BIOLOGICALLY ACTIVE COMPOUNDS CONTAINED IN GRAPE POMACE

A healthy lifestyle and gastronomic trends based on traditional and local foods accompanied by waste-free technologies are currently in the primary focus. One of the raw materials with properties in alignment with such requirements is grape pomace. This paper evaluates the antioxidant activity of grape pomace (which is homogenized into a brown powder) and selected commonly available commercial flours – wheat bread, rye plain, and rye whole grain flour – using DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) and total polyphenol content method, where was used Folin-Ciocalteaure agent and then it was determined by spectrophotometric method (the measure of absorbance). The total amount of polyphenols in grape pomace was measured of 47.94 mg GAE.g -1 , but the value 0.27 mg GAE.g -1 was measured in wheat bread flour. Grape pomace performed the antioxidant activity of 57.45 mg AAE.g -1 , whereas wheat bread flour of only 0.21 mg AAE.g -1 Compared to selected commercial flours, the total amount of polyphenols in grape pomace was up to 150 times higher and the ratio of antioxidant activity between grape pomace and wheat bread flour was even more than 280 times higher. This makes it possible to fortify commercial, commonly available flours with different amount of grape pomace so that products with a higher amount of biologically active substances can be prepared. Another benefit could be a reduction in health risks and a contribution to improving consumer health.


INTRODUCTION
The interest in healthy and balanced nutrition has been increasing. The society is striving to find suitable food beneficial to the human bodya diet rich in vitamins and minerals. The WHO recommends 600 g of fruit and vegetable, including cooked vegetables, as a daily intake (WHO, 2013). The ratio between fruit and vegetable should be 1:2 (The Czech Society for Nutrition, 2012). Unfortunately, in many European countries, their income is lower. However, fruit and vegetable are rich in biologically active substances (Juriková et al., 2016). These include antioxidants, such as vitamins C, E, provitamin A, minerals with antioxidant properties, polyphenols, tannins, and other groups of substances that can reduce health risks and contribute to health enhancement (Salter, Wiseman and Tucker, 2012; Tomás-Barberán and Gil, 2008). One source of these substances may be grapevine and products from it American grape type (Vitis labrusca L.), Muscadine type (Vitis rotundifolia Michx.) and Amurensis (Vitis amurensis). Out of these, the European type has spread worldwide and accounts for 71% of total grape production in the wine industry (Yang, Martinson and Liu, 2009). An example of producing of grapes can be mentioned in Brazil, where the production was approximately 534 million kg in the year 2009. This number increased by 24%, which means that the number was 709 million kg two years later (in the year 2011). Logically, when the production of grapes increased, automatically the amount of grape pomace increased as well (Casagrande et al., 2019). Grapes serve as a suitable source of phenolic compounds with antioxidant effects, such as anthocyanins, flavonoids, and resveratrol (Yang, Martinson and Liu, 2009;Hernández-Salinas et al., 2015).
It is a fruit consumed all over the world. It can be eaten in a form of fresh table fruit and also in a form of diverse processed products including wine, juice (Kim et al., 2017), grape flour, or grape pomace. Grape pomace, dried and homogenized grape seeds and skin, is a by-product of wine production applied for further processing. It can be used as a substrate to produce alcoholic beverages due to sufficient sugar content. The most famous liquor prepared from grape pomace is Italian Grappa. In France, brandy originated from grape pomace is known as Marc, in Spain it is named De orujo and in Portugal Bagaceira (Piras, 2008;Gasnier, 2005). Furthermore, the inedible components such as seeds and skins can be used as a food additive, feed, fertilizer, seed source for oil production, or fuel. Therefore, grape pomace is considered to be a favorable alternative product allowing waste-free management. Grape pomace is rich in polyphenols, especially flavonoids such as gallic acid, catechin, and epicatechin (Özvural and Vural, 2014 The number of phenolics which has been found in plants is over 8000. The particular profile of polyphenols depends also on specific cultivar within a species. An example can be given grape varieties where the composition can depend on factors, such as geographical region, soil composition, or terroir. This is the reason why are different polyphenolic compositions but also the success of growing specific species of wine (Bakota et al., 2015).
As a by-product, grape pomace is an inexpensive source of polyphenolic antioxidants The process of obtaining polyphenols from fruit or vegetable is usually extraction. It can be for example Soxhlet extraction, maceration, or hydrostillation. Nowadays there are also new non-conventional methods, due to the effort to use a method that is environmentally friendly to compare to the classical method. An example can be ultrasound, microwave, and pressure-assisted extractions (it can be applied alone or together with various solvents for the sake of reducing solvent requirements and the energy) (Milella et al., 2019).
Furthermore, apart from phenolic compounds with antioxidant effects, grape pomace contains other antioxidants neutralizing free radicals. In the human body, free radicals may harm compounds altering cell membranes, cause cell damage and cell inflammation, promote abnormal cell growth including certain types of cancer and even cause cell death. Free radicals are formed in the body in natural metabolic processes. In their formation, exogenous factors, including ultraviolet light, radiation, fumes, and air pollution, play an important role (Krishnaswamy et al., 2013). Antioxidants, comprising phenolic compounds, flavonoids, and carotenoids, can reduce the risk of oxidative damage by converting free radicals into inactive molecules (Štípek et al., 2000).
These beneficial properties of grape pomace are in accordance with current nutritional trends emphasizing the application of traditional, local and organic food in compliance with waste-free management (Burg, Masan and Ludin, 2017; Makris et al., 2008).
Due to the fear of the unfavorable effects and the safety of synthetic antioxidants, the studies started to be more interested in studies that were focused on natural products. Namely, it was for example herbs, vegetables, fruits, or agro-industrial waste. The reason for attention on these products is because they are rich in polyphenols and they can be used instead of synthetic antioxidants that are used in the pharmaceutical, food, or cosmetic industry (Casagrande et al., 2019).
It is assumed that polyphenol content is about 60 -70% in grape seeds, 30% in the skin, and merely 6% in the flesh (Kim et al., 2017).
Homogenized grape pomace has reached the market and allows fortification of common commercial flours or their complete replacement. The purpose of fortification is to increase the content of biologically active substances. First, it is necessary to identify the original content of biologically active substances in the individual basic materials.
This paper aims to compare polyphenolic substances in grape pomace and their antioxidant effects with commercial, commonly available flours on the market.

