Effect of Biofortification with Iodine by 8-Hydroxy-7-iodo-5-quinolinesulfonic Acid and 5-Chloro-7-iodo-8-quinolinol on the Chemical Composition and Antioxidant Properties of Potato Tubers (Solanum tuberosum L.) in a Pot Experiment

Abstract

Iodine deficiency impacts on the development of thyroid disease. Vegetables and fruits usually have a low iodine content; hence, it makes sense to increase their iodine content. Potato is consumed daily by millions of consumers and would, therefore, be a good target for biofortification with iodine programs. The aim of this study was to determine the effects of biofortification via the application of soil solutions of two iodoquinolines [8-hydroxy-7-iodo-5-quinolinic acid (8-OH-7-I-5QSA) and 5-chloro-7-iodo-8-quinoline (5-Cl-7-I-8-Q)] and KIO₃ (as an iodine positive control) on the iodine content and basic chemical composition, macro and micronutrient content, nitrogen compounds, vitamin C, and antioxidant potential of potato tubers Solanum tuberosum L. The biofortification process had no significant effect on the tuber weight in yield. The application of I in forms of KIO₃, 8-OH-7-I-5QSA, 5-Cl-7-I-8-Q resulted in an increase in the I content of tubers (1400.15; 693.65; 502.79, respectively, compared with control, 24.96 µg·kg⁻¹ d.w.). This also resulted in a decrease in elements that are harmful to consumers, such as: Al, Ni, Cr, Ag, Pb and Tl. The enrichment of tubers with 8-OH-7-I-5QSA and 5-Cl-7-I-8-Q resulted in a significant reduction in the content of ammonium ions (from 19.16 to 14.96; 13.52 mg∙kg⁻¹ f.w.) and chlorides (from 423.59 to 264.92; 265.31 mg∙kg⁻¹ f.w.). Biofortification with 8-OH-7-I-5QSA improved the polyphenolic profile of the potato tuber from 197.31 to 233.33 mg GAE·100 g⁻¹ f.w. A significant reduction in the carotenoid content of tubers after the enrichment of the plant with iodine in KIO₃, 8-OH-7-I-5QSA and 5-Cl-7-I-8-Q (from 3.46 to 2.96, 2.45, and 1.47 mg∙100 g⁻¹ d.w., respectively) was observed. It can be postulated that the production of potatoes enriched with iodoquinolines and/or KIO₃ is worthwhile, as it can provide a good source of I in the diet and simultaneously reduce the risk of developing deficiencies.

1. Introduction

It has been documented that dietary iodine deficiency among people living far from seas and oceans has a direct impact on the development of goitre and other thyroid diseases [1]. Although the iodisation of table salt with potassium iodide is effective in reducing the incidence of goitre, salt is an unhealthy carrier due to its induction of cardiovascular disease. Iodine deficiency can cause permanent mental and physical impairment and foetal defects. In addition, iodine deficiency increases the risk of premature death [2]. The main reason for this is the insufficient bioavailability of this element in soils. Moreover, there is an insufficient iodine flux in the soil–plant–consumer system, which ultimately results in a low dietary intake [3]. Plant biofortification, i.e., the enrichment of plants with iodine, may be a relatively inexpensive tool to effectively combat iodine deficiencies in the human diet and animal feed [3]. Previous studies of potato biofortification have involved tuber enrichment through soil fertilisation in soil cultivation [4], pot experiments [5] and the possibility of growing potatoes in hydroponic systems, including aeroponics [6,7]. The main methods used are selective breeding via conventional methods, and genetic modification. The agronomic approach focuses on the application of fertilisers and the concentration of minerals. Genetic engineering aims to improve the plant varieties and thus enable edible plant tissues to accumulate micronutrients more effectively. In addition, the aim is to increase their bioavailability through a higher concentration of promoter substances and a lower concentration of antinutrients [8].

Iodine fertilisers are known to improve the absorption and accumulation of this micronutrient, making it readily available in plant-based foods. The application of the fertiliser via foliar feeding is highly preferred since it requires much less fertiliser than soil application [9]. However, in the case of the potato tubers, this is not possible.

Researchers have found that not every fertiliser is beneficial for biofortified crops. Unfortunately, the application of phosphate and sulphate fertilisers results in a reduction in iodin and selenium herbage concentrations, through ion competition and increased yield [10]. Creating a fertiliser that does not affect the absorption of microelements is the subject of further research.

Mageshen et al. (2022) showed that biofortification with iodine using an iodate–chitosan complex increased the iodine content of tomatoes. Potassium iodate and chitosan in the form of soil, foliar and an chitosan–iodate complex were applied at different stages of plant growth. The results showed that soil fertilisation with potassium iodate alone resulted in a lower residual iodine uptake, as iodine was prone to high volatilisation and lower phytoavailability. However, the higher iodine accumulation was achieved by the combination of foliar and iodine forms of chitosan [11].

A new line of research is now being pursued regarding Arbuscular Mycorrhizal Fungi (AMF) and Plant Growth-Promoting Bacteria (GPB). AMF may also be beneficial to the accumulation of microelements and the enhancement of the plant antioxidant status. It is supposed that the resulting phenomenon is connected to the fact that sulfate and phosphate transporters are decoded also by the AMF genome, resulting in an improvement in sulfur, phosphorus, and selenium accumulation [12].

Vegetables, including potatoes, are healthy products that form the basis of the food pyramid, with high consumption preventing the development of many diseases, such as obesity, diabetes and cardiovascular diseases [2]. Potato is consumed every day of the year by millions of consumers and could be regarded, therefore, as the best vegetable in terms of frequency of consumption. In 2020, 161 countries were compared. It was found that China ranks first in terms of potato consumption, with 69.312 kt. India and the USA ranked second and third. China, which is the highest ranking country, accounted for 27.0% of global potato consumption. The Central African Republic (1.00 kt), Guinea-Bissau (1.00 kt) and Comoros (1.00 kt) were estimated to consume the least amount of potatoes. According to FAOSTAT, in 2020, the total world potato consumption reached 256.432 kt. This quantity has since increased by 1.74 percentage points compared to the previous year. A decade ago, the consumption was 10.6 percentage points lower. The consumption record was reached in 2020, when 256.432 kt of potatoes were consumed. Going back in history, the lowest recorded level of potato consumption occurred in 1962, with only 106.356 kt of potatoes consumed [13]. The population of Earth is steadily growing, and its adequate nutrition is now a priority task for the agri-food sector. Iodine is responsible for the normal growth, development and physiology of the human body [2]. Iodine deficiency disorders (IDDs) occur in almost every country in the world [14]. Currently, there is a global increase in the number of people who are overweight or obese with concomitant deficiencies of selected macro and micronutrients [15]. Therefore, promoting the importance of a well-balanced diet in the context of human health and ensuring the adequate nutrition of the population is important. According to the WHO, at the turn of the 20th century, almost 2 billion people living in areas far from the sea were at risk of iodine deficiency (200 million of whom had thyroid goitre) [15,16].

The potato is one of the world’s major crops [17], along with maize, wheat and rice. It follows that tubers are a valuable source of energy and compounds that are important in the human diet. Potatoes, moreover, are relatively easy to grow, which is why they are planted almost everywhere in the world. This famous tuber is grown in 80 percent of the world’s countries [13,17]. This shows that Solanum tuberosum L. is one of the most productive crops in the world. Potato can yield more nutritious food on less land and in harsher climates than most other major crops. In addition, the tuber can be harvested in as little as eight weeks [18].

