NUTRITIONAL COMPOSITION, BIOLOGICAL ACTIVITY AND TECHNOLOGICAL PROPERTIES OF NEW SLOVAKIAN BLACK OAT VARIETIES

Black and red cereal grains, rich in anthocyanins, have recently gained a lot of interest in the food industry due to health benefits. The aim of this study was to evaluate the nutritional composition (crude protein, starch, ash, fat, β-glucans), biological activity (antioxidant activity, total polyphenols, carotenoids and anthocyanins) and technological properties (farinograph, amylograph and extensograph) of new Slovakian black oat varieties Hucul and Norik. Technological properties were observed by incorporation of whole-grain oatmeal flour to standard wheat flour T-650 in amount 3, 6 and 9%. The grains of selected varieties were grinded and resulting whole-grain oatmeal flour was used for analyses. The crude protein content was in both varieties in amount ~ 11%; starch content ~ 44% and ash content in amount ~ 2.6%. The Norik variety showed a higher content of fat – 5.80%, β-glucans – 4.29%, and total carotenoids – 7.47 μg.g-1. In Hucul variety was detected a higher content of total polyphenols – 0.19 mg GAE.g-1(GAE – gallic acid equivalent) and anthocyanins – 22.00 μg.g-1. The antioxidant activity (DPPH method) was approximately at the same level in both evaluated varieties – 3 mg TEAC.g-1 (TEAC – Trolox equivalent antioxidant capacity). There were several differences in mineral composition between observed varieties. The Norik variety showed higher content [mg.100 g-1] of Fe – 5.94, K – 397.56, Na – 6.33 and P – 365.51, while in Hucul variety it was detected higher content of S – 142.89 and Zn – 2.05. Other mineral compounds were not significantly different. Amylograph device shown that with an increasing addition of black oat flour, the amylograph maximum fell down with the best value at 9% addition with compared to standard flour – 1593 AU. Farinograph values on the contrary, showed the good quality of standard flour but parameter water absorption was better with addition of oat flour due to non-starch polysaccharides presented in oat. Extensograph device showed that increasing addition of oat flour decreased resistance to extension. Colorant cereals generally can increase biodiversity in agriculture, and can be attractive raw materials for production foods with added value.

