Antinutritional Nitrogen Compounds Content in Potato (Solanum tuberosum L.) Tubers Depending on the Genotype and Production System

Abstract

This experiment analyzed the effect of genetic determinants, the production technology, and the location on the nitrate and nitrites content in potato tubers directly after harvest. Nitrates and nitrites are anti-nutritional compounds, which limit or prevent the use of nutrients from consumed products and have a detrimental effect on the human body. The study was conducted on seven tetraploid potato (Solanum tuberosum L.) clones (‘13-VIII-10’, ‘13-VIII-27’, ‘13-VIII-45’, ‘13-VIII-49’, ‘13-VIII-50’, ‘13-VIII-60’, ‘TG-97–403’), and three cultivars (‘Jelly’, ‘Satina’, and ‘Tajfun’) at four locations in Poland under either an integrated (Młochów, Boguchwała) or organic (Radzików, Chwałowice) production system. The results showed that the production system, location, and year significantly affected the nitrate and nitrites content in potatoes. Potato tubers originating from organic farming exhibited a lower content of the tested compounds (NO₃⁻—124.1 mg kg⁻¹ FW; NO₂⁻—2.4 mg kg⁻¹ FW) than that in the tubers obtained from an integrated production system (NO₃⁻—203.7 mg kg⁻¹ FW, NO₂⁻—4.0 mg kg⁻¹ FW). Under conditions of moderate stress as usual in the case of organic farming, the production of nitrates and nitrites are significantly lower than in the integrated production system. In our research potatoes from an integrated production system were characterised by a nitrate content at the limit of the established standard, i.e., 200 mg kg⁻¹ FW. Tubers from the locality Chwałowice were characterised by the lowest nitrates and nitrites content, due to adequate environmental conditions, in addition to the best agricultural practice. The nitrates content depended to a greater extent on the environmental conditions than the potato on the genetic conditions. A higher content of nitrates and nitrites was found in the following potato clone 13-VIII-10 in Boguchwała from an integrated production system (NO₃⁻—269.2 mg kg⁻¹ F; NO₂⁻—6.0 mg kg⁻¹ FW), and in the ‘Tajfun’ cultivar (235.8; 5.8 mg kg⁻¹ FW, respectively). Such a relationship was not found in integrated production in Młochów, which indicates the influence of the environment.

1. Introduction

Despite a significant reduction in the area under potato cultivation in recent years, due to the multidirectional use of tubers, the potato is still one of the most important crops [1,2,3]. Edible potatoes, apart from ingredients constituting the nutritional value of tubers such as starch, sugars and minerals, contain undesirable substances. These are referred to as anti-nutritional and toxic components that cause poisoning. They occur naturally in the plant or arise from metabolic disturbances. In the consumer’s body, they impede the absorption of nutrients. Potato safety depends on the amount of antinutritional and toxic substances found in the tubers. This indicator is a basic condition for allowing the potato to be sold [4,5,6]. The main anti-nutrients contained in potato tubers are nitrates. Tubers also contain small amounts of toxic nitrites. The presence of nitrates in excessive amounts is dangerous, as they are precursors of highly toxic nitrites that cause methemoglobinemia or vitamin A deficiency. Nitrites entering the blood bind hemoglobin to form methemoglobin, which in the case of excessive intake may cause acute poisoning, even leading to death. This is particularly dangerous in infants under six months of age due to the presence in the blood of so-called fetal hemoglobin, which is more susceptible to oxidation and does not contain the enzyme: methemoglobin reductase, which is capable of unlocking hemoglobin [7,8,9,10]. The accumulation of nitrates in plants is particularly important, as 70% to 90% of these compounds supplied during the day come from the consumption of vegetables with 32% of this coming from the consumption of potatoes. Consequently, standards have been set for their content in plants as well as daily human intake [7,9,11,12]. According to Commission Regulation (EC) No. 1822/2005 of 8 November 2005, the nitrates content of potatos should not exceed 200 mg kg⁻¹ fresh weight of tubers (food). Based on the results of toxicological studies, the FAO/WHO Committee of Experts set the maximum acceptable daily intake (ADI) of nitrates for humans at 0–3.7 mg kg⁻¹ body weight human. For toxic nitrites, the value is 0–0.7 mg kg⁻¹ body weight [13,14,15]. Nitrogen taken up by plants from the soil (in the nitrate and ammonium forms) is used by them to synthesize amino acids, proteins, chlorophyll or phytohormones, among others. However, when there is an excessive supply of nitrogen, such as from high nitrogen fertilization, plants are unable to metabolize all of it and may accumulate it in the form of nitrates [9,16]. Nitrates content is genetically controlled and can be a characteristic of a species or even a variety. Expanding biological progress in potato production mean that new varieties are constantly being brought to market. They require research as early as the breeding-clone stage, related to determining their quality in terms of interaction with environmental conditions. Also important are the criteria set by direct consumers and the processing industry [1,17,18]. In addition to the genetic factor, agronomic practices including nitrogen fertilizers, crop protection strategies, soil moisture, light intensity and temperature also play a decisive role in nitrates accumulation in plants [7]. For the safety of tubers, organic cultivation based on the principles of “Ecologization of agricultural crop production space” [19] is of particular importance. Its development is related to the great interest, in recent years, in healthy food. However, it should be noted that research results to date on the safety of organically harvested potato are often inconsistent or even contradictory [20,21,22,23]. In this review, we present some of the most recent findings on the impact of environmental factors—crop locations to reduce nitrate content in potato clones and varieties. We also highlight several perspectives for future research aimed at optimizing the quality of potato tubers grown with different technologies used in sustainable agriculture. High edible potato consumption, continuous development in cultivation, concern for the environment and consumer health, and the order for constant monitoring of food contamination constituted the basis for the formulation of an original research objective. It concerned the evaluation of the influence of genetic and variable environmental conditions on the content of nitrates and nitrites in the fresh weight of tubers of edible potato clones and varieties. The results of the study will provide valuable guidance to farmers, as they will indicate which clones can be considered safe for the consumer.

