*2.5. Atomic Absobrance Analysis of Microelements*

The stock atomic spectroscopy standard solutions (1000 mg/L) Cu, Zn, Mn, Fe, Mg, Ca, Se, and K were purchased from Fluka, Buchs, Switzerland. Spectra AA 220F and 220Z atomic absorption spectrometers (Varian, Mulgrave, Australia) equipped with a side-heated GTA-110Z graphite atomizer, a Zeeman effect background correction, and an integrated autosampler were used. Graphite tubes with coating and platforms made of pyrolytic graphite were used throughout the work. Argon of 99.99% purity (AGA, Helsinki, Finland) was used as the purge gas. Acetylene of 99.99% purity (AGA, Helsinki, Finland) was used as the fuel gas in flame atomic absorption spectroscopy. For the determination of total mineral element constituents ~1 g of potato tubers homogenate was mineralized with 4 mL of concentrated nitric acid and 1 mL of concentrated hydrogen peroxide in 50 mL plastic tubes at temperature 80 ◦C for 5 h. After cooling down, the solution was transferred to volumetric flasks (15 mL) with ultrapure water. All the experiments were made in triplicates. The concentrated nitric acid and hydrogen peroxide were from Sigma (Darmstadt, Germany).

#### *2.6. Statistical Analysis*

The statistical analysis was conducted using Microsoft Excel and R version 4.0.2. The significant variations were evaluated using the Excel built-in data analysis package (*t*-test two samples, *p* = 0.05) and the principal component analysis (PCA) was carried out and visualized in R 4.0.2 x64.

#### **3. Results and Discussion**

#### *3.1. Total Phenlos and Anthocyanins*

To evaluate the possible positive health influences of colored tubers, the total phenols (TPs) and anthocyanin concentrations were evaluated compared to sweet potato and two yellow-fleshed tubers. The total phenols were evaluated in all tubers and compared as concentration of gallic acid equivalents mg/g of dry weight (mg GAE/g of DW). All sample tubers (Sample 1–20) as well as the sweet potato sample (Sample 21) showed TP concentrations between 0.8 and 3.1 mg GAE/g of DW as shown in Figure 1.

**Figure 1.** The concentrations of anthocyanins and total phenols in samples 1–21. All concentrations are given for dry weight. \* *p* < 0.05 for total phenols and anthocyanin concentration between yellow-fleshed and colored potato tubers.

The biggest difference occurred between all colored tubers (Samples 1–18) and the yellow varieties (Samples 19–20), where the yellow-fleshed tubers had significantly (*p* < 0.05) lower TP concentrations. The values for all the colored potato tubers were from 1.4 to 3.1 mg GAE/g of DW, but for the yellow fleshed tubers only 0.8 mg GAE/g of DW, showing an increase of 75–175% of TPs for purple-fleshed varieties. These results are in accordance with previous studies of colored tubers, that show an increase of various phenolic acids, coumarins, and flavonoids in purple fleshed potatoes compared to yellow or white tubers [6,23]. The results also show that crossbreeding with potatoes with yellow flesh do not significantly decrease the variation of TPs in tubers, allowing for more versatile and specific breeding. The TP concentration in the purple sweet potato tuber did not have a remarkable difference compared to all other purple-fleshed tubers.

Another important evaluation criterion for the health-benefit of the purple-flesh potato is the content of anthocyanins. The same samples were analyzed for anthocyanins with results shown in Figure 1. As anthocyanins are directly linked to the coloration of the flesh, the concentrations of anthocyanins in yellow-fleshed sample tubers, samples 19–20, were the smallest, showing a statistical difference to all other tubers (*p* < 0.05). All colored potato tubers had anthocyanin concentrations from 138.6 to 588.5 mg CGE/kg of DW, compared to yellow fleshed tubers concentrations around 20 mg CGE/kg of DW. Such results are to be expected as Jansen and Flamme [24] have previously shown, that whole violet and violet/white fleshed tubers had anthocyanin concentrations from 181 to 1570 mg/kg of FW, while white and yellow fleshed tubers only had 4–82 mg/kg of FW of anthocyanins. The purple sweet potato had over six times higher concentration of 1613.4 mg CGE/kg of DW compared the average of 254.9 mg CGE/kg of DW from Blue Congo and its examined accessions varieties. Unlike TPs, the anthocyanins had a small increase in the average concentration in Blue Congo cross-breeds with Granola, implying the positive effect from the breed Granola.