Scientific hypothesis
Hypothesis No. 1: The total content of polyphenolic substances and antioxidants in grape pomace is higher than in the commercial flours.

MATERIAL AND METHODOLOGY Material for analysis
The analysis employed material samples from grape pomace (Figure 1), wheat bread flour, rye flour, and rye wholemeal flour. A presented grape pomace sample came from viticulture Ludwig, vineyard track "Zbavce", village Němčičky, region Velkopavlovická, area Moravia. It was homogenized from grapes of the Riesling variety which was manually harvested in October 2018. Next, the grapes were processed using a time-driven horizontal press. Drying and grinding were performed at 70 °C. The grinding was performed until the grape pomace reached similar size as the flours which were bought for the analysis.
The flours were bought in a supermarket as a commonly available commercial material: wheat bread flour -wheat flour "Babiččina volba", GoodMills Česko inc., Prague, the Czech Republic, with the following nutritional values per 100 g: energy 1461 kJ/345 kcal, fats 1.7 g, of which saturated fatty acids 0.2 g, carbohydrates 69 g, of which sugar 2.0 g, fiber 3.1 g, proteins 12 g, salt <0.01 g; rye plain flour -rye plain dark flour "Babiččina volba" GoodMills Česko inc., Prague, the Czech Republic, with the following nutritional values per 100 g: energy 1435 kJ/339 kcal, fats 1.0 g of which saturated fatty acids 0.2 g, carbohydrates 70 g, of which sugars 6.0 g, fiber 9.0 g, proteins 8.0 g, salt <0.01 g; rye wholemeal flour -"Pernerka" rye wholemeal fine flour, Mlýn Perner, Svijany, the Czech Republic, with the following nutritional values per 100 g: energy value 1449 kJ/342 kcal, fats 1.3 g of which saturated fatty acids 0.13 g, carbohydrates 72.1 g, of which sugars 1.8 g, proteins 6.1 g, salt 0 g. An extract (5 g of sample extracted in 50 mL of methanol (MERCI, s.r.o., Brno, the Czech Republic)) was prepared from all flour samples and used for further determination.

