**Piret Saar-Reismaa 1, Katrin Kotkas 2, Viive Rosenberg 2, Maria Kulp 1, Maria Kuhtinskaja <sup>1</sup> and Merike Vaher 1,\***


Received: 25 November 2020; Accepted: 12 December 2020; Published: 14 December 2020

**Abstract:** The use of colored tubers of *Solanum tuberosum* L. is growing worldwide due to their health benefits and attractive color. The positive health effects of purple-fleshed tubers are a result of anthocyanins and various phenolic compounds. The aim of this study was to evaluate and compare variety Blue Congo and its cross-breeds of Desiree and Granola to yellow-fleshed tubers. The concentration of total phenols, anthocyanins, sugars, and mineral elements were evaluated in all tubers. The results showed differences between all tested materials, with largest differences in sugar content. Moreover, the results confirmed the preservation of health improving compounds of Blue Congo when cross-bred with yellow-fleshed tubers. The total phenolic content and anthocyanin concentrations of all analyzed tubers were above the comparison yellow ones.

**Keywords:** colored potato tubers; total phenols; anthocyanins; antioxidants; saccharides; nutrition; microelements

#### **1. Introduction**

Worldwide, potatoes (*Solanum tuberosum* L.) are the fourth important food crop after wheat, rice, and maize. Potatoes are grown in cool-temperature regions, in mountainous areas as well as at higher altitudes in the tropic. Potato tubers are a rich source of high-value protein, carbohydrates, essential vitamins, minerals, and trace elements. The average range of a potato tuber composition is as follows: starch (10–18%) having 22–30% amylose content, total sugars (1–7%), protein (1–2%), fiber (0.5%), lipids (0.1–0.5%), vitamin A (trace/100 g of fresh weight (FW)), vitamin C (30 mg/100 g of FW), various trace minerals, and glycoalkaloids (1–3 mg/100 g of FW) [1].

Potato tubers are known to be naturally high in potassium, up to 400 mg per 100 g fresh tubers [2], or 1.7% of dry matter [3]. The potassium from potatoes can help lower blood pressure due to its vasodilation effect [4]. The tubers also contain other essential microelements like iron and calcium, that are responsible for bone structure and strength, in addition, zinc, manganese, copper, and magnesium are represented, that regulate cell renewal, energy production, and are responsible for over-all immunity [5]. Consumption of potato tubers with high nutritional content contributes to fulfilling of the daily recommended intake of several essential elements.

Over the past decade or so, colored-flesh potatoes have become more widely available to home gardeners, including potato tubers of blue, red, yellow, and white flesh. Purple-fleshed potatoes give the possibility to add color to the menu and additional nutrients to human diet. The addition of colored tubers adds the benefits from healthy antioxidants. Colored-flesh potatoes get their color from various pigments, which are antioxidants. Purple and rose-flesh potatoes contain the anthocyanin pigments, while yellow-colored flesh varieties contain carotenoids [6]. Purple-fleshed potatoes like Blue Congo get their color from common anthocyanins malvidin, peonidin, delphinidin, cyanidin, and petunidin [7]. Epidemiological evidence indicates health benefits from anthocyanins include improved eyesight and circulatory system function, benefits for diabetics, and anti-inflammatory, antiviral, and antimicrobial activity [8–10].

Potato tubers are also a great source of carbohydrates, which occur mainly in starch form [11] and are also used for industrial starch production. The starch content is directly related to the sugar content of tubers. As most potatoes are consumed after processing at high temperatures, the asparagine and glycose or fructose from the tubers can lead to acrylamide formation. This is due to Maillard reaction—a reaction between amino acid and sugar [12]. Moreover, higher levels of reducing sugars (glucose and fructose) as well as non-reducing sucrose in potato tubers may result in unfavorable browning or even a bitter taste [13]. Therefore, it is important to evaluate the sugar content in raw potato tubers, to determine if new varieties would produce high levels of acrylamide in processing. Moreover, potato tubers additionally contain myo-inositol, which is a sugar-like carbohydrate produced by most plants. Myo-inositol is important for phosphate storage and normal cell-to-cell communication and its metabolism is associated to diabetes [14].

The aim of the study was to evaluate the content of total phenols (TPs), anthocyanins, mineral elements, and sugars in tubers from variety Blue Congo, its seedlings, and cross-breeds with Granola and Desiree using various selective methods to create the plant material.

### **2. Materials and Methods**

#### *2.1. Studied Plant Material*

The plant material, created by different methods in vitro was grown in a test-field in Saku, Estonia (local latitude 57◦25 ). The soil type of the experimental area was Calcaric cambisols according to the World Reference Base classification (EAO 2014) where the agrochemical indicators were as follows: pH 6.3 (ISO 10,390 [15]); soil carbon content Corg 3.3% (Tyurin method [16]) and concentration of soluble P and K being 114 and 161 mg/kg (Mehlich III method [17]). In spring time, before the cultivation, in the field the complex fertilizer Cropcare 8-11-23 500 kg/ha was used.

Detailed description of tubers is given in Table 1. Initial mini-tubers of the variety Blue Congo were received from Sweden by the potato grower in 1991. Mini-tubers grown in green-house were eradicated on virus infection by using thermotherapy and meristem-plants were created in vitro [18].


**Table 1.** Descriptors for tubers of the variety Blue Congo seedlings and cross-breeds between Blue Congo with Desiree and Blue Congo with Granola.