#### *3.2. Analysis of Sugars*

The sugar content of potatoes is an important quality indicator. It has been shown that the tuber sugar content can vary by genotype, but is largely influenced by the storage and treatment, even more so at temperatures below 10 ◦C [13]. All analyzed samples were therefor stored at similar temperature to avoid any environment factor as a variable.

The analysis was carried out using a CE with UV detection and lactose was used as an internal standard (IS). All samples were analyzed for myo-inositol, sucrose, maltose, glucose, and fructose. Examples of analyzed tuber electropherograms are shown in Figure 2.

**Figure 2.** Electropherograms of sugar analysis from sweet potato tuber Sample 21 (blue), purple-fleshed cross-breed with Granola Sample 14 (red) and a meristem clone of Blue Congo sample 16 (green). Identification 1—myo-inositol, 2—sucrose, IS—internal standard, 3—maltose, 4—glucose, 5—fructose.

None of the potato tuber samples contained maltose above the detection limit. The only sample to contain maltose was the purple sweet potato tuber, which was to be expected as the sweet taste of the tuber is due to maltose concentration [25]. To better evaluate the overall sugar content of all analyzed tubers, the fructose, glucose, sucrose, and myo-inositol concentrations were summed in a bar chart shown in Figure 3.

The overall sugar content in all potato tubers ranged from 10.3 mg/g to 47.1 mg/g with an average of 21.6 mg/g. These results are in accordance with other published reports of total sugar concentrations in potato tubers that ranged from 7.5 to 74.1 mg/g of DW [26], and are also in accordance to climatically similarly grown potato tubers, where sucrose, glucose, and fructose ranged from 6.4 to 21.8, 2.3 to 29.7, and 1.2 to 25.4 mg/g of DW, respectively [27]. The sweet potato tuber total sugar concentration was 95.0 mg/g, which was mainly due to high concentration of sucrose. The lowest sugar concentrations were observed in the Blue Congo meristem clones, that averaged only 11.0 mg/g of total sugars.

**Figure 3.** The total content of sugars from analyzed potato tubers Samples 1–18 correspond to purple-fleshed tubers, samples 19–20 to yellow-fleshed tubers and sample 21 to a purple sweet potato tuber. \* *p* < 0.05 for total sugars between cross-breeds of Blue Congo with Granola compared to all other potato tubers.

Additionally, there was a significant difference (*p* < 0.05) in the samples 9–15 corresponding mainly to the cross-breeds of Blue Congo and Granola with an average sugar content of 34.1 mg/g, while all other potato tubers had an average of only 16.3 mg/g. As the sugar content is used as a quality factor, the higher sugar concentration may result in unacceptably brown and bitter food products. Thus, the higher amounts found in Granola cross-breeds make such cultures less-favorable compared to the original Blue Congo variety for fried products. The cross-breeds with Desiree did not show such tendencies.

#### *3.3. Microelements in Tubers*

The dispersion of various elements throughout the potato tubers is mixed, as some elements have higher concentration in the skins, while others in the flesh; moreover, there is some research that shows heterogeneous distribution between the stem and distal end of the tubers [28]. To better evaluate overall concentrations, whole tubers were washed and grounded for the analysis. Selected microelements essential for living organisms were evaluated using atomic absorbance spectroscopy (AAS). In total, eight elements were measured from all samples including copper, zinc, manganese, iron, magnesium, calcium, potassium, and selenium. All levels of Se were below the detection limit of the method (<20 mg/kg of DW). The results for all other elements are presented in Table 2 below.

Potato tubers are best-known for their high potassium content as it is an essential element for the acid–base regulation as well as heart, liver, nerve, and muscle functioning [29]. The potassium concentration was the highest, ranging from 10.35 to 22.83 g/kg of DW, with an average of 16.4 g/kg of DW. These results are comparable to non-organic K levels determined in potato tubers [30].

The zinc concentration varied from 9.8 to 26.0 mg/kg of DW, matching with previous results of fertilized and organic Zn concentrations [5,31,32]. The concentrations did not vary depending on the flesh coloration of the tuber, but the purple sweet potato tuber had a significantly lower Zn concentration of only 8.2 mg/kg of DW.

The Fe concentrations of analyzed tubers varied from 48.3 to 133 mg/g of DW, which is similar to works by Andre et al. [28]. The Fe levels in samples 19 and 20 as well as in the sweet potato were the lowest compared to colored-flesh tubers. The Fe found in potato tubers is considered to be non-heme iron and therefor considered a valuable source of iron for the human diet. The lack of iron can cause severe health problems, including impaired development in adolescence and reduced work capacity, making potatoes useful sources for such nutrients. Moreover, as potatoes also contain vitamin C, which increases the iron uptake from potatoes, the health benefits are significant against anemia.