2. Materials and Methods

2.1. Material

The experiment was conducted on ten genotypes of the potato (Solanum tuberosum L.); three cultivars: ‘Jelly’, ‘Satina’, and ‘Tajfun’ (with yellow flesh), and seven tetraploid breeding lines (four clones with yellow flesh, and three clones with white flesh). The potato clones were obtained from a crossing programme performed at the Plant Breeding and Acclimatization Institute—National Research Institute, Młochów Research Centre. All lines are complex hybrids that include introgression from multiple sources of tuber bearing Solanum species [24].

2.2. Field Experiment

The field experiment was conducted in three consecutive years (2014, 2015, 2016) as a randomized complete block design with two biological repeats (blocks) and ten-plant plots (plus two plants at the margin of each side of the field).The experiment was carried out at four locations: in Boguchwała (49°58′59″ N 21°56′23″ E; type of soil cambisol) and Młochów (52°3′0″ N 20°46′7″ E; type of soil podzol), under integrated cultivation technology, and in Radzików (52°13′38″ N 20°36′55″ E; feozem) and Chwałowice (51°10′56″ N 21°18′17″ E; cambisol), under organic cultivation. The cultivation sites where the organic technology was applied were certified by a Polish company (AGRO BIO TEST) in accordance with standard PN-EN ISO/IEC 17065:2013–03 [25]. The tubers were planted in the third decade of April, and the potato tubers were harvested after 130 days.. Temperature differences among localities were small with the mean values of the 3 years lowest at Chwałowice (14.4 °C), intermediate at Boguchwała and Młochów (14.5 °C) and highest at Radzików (14.7 °C). Precipitation varied among localities and years. The highest was recorded in Chwałowice, followed closely by Boguchwała and Młochów and lowest at Radzików. Detailed the conditions of potato cultivation including weather conditions from the locations, fertilisation and individual years are described in previous publications on this experience [24,26].

2.3. Sample Preparation for Laboratory Testing

Immediately after the harvest, potato tuber samples were placed in a storeroom at a temperature of 10 °C and RH of 80%. After three days of storage, the part of potato tubers were washed. Raw tubers were cut into 1 × 1 × 1 cm cubes and frozen in liquid nitrogen. Frozen potato samples were stored at a temperature of −18 °C. The samples were then lyophilised (CHRIST ALPHA 1–4 LSC, Osterode am Harz, Germany) and ground (particle size of 0.3–0.5 mm) using a laboratory mill Ultra-Centrifuge Retsch mill ZM 100 (Retsch, Haan, Germany). The ground samples were stored in the dark in tightly sealed bags in a desiccator until laboratory testing. In this way, the nitrates and nitrites content were determined in the prepared material. Prior to nitrates and nitrites determinations samples were prepared as described above.