**Table 1.** *Cont.*

Botanical seeds of the variety Blue Congo (1–7 in Table 1) were cultivated into the test-tubes in sterile condition, regenerated plants were multiplied in vitro and grown in the test-field. In 2013, the plants of the variety Blue Congo were pollinated in field conditions with the variety Desiree (8, 9) and with the variety Granola (10–15). The tubers of the variety Desiree are red skinned with yellow flesh, and the tubers of the variety Granola are yellow skinned with blanched yellow flesh.

New tissue cultures from the plants preserved in vitro were created on years 2004–2007. On the base of field results, three best meristem clones were selected (16–18).

As a comparing variety group, two commercial varieties were included. Variety Teele tubers are with bright yellow skin and yellow flesh and variety Laura tubers are with red skin with dark yellow flesh. Comparison with super-market purple-fleshed sweet potato tubers (*Ipomoea batatas* L.) were also included.

#### *2.2. Extraction and Dry-Weight*

From each genotype (plant material type) (Table 1) 5 tubers were selected. The tubers were washed with distilled water, dried at room temperature and homogenized (Nutribullet, Los Angeles, CA, USA). The homogenates were divided into aliquots and stored at 4 ◦C for further analysis. The aliquots were used for determination of dry weights (DW), microelements, and for making extracts for analysis of total phenols (TPs), anthocyanins, and naturally occurring sugars.

For evaluation of TPs and anthocyanins the following extraction procedure was conducted: 5 g of tuber homogenate was mixed with 25 mL 80% (*v*/*v*) methanol in a 50 mL graduated tube. The mixture was allowed to stand with intermittent shaking for 1 h at room temperature in the dark. After that the mixture was subjected to ultrasonication (Sonorex digital 10P, Bandelin, Berlin, Germany) for 30 min at 30 ◦C. After which it was centrifugated (EBA 200S, Hettich, Westphalia, Germany) at 8000 rpm/min for 15 min and the supernatant was filtered through 0.45 μm Minisart® Syringe Filter (Sartorius, Goettingen, Germany) and stored at 4 ◦C. The carbohydrate extraction was achieved in the ultrasonic bath with 50% (*v*/*v*) aqueous methanol from 5 g of homogenate using the same procedure as in case of extraction of polyphenols.

The dry weight of the tubers was measured by drying ~1 g of the homogenate at 105 ◦C until constant weight using an Ohaus Moisture Analyzer MB90 (Parsippany, NJ, USA) in triplicates. The content of dry matter ranged from 15.8 to 31.5% for examined potato tubers and was 34.9% for the purple sweet potato tuber.

#### *2.3. Determination of Total Phenols and Anthocyanins*

The concentration of the total phenolic compounds was determined for each extract by an adapted micro-scale protocol for the Folin–Ciocalteu colorimetric method [19,20]. In brief, phenolic groups are oxidized by phosphomolybdic and phosphotungstic acids in Folin–Ciocalteu reagent, forming a green–blue complex detectable at 765 nm. 50 μL of each tuber extract solution of an appropriate concentration was mixed with 1350 μL of water, 100 μL Folin–Ciocalteu reagent and 500 μL of Na2CO3 (20% *w*/*w*). The absorbance at 765 nm was measured after2hreaction at room temperature (in the dark) with a Cary 50 Bio UV–vis spectrophotometer (Palo Alto, Varian, CA, USA). The hydro-methanolic gallic acid solution was freshly prepared in a series of concentrations (0.3–3 mM) and tested in parallel to establish the calibration curve. The total phenolic content of each potato extract was calculated as milligrams of gallic acid equivalent per g dry sample (mg GAE/g of DW).

The total content of non-hydrolyzed anthocyanins was measured using the pH differential method described by Albishi et al. [21] with minor modification. Monomeric anthocyanin pigments reversibly change color with change in pH. The colored oxonium form exist at pH 1.0, and the colorless hemiketal form predominates at pH 4.5. The difference in the absorbance at 520 nm is proportional to the pigment concentration. 400 μL of potato tuber or 200 μL of sweet potato tuber extract and 600 or 800 μL of 25 mM potassium chloride buffer (pH 1.0) and 400 mM sodium acetate buffer (pH 4.5) respectively were mixed. The mixtures were left at room temperature for 1 h (in the dark). The absorbance was measured at 520 nm and 700 nm against a blank cell filled with 80% MeOH in buffer. The results were expressed as mg of cyanidin-3-glucoside equivalents per kg dry sample (mg CGE/kg of DW).

The experiments were carried out in triplicates, and the results are reported as the mean ± standard deviation.

#### *2.4. Capillary Electrophoretic Analysis of Natural Sugars*

Capillary electrophoresis (CE) was performed using an Agilent 3D CE instrument (Agilent Technologies, Santa Clara, CA, USA) equipped with a diode array UV/Vis detector. Uncoated fused silica capillary with effective length of 71.5 cm and i.d. of 50 μm was employed. The optimized conditions for the analysis: temperature of the capillary was 16 ◦C, applied voltage was +17 kV and samples were injected under 35 mbar pressure for 10 s. 130 mM NaOH containing 36 mM Na2HPO4 (pH 12.6) was used as a background electrolyte (BGE). The wavelength for detection was 270 nm [22]. Identification of the sugars was done by standard addition method, the standard solutions of sucrose, D-(+)-maltose, D-(+)-glucose, D-(−)-fructose, sugar alcohol myo-inositol, and sodium hydroxide were from Sigma (Darmstadt, Germany). Milli-Q water (Millipore S. A, Molsheim, France) was used for all solutions of standards, background electrolyte (BGE), and dilution of samples.