from many researchers and also have increased interest of food industry in using oats as food ingredient in various food products as infant foods (Valle et al., 1981), bread (Zhang et al., 1998) Oat is well known for its unique protein composition with good nutritional value; their content is between 11 -15%. Compared to other cereal proteins, oats are special in the structural properties and also in distribution of protein fractions (Klose et al., 2009). Their proteins are mainly comprised of globulins and therefore have a more balanced amino acid profile than other cereals. Oat proteins were thoroughly studied mainly because of their importance in horses' nutrition (Shewry, 1999;Shotwell, 1999). It was reported that thanks to its unique protein composition oats can be tolerated for most individuals with the celiac disease (Størsrud et al., 2003;Pulido et al., 2009). Although it has some wheat-like sequences in its protein structure, these are less frequent in oats. On the other side, there is no unambiguous consensus among scientists and nutrition experts on whether oats can be definitely recommended for patients with celiac decease (Kaukinen et al., 2013). Oat is also highly valued cereal due to its high energy and nutritional value resulting from high fat content and the unsaturated fatty acids, in particular. On the other side, oat products are more susceptible to oxidation and swelling (Vollmanová et al., 2018). The positive benefits of consumption, especially in whole grain forms, are partly attributed to β-glucans, which have excellent functional and nutritional properties (Gracia et al., 2017). β-glucans are indigestible fibres that form a viscous gel in the gastrointestinal tract. They are approved by the US Food and Drug Administration as LDL cholesterol lowering agents at dosage 3 g.day -1 (Jenkins et al., 2002). High viscosity of oat β-glucans, even at low concentrations (1%), is believed to delay gastric emptying, slow intestinal transit and thereby delay glucose and sterol absorption, which may contribute to lowering plasma glucose and insulin levels as observed in individuals with type 2 diabetes (Butt et al., 2008). However, in study of Frid et al. (2017), general positive effect of βglucan before meals on glucose control or variability in type 2 diabetes was not demonstrated. Nevertheless, adequate intake of dietary fibre is good prevention of obesity and World Health Organization recommends a daily intake of at least 25 g; however average intake is only 12 -18 g in the USA and 15 -20 g in Europe, but 40 -60 g in Africa (Sterna, Zute and Brunava, 2016). In the oat, several substances with antioxidant activity are present in larger quantities. The soluble fibre components, the high content of butyric acid in oat and the antioxidant properties of the tocorienols and phenolic compounds present in its grain can decisively inhibit the development of colon tumors. In addition, the oat contains a wide range of other antioxidants with a beneficial effect. These components, which include, in particular, avenantramides, may have other substances in addition to the antioxidant effect. Bioactive agents in oat also include lignins, saponins, polyphenol substances and isoflavonoids, which may reduce the risk of cancer (Prugar et al., 2008). Avenantramides are phenolic compounds that are found only in oats with anti-inflammatory effects and the initiation of natural cell deathapoptosis (as opposed to necrosis where a cell is violently destroyed and its contents may endanger surrounding cells) (Pridal et al., 2018). In the study of healthy people, avenantramides in conjunction with polyphenols in oats caused an increase in serum superoxide dismutase and reduced glutathione hormones by 8.4, respectively 17.9% and potentially mutagenic malondialdehyde reduction of 28.1%. Total cholesterol, triglycerides, and LDL cholesterol decreased by 11.1%, 28.1%, respectively. 15.1% while HDL cholesterol increased by 13.2% (Liu, Zou and Wang, 2011). Black oat is a potential source of natural colorants such as carotenoids and anthocyanins. Carotenoids are fat-soluble bioactive ingredients responsible for yellow, orange and red colors in various fruits, flowers and vegetables. Generally they are less important ingredients of cereals, but their high consumption covers this shortage. Some cereals have even higher amounts of carotenoids than found in fruits and vegetables (Hussain et al., 2015). Zeaxanthin is the dominant carotenoid in corn, while lutein is a major component of oat, barley, spelled and durum wheat. However, there are few studies on carotenoid content in oat and barley (Ndolo and Beta, 2013). Anthocyanidins and their glycosides anthocyanins, are among the most widespread plant pigments. These include e.g. pelargonidine, cyanidine, peonidine, delfinidine, petunidine and malvidine, which are responsible for the color of the flowers, fruits, cereals and products made from them. Anthocyanin pigments are characterized by high biological activity (Vollmanová et al., 2018). The objective of thisstudy was to observe the new Slovak black oat varieties (Hucul and Norik), their nutritional composition, biological activity and technological propertiesespecially rheological.

Biological materials
Two samples of the first Slovak black oats bred in the Research and Breeding Station Vígľaš -Pstruša were thoroughly analyzed. These are specifically varieties named related from Slovak breeds of horses: Hucul (PS -212) and Norik (PS -213 e -). Hucul -it excels in high one thousand grain weight (39.9 g) and bulk density (57 kg.hl -1 ). The length of the husk is short to medium, moderately radiant. Resistance to lodging is medium to good. The variety has no special requirements for agricultural technology. It is preferred in the nutrition of sport and racing horses for higher protein content and finer, less lignified husk.
Year of registration: 2017. Norik -it excels in high one thousand grain weight (40.6 g) and bulk density (56 kg.hl -1 ). The length of the husk is medium, slightly to moderately serrated. The resistance to lodging is medium to good. It has no special requirements for agricultural technology. It is preferred in the nutrition of sport and racing horses for higher protein content and finer, less lignified husk. Year of registration: 2017 (VURV, 2017). The grain of the samples was homogenized using an Osttiroler Getreidemühlen laboratory mill (Green, Germany) and the wholemeal flour was used for analyzes. Wheat flour T-650 was purchase from mill Vitaflóra (Kolárovo, Slovak Republic). To prepare the extract 0.5 g of the homogenized sample was extracted for 2 h in 40 ml of 80% ethanol on a shaker (GFL 3005, Germany) at room temperature. Subsequently, the extract was centrifuged (Rotofix 32A, Hettich, Germany; 4000 RPM, 10 minutes) and the supernatant was used for measurement (DPPH method, total polyphenol content). An amount of 0.5 g of sample was homogenized in the mortar with sea sand, and repeatedly extracted with 10 mL acetone until the sample became colorless. The extract was filtered using a Whatman filter paper and used for detection of total carotenoid content. An amount of 1 g of sample was extracted with 40 ml of acidified ethanol (96% ethanol acidified with 0.1 M HCl) until the sample became colorless. The extract was filtered using a Whatman filter paper and used for detection of total anthocyanin content.

