In ﬂ uence of potato variety on polyphenol pro ﬁ le composition and glycoalcaloid contents of potato juice

: The results of studies published in recent years indicate the broad biological activity of potato juice ( PJ ) , which is a byproduct of the starch production process. Among the most frequently described activities are anti - inflammatory, antioxidant, and cytotoxic effects. Nevertheless, this waste juice is produced by the pro cessing of many varieties of potatoes with different proportions, which does not allow to conclude on the biological activity of individual varieties. This article is a report on the antioxidant activity of PJ from seven selected potato varieties, their profile of polyphenolic compounds, and the content of glycoalkaloids ( GAs ) . The use of similar cultivation conditions allowed to eliminate the influence of environmental factors on the content of the analyzed compounds. The influence of PJ on the growth of probiotic, commensal, and pathogenic bacteria was also assessed. It was shown that the varieties significantly influenced the differ ences in antioxidant activity as well as the content of GAs, but despite the observed differences, none of them showed antimicrobial activity. Therefore, it can be concluded that an appropriately selected variety will make it possible to obtain PJ that will be charac terized by high antioxidant activity and, at the same time, will be safe from the toxicological point of view.


Introduction
Among the factors reducing the risk of several types of cancer and cardiovascular diseases, a proper diet rich in plants, being a source of biologically active compounds, is indicated [1]. Plant phenols are a complex group of various secondary metabolite compounds, produced mainly via the shikimic acid pathway from L-phenylalanine and L-tyrosine, they have protective functions in the plant, for example, against UV radiation, competitive warfare against viruses, bacteria, insects, and other plants [2,3]. Their synthesis takes place more intensively in plants subjected to stress factors, including abiotic and biotic stress [4][5][6]. In recent years, the interest of researchers from around the world has been focused on the influence of free radicals on the human body, in particular reactive oxygen species. It is believed that oxidative stress can cause many chronic diseases. Free radicals can react with proteins, fats, and DNA. The consequence of these reactions is permanent DNA damage that can contribute to cardiovascular diseases, in particular atherosclerosis, age-related macular degeneration, cataracts, Parkinson's and Alzheimer's diseases, as well as cancer [7,8]. Numerous literature data [3,9,10] indicate that there are powerful natural antioxidants that play a key role in a wide range of biological and pharmacological properties, such as anti-inflammatory, anticancer, antimicrobial, antiallergic, antiviral, anticoagulant, and hepatoprotective activities.
Potatoes (Solanum tuberosum L.) are one of the most important cultivated plants intended for food and feed purposes. Easy cultivation and reasonable climate requirements make it grown all over the world [11]. The plant in its root system produces tubers rich in starch, which is why often high-starch varieties are used in the starch process, which results in pure potato starch [12]. The first mentions of potato cultivation date to 8,000 years ago, and today more than 5,000 varieties of this plant are cultivated [13]. The dry mass of potato tubers reaches 20-25%, and their chemical composition, determining the nutritional value, depends not only on the variety but also on the cultivation and harvesting conditions [14]. Literature sources indicate a significant impact of storage conditions on the content of dry matter of potato tubers, content of reducing sugars, or amino acid composition [15][16][17]. Among the nutrients forming the dry mass of potato tubers, starch, protein, vitamins, minerals, and fiber should be distinguished. Potato tubers contain, among other vitamins C, PP, B1, B2, and B6, iodine, calcium, chlorine, and sulfur. Consumption of one medium-sized potato on skin (approximately 150 g) provides the recommended daily dose of vitamin C for an adult (100 mg) [18]. It is worth adding that potatoes are vegetables with a high iron content [19,20]. Potatoes are a low-fat raw material (about 0.5% of fresh weight), containing mainly linoleic (omega-6) and linolenic (omega-3) acids [21].
Potato juice (PJ) is formed in the production of potato starch as one of the byproducts. Currently, acidic thermal coagulation is the most commonly used process of PJ management. Unfortunately, the protein preparation obtained in this way is insoluble and enzymatically inactive [22]. Low emulsifying, foaming, and water-binding abilities significantly limit the potential use of potato protein in food production [23]. In starch production factories, acid thermal coagulation of the PJ protein is commonly used, and the protein mass is then separated, dried, and used as a feed ingredient [24]. Therefore, new methods of PJ management are sought, and the directions for the use of PJ described in the literature include microbiological mediums [25][26][27] and prohealth food [28][29][30]. PJ is gaining increasing interest as a source of nutritional and bioactive compounds. On the one hand, the proteins present in PJ are characterized by high nutritional value and interesting technological properties [20,[31][32][33]; on the other hand, nonprotein compounds with anti-inflammatory, cytotoxic, and antioxidant activities are also present [18,34]. Among the compounds responsible for the antiproliferative properties of PJ against human skin [35], liver [36], prostate, and breast [37] cancer cells, glycoalkaloids (GAs) are indicated, mainly α-chaconine and α-solanine. Isolating the biologically active ingredients of PJ may allow them to be used in pharmacy and medicine.
PJ is a seasonal product obtained between August and December, during the starch production season. Many different varieties of potatoes are used for processing, so the described biological activity of the juice obtained may differ depending on the varieties being processed at the time. Therefore, this article is a report on antioxidant activity, polyphenols profile composition, and GA content in PJ from selected seven potato varieties, commonly cultivated in Poland.

