Tubers of ulluco (U. tuberosus) and arracacha (A. xanthorrhiza) were procured from a market in Popayán City, Colombia. The tubers were then selected and washed to remove any solids and impurities, and subsequently stored at room temperature until further processing.

To produce flour from U. tuberosus and A. xanthorrhiza, the tubers were thoroughly washed using friction with water. The samples were then chopped and subjected to convection drying at 45°C for 24 hours in a BINDER convection oven, until a moisture content of approximately 8% was achieved. The dehydrated samples were then ground using an IKA™ mill (basic microfine grinding unit MF10) and sieved to a particle size of 0.5 μm. The resulting flours were stored in zip-lock bags at room temperature.

For species identification and phenolic compound analysis, 0.5 kg of fresh roots were processed. The outer shells were removed using an ILKO vertical vegetable peeler. The shells were then dried at 50°C a BINDER convection oven for 12 hours. These dried samples were used for species identification and phenolic compound extraction. The identification of the tubers was conducted by sequencing the chloroplast genes ribulose-1,5-bisphosphate carboxylase (rbcL) and Maturase K (matK). For this purpose, the tuber shells were first frozen in liquid nitrogen and then ground to a fine powder for DNA extraction using the DNeasy Mericon kit (Qiagen). The polymerase chain reaction (PCR) reaction was performed in a final volume of 25 μL, containing 12.5 μL of DreamTaq Green PCR Master Mix (2X) (ThermoFisher Scientific), which included DNA polymerase DreamTaq, DreamTaq Green 2X buffer, deoxynucleoside triphosphate (dNTP) and 4 mM MgCl₂, and 0.5 μL of each primer at a concentration of 10 mM, 10.5 μL of water, and 1 μL of DNA template (100 μg/μL). For the amplification of the rbcL gene, the following primers were used: Rbcla-F 5’-ATGTCACCACACAAACAGAGAGAGACTAAAGC-3’ and Rbcla-R 5’-GTAAAATCAAGTCCACCRCG-3’. The PCR conditions were as follows: an initial denaturation at 95°C for 2 minutes, followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute, with a final extension at 72°C for 2 minutes. For the amplification of the matK gene, the same reagent concentrations were used. The primers employed were: matK-F 5’-CGTACAGTACTTTTTTGTGTGTTTACGAG-3’ and matK-R 5’-ACCCAGTCCATCTGGAAATCTTGGTTC-3’. The PCR conditions included an initial denaturation at 94°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 1 minute, with a final extension at 72°C for 2 minutes. Gel electrophoresis images were captured using a Transilluminator Ultraviolet Spectroline UVP mini Darkroom (Spectroline), with image analysis performed via VisionWorksTMLS software. Sequencing of the amplified DNA fragments was carried out using an Applied Biosystems Model 3730 sequencer (ThermoFisher Scientific), employing the Taq FS Dye Terminator Cycle Sequencing method. All obtained DNA sequences were compared with those in the GenBank database of the NCBI (National Center for Biotechnology Information) using the BLAST tool (Basic Local Alignment Search Tool) to identify highly similar sequences.

The physicochemical properties of the two tuber flours were analyzed using standard methods recommended by the Association of Official Analytical Chemists (AOAC, 1990). The analyses included the determination of total dry matter (TDM) and moisture percentage (AOAC method 920.39), total ash content (AOAC method 923.03), fat percentage (AOAC method 920.39), and crude fiber content (AOAC method 985.29). Crude protein content was determined using the Kjeldahl method (AOAC method 962.10), which involves converting nitrogen to ammonium sulfate and applying a conversion factor of 6.25. The carbohydrate content was calculated by difference, following the equation recommended by de Camargo et al. [22] (2014).

Mineral identification and quantification were conducted using the methodology described by Michel-Michel et al. [23] (2020). The analyses were performed with an Epsilon 1 X-ray fluorescence (XRF) spectrometer from Malvern Panalytical, located in Madrid, Spain.