Total phenolic content analysis
To determine the total phenolic content (TPC) Folin-Ciocalteaure agent (Sigma Aldrich, the Czech Republic) was used. The extract of the amount of 0.1 mL was mixed with water in a 10 mL volumetric flask. Then, 0.5 mL of Folin-Ciocalteaure agent and 1.5 mL of 20% solution of Na 2 CO 3 (Sigma Aldrich, the Czech Republic) were added to the solution. Specord 50 Plus (Analytik Jena, Jena, Germany) was used to measure absorbance at a wavelength of 765 nm. Pure water was used as a reference (Thaipong et al., 2006). To express the results, unit milligrams of Gallic acid (GAE) per grams of fresh mass (FM) were used. TPC was measured four times.
The stock solution was prepared by the solution of 24 mg of DPPH in 100 mL of methanol (MERCI, s.r.o., Brno, the Czech Republic) which was stored at -20 °C until it was needed. The absorbance of DPPH radical without any added extract was recorded and corrected every day. The sample solution used for measuring was prepared by mixing 10 mL of the stock solution with 45 mL of methanol to obtain the absorbance of 1.1 ±0.02 units at 515 nm using spectrophotometer Specord 50 Plus (Analytik Jena, Jena, Germany). The extract (210 μL) was allowed to react with a 4 mL DPPH solution for 1 hour in the dark. Then, the absorbance was recorded at a wavelength of 515 nm. TAC was measured three times.
For the calculation of antioxidant capacity, a decrease of the absorbance value was used following the formula: Antioxidant capacity (%) = (A 0 -A i /A 0 ) x 100, where A 0 is the absorbance of a blank without the sample and A i is the absorbance of the mixture containing the sample. The calculated antioxidant capacity was converted using a calibration curve of the standard and expressed in ascorbic acid equivalents (AAE) (Vasantha Rupasinghe, Jayasankar and Lay, 2006).

Statistical analysis
Excel 2013 (Microsoft Corporation, USA) and STATISTICA CZ version 12 (StatSoft, Inc., USA) were used for data analysis. The results were expressed by mean ± standard deviation. The comparison of TPC and total antioxidant activity (TAA) of grape pomace with those of flour samples were calculated by non-parametric tests -Kruskal-Walllis test Wald-Wolfowitz test, Kolmogorov-Smirnov test and Mann-Whitney U Test (α = 0.05).