Potatoes are a good source of easily digestible carbohydrates, which account for approximately 80% of their dry matter, as well as wholesome protein (6–10% of dry matter), which is rich in endogenous amino acids and especially lysine, increasing its importance as a component in the daily human diet. Potato also contains an optimum amount of dietary fibre (approximately 10% of dry weight), but a low level of fat (0.1% of dry weight). The nutritional value of potato protein is comparable to that of an egg white and is superior to that of other plant proteins [19]. Health-promoting compounds in potatoes with a traditional flesh colour include phenolic acids, ascorbic acid, carotenoids, tocopherol and also selenium; potatoes with coloured flesh also possess anthocyanins, which are colour compounds with a high antioxidant potential [20,21,22,23]. Such varieties represent a potential raw material for the production of foods with an increased content of bioactive compounds. In the human diet, phenolic compounds and carotenoids have antioxidant effects. A diet rich in antioxidants is associated with a lower incidence of atherosclerosis of the heart, certain cancers, macular degeneration, as well as cataracts [24].

Potato, nevertheless, is not counted as an antioxidant-rich food, but there is some indication that breeding work may lead to increased concentrations of these compounds [25]. Furthermore, Tatarowska et al. [26] indicated that organic growing conditions may favour more active carotenogenesis in potato tubers. Increased antioxidant levels in potato tubers may be important to regions of the world in which potato production is increasing, due to it being a crucial element of the daily diet. On the other hand, obtaining potatoes with a higher content of antioxidant compounds could be particularly beneficial for organic production, as the resulting product would be even more nutritionally valuable. Furthermore, the implementation of biofortification would increase the nutritional value of the vegetable even more.

Biofortified vegetable crops, including potatoes, could also become functional foods; these generally show a higher mineral content than conventional products. In addition, a sufficiently high intake of biofortified vegetables can reduce the incidence of lifestyle diseases such as obesity, cancer, diabetes, osteoporosis and cardiovascular disease, and prevent mineral deficiencies [2]. Functional foods, or FOSHUs (Foods for Specified Health Use), are normal foods from which harmful ingredients (e.g., allergens) have been removed or that have been enriched with physiologically active substances to produce a product with sufficient nutritional value to improve human health [27]. The main bioactive compounds include dietary fibre, chitosan, plant polyphenols, artificial sweeteners, probiotics, prebiotics, amino acids, peptides, proteins, glycosides, carnitine, stanols and plant sterols, including conjugated linolenic acid diene (CLA), probiotics, prebiotics, synbiotics, phytochemicals, vitamins and minerals, choline and lecithin. The term ‘functional food’ first appeared in Japan in 1984 [27]. To this day, the country is still a leader in the production of this type of food and is promoting new possibilities for its use. Biofortified crops could become the primary source of food for the world’s population in the future, providing coverage for all essential dietary components.

The aim of this study was to determine the effects of biofortifying potato (Solanum tuberosum L.) tubers with iodine in the form of two iodoquinolines [8-hydroxy-7-iodo-5-quinolinic acid (8-OH-7-I-5QSA) and 5-chloro-7-iodo-8-quinoline (5-Cl-7-I-8-Q)] and KIO3 (as iodine positive control) on the chemical composition of their antioxidant properties. The plants were cultivated via soil fertilisation (by periodical fertigation) in a pot experiment.

2. Materials and Methods

2.1. Plant Material and Cultivation

In the spring season of 2022, early potatoes Solanum tuberosum L. ‘Denar’ c.v. were grown in a pot experiment. The experiments were located in a foil tunnel with a one-side vent at the campus of the Faculty of Biotechnology and Horticulture of the University of Agriculture in Kraków (50°05′03″ N 19°57′01″ E). Seed potato tubers were planted on 8 March 2022 in 7.5 L pots filled with heavy mineral soil.

To characterise the chemical properties prior to the experiment (

Table 1. Selected chemical properties of the soil prior to the experiments and physicochemical soil characteristics.

Physicochemical Soil Characteristic	Peat Substrate—Experiment 2
pH(H2O)	6.00
EC (mS∙cm−1)	580.5
Eh(mV)	260.6
Macroelements:
N-NH4 (mg∙dm−3)	14,6
N-NO3 (mg∙dm−3)	10,7
N-NH4+ N-NO3 (mg∙dm−3)	25,3
P (mg∙dm−3)	10.9
K (mg∙dm−3)	43.2
Mg (mg∙dm−3)	158.5
Ca (mg∙dm−3)	4 363.5
S (mg∙dm−3)	73.9
Na (mg∙dm−3)	17,8
Iodine (mg I∙kg−1)	1.2
Al-hydroxides (mg∙kg−1)	1251.0
Fe-hydroxides (mg∙kg−1)	2345.6
Mn-hydroxides (mg∙kg−1)	235.4
Soil organic matter (%)	2.49
Soil texture (according to the ISSS classification).	Loam soil 35 sand 28% silt 37% clay

), the following chemical analyses were carried out on soil samples taken before potato planting: soil texture, acidification (pH(H₂O)), salinity (EC) (mS∙cm⁻¹), soil oxyreduction potential (Eh mV), organic matter content and mineral composition. The content of macroelements, including N-NH₄, N-NO₃, N-NH₄⁺N-NO₃, P, K, Mg, Ca and S, was analysed using the methods and technologies presented by Smoleń et al. [28]. The soil content of hydroxides, Al, Fe, and Mn was analysed according to methods proposed by Kostka and Luther [29], and Anschutz et al. [30]. Three-tenths of a gram of dry soil were weighed into 30 mL falcon tubes. Then, 25 mL of extraction solution was added. The extraction solution was composed of 0.22 M trisodium citrate, 0.11 M sodium bicarbonate and 0.1 M sodium dithionite. The samples were shaken at 45 °C for 22 h. Then, they were centrifuged for 20 min at 3000 rpm and filtered using medium qualitative filters. Al, Fe, and Mn were analysed using ICP-OES (Prodigy Spectrometer, Leeman Labs, New Hampshire, MA, USA).

Before planting the tubers, Polifoska 10, 5, 15, 5, and 14 (N, P, K, Mg, S), produced by the Polish granular fertiliser manufacturer Grupa Azoty Police, were applied to the soil in the amount of 0.55 g/kg of soil. Polifoska 5 is a complex multi-nutrient fertiliser that is particularly recommended for use before sowing, but its late application in early spring is also possible. This represents the only instance of the fertilisation of the plants via the soil for the entire cultivation period. Plants were grown in four replicates for each treatment in a randomised design. There were 12 plants per one replication (in each treatment), which equalled 48 plants per each treatment; there was a total of 192 plants per experiment.

The subject of the study was plant fertilisation with iodine in the form of organic compounds; this included iodoquinolines, and an inorganic form of iodine that was presented as KIO₃, which was used as a reference treatment and as a positive control against the iodoquinolines. The following treatments were tested in the studies: (1) Control (without iodine application); (2) KIO₃, (3) 8-OH-7-I-5QSA and (4) 5-Cl-7-I-8-Q. All the iodine compounds were applied to soil once a week through manual fertigation (manual watering) with 50 µM (molar mass equivalents) of the solutions of the studied compounds, at a dose of 500 mL/pot-1 (one plant-1). During the experiment, nine applications of these compounds were made every seven days; the first application was made after the emergence of plants (26 April 2022) and the last one was made on 7 June 2022, one week before the harvest. Finally, 1.77 µmol of I per pot was applied. The working solutions of the tested iodine compounds used for the fertigation were prepared in time intervals of every two weeks. On the days between the application of iodine compounds, the plants were watered via a drip irrigation system with tap water in the amount of approximately 200–500 mL/pot-1 (depending on the growth phase and weather conditions). Plants were harvested on 15 June 2022. During the plant harvesting, the number of tubers from one plant and the total weight yield of tubers from one plant were measured separately for each replication. Both iodoquinolines are commercial compounds and are commercially available for purchase. Our facilities were purchased from Maybridge (Thermo Fisher Scientific, Waltham, MA, USA), and 5-chloro-7-iodo-8-quinolinol (5-Cl-7-I-8-Q) and 8-hydroxy-7-iodo-5-quinolinesulfonic acid (8-OH-7-I-5QSA) were purchased from Sigma-Aldrich (Merck, St. Louis, MO, USA).