2.4. Procedure for Nitrates and Nitrites Determination

Two grams of freeze-dried potatoes were mixed with 50 mL of 1% KAl(SO₄)₂ solution (Merck, Darmstadt, Germany) and well extracted. The extraction was carried out for 1 h using a shaker (IKA KS, Model 130 Basic—Staufen, Germany). The samples were filtered through Whatman No. 4 filter paper. Ten millilitres of 60% Al₂(SO₄)₃ solution (Acros Organics, Carlsbad, CA, USA) was added to the filtrate and mixed immediately before the assay. The nitrate content was determined based on the KNO₃ (Merck, Darmstadt, Germany) standard curves. At each stage of the analytical testing, deionised water was used.

A CX-721 multifunctional computer apparatus (Elmetron, Zabrze, Poland) was used to determine the nitrate content by the ion-selective potentiometric method [27]. The principle of measurement is based on the linear dependence of the electrode potential on the logarithm of ion activity in solution. It is expressed by the Nernst equation.

$$E=E_o+2.303\frac{RT}{nF} log \left(aj\right)$$

where:

• E—SEM measuring cell composed of ionoselective electrodes and references in the test solution (V)
• E_o—normal potential of the ion-selective electrode depends mainly on the activity of the internal electrode solution and the type of reference electrode (V)
• aj—activity of the determined ion
• n—value of the measured ion
• R—is the universal gas constant: R = 8.31446261815324 J K⁻¹ mol⁻¹
• T—is the temperature in Kelvins
• F—is the Faraday constant, the number of coulombs per mole of electrons: F = 96,485.3321233100184 C mol⁻¹

The apparatus was equipped with a nitrate electrode, double-junction reference electrode (fill outer chamber with 0.02 M (NH₄)₂SO₄ solution (Merck, Germany)), specific ion meter, and a pH/millivolt (mV) meter with a 0.1 mV readability. The determination limit was established at 30 mg kg⁻¹, and the measurement error was approx. 15%, depending on the sample matrix that was measured. The nitrate content was expressed in FW (mg kg⁻¹ FW).

2.5. Statistical Analysis

The data were analysed using the Statistica 13.1 software (StatSoft, Tulsa, OK, USA). For quantitative variables, the mean value and the standard deviation were calculated. Each year of study was analysed separately. In order to investigate the significant correlation between the nitrates and nitrites levels in potato tubers, one- and two-way ANOVA variance analysis was applied while determining the differences between mean values at the significance level at p ≤ 0.05, Tukey’s test was employed. Categorised 3W diagrams were used for data subsets to present the results. The significance of the correlation between different parameters was considered with reference to a 5% error level and 95% confidence level.

3. Results and Discussion

The study confirmed the thesis that the potato is a plant with a medium tendency to accumulate nitrates and a low capacity to accumulate nitrites in the tubers [7,28,29]. The nitrates content in the tubers, determined immediately after the harvest, ranged from 133.6 to 203.0 and the nitrites content ranged from 2.0 to 4.5 mg kg⁻¹ fresh weight (FW) (Figure 1A,B). However, in studies by Murawa et al. [30] and by Tietzwe et al. [31], the nitrates content in potatoes was at a higher level (from 167.1 to 259.6 mg kg⁻¹ FW). Moreover, Marks [32] and Wadas et al. [33] noted the nitrates content in potato tubers ranging from 77.0 to 102.0 mg kg⁻¹ FW and from 72.3 to 94.7 mg kg⁻¹ FW, respectively. According to [5,34,35], the highest concentration of nitrates is found in the peel or right under it in the tuber. Pretreatment (washing, hand peeling and rinsing) has a significant impact on changes in nitrates and nitrites content in potato tubers. Mozolewski and Smoczynski [36] report that losses of nitrites after pretreatment are higher than for nitrates, ranging from 25 to 75% and 18 to 40%, respectively. Moreover, losses of these compounds are observed during heat treatment of potato tubers. For nitrates they are 16–62% and for nitrites 61–98%. According to the authors, the differences in losses of these compounds depend on the variety [36].