Chemicals
All chemicals used were of analytical grade and were purchased from Sigma-Aldrich (USA) and CentralChem (Slovakia).

Nutritional evaluation
Moisture content, protein, starch and ash content were determined following the standard AACC method 08-01 (AACC 1996). Nitrogen content was measured by the semi-micro Kjeldahl method, in which protein was calculated by using a factor of 6.25. Fat content was detected with Ancom XT15 Fat Extractor (USA) in line with producer instructions. The Streamlined β-glucan method (Megazyme method) has been successfully evaluated by AACC (Method 32-23.01) and ICC (Method no. 166, approved 1998). This method determines quantitative (1,3),(1,4)-β-D-glucan (β-D-glucan, mixed-linkage β-D-glucan) and is applicable to cereal grains (e.g. barley, oat, rye), their milling products and cereal based foods containing high level of glucose, after preextraction with aqueous ethanol. The method is a rapid procedure for direct, quantitative measurement of (1,3),(1,4)-β-D-glucan (β-D-using highly purified lichenase and β-glucosidase). β-D-Glucan is specifically hydrolysed by lichenase to oligosaccharides, which are then quantitatively cleaved to glucose by β-glucosidase. Glucose is measured using glucose oxidase-peroxidase-buffer mixture (Cauvain and Young, 2009). The absorbance is measured at 510 nm. The amount of mineral elements was analyzed by the ICP-OES method (ICP-OES spectrophotometer, Thermo iCAP Dual 6500, USA)the samples (0.2 g) were subjected to mineralization under high pressure, in HNO3 65%, super pure. All nutritional measurements were repeated six times.

Total polyphenol content
Total polyphenol content was measured in accordance to Singleton and Rossi, (1965) using Folin-Ciocalteu reagent. All measurements were repeated six times.

Determination of total carotenoid content
Total carotenoid content was determined according to STN 56 0053: 1986 at the wavelength of the absorption maximum for the dominant carotenoid which was β-carotene. All measurements were repeated six times.

Determination of total anthocyanin content
For determination ot total anthocyanin content extract of sample was measured at the wavelength of the absorption maximum for the dominant anthocyanin, which was cyanidine-3-glucoside (Fuleki and Francis, 1968). All measurements were repeated six times.

Technological/rheological evaluation
Technological/rheological measurements were made of prepared mixture of wholemeal oat flour of each variety in amount 3, 6 and 9% with wheat flour (T 650). Control (100% T650 wheat flour) without addition of oat flour was also analysed.

Statistical analysis
All experiments were carried out six times and the mean of replications with standard deviations were reported. The experimental data were subjected to analysis of variance (ANOVA), using Duncan's test to determine significances between experimental groups at the significance level of 0.05 (Software XLSTAT, Addinsoft, France, 2020).