Approximately 5 kg of tubers with a diameter > 3 cm were collected for chemical analyses. The tubers were washed several times in tap water. Further, for the analysis, each tuber was cut in half and crushed using a kitchen mixer. Next, chemical analyses were performed on fresh tubers. The second half of the tubers were used for the lyophilisation process and other types of chemical analyses on dry samples; the individual analytical methods are described below.

2.2. Determination of Total Iodine

The iodine content in freeze-dried potato tuber samples was analysed via the alkaline extraction of 0.2 g of the samples with tetramethylammonium hydroxide (TMAH), with the use of amylase to digest starch (during the extraction process), and by using inductively coupled plasma mass spectrometry (ICP-MS/MS) with a triple quadruple spectrometer (iCAP TQ ICP-MS). The analysis was based on research published by Smoleń et al. [31], based on PN-EN 15111:2008 [32]. In total, 0.5 g of air-dried potato tuber samples, 10 mL of double distilled water and 1 mL of 25% TMAH (Sigma-Aldrich) were put into 30 mL Falcon tubes. After mixing, the samples were incubated for 3 h at 90 °C. After incubation, the samples were cooled to a temperature of approximately 20 °C and filled to 30 mL with double distilled water. After mixing, the samples were centrifuged for 15 min at 4500 rpm.

2.3. Determination of Iodoquinolines

The contents of 5-Cl-7-I-8-Q and 8-OH-7-I-5QSA in potato tubers were analysed by LC-MS/MS. They were determined in extracts prepared with 75% ethanol: samples of 4 g of fresh mixed plant material were weighed into 50 mL PP falcon tubes and treated with 20 mL of 75% ethanol with internal standard dissolved in it (50 ng/mL of hydroxychloroquine sulfate/Merck KGaA, Darmstadt, Germany/was used). Then, the samples were sonicated in an ultrasonic bath at 50 °C for 1 h. After that, the samples were homogenised and centrifuged at 4500 RPM (RCF = 4188× g) for 15 min at 5 °C. Next, approximately 4 mL samples of the extracts were filtered through a syringe filter (FilterBio NY Syringe Filter, nylon, pore size of 0.22 μm) to a 7 mL PP test tube and 1 mL was transferred into a chromatographic vial for analysis.

The analysis was performed using LC-MS/MS (HPLC: Ultimate 3000, Thermo Scientific; spectrometer 4500 QTRAP, Sciex). Chromatographic separation was carried out on a Luna 3 µm PhenylHexyl 100 Å column (150 mm × 3 mm, id 3 µm; Penomenex, Torrance, CA, USA). The column temperature was set to 40 °C and the autosampler temperature was set to 10 °C

For the determination of 5-Cl-7-I-8-Q, demineralised water (eluent A) and 0.3% formic acid (FA) in 100% methanol (eluent B) were used as eluents (Table S1). For the determination of 8-OH-7-I-5QSA, a separate chromatographic analysis (as well as a re-extraction process) was conducted: eluent A was the same and 0.3% FA in acetonitrile (eluent B) was used instead of methanol (other parameters of the chromatographic process were the same as for 5-Cl-7-I-8-Q). For both analytes, a gradient of 0.5 mL/min was used. The gradient steps used are listed in Table S1. [33].

2.4. Potato Analysis after Sample Drying

Half of the tubers samples (separately for replication and treatments) were frozen at −20◦C and lyophilised with the use of a Christ Alpha 1–4 lyophiliser (Martin Christ Gefriertrocknungsanlagen GmbH, Harz, Germany). The samples of lyophilised tubers were ground in a laboratory hurricane grinder (WŻ-1, Sadkiewicz Instruments, ZDPP, Poland) and stored in tightly closed polyethylene bags (at a temperature of 2–8 °C) until the analysis. The samples were subsequently analysed to determine the concentrations of the following elements: iodine, by using the ICP-MS/MS technique (Inductively coupled plasma–triple quadrupole mass spectrometry; iCAP TQ ICP-MS; ThermoFisher Scientific, Bremen, Germany); total nitrogen (N-total), by using the Kjeldahl method; P, K, Mg, Ca, S, Na, Al (by using a ICP-OES spectrometer, Prodigy Teledyne Leeman Labs, Mason, OH, USA); and B, Cu, Fe, Mn, Zn, Mo, Ni, Ag, As, Cd, Pb, Sb and Tl, by using the ICP-MS/MS technique.

2.5. Determination of Macro, Microelements and Trace Elements

The Kjeldahl method was applied to determine N-total [34] using a Foss Digestor 2020 oven digester from the Tecator TMi Velp UDK 139 Semi Automatic Distillation Unit. For the determination of other macro, microelements and trace elements, the samples (0.5 g) were placed in 55 mLF TM (TFM—Modified Poly-Tetra-Fluoro-Ethylene (PTFE)) vessels and mineralised in 10 mL of 65% super-pure HNO₃ (Merck No. 100443.2500) in a Mars 5 Xpress microwave mineralisation system (CEM, Matthews, NC, USA). Mineralisation was carried out according to the following method: a temperature of 200 °C was reached for 15 min and this temperature was maintained for 20 min. After cooling, the samples were quantitatively transferred to 25 mL volumetric flasks with redistilled water.

The contents of P, K, Mg, Ca, S, Na, Al were determined using a high-dispersive inductively coupled plasma optical emission spectrometer (ICP-OES, Prodigy Teledyne Leeman Labs, Mason, OH, USA) [35]. For the determination of B, Cu, Fe, Mn, Zn, Mo, Ni, Ag, As, Cd, Pb, Sb and Tl contents, inductively coupled plasma mass spectrometry (ICP-MS/MS) with a triple quadrupole (iCAP TQ ICP-MS ThermoFisher Scientific, Bremen, Germany) was used.

2.6. Determination of Nitrogenous Ions, and Chlorides

The content was analysed in samples extracted with ethanol. To analyse NH₄⁺, Cl and NO₃⁻ levels in the potato tuber, samples were extracted with 2% acetic acid, using an AQ2 Discrete Analyzer (Seal Analytical, Mequon, WI, USA), and the protocols provided by the manufacturer of this analyser.

2.7. Determination of the Antioxidant Capacity

Preparation of Ethanol Extracts

Ethanol extracts (20 g of raw potato in 80 mL of a 96% ethanol solution) were placed in a bath heated to 95 °C and incubated for 15 min. They were then cooled and homogenised. The ground material was filtered on a medium quality filter. The resulting filtrate was transferred to a 100 cm³ flask and made up to the mark with 96% ethanol solution. They were then filtered using filter paper and finally stored at −22 °C. The polyphenol content and ABTS radical quenching activity were determined in the extract thus prepared.

2.8. Determination of the Polyphenols Content

The ethanol extracts described above were used to determine the total content of polyphenolic compounds, using the Folin–Ciocalteu reagent (Merck, St. Louis, MO, USA). Ethanolic extracts were diluted 1:20 with distilled water. The total phenolic content of the diluted 5 mL extracts was measured after 20 min by spectrophotometry at 760 nm (blank = ethanol), using 0.5 mL of Folin–Ciocalteu reagent and 0.25 mL of 25% sodium carbonate and a RayLeigh UV-1800 spectrophotometer (China). Results were expressed as milligrams of gallic acid equivalents per 100 g of fresh weight, based on the standard curve for the aforementioned acid (mg GAE 100 g⁻¹ f.w.) [36].