The literature indicates that the nitrates content is genetically controlled and may be a characteristic trait of the cultivar [7,37,38]. Tajfun was characterised by the greatest content of nitrates (Figure 1A) of all the tested cultivars. However, no significant differences were demonstrated in this regard for other cultivars. Similar results were noted by [39], who found no significant differences in the accumulation of nitrates in three tested potato cultivars (‘Bryza’, ‘Sokół’, and ‘Irys’). Lachman & Hamouz [40] and Hamouz et al. [41], when carrying out studies on cultivars, achieved nitrates contents ranging from 70.2 for ‘Agria’ to 199.2 mg kg⁻¹ FW for ‘Impala’. On the other hand, Wierzbicka et al. [42] determined the NO₃⁻ content to be 132.0 for the ‘Denar’ cultivar and 237.0 mg kg⁻¹ FW for ‘Karatop’. Moreover, the tested Tajfun cultivar contained the significantly highest amount of nitrites (Figure 1B).

Three of the seven tested potato clones (TG-97–403, 13VIII-45, and 13VIII-49) were qualified as having a low tendency to accumulate nitrates content in the tubers (Figure 2A,B). However, a high tendency to accumulate was exhibited by the clone 13VIII-10 in which, irrespective of other factors, the nitrates content was 203.0 mg kg⁻¹ FW, thus exceeding the acceptable content limit (Figure 2A). The other clones accumulated nitrates at an average level of 160 mg kg⁻¹ FW. As regards nitrites, similar relationships were noted (Figure 2B). Therefore, the basic aim of potato cultivation is to obtain cultivars that meet producers and consumers’ needs. Many authors believe that it is necessary to obtain information on the differences in tolerance and the accumulation of contaminants, including nitrates in potatoes as early as the cultivation stage [43,44]. This enables obtaining cultivars that meet the needs of producers and consumers. It was pointed out that the average nitrates and nitrites content, irrespective of the location and cultivation system, was at a similar level for the clones and cultivars (Figure 3). In addition to genetic variability, the environmental variability occurring in parallel also determines (up to 85%) the nitrates and nitrites accumulation in the tubers [33,45]. The most important environmental factors include the imbalance between nitrates intake and nitrates reduction, which is determined by the nitrates reductase activity (NRA) [7,46].

The results of the authors’ own research (Figure 4A,B) indicate a higher nitrates and nitrites content in potato tubers produced under an integrated system, as compared to an organic system. This was also confirmed by research conducted by other authors who, in their studies, also took into account conventional production [11,47,48]. This results from the application of high nitrogen fertilizer doses in both conventional and integrated systems. However Hamouz et al. [41] demonstrated no significant effect of the cultivation technology (organic and conventional) on the nitrates content in the tubers of eight potato cultivars. It should be noted, however, that they observed a tendency to a higher nitrates content in traditional agriculture (153.9 mg kg⁻¹), as compared to organic farming (136.9 mg kg⁻¹).

According to reports by authors [7,49,50], there are no significant differences in the nitrates contents in the tubers of potatoes cultivated under the conventional production system “CPS” and under the integrated production system “IPS”. The nitrates or nitrites content in organic raw material averaged 40% compared to the integrated system (Figure 4A,B). In a study by Zarzyńska et al. [23], this value was approx. 27%, and in a study by Rembiałkowska [51] it reached 48%. As reported by Nurmanov et al. [52], and Pobereżny et al. [53], a significant reduction in the nitrates content in the tubers can be achieved by introducing the limitation of mineral fertilisation. This provides a basis for the conclusion that organic cultivation using no mineral fertilisers and chemical protection products may even reduce the nitrates content in agricultural product to 50%, which has a direct effect on the reduction in the intake of harmful nitrates and nitrites by the human body. Under the conventional and integrated production, either the acceptable standard for the nitrates content in potato tubers being exceeded or an overestimated nitrates content in potato tubers is often observed [30,41,54]. In the authors’ own research, the tubers cultivated under the integrated system contained a high nitrates content of an average of 203.7 mg kg⁻¹, i.e., oscillating at the limit of the established standard content in the FW (Figure 4A). The environmental variability factors that affect the nitrates and nitrites content in the tubers also include the heterogeneity of the soil environment and meteorological conditions (precipitation, temperature, light) [7,55]. The study demonstrated that irrespective of genetic determinants or the cultivation system, the nitrates content was significantly affected by the location and the year of potato production (Figure 5). The tubers originating from the cultivation in Boguchwała, where a high average annual total precipitation (390.3 mm ± 68.2 mm) was noted, were characterised by the highest nitrates content of 220.7 mg kg⁻¹ FW, irrespective of the technology applied and genetic determinants (Figure 5). Periodic precipitation deficiency and a high air temperature, excess precipitation and a low temperature during the growing period result in a reduction in the nitrates content in the tubers [7].