Results of nutritional evaluation
Dry matter content in observed black oats was in amount ~91.60% (Table 1). Determination of the dry matter and moisture content in cereals and related food is of great importance in order to ensure a proper technological quality and hygienic compliance related to durability due to a long shelf-life of cerelas. Biel, Jacyno and Kawęcka (2014) observed husked, dehusked and naked oats and indicated that the dry matter was highest in the husked oats -90.25%. By removing husks from the same varieties, the dry matter was reduced by almost 0.5%. The average dry matter in naked oats was lower -89.34%. Their dry matter values are slightly lower compared to our husked black oats. Starch content (Table 1)  compared ten varieties of oats grown in sandy and loamy soils with the finding that oats grown in sandy soil had more starch (58%) than that grown in clayey soil (48.1%). Agro-ecological condition can have strong effect to nutritional and biological properties of plants.
Oat is a rich source of beta-glucans (β-D-glucans) with bioactive and healing effects, e.g. immunostimulatory (activating immune cells macrophages), antiinflammatory, antimicrobial, antifibrotic, antidiabetic, hepatoprotective, or lowering blood glucose and cholesterol levels (Vollmanová et al., 2018). Prugar et al. (2008) reported that naked oat varieties contain the highest value of βglucans from all cereals, ranging from 3.1 to 5.8%. This is well comparable to our samples (Table 1); significantly higher (P≤0.05) β-glucans content was found in Norik variety -4.29%, while in Hucul it was 3.15%. Similar amount of βglucans was described by several authors which reported amount ~3.20% (Chapell et al, 2017), ~3.30% (Kriger et al. 2018). Biel, Jacyno and Kawęcka (2014) determined that β-glucans is lower in husked varieties -3.12%, compared to dehusked -4.48% and naked -4.42% oats. Oat β-glucans have an important role in hypertension, dyslipidemia, insulin resistance and obesity. The basis for their health effects is their ability of intestinal bacteria to ferment them, making them more accessible to human body. They stimulate the defence system by supporting cellular immunity, thereby providing support against infections. Their anticytotoxic, antitumor and antimutagenic effects have also been demonstrated (Jamil et al., 2016). The quantitative content of proteins and amino acids in oat grain is high and exceeds the content of other cereals (third place after wheat and winter rye) (Kriger et al., 2018). The protein content of oat grain is usually in the range of 11 -15%, but the advantage is high biological value as well as good digestibility (Särkijärvi and Saastamoinen, 2006). However, we found lower crude protein content, with significantly higher (P≤0.05) amount in Hucul variety -10.94% (Table 1). Also Biel, Bobko and Maciorowski (2009) found higher protein content in husked oat of variety Bohun -11.44% and even more in naked oat of variety Polar -12.75%. Similar protein content in husked varieties was described by Sterna, Zute and Brunava (2016) -10.58% what was markedly lower compared to naked varietes -15.71% Proteins are not evenly distributed in the grain; about half of them is in endosperm and half in the aleurone layer. Nitrogenous substances in the examined oat grains ranged from 12.2 to 14.7%, of which up to 87 -90% are protein fractions and the others are non-protein nitrogenous substances. Protein fractions were in the samples examined represented as follows: glutelins 27.98 -41.88%, globulins 26.22-32%, albumins 10.16 -23.37% and prolamins 10.63 -23.37% (Kriger et al., 2018). Avenins are oat stock proteins, prolamins, which contain from 10 to 13% of the total protein content, while the main stock proteins in oats are globulins, which make up approximately 55%. Proteins in oats are generally higher lysine content and lower content of glutelins and prolamins compared to other cereals, therefore oat proteins suitably complement human amino acid requirements (Lásztity, 1998). Avenin content was higher in the examined oat varieties described by Van den Broeck et al. (2015), when they grew in clay soil compared to sandy soil and correlated with the total protein content. Prolamins of oats may contribute to the technological properties in relation to the quality of baking. Also they describe the absence of epitopes that stimulate celiac disease in contrast to prolamins from wheat, rye and barley. The husks also affect the crude protein content. According to Biel, Jacyno and Kawęcka (2014) observed husked varieties contain on average only 10.49%, what is comparable to our black oats. Naked varieties contain on average 12.67% of crude protein. However, naked oats have higher values of all essential amino acids by 11.6% compared to husked and by 11.8% compared to dehusked varieties. Fat content (Table 1)  Ash positively affects not only the colour but also the enzymatic activity of the flours. In addition, their content is related to the flours type number. The ash content (Table 1) was 2.48% in the Hucul variety what was significantly lower (P≤0.05) compared to Norik variety (2.64%). Comparing the mineral composition (Table 2) of observed black oats, we can state, that Norik variety significantly exceeded (P≤0.05) in most minerals content: Al -0.41, Cr -0.27, Fe -5.94, K -397.56, Mo -0.05, Na -6.33 and P -365.81 mg.100 g -1 compared to Hucul variety: S -142.89 and Zn -2.05 mg.100 g -1 . The content of other microelements was not significantly different. Black oats differ substantially from other standard oat species in their high ash content. For example, Gambuś, Gambuś and Pisulewska (2006) reported the ash content in yellow of 3.35% and black oats up to 3.66%. The ash content of black oats (2.16 to 2.53%) measured by