2.9. Determination of the Total Carotenoids Content

The total carotenoid content was analysed by extracting carotenoids from samples using a mixture of acetone and hexane (4:6 v/v) (PureLand, Chemland, Stargard, Poland) according to the Polish Standard, with minor modifications [37,38]. The samples from 0.5 g of freeze-dried potato tuber were extracted in a porcelain mortar with a mixture of acetone and hexane, with the addition of approximately 0.5 g of roasted sand. The extracts were transferred to a cylinder and the volume of the extract was measured. Absorbance was examined after 30 min, at a 1:10 dilution, and at 450 nm using a spectrophotometer (UV-1800, Rayleigh, Beijing, China). The resulting values were assessed using a β-carotene standard curve (Merck, St. Louis, MO, USA).

2.10. Determination of the Antioxidant Activity Using the ABTS Method

The antioxidant potential was determined in ethanol extracts from potato tubers using the ABTS radical quenching capacity method and was expressed as the Trolox equivalent in mg/g of the sample. Trolox was purchased from Merck (Merck, St. Louis, MO, USA). The colour change, monitored by the change in absorbance at 734 nm via a spectrophotometer (UV-1800, Rayleigh, Beijing, China) after a specified time and temperature (6 min at 30 °C), is proportional to the antioxidant’s concentration [39].

2.11. Determination of L-Ascorbic Acid

The vitamin C content was determined by capillary electrophoresis (dehydroascorbic acid (DHA)) and (ascorbic acid (AA)) in fresh tuber samples. After the homogenisation of 2 g of potato tubers in 8 mL of 2% oxalic acid, the homogenates were centrifuged for 15 min at 4500 rpm and at 5 °C. Supernatants were collected, centrifuged for a further 10 min at 10,000 rpm and analysed using a Beckman PA 800 Plus capillary electrophoresis (CE) system with DAD detection. Capillaries of i.d. 50 µm, o.d. 365 µm and a total length of 50 cm (40 cm to detector) were used. The negative power supply of −25 kV was applied. A running buffer solution containing 30 mM of NaH₂PO₄, 15 mM of Na₂B₄O₇ and 0.2 mM of CTAB (pH 8.80) was used. The method was described in an earlier publication [37].

2.12. Basic Chemical Composition

The total protein content was measured using the Kjeldahl method (PN-EN ISO 8968-1: 2014-03). The sample was previously lyophilised and mineralised in concentrated sulphuric acid (VI). Then, the ammonia formed from the nitrogenous bonds contained in the protein was distilled off and titrated with 0.1 M hydrochloric acid in the presence of a Tashiro indicator to a light purple colour (AOAC procedure No. 950.36) [40].

The fat content was determined by the Soxhlet method (PN-A-79011-4: 1998) [40]. The sample was subjected to continuous multiple extractions with a Foss Soxtec 2050 Solvent Extraction System apparatus. The organic solvent in the form of petroleum ether was driven off and the mass of the extracted fat was determined (AOAC procedure No. 950.38).

The mineral content in the form of ash was determined using the method (PN-A-79011-8: 1998) [40]. Samples were weighed, ashed over a burner, and finally ashed in a muffle furnace. The time and rate of the process were adjusted accordingly so that the organic compounds were ashed and only the minerals forming the crude ash remained (AOAC procedure No. 930.05).

The determination of the total dietary fibre was based on the AACC 32-05.01 method and the AOAC 985.29 method [41]. A sample of the potato was subjected to enzyme digestion (including thermostable a-amylase, purified protease and purified amyloglucosidase).

The total carbohydrates are expressed using the following formula: 100 is the sum of the total fat (g), total protein (g), minerals in the form of ash (g) and dietary fibre (g) [42].

2.13. Assessment of Iodine-Fortified Potato Tubers for Consumer Health Safety

The daily intake of I (D-I), as well as the percentage of the Recommended Daily Allowance of I (% RDA-I), were calculated from 100 g of potato tubers. The value of RDA-I was based on the World Health Organization’s (WHO) recommendation for children above 12 years old and adults, i.e., 150 μg of I [2,43]. The consumer safety of iodine-enriched potato tubers was evaluated on the basis of the HQ (hazard quotient) values that describe the risk to human health that results from the intake of I through the consumption of fresh potato tubers. The calculation of the HQ-I values was performed according to the following equation: HQ = ADD/RfD, where ADD is the average daily dose of I (mg of I per kg body weight per day) and RfD represents the recommended dietary tolerable upper intake level of I [44]. The average daily dose (ADD) was determined using the following equation: ADD = (MI·CF·DI)/BW, where MI is the iodine concentration in potato tubers (mg·kg⁻¹ d.w.), CF is the fresh to dry weight conversion factor for plant samples (the dry weight to fresh weight ratio is 0.162 on average), DI is the daily intake of iodine (0.1 kg) and BW is the obtained body weight measurement (70 kg). The RfD value obtained for I was 1100 μg of I·day⁻¹ [2,45]. The presented method of HQ calculation only represents I intake from fresh potato tubers. The method of calculation was described in an earlier publication [45].

2.14. Statistical Analyses

All data were statistically verified using two-way analysis of variance (ANOVA) in the Statistica 13.0 PL (https://www.tibco.com/products/data-science, acceseed on 31 January 2023, StatSoft Inc., Tulsa, OK 74104, USA)(2022) program at a significance level of p < 0.05. In the case of significant effects, homogenous groups were distinguished on the basis of a post hoc Duncan’s test.

3. Results and Discussion

Péter Dobosy et al. (2020) showed that the biofortification of potatoes was not effective. The amount of accumulated iodine was too low to cover the daily iodine requirement of an adult (150 μg/day) [46]. The lack of a positive iodine accumulation in the potato for these authors could have been due to the use of an inappropriate chemical analysis procedure. The method used could result in iodine loss during mineralisation in nitric acid. The use of alkaline extraction in TMAH (Tetramethylammonium hydroxide), according to the European Union analytical standard, should have a positive effect on the accumulation of iodine in potatoes. Ledwożyw-Smoleń et al. (2020) evaluated the effectiveness of the iodine biofortification of potato tubers. The application of KI via soil and the application of KIO₃ via foliar feeding were tested in a three-year field experiment. Both the soil and foliar feeding iodine application methods resulted in potato tubers with an increased content of this element without a decrease in starch and sugar content. The highest efficiency of iodine biofortification was recorded for the foliar spraying of KIO₃. The obtained iodine level in 100 g of potatoes could be sufficient to cover up to 25% of the recommended daily intake of this element [47].

Our research confirms that the applied biofiltration method using inorganic and organic forms of iodine, i.e., iodoquinolines, can cover approximately 46.02% (KIO₃), 18.88% (8-OH-7-I-5QSA) and 16.02% (5-Cl-7-I-8-Q) of the daily iodine requirement (RDA) if a 260 g daily portion of potatoes is consumed.

3.1. Description of Potato Tuber Yield

Table 2. Average weight of tubers per plant and average number of tubers per plant.