All experiments were conducted following a randomized complete block design with three replications. Statistical analysis was performed using a two-sample T test at a significance level of 5%. Data analysis was conducted using InfoStat software version (2020). In addition, a principal components analysis was carried out, where the species were considered as classification variables, using the OriginPro 2021 software.

Upon comparison of the obtained sequences from the rbcL (RuBisCo) and matK genes of the tubers with sequences deposited in GenBank, it was verified that the tubers used in this study corresponded to U. tuberosus (Figure 1A) with 100% identity with sequence HQ620896.1 (U. tuberosus) and A. xanthorrhiza to (Figure 1B) with 100% similarity with sequence NC-032364.1 (A. xanthorrhiza).

The physicochemical composition of U. tuberosus and A. xanthorrhiza flours is presented in Table 1, showing percentages of moisture, total ash, lipids, and crude fiber that align with previously reported values for these tubers [12, 28]. Both tubers exhibit starch content exceeding 50%, confirming starch as the predominant carbohydrate in their structure [29, 30]. This characteristic suggests their potential application in the food industry, particularly in baking, where starch serves as a significant energy source [31–33].

Proximate characterization reveals significant statistical differences (p < 0.05) in fiber content among tubers, with U. tuberosus exhibiting the highest value (14.0%), surpassing reported values for tubers from the Andean region. Lipid content in these tuber flours is notably low (Table 1), consistent with previous reports ranging from 0.1% to 1% [9–12, 29], making them suitable for developing foods aimed at maintaining healthy cholesterol levels [34].

Potassium (K) is the most abundant mineral in both U. tuberosus (4.12%) and A. xanthorrhiza (3.32%) (Table 2), likely due to their role as reservoirs for K in flowers, developing fruits, and tubers [35], consistent with findings from other studies [8]. Studies have indicated higher proportions of Ca, P, and K, averaging around 2%, with slightly lower levels observed in Arracacia. K plays essential roles in fluid balance, nerve function, and muscle and cardiac contractions [36].

Additionally, both flours exhibit high magnesium (Mg) content (1.22% and 1.12% for U. tuberosus and A. xanthorrhiza, respectively), followed by Ca, P, sulfur (S), chlorine (Cl), Fe, and manganese (Mn).

The functional properties assessed in tuber flours establish parameters for their diverse applications in food [37]. Parameters such as WHC and SC indicate the flours’ ability to retain water or other substances upon partial rehydration [34]. U. tuberosus flour exhibited significantly higher WHC compared to A. xanthorrhiza flour (see Table 3). Both flours also demonstrated a high capacity to increase volume upon water addition (1.42 and 3.86 g H₂O/g flour for U. tuberosus and A. xanthorrhiza, respectively), potentially enhancing satiety due to their hydrophilic nature and influencing starch behavior related to water absorption and gelation processes [34]. In terms of OHC, A. xanthorrhiza flour exhibited higher values than U. tuberosus flour, suggesting its potential in structural interactions within foods, particularly in retaining flavors, aromas, improving quality, and extending shelf life [17].

The principal component analysis indicates that the amount of described information is substantial, as the first component represents 87.53% and the second component 6.34%, summing up to a total of 93.87%. The Biplot displays the grouping of variables and their relationship with each of the tubers, indicating that certain variables are associated with specific species (Figure 2). K, P, Mn, S, Mg, and Cl are the minerals directly associated with U. tuberosus. Additionally, the inorganic matter content (ash percentage), oil retention capacity, and fiber content are related to this tuber.

A. xanthorrhiza is observed to be related to the variables of moisture, carbohydrate content, protein, and fat. Additionally, water retention capacity and SC are related to this tuber. It is important to highlight that the variables associated with U. tuberosus and the variables associated with A. xanthorrhiza exhibit a negative correlation.