Biological activity
The values of the total antioxidant activity in the flours from our examined oat varieties were almost the same (Table 3), ranging around ~ 3 mg TEAC.g -1 .
Slightly lower values were also measured in the research by Chmelová et al. (2015), when the highest total antioxidant activity determined by methanol extraction was in the black-husked variety Paddock -2.81 mg TEAC.g -1 .
According to them oats are used as a healthy food due to their significant content of antioxidantslarge number of phenolic compounds (ferulic, coffee, phydroxybenzoic, p-hydroxyphenylacetic, vanillic, protocatechuic, p-coumaric (2018), polyphenols are bioactive secondary metabolites present in fruits, vegetables and cereals. In food, these metabolites can contribute not only to antioxidant activity, but also to colour or taste. Consumption of some cereal products protects human body against the development of chronic diseases such as diabetes, cancer and cardiovascular disease. As antioxidants, phenolic compounds prevent oxidative damage to cellular organelles, proteins, lipids, DNA and RNA. This authors found ~0.61 mg GAE.g -1 in observed oat samples. Higher amount of total polyphenols in oat samples was measured by Among the important biologically active phenolic substances of oats, the determination of which, however, is not the aim of this work, are avenanthramides. It was reported that they have various bioactive properties, such as antioxidant, antiinflammatory and antiproliferative effects. In addition, they can also regulate cellular responses during inflammation through their interactions with molecular and signalling pathways. Moreover, these compounds can inhibit the growth of four different human colon cancer-causing cells (Wang et al., 2015).They are not to be found in any other cereals . Hitayezu, et al. (2015) and Walters et al. (2018) found that enrichment of food with oat bran can substantially increase the amount of these substances. Based on the concentration of natural colorants (anthocyanins, carotenoids), the colors of individual grain parts (endosperm, aleurone layer, pericarp, husks) range from white through yellow, gray, red to black (Chmelová et al., 2015). We confirmed that while Norik variety contained significantly higher (P≤0.05) amount of carotenoids (7.47 µg.g -1 ), Hucul variety had significantly more (P≤0.05) anthocyans (22 µg.g -1 ). Carotenoids play an important role in the human body, for example, αand β-carotene have a primary function in vitamin A biosynthesis, important for proper visual function, embryo and fetal development. Lutein and zeaxanthin, in turn, play an important role in promoting eye and skin health as well as in reducing the incidence of cancer and cardiovascular disease (Hussain et al., 2015). Anthocyanins are attributed positive antioxidant, anticancer effects, protect the retina, retard aging and have a positive effect on intestinal health.