Treatment	Tuber Yield/Plant (g)	Total Number of Tubers/Plant (Pcs.)
Control	239.17 a ± 18.60	8.17 a ± 0.92
KIO3	244.38 a ± 18.46	8.23 a ± 0.44
8-OH-7-I-5QSA	250.63 a ± 32.30	8.50 a ± 0.57
5-Cl-7-I-8-Q	243.54 a ± 13.17	8.60 a ± 1.07

Results are shown as mean ± standard deviation (SD); n = 4; means followed by the same letter are not significantly different (p < 0.05).

shows the effect of biofortification on the total number of potato tubers per plant and the average yield weight of the whole plant. Biofortification with iodine compounds had no significant effect on tuber weight in terms of yield. The potato tuber weight ranged from 239.17 to 250.63 g per plant.

There were no statistically significant differences after biofortification regarding the total number of tubers per plant. The average number of tubers per plant was 8–9 per crop.

3.2. Determination of Total Iodine

The content of iodine in the potato tubers biofortified with the control, KIO₃, 8-Hydroxy-7-iodo-5-quinolinesulfonic acid and 5-Chloro-7-iodo-8-quinolinol treatments is presented in Figure 1A. The iodine content of the potato tubers biofortified with KIO₃, 8-OH-7-I-5QSA and 5-Cl-7-I-8-Q acid was significantly higher (compared to the control 24.96 µg·kg⁻¹ d.w.). The highest iodine content was in the tuber enriched with KIO₃, with a value of 1400.15 µg·kg⁻¹ d.w. In the case of the organic forms of iodine, the higher iodine accumulation in potato tubers was shown by 8-OH-7-I-5QSA acid at 693.65 µg·kg⁻¹ d.w., when compared to the application of 5-Cl-7-I-8-Q acid (502.79 µg·kg⁻¹ d.w.). Thus, it can be concluded that biofortification is effective in increasing the iodine content of the potato tuber Solanum tuberosum L. Several techniques for the biofortification of plants with iodine have been developed in studies carried out worldwide by various research centres. Iodine accumulation through the use of biofortification has been demonstrated mainly in studies on vegetables, but also on fruit. So far, it has been possible to enrich strawberries, cucumbers, kohlrabi, lettuce, potatoes, carrots, spinach, beans, pak choi, cabbage, Chinese cabbage, tomatoes, rice, celery and radish [48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65]. The high level of research sophistication and the large number of results that demonstrate the positive effects of iodine biofortification provide a good basis and opportunity for this agrotechnology to be used in the future.

3.3. Determination of Iodoquinolines

The content of iodoquinolines in the potato tubers biofortified with the control, KIO₃, 8-Hydroxy-7-iodo-5-quinolinesulfonic acid, and 5-Chloro-7-iodo-8-quinolinol treatments is presented in Figure 1B,C. There was a statistically significant higher accumulation of 8-OH-7-I-5QSA (24.48 µg kg⁻¹ d.w.) in potato tubers biofortified with this form of iodine compared to the control (2.98 µg kg⁻¹ d.w.). There were no statistically significant differences in the amount of accumulated iodine in the 8-OH-7-I-5QSA form in the other enrichment types (KIO₃, 5-Cl-7-I-8-Q) compared to the control. Therefore, the biofortification of potatoes with 8-OH-7-I-5QSA showed the highest accumulation of this form of iodine in the tuber.

When analysing the iodine accumulation of 5-Cl-7-I-8-Q in biofortified potato tubers, the content of the 5-Cl-7-I-8-Q form of iodine (15.94 µg kg⁻¹ d.w.) was higher compared to the control (5.68 µg kg⁻¹ d.w.); this difference was statistically significant. A statistically significant decrease in the accumulation of 5-Cl-7-I-8-Q in potato tubers biofortified with the KIO₃ form (1.45 µg kg⁻¹ d.w.) and the 8-OH-7-I-5QSA (0.91 µg kg⁻¹ d.w.) treatment was observed compared to the control (5.68 µg kg⁻¹ d.w.) As a result, potato biofortification with 5-Cl-7-I-8-Q showed the highest accumulation of this form of iodine in the potato tuber.

According to our study, both iodoquinolines (8-OH-7-I-5QSA and 5-Cl-7-I-8-Q) are endogenous compounds. Unfortunately, the metabolic pathway is not known at this time. Our next research objective will be to know and describe the metabolic pathways of the endogenous iodoquinoline compounds that occur in the potato tuber. The amounts of endogenous compounds detected are small in the potato tubers, but they are there. What is more, research conducted by the team at the University of Agriculture in Krakow on another vegetable, kale roots, also found the presence of both iodoquinolines. Small peaks were observed in the kale roots, demonstrating that endogenous compounds were detected.

3.4. Determination of Macro- and Microminerals

The contents of various minerals (N, Mg, Ca, S, Na, Al, B, Cu, Fe, Mn, Zn, Mo, Ni, Ag, As, Cd, Pb, Sb and Tl) in potato tubers biofortified with each treatment are presented in

Table 3. The contents of various minerals in potato tubers biofortified with the control, KIO_3, 8-Hydroxy-7-iodo-5-quinolinesulfonic acid, and 5-Chloro-7-iodo-8-quinolinol treatments.

Type	Chemical Element	Treatment
		Control	KIO3	8-OH-7-I-5QSA	5-Cl-7-I-8-Q
Macrominerals (% d.w.)	Nitrogen (N)	1.01 a ± 0.06	1.04 a ± 0.15	0.93 a ± 0.12	1.09 a ± 0.05
	Phosphorus (P)	0.26 b ± 0.02	0.25 a,b ± 0.01	0.24 a ± 0.00	0.25 a,b ± 0.00
	Potassium (K)	2.12 a ± 0.08	2.09 a ± 0.08	2.03 a ± 0.02	2.07 a ± 0.01
	Magnesium (Mg)	0.13 b ± 0.01	0.12 a,b ± 0.01	0.12 a ± 0.00	0.12 a,b ± 0.00
	Calcium (Ca)	0.06 a ± 0.00	0.06 a ± 0.00	0.06 a ± 0.00	0.06 a ± 0.00
	Sulphur (S)	0.17 a ± 0.01	0.23 a ± 0.11	0.16 a ± 0.01	0.16 a ± 0.00
	Sodium (Na)	0.01 a ± 0.00	0.01 a ± 0.00	0.01 a ± 0.00	0.01 a ± 0.00
Trace elements (mg kg−1 d.w.)	Aluminium (Al)	42.83 b ± 9.47	32.58 a ± 6.33	30.42 a ± 3.52	23.47 a ± 4.27
	Boron (B)	2.72 a,b ± 0.05	2.80 a,b ± 0.53	2.42 a ± 0.33	3.35 b ± 0.88
	Copper (Cu)	8.00 a ± 0.61	9.38 b ± 0.59	8.61 a ± 0.31	8.55 a ± 0.42
	Iron (Fe)	62.35 b ± 22.95	49.25 a,b ± 11.30	41.27 a,b ± 1.88	32.81 a ± 10.53
	Manganese (Mn)	8.16 a ± 0.74	9.48 b ± 0.51	8.66 a ± 0.29	8.44 a ± 0.33
	Zinc (Zn)	19.88 a ± 1.08	25.01 b ± 1.76	21.92 a ± 1.98	21.70 a ± 1.72
	Molybdenum (Mo)	0.63 b ± 0.11	0.49 a ± 0.04	0.51 a ± 0.02	0.51 a ± 0.03
Trace elements (µg kg−1 d.w.)	Nickel (Ni)	182.11 b ± 43.61	129.23 a,b ± 67.69	77.12 a ± 5.15	69.99 a ± 4.05
	Silver (Ag)	5.61 b ± 1.54	4.21 a,b ± 1.43	3.75 a ± 0.68	2.85 a ± 0.41
	Arsenic (As)	31.56 a ± 2.67	29.60 a ± 1.47	26.79 a ± 3.48	22.39 a ± 10.72
	Cadmium (Cd)	433.20 a ± 16.39	426.44 a ± 34.73	402.21 a ± 35.08	411.03 a ± 27.80
	Lead (Pb)	217.02 c ± 19.12	144.25 b ± 9.21	132.11 a,b ± 14.11	113.18 a,b ± 22.38
	Antimony (Sb)	8.80 b ± 0.35	7.20 a,b ± 1.93	5.87 a ± 0.84	5.27 a ± 1.53
	Thallium (Tl)	3.99 b ± 0.26	1.96 a ± 0.20	1.93 a ± 0.36	1.76 a ± 0.41

Results are shown as mean ± standard deviation (SD); n = 4; means followed by the same letter are not significantly different (p < 0.05); d.w. dry weight.