Various phenolic compounds were identified in both the skin and flour of U. tuberosus and A. xanthorrhiza tubers, employing HPLC-MS due to the raw material composition of skin mixed with pulp. Table 4 presents the identified compounds, highlighting hydroxycinnamic acids (caffeic acid 4-O-glucoside and p-coumaroyl glucose) and methoxyflavonols (3,7-dimethylquercetin), which were consistently found in all samples. U. tuberosus also exhibited flavonols (kaempferol 3-O-xylosyl-rutinoside) in both peel and flour. In addition, catechol was identified in the peel of U. tuberosus, scopoletin and tectorigenin 4’-sulfate were also detected.

For A. xanthorrhiza, 1-caffeoylquinic acid was identified in both the peel and flour. Additionally, d-viniferin was found in the peel of this tuber, a dihydro derivative of resveratrol with significant implications in the food industry. Also, lignans such as secoisolariciresinol and sesamolinol were identified in A. xanthorrhiza flour, while lariciresinol was found in U. tuberosus peel.

Both tubers are known for their high content of resistant starch and FOS [38]. Different rheological studies indicated that U. tuberosus starch could serve as an effective thickening or gelling agent in various food and biomaterial applications [14]. Variability in fiber content can be attributed to factors such as variety, geographic location, climate, soil type, and cultivation practices [39]. The fiber contents reported in these tubers typically range from 1% to 9% [9–12, 29]. The fiber content of ulluco and arracacha flours positions them as promising raw materials for food development [40], capable of meeting dietary fiber requirements, moderating glycemic response, and promoting gastrointestinal health due to their association with FOS and prebiotic activity, beneficial to probiotic bacteria [41, 42].

The nutritional composition of these tubers plays a crucial role in conserving Andean tuber biodiversity and promoting their use as novel raw materials for developing functional snacks. This approach not only supports socioeconomic sustainability in the region but also addresses current consumer demands, demonstrating the potential to diversify the utilization of these Andean tubers rich in primary metabolites such as starch, protein, fat, ash, and fiber.

U. tuberosus and A. xanthorrhiza are rich in various minerals essential for human nutrition, including Ca, Mg, Cl, P, and sodium (Na), among others. The contribution of K from these flours can meet recommended daily intake levels and benefit individuals with hypertension, as K intake has been linked to reduced blood pressure and antihypertensive effects [23, 43, 44].

Variations in macro and micronutrient content are influenced by soil fertility practices, with organic cultivation typically resulting in higher levels of P, K, Mg, S, Fe, Mn, and Cu, while conventional methods may yield higher nitrogen and Mn levels. Agroecological conditions also impact mineral absorption and content, as well as the specific plant species [45, 46].

The presence of these minerals in Andean tubers positions them as potential sources for developing functional foods that can promote human health. Ca, for example, exhibits hypoglycemic effects and stimulates insulin secretion, while other minerals contribute to bone health, hormone production, and carbohydrate metabolism, thereby supporting blood sugar regulation and appetite control [12, 34, 47].

The functional properties determined for each of the tubers, the WHC of U. tuberosus flour makes it suitable for industrial use as a thickening or gelling agent [14]. This characteristic is attributed to the hydrogen bonding between water molecules and matrix proteins [17].

The SC found in both flours makes them suitable for bakery products, pastries, pastas, or other high viscosity foods [17]. These values ​​contrast with the low swelling power reported for three other Andean tubers [48], and with the negative correlation presented in the principal component analysis. Some authors have reported that when the flour has a lower water retention capacity, it has a greater oil absorption capacity [17].

Based on the results from U. tuberosus and A. xanthorrhiza flours, the development of foods using these raw materials could offer beneficial effects for human health [34]. Furthermore, promoting their use as novel sources in the food industry is encouraged [49].

The hydroxycinnamic acids, methoxyflavonols, and flavonols identified are known for their cardioprotective effects, antioxidant properties, and potential benefits in glycemic control, neuroprotection, anti-inflammatory, antidiabetic, antimicrobial, and anticancer activities. Consumption of these compounds may help reduce the risk of several types of cancer, such as skin, liver, colon, ovarian, pancreatic, and bladder cancers [50, 51].