RESULTS AND DISCUSSION
The comparison of total polyphenol content of grape pomace and selected commercial, commonly available flours are shown in Table 1. The lowest value of polyphenols was established in wheat bread flour with a value of 0.27 mg.g -1 . Rye flour showed a slightly higher value in both plain and whole grain flour. The total amount of polyphenols in grape pomace was measured to be 150 times higher, namely 47.94 mg.g -1 . This indicates a statistically significant difference between grape pomace and commercial flours.
Due to the non-compliance with the prerequisites for the ANOVA type statistical evaluation, the Kruskal-Walllis test based only on the order of the individual samples had to be applied to compare obtained data as can be seen in Table 2. For this reason, a statistically significant difference was observed only between grape pomace and wheat bread flour, although the measured values indicated a statistically significant difference between grape pomace and all the commercial flours. In case that only two individual samples are compared, there was a statistically significant difference (p <0.05) between all flour samples both using non-parametric tests (Wald-Wolfowitz test, Kolmogorov-Smirnov test, Mann-Whitney U Test) and using the parametric Welch t-test. However, the homogeneity condition is not met in this test.  Interesting results for comparing were also analyzed in Ky and Teissedre (2015) where was measured total phenol content in grape pomace seed and skin extracts. The measured amount was 128.22 -215.93 mg GAE g -1 of dry weight in grape seed pomace extract and 71.88 -196.71 mg GAE g -1 of dry weight in grape pomace skin extracts when for the measuring was used the extraction method which is appropriate for the preparation of edible extracts.     As can be seen, there are differences between wine (the liquid product of grapes) as well, which shows the variety of polyphenols which can be seen in the different types of grapes, that were grown in the different conditions. The amount of TPC is affected by a number of factors, so the results presented in this article may be different compared to the others. The content of polyphenols can be affected by environmental factors such as soil type, sunlight, climate, etc., TPC is further affected by storage, during which polyphenols oxidize easily. Concentrations of phenolic acids generally decrease during maturation, while concentrations of anthocyanins increase. Another parameter that affects the content of polyphenols is culinary treatmentgrinding of plant tissues degrades polyphenols and maceration can increase the content of polyphenols due to diffusion in the juice (Manach et al., 2004; Pandey and Rizvi, 2009). Table 3 summarizes antioxidant activities which were up to 280 times higher in grape pomace than in other commercial flours. Grape pomace performed the antioxidant activity of 57.45 AAE mg.g -1 , whereas wheat bread flour of only 0.21 AAE mg.g -1 . Samples of rye flour differed from wheat bread flour by only 0.20 AAE mg.g -1 , which is a negligible difference if compared to grape pomace.
Similarly to the determination of the total amount of polyphenols, it was not possible to apply the ANOVA test. Therefore, a comparison of the obtained data was performed using the Kruskal-Walllis test as displayed in Table 4. The results follow the same trend as for total polyphenols content. As with the TPC determination, nonparametric tests (Wald-Wolfowitz test, Kolmogorov-Smirnov test, Mann-Whitney U test) and parametric Welch t-test between individual pairs of samples were performed again. There was a statistically significant difference (p <0.05) between all pairs in all tests.
Costa et al.
Even though more studies are examining this issue, data comparison is very problematic as different methodologies and their modifications, different grape varieties, and sample processing techniques of TPC and TAA determination were applied. It is essential to consider specifics of biological material that may be influenced by the ambient conditions (climate, temperature, sunshine), cultivation technologies (soil fertilization, harvest time, harvesting method), and by post-harvest processing and storage.
For future practical applications, it would be appropriate to monitor TAA and TPC in the mixtures of common commercial meals and grape pomace in various ratios to enable fortification of traditional flour with a small percentage of grape flour. Furthermore, it is necessary to identify specific characteristics of such mixtures, including sensory properties and leavening, and subsequently, to examine properties in baked goods, such as shelf life, to distinguish their mutual influences.

CONCLUSION
This research has examined the content of antioxidants and polyphenolic substances in grape pomace which were gained from the local farmer and it was thoroughly compared with three different commercials, commonly available flours, where two of them were from one company and the last one was from the different one for more valuable comparison. It evaluates differences in existing products and products containing non-traditional alternatives to commercial ones. The results indicate that grape pomace performed magnitude higher values of both polyphenolic contents and antioxidant properties than commercial flours.
As this study has revealed, grape pomace provides a diverse array of applications in the food industry; it plays a role as a food additive improving sensory and technological properties and works as a dietary supplement reducing the risk of civilization diseases including cardiovascular diseases, diabetes mellitus, and obesity. Newly, further advantageous attributes still required to investigate more is the possibility to employ grape pomace in gluten-free and lactose-free products.