Test material
The study used seven potato varieties, grown at the Experimental Station Gorzyń (52°57′N; 15°89′E), belonging to the Poznań University of Life Sciences (Poland). The juice was obtained from seven varieties and denoted as follows: VR 808denoted as PJ1, Saturnadenoted as PJ2, Toscanadenoted as PJ3, Dittadenoted as PJ4, Lorddenoted as PJ5, Denardenoted as PJ6, and Lady Clairedenoted as PJ7.
The potatoes were thoroughly washed and peeled, and then the juice was squeezed out. The obtained juice was left for an hour in a refrigerator (4°C) for starch sedimentation, and then the juice was decanted, frozen, and lyophilized. Three times 0.5 L of juice was obtained from each variety of potatoes, which was then freeze-dried and unified. The freeze-drying process was performed using Alpha 2-4 LD plus lyophilizer (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). The process was initiated with a freezing stage at −35°C for 20 h, followed by the main drying step at a shelf in 5°C for 12 h, and the final drying up at 20°C for 2 h. The obtained dried material was packed in laboratory glass and stored frozen until use.

Antioxidant activity and polyphenol
profile composition

Extraction process of antioxidants
The extraction process was carried out using an 80% solution of methanol using a double extraction from lyophilized samples. The lyophilizate (1 g) was mixed with 20 mL of methanol solution, and then shaken for 15 min using a laboratory shaker S50 (CAT Germany GmbH, Lehrte, Germany). Subsequently, the samples were centrifuged at 6,000×g for 10 min. The supernatant was decanted, and another 10 mL of methanol solution was added to the tube. It was shaken again and centrifuged under the conditions described above. The supernatant was decanted again and mixed with previously collected, then filtered through a 0.22 μm filter, and stored in the freezer at −80°C in a glass flask.

Total polyphenol content and antioxidant activity
The total antioxidant activity was assessed against the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical using methods described by Re et al. [38]. Results were presented as Trolox equivalent antioxidant capacity per 1 g of dry matter of the examined sample. The total content of phenolic compounds was determined by the standard Folin-Ciocalteu colorimetric method [39] using a spectrophotometer (Multiskan GO, Thermo Fisher Scientific, Vantaa, Finland) and expressed as chlorogenic acid equivalent per 1 g of dry matter.

Polyphenols profile composition determined by high-performance liquid chromatography (HPLC)
Chromatographic analyses of phenolic compound content were carried out on an Agilent 1260 Infinity II (Agilent Technologies, Inc., Santa Clara, CA, USA) liquid chromatograph equipped with an automatic sample feeder (G7129A), a pump (G7111A), and a diode detector (G7115A) with an overview of the spectrum (190-400 nm

