some valuable indicators, such as 4-OH benzoic acid content, total polyphenol content, total anthocyanin content, and K, Zn, Al and rutin content in some fruit species. The solution for species where the content of mineral elements has decreased is additional fertilization with these elements, of course after evaluating the profitability of such fertilizing. According to a dose of 150 g sodium selenate per hectare, the price for such a treatment is 260 EUR.

In terms of impact on practice, farmers or beverage producers must recognize that the foliar application of Se does not necessarily lead to an increase in yields. However, a significant marketing advantage is gained when a demonstrably increased and non-toxic content of organic Se in juice or other beverages can be presented, contributing to the enrichment of the beverage market in a highly acceptable and healthy form. From the perspective of practical application and the consumption of such beverages, the following amounts of undiluted pure juice would need to be consumed to reach the daily recommended dose of selenium, after converting the values of dry matter into juice (fresh weight): 183 mL of raspberry juice, 90 mL of blueberry juice, 19 mL of currant juice, 80 mL of honeysuckle juice and 230 mL of apple juice. Producers can highlight the organic Se content as a nutritional benefit, creating new opportunities for innovation in the functional beverage market.