The years of the experiment also affected the content of the tested compounds. Potatoes of all the studied cultivars and clones were characterised by the highest nitrogen (V) content in 2014 and by the lowest in 2016 (Figure 5). The year 2014 was characterised by the greatest number of long rainy periods during cultivation. The nitrates content in that year exceeded the established standard contents in the fresh mass of the tubers for as many as three clones, i.e., 13-VIII-10, 13-VIII-27, and 13-VIII-60, and for one cultivar, i.e., Tajfun (

Table 1. Mean values of nitrates and nitrites content (mg kg⁻¹ FW) of potato tubers from seven tetraploid potato clones and three cultivars at four localities depending on the year of cultivation.

Genotype	Nitrates			Nitrites
	2014	2015	2016	2014	2015	2016
13-VIII-10	231.5 ± 57.9 e	223.9 ± 57.0 e	154.1 ± 81.8 bcde	3.4 ± 0.9 abc	3.7 ± 1.2 abc	6.3 ± 2.7 d
13-VIII-27	211.5 ± 52.1 e	211.5 ± 60.0 e	79.6 ± 67.8 ab	4.1 ± 1.8 bcd	4.2 ± 2.0 bcd	3.2 ± 1.9 abc
TG-97–403	174.5 ± 58.4 cde	171.6 ± 69.8 cde	54.4 ± 40.9 a	2.1 ± 0.8 ab	1.8 ± 1.1 a	2.2 ± 1.4 abc
13-VIII-45	190.4 ± 51.2 de	193.4 ± 64.5 de	67.5 ± 49.0 ab	2.6 ± 1.1 abc	2.7 ± 1.2 abc	3.4 ± 1.5 abc
13-VIII-49	195.4 ± 24.5 de	194.8 ± 26.6 de	91.2 ± 68.2 abc	2.3 ± 0.7 ab	2.3 ± 0.8 ab	4.2 ± 1.7 abc
13-VIII-50	199.1 ± 77.0 e	194.5 ± 88.6 e	86.8 ± 44.9 abc	3.2 ± 1.1 abc	3.3 ± 1.2 abc	3.6 ± 1.0 abc
13-VIII-60	202.7 ± 52.3 e	205.8 ± 65.6 e	76.8 ± 45.5 ab	2.7 ± 1.6 abc	2.8 ± 1.8 abc	2.8 ± 1.8 abc
Jelly	184.3 ± 63.8 de	186.1 ± 75.3 de	79.4 ± 68.7 ab	2.8 ± 2.5 abc	2.8 ± 2.5 abc	2.9 ± 1.0 abc
Satina	196.0 ± 86.0 de	194.6 ± 97.6 de	89.0 ± 18.6 abc	2.0 ± 1.3 ab	1.9 ± 1.6 ab	3.4 ± 1.8 abc
Tajfun	218.2 ± 60.3 e	221.8 ± 69.2 e	110.8 ± 70.8 a–d	3.9 ± 1.9 abc	4.1 ± 2.0 a–d	4.7 ± 1.6 cd

a–e—Means sharing the same letter are not significantly different (p < 0.05); LSD test nitrates: year—14.93, genotype 36.00; LSD nitrites: year—0.431, genotype—0.444; a—is the lowest value.

). However, the demonstrated lowest nitrates content in 2016 results from the even distribution of precipitation throughout the growing period, which was favourable for potato cultivation and at all locations. It should be noted that this study found no significant differences in temperature between the localities, with an average of 16.4 °C.

It should also be noted that the above-mentioned clones and cultivars contained the most nitrates and nitrites in the year 2016, which was favourable for potato cultivation (Table 1). This may indicate a high genetic tendency to accumulate these compounds, which is consistent with the results of studies by other authors [38,56]. The nitrates content was determined by the interactions between the environment (location) and the cultivar, which indicates the significant effect of weather conditions on the nitrates content in the tubers (Figure 6A). No such response was found for the nitrites content (Figure 6B). This indicates a greater effect of weather conditions during the growing season on the accumulation of nitrates compared to nitrites.

4. Conclusions

The genetic potential did not significantly differentiate the nitrates content in the tubers, while significant differences were noted for the nitrites content. Of all the tested genotypes, the clones 13-VIII-10 and 13-VIII-27 and the Tajfun cultivar were characterised by the highest nitrates and nitrites content. Therefore, these clones should not be recommended for further cultivation.