UHPLC-MS/MS analysis
The quantitative and qualitative determination of α-solanine and α-chocaine in PJ extracts were performed using the chromatographic system UltiMate 3000 RSLC (Dionex, Thermo Fisher Scientific Inc., Bartlesville, OK, USA) coupled to an API 4000 QTRAP triple quadrupole mass spectrometer with electrospray ionization (from AB Sciex, Foster City, CA, USA) in positive ionization mode ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Chromatographic separation was done on a Kinetex 1.7 µm C18 (100 mm × 2.1 mm I.D.) column from Phenomenex (Torrance, CA, USA). A total of 0.1% formic acid (A) and acetonitrile (B) were used as the mobile phase. Elution was performed using a gradient: 25% B at 0 min, 32% at 3 min, increased to 100% B in 3 min and held for 0.5 min. The flow rate was 0.20 mL/min. The column temperature was maintained at 35°C, and the injection volume was 10.0 μL. A postrun time was set at 4.0 min for column equilibration before the next injection. The operating conditions for mass spectrometry for α-solanine and α-chocaine were as follows: curtain gas 10 psi, nebulizer gas and auxiliary gas 40 psi, source temperature 600°C, ion spray voltage 5,500 V, and collision gas set to medium. Quantitative analysis of the compounds was performed in multiple reaction monitoring (MRM) mode. For analytes was chosen one transitions of the protonated molecular ion and their respective ion product. The first MRM transition was used to quantitate, and the second was used to confirm. These transitions (m/z) with associated declustering potentials (V) and collision energies (V) were as follows:

Microbiological tests
Antimicrobial activity was determined by the disc diffusion method. In antimicrobial assays, the following strains types were used: Listeria, Salmonella, Yersinia, Clostridium, Escherichia, Enterococcus, Lactobacillus, Bacteroides, and yeast Candida. Specifics about species as well as conditions for the growth of microorganisms are presented in Table S1. The microorganisms were obtained from American Type Culture Collection (Rockville, USA). Antimicrobial activity was determined by point-diffusion method. Agar media suitable for the growth of the investigated microorganisms were inoculated in the logarithmic growth phase in such an amount as to obtain a cell concentration of approximately 10 6 CFU/mL. Then, 20 μL of dried PJ at a concentration of 100 mg/mL was applied to solidified agar media. After 24 h incubation, the degree of inhibition of the growth of indicator microorganisms was assessed.

Statistical analysis
Statistical analysis of the data was performed with Statistica 13 (Dell Software Inc., USA) software. All measurements were studied using one-way analysis of variance independently for each dependent variable. Post hoc Tukey honest significant difference multiple comparison tests were used to identify statistically homogeneous subsets at α = 0.05.

Antioxidant activity and polyphenol profile
The mentioned previously published data indicate that there are also antioxidants in PJ, mainly phenolic compounds, which exhibit antioxidant properties. To compare the properties of the juice obtained from a particular variety, potatoes were grown under similar conditions to eliminate the differences resulting from stress factors. The content of phenolic compounds in the analyzed juices differed between the cultivars (Figure 1). Their lowest content was recorded for PJ7 (8.09 ± 0.19 mg/g), and the highest content was recorded for PJ6 (12.85 ± 0.20 mg/g). This is much higher than the average content in potato tubers of 0.74 mg/g fresh weight [18]. Nevertheless, the polyphenol content is in each form lower than that of the waste PJ obtained in the starch production process [41]. However, the high content of phenols is not associated with high antioxidant activity. The highest total antioxidant activity was recorded for PJ5 (3.04 ± 0.11 mM/g) and the lowest for PJ2, PJ1, and PJ7 (1.45 ± 0.27, 1.50 ± 0.03, and 1.63 ± 0.76 mM/g, respectively). It can, therefore, be assumed that PJ contains phenolic compounds in bound form that are linked to cell wall polysaccharides by ester bonds. They are not detected by a simple colorimetric method, but when released in the digestive tract, they can have a beneficial effect on our body. Also, simple heat treatment can release the bound phenols [42][43][44] and thus increase their bioavailability.
Among the free phenolic acids and polyphenols present in potatoes, the most frequently described are catechin, chlorogenic, caffeic, ferulic, gallic acids, and malvidin [45][46][47]. However, the analyzed juices showed the presence of chlorogenic, sinapic, coumaric, hydroxybenzoic acids, niacin, rutin, and vanillin. Importantly, PJs from different potato cultivars were characterized by a different profile of the analyzed compounds. PJ4 was the only one that contained chlorogenic acid, which has many health-promoting properties, most of them related to the treatment of metabolic syndrome, including antioxidant, anti-inflammatory, antilipidemic, antidiabetic, and antihypertensive effects [48], and PJ4 was the only one that did not contain hydroxybenzoic acid. PJ4 also contained the greatest amount of rutin (3,30,40,5,7-pentahydroxyflavone-3-rhamnoglucoside), which demonstrated a number of pharmacological activities, including antioxidant, cytoprotective, vasoprotective, anticarcinogenic, neuroprotective, and cardioprotective activities [49]. Detailed data on the content of individual phenols are presented in Table 1.