U. tuberosus, suggesting potential applications in pharmaceuticals, plastics, dyes, and cosmetics. Catechol is recognized for promoting skin wound healing and possessing hydrating and antimicrobial properties due to its physicochemical and biological characteristics [52, 53]. Of the compounds scopoletin and tectorigenin 4’-sulfate are reported to have antioxidant, antidiabetic, hepatoprotective, neuro-protective, and antimicrobial activities mediated through various intracellular signaling mechanisms [54, 55], which enhances the use of these tubers in different industries.

Hydroxycinnamic acids identified in arracacha are beneficial for health as they mitigate oxidative damage by enhancing the endogenous antioxidant system through scavenging free radicals, chelating metals, and modulating enzymatic activity [56]. So far, it has been reported that d-viniferin exhibits anticancer, neuroprotective, antioxidant effects, serves as a modulator of lipid and lipoprotein metabolism, functions as a platelet antiaggregant, and shows estrogenic activity [57, 58].

Quercetin, one of the flavonols found in U. tuberosus peel and A. xanthorrhiza flour, plays a crucial role in human health due to its antioxidant properties, which neutralize free radicals [34]. These flavonols are significant as their consumption has been linked to reduced risks of certain cancers, neuronal damage, and cardiovascular diseases [59]. These findings align with previous research using HPLC-diode array detection (DAD)-electrospray ionization (ESI)/MS on three Andean tubers, which identified flavonols such as quercetin-3-O-rutinoside [40.6 μg/g dry matter (DM)], kaempferol-O-dirhamnosylhexoside (29.5 μg/g DM), and kaempferol-O-dihexoside (46.22 μg/g DM). These tubers are thus suggested as natural antioxidant sources for both food and non-food applications [60].

The lignans can be metabolized by human intestinal microflora, potentially reducing the risk of breast, prostate, and colon cancers due to their (anti)-estrogenic and antioxidant activities [61, 62]. Furthermore, 5-5’-dehydrodiferulic acid, known for its inhibitory effects on P-selectin expression compared to its phenolic precursors, suggests enhanced efficacy in modulating platelet activation through phenolic compound metabolism [63]. Tetramethylscutellarein, identified for its bioactive role in blood coagulation and anti-inflammatory activity, was also detected [64].

Andean tubers are known for their richness in phytochemicals such as carotenoids, anthocyanins, phenolic acids [14], and glucosinates [9], all of which possess potential antioxidant properties [65]. U. tuberosus has been reported to contain betalains in the form of betaxanthins and betacyanins [9, 15], including betanidin- and isobetanidin-5-O-(4’-O-malonyl-β-glucoside), 2-decarboxyphyllocactin, betanidin- and isobetanidin-6-O-(6’-O-feruloyl)-β-glucoside (gomphrenin and isogomphrenin III), dehydrophilocactin and isophilocactin, as well as arginine and glycine-betaxanthins (portulacaxanthin III). A. xanthorrhiza contains limonene and myrcene [66], yet a comprehensive phenolic profile of these tubers remains to be fully documented. The presence of these diverse compounds underscores Andean tubers’ potential as raw materials for developing healthy snacks with functional properties [10, 21, 67–69].

The characterization of flours from two Andean tubers, U. tuberosus and A. xanthorrhiza, revealed them to be significant sources of starch, dietary fiber, and minerals such as K, Mg, Ca, and P. Moreover, these tubers exhibit functional properties that facilitate their application in both the food and non-food industries. Phenolic compound characterization identified primarily hydroxycinnamic acids, highlighting the potential of these tubers as sources for developing functional foods known for their health-promoting effects. Additionally, their content of FOS, related to fiber and starch structures, suggests potential prebiotic activity, as supported by literature findings.