. The study shows that the contents of the macrominerals N, K, Ca, S, Na, and the trace elements As and Cd, in potato tubers biofortified with KIO₃, 8-OH-7-I-5QSA acid and 5-Cl-7-I-8-Q did not change compared to the control. For phosphorus (P) and magnesium (Mg), biofortification with 8-OH-7-I-5QSA decreased the content of these elements in potato tubers in a relatively small but statistically significant way. This is not beneficial, as these elements have a positive effect on the functioning of the bones (P) and the muscular and nervous systems (Mg) [2]. Most of the daily requirements of these elements are covered by potato consumption.

Biofortification reduced the aluminium (Al) accumulation in potato tubers enriched with KIO3, 8-OH-7-I-5QSA, 5-Cl-7-I-8-Q. Aluminium, like other metals, can act as a substitute for magnesium, calcium or iron ions, and thereby block the action of enzymes that are dependent on them. This results in changes in the synthesis of essential proteins, in the function of genetic material and the permeability of cell membranes [2]. Its excess in the diet is not beneficial, and thus a reduction in the aluminium content after biofortification is a good effect.

When compared to the control, the boron (B) content did not change in a statistically significant way after the application of all the tested iodine compounds. Boron protects against excessive fluoride accumulation and aids in its removal, facilitates vitamin D absorption and is responsible for maintaining adequate calcium levels in the human body [2].

It was observed that the application of KIO₃ had a statistically significant effect on the increase in copper (Cu) and manganese (Mn) content. However, compared to the control, the application of 8-OH-7-I-5QSA and 5-Cl-7-I-8-Q did not affect the concentrations of Cu and Mn in potato tubers. Copper in the body performs the role in maintaining normal levels of collagen and elastin, which are the main structural components of our body. Manganese, on the other hand, regulates bone metabolism and promotes bone growth and healing, which is particularly important during post-traumatic recovery and in the prevention of osteoporosis [2]. Their higher accumulation in the potato tuber is a positive consequence of biofortification.

This study shows that only biofortification using 5-Cl-7-I-8-Q had a statistically significant effect on the reduction in the iron (Fe) content of tubers. However, in comparison with the control, in each treatment that applied iodine compounds, there was a decrease in the iron content of the tubers. Some varieties of Solanum tuberosum L. contain iron in amounts that are comparable to those found in some cereals (rice, maize, wheat). The iron of Solanum tuberosum L. should be bioavailable because, unlike cereals, it contains very little phytic acid [18].

The zinc (Zn) content of the tubers that were enriched only with KIO₃ was statistically significantly higher than that in the control. Solanum tuberosum L. of different potato varieties contains zinc in the amount of 0.5–4.6 μg g⁻¹ of fresh weight. Zinc is essential for the proper functioning of the body’s immune system and is involved in cell division, growth and wound healing [2,18]. An increasing accumulation of this element after biofortification with KIO₃ is a positive effect of our experiment because it improved the nutritional value of the tubers.

Based on the results, biofortification was shown to significantly reduce molybdenum (Mo) in potato tubers after the application of KIO₃, 8-OH-7-I-5QSA and 5-Cl-7-I-8-Q. Among other things, molybdenum enables the production of enzymes that are necessary for the assimilation of fats and sugars, i.e., it is needed to provide cells with energy. Molybdenum also influences the absorption of iron and thus indirectly protects the human body from becoming anaemic [2,18].

A decrease in nickel (Ni) and silver (Ag) content was demonstrated in potato tubers biofortified with 8-OH-7-I-5QSA, 5-Cl-7-I-8-Q and also with KIO₃, but with less intensity. Nickel performs an important role in lipid metabolism and is also involved in enhancing hormonal activity and stabilising nucleic acid structures. Chromium stimulates pancreatic activity, increases the effectiveness of insulin, facilitates its binding to receptors, increases the number of these receptors, and improves the efficiency of glucose uptake and utilisation by cells. Chromium is also involved in the metabolism of proteins and fats, particularly cholesterol compounds. The reduced accumulation of nickel is not beneficial for consumers [2]. However, the opposite is true for silver. Silver has no physiological role in the human body. The element is absorbed into the human body and enters systemic circulation in the form of a protein complex. This complex is metabolised and eliminated by the liver and kidneys; thus, its reduced accumulation is not dangerous to the body and is not among the negative effects of biofortification [2].

Biofortification with KIO₃ acid significantly increased the cobalt (Co) content of potato tubers, in contrast to organic forms of enrichment with 8-OH-7-I-5QSA and 5-Cl-7-I-8-Q, where no statistically significant changes in the cobalt content were shown in potato tubers. Cobalt has a regulatory function in the production of red blood cells, as well as in the metabolism of nucleic acids and proteins. It is also an essential element for pregnant women, as it is involved in the production of vitamin B9 (folic acid) [2].

A statistically significant decrease in the lead (Pb), antimony (Sb) and thallium (Tl) content of potato tubers after biofortification with KIO₃, 8-OH-7-I-5QSA, 5-Cl-7-I-8-Q was demonstrated. Pb, Sb and Tl in the human body are completely redundant, as they have no biologically relevant functions. The reduced accumulation of these elements following biofortification is a beneficial effect that improves the nutritional value of the potato tuber [2,66,67].

The analysis showed a significantly higher selenium (Se) content after KIO₃ biofortification. Unfortunately, this was not observed after the treatment with 8-OH-7-I-5QSA and 5-Cl-7-I-8-Q. In addition, no reduction in selenium accumulation was observed after biofortification with 8-OH-7-I-5QSA and 5-Cl-7-I-8-Q. Selenium comprises part of the amino acids that build key enzymes for many metabolic processes. It protects cells from oxidative stress and exerts effects on the immune system. It inhibits the division of cancer cells and has a protective effect on neurons and cardiac muscle cells [2].

3.5. Determination of Nitrogenous Ions, and Chlorides

The contents of ammonium ions, nitrate(V), nitrate(III) and chlorides in the potato tubers biofortified with the control, KIO3, 8-Hydroxy-7-iodo-5-quinolinesulfonic acid and 5-Chloro-7-iodo-8-quinolinol treatments are presented in

Table 4. The content of nitrogenous compounds and chlorides.

Treatment	NH4+ (mg∙kg−1 f.w.)	Nitrate (V) NO3− (mg∙kg−1 f.w.)	Nitrate (III) NO2− (mg∙kg−1 f.w.)	Cl− (mg∙kg−1 f.w.)
Control	19.16 a ± 1.41	4.77 a,b ± 1.42	0.30 a ± 0.19	423.59 b ± 76.48
KIO3	20.04 a ± 2.03	3.81 b ± 0.96	0.32 a ± 0.14	333.12 a,b ± 86.80
8-OH-7-I-5QSA	14.96 b ± 0.88	6.21 a ± 1.23	0.32 a ± 0.05	264.92 a ± 13.26
5-Cl-7-I-8-Q	13.52 b ± 2.00	6.43 a ± 0.99	0.20 a ± 0.08	265.31 a ± 31.25

Results are shown as mean ± standard deviation (SD); n = 4; Means followed by the same letter are not significantly different (p < 0.05); f.w. fresh weight.