Glycoalcaloid contents
Despite the high nutritional value of potatoes, concern is raised by the presence of GAs, compounds that are widely recognized as toxic [50]. They are formed by combining alkaloids with one or more sugar molecules. GAs are present throughout the plant, but the greatest risk comes from their presence in the edible part, that is, tubers, from which they then pass into the PJ in the starch production process. Almost 95% of the GAs of potatoes are α-solanine and α-chaconine [51]. Due to slight differences in structure, and thus similar physicochemical properties, publications often include the total content of GAs, without distinguishing individual compounds that make up this group of compounds [52]. The use of mass spectrometry allowed to determine the content of both main GAs in the analyzed juices, and the results are presented in Figure 2. It was shown that the juices from PJ4 and PJ3 varieties had the lowest GA content, and the highest levels were observed for PJ1 and PJ6. GAs are essential for the proper growth of the plant. Like phenols, they are secondary metabolites that protect plants against pests and pathogens [53]. However, no relationship was found between the GA and phenol contents. Due to the high toxicity of GAs, an acceptable limit of their content in potatoes has been established, which is 200 mg/kg of fresh potato tubers [54]. It is, therefore, important to monitor the GA content and use varieties with the lowest possible content. For this reason, the PJ4 and PJ3 varieties can be recommended for the production of PJ, on the one hand, due to the low content of GAs and, on the other hand, due to the high content of biologically active phenolic compounds.

Microbiological tests
Substances present in plants, as well as plant extracts, often exhibit antimicrobial properties. The antimicrobial properties of essential oils are the most widely described [23,[55][56][57]. Also, so far few sources indicate the antimicrobial activity of substances present in potato tubers. Among them, proteins and protease inhibitors are most often described [58,59] but also polyphenolic compounds and GAs [53,[60][61][62]. Strains of microorganisms selected for research represent many groups of microorganisms that are important from the point of view of human physiology, both positively influencing our body, but also pathogenic. However, despite the different contents of both phenols and GAs, no antimicrobial activity was found for any of the analyzed juices (data not shown). Therefore, it can be concluded that although PJ will not protect us against the development of pathogenic microflora, its consumption will not interfere with the growth of commensal microflora.

Conclusion
PJ is of increasing interest as a raw material with a high biological activity. Literature data describe the broad activity of PJ as antioxidant, anti-inflammatory, or even cytotoxic. In this study, the juices obtained from seven potato varieties, grown under the same conditions, were analyzed. It has been shown that both the content and the profile of polyphenolic compounds depend on the variety from which the juice was obtained. The highest content of polyphenols was found in the juice obtained from the Denar, Ditta, and Toscana varieties, and the lowest content was found in the juice obtained from the Lady Claire variety. However, the use of PJ raises toxicological concerns due to the presence of GAs in potatoes. Also in this case, a significant influence of the potato variety on the GA content in the juice was observed. The lowest levels of solanine and chaconine were recorded in juices from Toscana and Ditta. None of the analyzed juices inhibited the growth of the analyzed microorganisms. It can therefore be assumed that the Ditta and Toscana varieties, due to the high content of polyphenols and the low content of GAs, can be recommended for the industrial production of PJ, which is further used in the production of functional food or dietary supplements. P.Ł.K.; resources, D.R. and Z.S.; supervision, P.Ł.K.; visualization, P.Ł.K. and P.J.; writingoriginal draft, P.Ł.K., K.S., and P.J.; and writingreview and editing, P.Ł.K. All authors have read and agreed to the published version of the manuscript.

Conflict of interest:
The authors declare no conflict of interest.
Ethical approval: The conducted research is not related to either human or animal use.
Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.