Technological/rheological properties
In the control sample (wheat flour T 650) it was recorded the highest amylographic maximum -1593 AU (Table 4). With the addition of whole-grain oatmeal flour in the selected percentages, the amylographic maximum decreased; most notably in variant 9% addition of whole-grain oatmeal flour variety Hucul -1404 AU. From results it can be concluded that the Hucul variety had better amylolytic activity with compare to Norik variety. This finding indicated that observed variety is richer for α-amylase, which is important in bakery industry, because amylolytic activity very strongly influenced character of bread crumb (porosity, flexibility, taste). Maximum gelatinization temperature was not affected, and ranging around ~92.09 ˚C value (Table 4) In their study amylographic maximum was higher, and ranged from 460.33 to 489.33 AU, and the gelatinisation maximum temperatures was also higher with the value ~ 95 °C. It was found the significantly highest farinographic water absorption (Table 5) in flour mixture after addition of 9% Hucul variety whole-grain flour -59.97% especially compared with to control sample. It was no surprise that the significantly lowest development time (Table 5) was determined in control wheat flour (T 650) -2.33 min. and increase with black oats whole-grain flour addition to maximum (8.27 min.) in variant 6% of variety Norik. As expected, addition of black oats whole-grain flour reduced dough stability of most enriched flour mixtures. However the addition of 3% whole-grain flour variety Norik exceeded even control flour (11.97 min.) with dough stability of 14.63 min. The highest level of degree of softening (Table 5) was found in variant with addition of 6% Hucul whole-grain flour -61 FU with compared to lowest in control sample -38.33 FU. In our study was determined different farinographic quality number (Table 5)  Replacing wheat with 40% of oats or barley significantly increased water absorption by 8 -11.8% with compared to pure wheat flour. This is probably due to the high fibre content in oat and barley flour. Interestingly, oat bran showed significantly higher water absorption than barley, although the total amount of fibre was lower. They also found that the content and molecular weight of β-glucans was more important for water absorption than other types of fibre, because oat had much higher β-glucan content than barley. Their findings are comparable to our results; we supposed that mainly β-glucans and also other non-starch polysaccharides can positively influenced the farinographic characteristic of dough. The control sample had an average resistance to extension of 259.33 EU (Table  6) while the lowest resistance was observed in variant with addition of 6 and 9% Hucul variety whole-grain flour (224.67 and 222.33 EU, respectively). All observed flours reached the high extensibility values characteristic of "weak" flours ranged from 184 to 205 mm (Table 6). Extensographic energy is the factor that was the most affected with the addition of whole-grain oat flour. While in the control flour and flour mixtures (Hucul 3% and Norik 3%) it was still in the value around ~ 100 cm 2 , the addition of 6 and 9% caused its significant decrease to values below 90 cm 2 . Londono et al. (2015a) used pure oatmeal flour and with the addition of gluten as a base and addition of medium and highly viscous βglucan. They found that gluten-enriched dough was more extensible -61 mm, while its equivalent without gluten addition was only 15 mm. The addition of 3.8% of β-glucan significantly reduced the extensibility of gluten-added samples, namely by 22.8 mm for medium viscosity up to 34.9 mm for high-viscous βglucan. For samples that did not contain gluten, the decrease extensibility was not so significant. Londono et al. (2015b) discussed in other research the effect of roasting and grinding (particle size) on dough extensibility. The untreated oatmeal dough (control) and steam-dried dough gave low stretching resistance and their extensibility was about 85 mm, while in infrared drying dough was short with high resistance. The size of the particles and the addition of bran also played an important role; fine oatmeal with the addition of fine bran (<0.25 mm) gave a tough dough with low resistance, while coarser fractions with the addition of coarser bran gave a rather short dough with higher resistance.

CONCLUSION
According to the obtained results, it can be concluded that selected varieties of black oats Hucul and Norik had a different nutritional composition. With almost the same content of dry matter, the Hucul variety contained significantly more crude protein. Norik, variety on the other hand, had more starch, fat and ash content. In Norik variety was also significantly higher content of several mineral compounds especially Fe, K, Mo, Na and P.In Hucul variety was observed significantly higher content of S and Zn. Biological activity of both varieties was similar; they differed mainly in the content of carotenoids and anthocyans. While Hucul contained significantly more anthocyanins, Norik contained more carotenoids. Based on amylograph results, wheat flour as control sample reached high values of an amylographic maximum of almost 1600 AU and the addition of whole-grain oat flour was able to reduce this high value by almost up to 200 AU with a 9% addition of the Hucul variety. Farinographic indicators were generally good for control flour and experimental flour mixtures. As important may be significant increase of water absorption which is very important parameters for bakery industryhigher water absorption=higher dough yield. The extenzograph results pointed to the higher extensibility of the dough at low resistance in experimental flour mixtures. Overall, we can concluded that observed new black oat varieties are nutritionally very interesting, especially due to the fat content and the content of biologically active substances. Their addition into flour mixtures can improve some technological parameters, however we recommend further research and practical application.

Conflicts of interest:
All authors declare no conflicts of interest.