. The ammonium ion content was significantly higher and the chlorides content was lower than that in the control for potato tubers biofortified with 8-OH-7-I-5QSA and 5-Cl-7-I-8-Q. Biofortification with all the tested iodine compounds had no statistically significant effect on the nitrate(V) and nitrate(III) content of the tubers relative to the control. These are surprising results. In the research conducted by Smoleń et al. [68], the application of IO₃⁻ (as opposed to I⁻) stimulated reductions in NO₃⁻, increased NH₄⁺ assimilation in the GS-GOGAT cycle and improved photosynthesis in lettuce plants. However, in our study, the application of both iodoquinolines, 8-OH-7-I-5QSA and 5-Cl-7-I-8-Q, reduced the content of ammonium ions and increased the content of nitrate(V), in contrast to KIO₃. However, in our study, the nitrate content of the tubers was at a relatively low level, rising from 3.81 after KIO₃ application to 6.43 in the 5-Cl-7-I-8-Q treatment. Nitrate(V) is among the compounds found in food products. The main sources of Nitrate(V) in the daily human diet are vegetables and their preparations, and potatoes. Many factors are considered to influence the nitrate(V) levels, including the species, cultivar, whether the plant is fertilised with nitrogen compounds, the soil and climatic conditions during the growing season, the harvest date, storage conditions and time, and technological processes. Nitrate(V) is not directly harmful to humans or animals, but when it is reduced to nitrate(III), it may pose a health risk. Potato is classified as a plant with a medium tendency to accumulate nitrate(V) and nitrate(III) in tubers. In our study, biofortification with iodine compounds did not increase nitrate(III) content, and the concentration of nitrate(V) was relative low in the potato; as such, the tuber used in the experiment can be considered safe for consumers in terms of nitrate(V) and nitrate(III) content.

3.6. Determination of the Polyphenols Content

The total polyphenol content of the control potato was significantly lower (197.31 mg GAE∙100 g⁻¹ f.w.), but only when compared to the potato biofortified with 8-OH-7-I-5QSA acid (233.33 mg GAE∙100 g⁻¹ f.w.) (

Table 5. The antioxidant activity and content of total poliphenols, total carotenoids, and vitamin C in two forms (ascorbic acid (AA)) and (dehydroascorbic acid (DHA)) in fresh potato tubers.

Treatment	Total Phenolics (mg GAE∙100 g−1 f.w.)	ABTS (µmol Trolox∙g−1 f.w.)	Ascorbic acid (AA) (mg∙100 g−1 f.w.)	Dehydroascorbic acid (DHA) (mg∙100 g−1 f.w.)	Total Carotenoids (mg∙100 g−1 d.w.)
Control	197.31 a ± 7.25	19.07 a ± 0.13	25.04 a ± 1.69	1.99 a ± 0.58	3.46 d ± 0.07
KIO3	213.44 a,b ± 8.96	15.00 a ± 3.43	24.37 a ± 1.74	3.65 b,c ± 0.85	2.96 c ± 0.13
8-OH-7-I-5QSA	233.33 b ± 9.97	15.87 a ± 2.67	24.76 a ± 1.47	4.81 c ± 1.55	2.45 b ± 0.02
5-Cl-7-I-8-Q	197.31 a,b ± 8.77	14.10 a ± 2.52	23.98 a ± 1.98	3.00 b ± 0.52	1.47 a ± 0.06

Results are shown as mean ± standard deviation (SD); n = 4; means followed by the same letter are not significantly different (p < 0.05); f.w. fresh weight; d.w. dry weight.

). Biofortification with iodine using the organic form of 8-OH-7-I-5QSA acid improved the polyphenolic profile of the potato tuber. It is worth noting that there was no statistically significant difference between the three different iodine treatments with respect to the polyphenol content of the tubers. Polyphenols are organic compounds that naturally occur in various parts of many plant species: flowers, fruits, seeds, leaves, roots, bark and wood parts. They have a diverse structure, molecular weight, and physical, biological and chemical properties. The most important source of polyphenols (in the human diet) after apples and oranges is potatoes, which contain an average of 160 mg∙100 g⁻¹ of these compounds in fresh weight [69].

Phytochemicals perform important antioxidant functions that contribute to the delay of ageing and the prevention of diseases, including cancer. Their antioxidant properties help inhibit the formation of reactive oxygen species (ROS) or nitrogen species (RNS) that form free radicals, help neutralise them, chelate oxidative Fe and Cu metal ions, and reduce the activity of enzymes that catalyse the oxidation reaction. In addition, polyphenols prevent blood clots, protect the vessels from harmful “bad” LDL cholesterol, limit the absorption of sugars in the gastrointestinal tract, accelerate the burning of fats accumulated in the tissues, act as an antibacterial and antioxidant in tissues, have antibacterial, antiviral and antifungal effects, and are responsible for the yellow, red, green and blue colouring of vegetables and fruit (flavonoids), including potatoes [70,71].

Given the above-mentioned properties, the high polyphenol content of the potato is important for the consumer because it has a health-promoting effect on the body. Biofortification does not adversely affect the potato’s antioxidant properties, and thus the process is safe. Furthermore, biofortification with 8-OH-7-I-5QSA can improve the polyphenol profile, which increases the nutritional value of the potato.

3.7. Determination of the Total Carotenoids Content

The carotenoid content of the tubers depended on the tested treatments, varying between 1.47 and 3.46 mg∙100 g⁻¹ d.w. (Table 5). On average, the control contained the most total carotenoids, with 3.46 mg∙100 g⁻¹ d.w., and the tuber enriched with 5-Cl-7-I-8-Q contained the least, with 1.47 mg 100 g⁻¹ d.w. A statistically significant decrease in the total carotenoid content was found in potato tubers biofortified with all the tested forms of iodine. The occurrence of carotenoids is characterised by high variability. This is due to genetic and environmental factors (light, temperature), as well as tuber maturity. The colour of fresh potato flesh is determined by the varying content of blue or red anthocyanin pigments or is a result of the accumulation of yellow and orange carotenoids [25]. Potato carotenoids are mainly oxygen carotenoids, which are also called xanthophylls. Potatoes with white flesh have 5–10 mg∙kg⁻¹ f.w. of the so-called total carotenoids, potatoes with yellow flesh have 10–35 mg kg⁻¹ f.w., and dark yellow potatoes can contain up to 100 mg∙kg⁻¹ f.w.; the highest recorded content reached up to 260 mg kg⁻¹ f.w. [25]. Lutein, zeaxanthin, violaxanthin and neoxanthin are the main carotenoids present in potatoes, while β-carotene is present in trace amounts [72]. Lutein and zeaxanthin are components of the human retina [25] that must be obtained from the diet to prevent age-related macular degeneration (AMD). Green leafy vegetables are a good source of lutein, but a poor source of zeaxanthin, which is found in significant concentrations in a small number of foods. Therefore, the potato could be a strategic source of lutein and zeaxanthin, especially as a staple food. The concentration of the total and individual carotenoids in potato tubers depends on several factors, such as the genotype, agronomic factors, post-harvest storage, and cooking and processing conditions [73].

Potato biofortification caused a reduction in the total carotenoid content. This is an unfavourable consequence of the application of this process, as it reduces the attractive nutritional value of potato tubers. However, our research was carried out in a pot experiment, and thus the effect of the use of iodoquinolines on this parameter should be verified in field cultivation.