**Table 2.** Mineral composition of the tubers as determined by atomic absorbance spectroscopy (AAS) per dry weight (*n* = 3).

Calcium is needed for skeletal and neural functioning, as well as metabolism. Although, the Ca content is insufficient for marginal dietary benefits, the tuber quality and storage capacity is evaluated on the basis of it [33]. The Ca levels averaged at 470 mg/kg of DW, being similar to previously reported values, with the highest concentration of 729 mg/kg of DW and lowest of 270 mg/kg of DW. There were no significant variances between different breeding varieties or colored and yellow-fleshed tubers, nor sweet potato tuber.

Mn concentrations were relatively low, similar to organic cultivars, with the levels being from 5.3 to 12.0 mg/kg of DW. In general, higher magnesium levels were detected in the tubers of cross-breeds of Blue Congo and Granola. The overall levels varied from 629 to 1264 mg/kg of DW with an average of 1015 mg/kg of DW, which is comparable to both conventional and organically cultivated potato tubers, that averaged from 1183 to 1646 mg/kg of DW [5,32].

No obvious differences were seen between breeding varieties and cross-breeds of Blue Congo tubers. The differences were observed with a purple sweet potato tuber as well as yellow-fleshed tubers compared to purple-fleshed tubers. Most of the results correlate to each other as the main source of mineral concentrations is due to the soil and fertilization processes used, which were similar for all the tubers analyzed, except for the purple sweet potato tuber and store-bought varieties.

#### *3.4. Principal Component Analysis*

A principal component analysis (PCA) was done to better evaluate the correlations between different breeding varieties and concentrations of mineral elements, natural sugars, and total phenols as well as anthocyanins. The sweet potato tuber results were excluded from the PCA analysis sweet potato is from another genus and it would be an outlier due to too different profile. The results of principal components 1 and 2 (PC1 and PC2) showed obvious groupings of all varieties as is seen in

Figure 4. As the PC1 and PC2 had a total explained variance of 65.5%, the PCA was determined to be successful.

**Figure 4.** Principal component analysis (PCA) results of all analyzed 20 potato varieties and their mineral composition, natural sugars, total phenols, and anthocyanin content. Pink—meristem clones of Blue Congo, green—Blue Congo cross-breeds with Granola, blue—yellow-fleshed tubers, orange—Blue Congo botanical seeds, yellow—Blue Congo cross-breeds with Desiree.

There were five main groups of potato tubers: botanical seeds of Blue Congo, meristem clones of Blue Congo, Blue Congo cross-breeds with Granola, Blue Congo cross-breeds with Desiree, and commercial varieties (Laura, Teele). The main distinguishable groupings were of botanical seeds of Blue Congo, which showed similarities to the meristem clones, but had almost no overlapping with cross-breeds with Granola. The main differences in the meristem clones and cross-breeds can be attributed to the reducing sugars, which make the Granola cross-breeds sugar rich as previously determined. Unfortunately, the high sugar content has links with higher copper and calcium concentrations, meaning that the possible benefits of Ca and Cu will not be obtained if the variety is deemed unsuitable as the sugar content may lead to cancerogenic acrylamide. To overcome this, there are studies that demonstrate the possibility to reduce the concentration of acrylamide formation even up to 93% by pretreatment of tubers [34]. The multi-step pretreatment may unfortunately diminish other nutrients. Moreover, the anthocyanin concentration is also in correlation with total saccharides, proving the value of such variety.

Additionally, there was a correlation between higher concentration of Fe and total phenol concentration, which is in accordance with Brown's previous results [31] where colored-flesh tubers have higher concentrations of TPs and Fe. The correlation of Fe and TPs also includes the higher concentration of Mg. The concentration of potassium was slightly correlated, showing cross-breeds with Desiree and some botanical seeds presenting with both positive characteristics. Interestingly, the botanical seeds of Blue Congo showed differences to the meristem clones of Blue Congo, with the botanical seeds having higher levels of nutrients. The yellow-fleshed tubers of Teele and Laura separated from all other tested material and had the smallest levels of anthocyanins, total phenols and additionally microelements, proving further that the purple-fleshed tubers have higher potential for a healthier alternative in the human diet.