3.8. Determination of the Antioxidant Activity Using the ABTS Method

Antioxidant activity was measured by the ability to quench the ABTS cation radical. Based on the performed analyses, there were no statistically significant differences in the antioxidant properties measured by ABTS between the applied iodine compounds (Table 5.). High antioxidant activity for the consumer is important as it reduces oxidative stress in cells and inflammatory processes in the body [74]. The tested iodine application treatments did not negatively affect the antioxidant activity of the potato; thus, the agronomic biofortification process can be considered as neutral for this parameter and as safe for future use.

3.9. Determination of L-Ascorbic Acid

Compared to the control, no statistically significant differences were found between the applied iodine compounds and the increase in the AA content of potato tubers (Table 5). However, a statistically significant increase in the DHA content was found in potato tubers that were biofortified with 5-Cl-7-I-8-Q > 8-OH-7-I-5QSA > KIO₃, relative to the control.

AA and DHA perform an oxidoreduction function in plant metabolism. Tubers of Solanum tuberosum L. have been found to contain between 22.2 and 121.4 mg∙100 g⁻¹ on a dry weight basis and between 6.5 and 36.9 mg∙100 g⁻¹ on a fresh weight basis of AA. The availability of AA depends on the cultivar, the state of maturity and the environmental conditions in which the plant is grown. The AA content decreases with the storage period of all varieties of tuber [18].

In Poland, due to the high consumption of potatoes, at 260 g day⁻¹ [75], half of the daily requirement of vitamin C is covered at the same time [2]. Therefore, for consumer reasons, a reduction in the content of vitamin C through biofortification would have a major negative impact on the ability of potato to cover the daily requirement of this vitamin. This study showed that biofortification has a positive effect on the total AA + DHA content of tubers; thus, the process of potato biofortification with iodine has a positive effect.

3.10. Basic Chemical Composition

There was a statistically significant increase in the dry matter content only in potato tubers enriched with 5-Cl-7-I-8-Q (Figure 2). The ash content of the potato tubers was statistically significantly different in tubers and ranged from 0.16 to 0.23 g∙100 g⁻¹ d.w. The lowest ash content was in tubers biofortified with the 8-OH-7-I-5QSA treatment and the highest content was found after the KIO₃ application. Biofortification had no statistically significant effect on the fat content of potato tubers enriched with 5-Cl-7-I-8-Q. However, there was a statistically significantly higher fat content in tubers enriched with KIO₃ and 8-OH-7-I-5QSA compared to the control. The results of the analyses showed that the protein content of potato tubers varied and ranged from 5.79 to 6.13 g∙100 g⁻¹ d.w. There was a statistically significantly lower protein content in tubers biofortified with the 8-OH-7-I-5QSA and 5-Cl-7-I-8-Q treatments relative to the control. Biofortification with iodine compounds had no significant effect on the dietary fibre content of tubers. The fibre content ranged from 10.5 to 11.2 g∙100 g⁻¹ d.w. The assimilable carbohydrate content ranged from 76.65 to 78.24 g∙100 g⁻¹ d.w. All results were relatively similar to each other.

Biofortification with KIO₃, 8-OH-7-I-5QSA, 5-Cl-7-I-8-Q did not engender changes in the basic composition of the potato tubers (dry matter, protein, fat, fibre and digestible carbohydrates). In conclusion, this process in no way reduces the nutritional attractiveness of potato tubers.

3.11. Assessment of Iodine-Fortified Potato Tubers for Consumer Health Safety

The enrichment of potato tubers with iodine compounds increased the percentage of the recommended daily intake of I (RDA-I) and HQ for iodine intake through the consumption of 100 and 260 g portions of potatoes by adults (

Table 6. Percentage coverage of Recommended Daily Allowance for iodine (% RDA-I) and the hazard quotient (HQ) for the intake of iodine via the consumption of 100 g and 260 g portions of fresh potato tubers by adults of 70 kg body weight.

Treatment	% RDA I (in 100 g Portion of Potato)	% RDA I (in 260 g Portion of Potato)	Daily Intake of I with 100 g of Potato (mg I∙day−1)	Daily Intake of I with 260 g of Potato (mg I∙day−1)	HQ for 100 g Portion of Potato	HQ for 260 g Portion of Potato
Control	0.31 b ± 0.05	0.82 b ± 0.12	0.001 b ± 0.00	0.001 b ± 0.00	0.001 b ± 0.00	0.003 b ± 0.00
KIO3	17.70 c ± 0.67	46.02 c ± 1.75	0.027 c ± 0.00	0.069 c ± 0.00	0.065 c ± 0.00	0.170 c ± 0.01
8-OH-7-I-5QSA	7.26 a ± 1.86	18.88 a ± 4.83	0.011 a ± 0.00	0.028 a ± 0.01	0.027 a ± 0.01	0.070 a ± 0.02
5-Cl-7-I-8-Q	6.16 a ± 0.56	16.02 a ± 1.45	0.009 a ± 0.00	0.024 a ± 0.00	0.022 a ± 0.00	0.057 a ± 0.01

Results are shown as mean ± standard deviation (SD); n = 4; means followed by the same letter are not significantly different (p < 0.05).

). The hazard quotient (HQ) value is intended to inform consumers about the safety of consuming food. If the HQ value is higher than 1, adverse health effects are possible. The HQ value for the consumption of potato tubers ranged from 0.001 to 0.065 and from 0.003 to 0.17 for 100 and 260 g portions, respectively, which is a harmless intake for consumers. The highest RDA-I percentage was observed in potato tubers that had been biofortified KIO₃, which was statistically significant compared to other treatments. The recommended daily intake of iodine for adults is 150 μg [2].

On average, a typical Pole eats 260 g of potatoes per day throughout the year [75]. This means that consuming potatoes enriched with KIO₃ would cover almost 46% of the daily iodine intake requirement. The consumption of potatoes enriched with the organic forms of iodine, 8-OH-7-I-5QSA and 5-Cl-7-I-8-Q, would cover about 19% and 16% of the daily iodine requirement, respectively. The intake of this micronutrient from KIO₃, 8-OH-7-I-5QSA and 5-Cl-7-I-8-Q enrichments are not higher than the tolerable upper intake level of iodine, which is 1100 μg∙day⁻¹ [76].

4. Conclusions

In summary, the results of the above studies showed an increase in the iodine content of the potato tubers Solanum tuberosum L. that was within a safe level for consumers (1 < HQ). Biofortification did not affect, in any negative way, the reduction in the yield weight relative to the whole plant. Biofortification with 8-OH-7-I-5QSA was shown to improve the polyphenolic profile of the potato tuber. In addition, it was shown to reduce the accumulation of the heavy metals Al, Ag, Pb and Tl in the potato tuber. Biofortification did not generate adverse changes in the basic composition of the potato tuber (dry matter, protein, fat, fibre, and digestible carbohydrates). Unfortunately, the only unwanted effect of this agrotechnology is a reduction in the carotenoid content of potato tubers. In the future, the research field of the cultivation of potato could be extended from the application of this process to research regarding the impact on the total carotenoid content. The refinement of this agrotechnology in practice may one day increase the content of iodine for consumers. The biofortification of Solanum tuberosum L. with inorganic and organic forms of iodine compounds may, therefore, become an additional preventative measure against iodine deficiencies in humans and may become, in part, a healthier alternative to iodine table salt (potassium iodide). The combination of the two concepts, taking into account the positive health-promoting properties already present and the biofortification used to further enhance the potato’s nutritional qualities, could create a vegetable with outstanding properties, the appropriate consumption of which will benefit the health of all consumers.