The values of biologically active substances are monitored by biochemical analysis in connection with the determination of the oenological, microbiological and other characteristics. Critical points of instability of the biologically active substances value are determined, possibilities of restraint until the causes of this instability are eliminated. Values of endogenous sulfur dioxide content and possibilities of reduction until its elimination (Wells and Osborne, 2011; Ivanova, Petruseva and Mitrev, 2015). Correlation of concurrent laboratory and organoleptic values, which create the first sense of customer interest and interest in manufacturing and commercial applications. The equilibrium of the total polyphenol values demonstrates the stability of the biologically active substances values, thus demonstrating the correctness of the design of the technological processes (Mlček et al., 2016; Tarko et al., 2015). The protein content test proves their stability, which is a measure of protection against sunburn.

In this case, however, completely different technological processes, due to the prepared conditions for yeast activity, are also of completely original origin (Hernández et al., 2018). The yeast for the preparation of the fermentation support comes from the same variety and locality, including agrotechnical practices and technology for the cultivation of grapevine (Lachman et al., 2009). The fermentation process is prepared from grapes harvested 5 to 7 days before the harvest for the entire production process. The exact start date of the preparation of the fermentation support is determined by the temperature relations of the habitat and the duration of the preparatory processes for the technological process of fermentation and its course (Yu et al., 2018). The temperature sessions need to be regulated in the range of 18 – 28°C and YAN conditions (assimilable N). In the case of unfermented must in the range of 120 – 200 mg.L^-1 (on the experimental parcels of the submitted project, it ranged in three sampling times at 177 – 166 – 134 mg.L^-1, the yeasts were not referred to as "life", they did not consume nitrogen). In fermentation, nitrogen value has fallen further since the beginning of the fermentation process and reached 50 – 44 – 40 mg.L^-1. However, it is necessary to emphasize that the resources for obtaining the raw material needed for its processing are consistent but the technological procedures for the respective goals are quite varied.

The technological bond of the basic raw material, ie the apple pulp, has been created to obtain apple juice and the sources of tripenten biological substances contained in apple peel. Apples contain a range of substances that the technologist must register and monitor their development (Baron, Dénes and Durier, 2006; Valdramidis et al., 2009). Mutual relationships of organic acids and sugar content, which is represented by fructose, glucose. During fruit maturation, sucrose passes gradually into fructose. Ripening also creates conditions for the transformation of starch (in immature fruit) into sugar. Furthermore, it is necessary to monitor proteins, pectin substances, fiber and others, especially vitamins and aromatics (Qin, Petersen and Bredie, 2018). Biological activity is heavily enriched with biologically active substances from grape vines, which which belongs to high-incidence plants. The combination of both components – i.e. apples and natural additives, i.e. biologically active substances from grapevine products, creates a value that is purposefully controlled on the basis of the relevant analyzes (Vrancheva et al., 2018).

Samples of natural beverages from grape vines and apple juice were supplemented with biologically active substances from grapevine and free sulfur dioxide, total antioxidant activity (TAA) by methods using DPPH and ABTS, total polyphenol content (TPC), total anthocyanins content (TAC), sugar (fructose, glucose and sucrose), alcohol (ethanol), free and volatile acids and yeast assimilable nitrogen (YAN) were determined at regular monthly intervals during beverage storage under the prescribed conditions at 5 ° C.

Free sulfur dioxide in test samples of natural beverages (Table 1 – 4) ranges from 1.84 to 15.05 mg.L^-1. The smallest content was found in the natural beverage from apple juice with values of biologically active substances from vine products without alcoholic fermentation after 3 months of storage, on the contrary, the highest value was in natural beverage from white grape must with alcoholic fermentation after the first determination. If we compare the achieved values for free SO₂ (Table 1 – 4) with a study of sulfur dioxide (Snopek et al. 2018) the measured values are compared to those in organic quality. Table 2, 3

To determine the total polyphenols content, a spectrophotometric method using Folin-Ciocalteau reagent was used in samples of a natural beverages of grape vines and fortified beverages of apple juice, using gallic acid as a standard. For beverages tested, the higher value of total polyphenols in fortified apple beverages was made by biologically active substances from grapevine and its change during storage was only fractionally. For alcoholic beverages, they exceeded 677.26 mg GAE.L^-1 after first measurement to 619.54 mg GAE.L^-1 after 3 months of storage.

The least total polyphenols were determined for a natural drink of white grape vines with alcoholic fermentation. It reached from 305.03 mg GAE.L^-1 after the first measurement to 306.58 mg GAE.L^-1 after three months of storage.

Paixao et al. (2007) evaluated the content of polyphenols in a fermented beverage of white grape vines. The average measured content was 369 mg GAE.L^-1, another study (Hurtado et al., 1997) reported the TPC of the beverage of 292 mg of GAE.L^-1. These published values are comparable to our values. The results of the work by Ricci, Parpinello and Versari (2017) for a TPC were reported at 222 mg GAE.L^-1 which is in correlation with our results. Fortified beverage achieved above-average results and can be evaluated very positively. Tarko et al. (2015) published comparable values for apple juice, indicated as raw materials with high TPC.

To determine the antioxidant activity of natural beverage samples, the DPPH method was used, based on the reaction of the test substance with a stable 1,1-diphenyl-2-picrylhydrazyl radical. In addition, the ABTS method was used. It is characterized by reacting the test substance with 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid). Both methods used trolox as standard. The highest antioxidant activity in natural beverages (Table 1 – 4) was determined by both methods in non-alcoholic natural beverage from apple juice with fortified bioactive substances from white grape vines. The value ranged from 655.58 – 644.48 mg TE.L^-1 determined by the DPPH method and 975.73 – 956.89 mg TE.L^-1 using the ABTS method during storage. The smallest antioxidant activity was determined in a beverage sample of white grapes without alcohol.

The determination of total anthocyanins content was another of analyzes that support the high content of biologically active substances in a natural beverage based on fortified apple juice. Its content ranged from 9.51 mg C3G.L^-1 to 8.29 mg C3G.L^-1. There followed a non-alcoholic natural drink of white grape vines with values ranging from 5.95 mg C3G.L^-1 to 6.14 mg C3G.L^-1. A decrease in total anthocyanin values was observed for alcohol drinks during a three-month storage period with a white grape drink from 4.35 mg C3G.L^-1 to 3.23 mg C3G.L^-1 and for fortified apple juice with alcohol then 3.31 mg C3G.L^-1 to 2.66 mg C3G.L^-1. Our measured values of the content of anthocyanin in apples reached the published values by Wolfe, Wu and Liu (2003). Losses were attributed primarily to the technological modifications that took place during the production of the beverage.

Chromatographic techniques were most suitable and accurate for the identification and quantification of mono- and oligosaccharides in food (Duarte-Delgado et al., 2015) and according to the guidelines of the Association of Official Analytical Chemists (AOAC, 1993; Sims, 1995) for quantification of sugars. Table (1 – 4) lists the sugars (fructose, glucose and sucrose) content in natural beverages samples at the beginning of storage determined by the HPLC method. Figure 1 shows the chromatogram of determination of sugars and alcohol in a sample of white grapes (HPLC – RID). When the highest peak is ethanol, the other peaks represent the individual sugars. Table 1 – 4 shows that alcohol-free beverages contain up to ten times more sugars than alcohol drinks. Most of the sugars were determined for a grape wine without alcohol 226.64 g.L^-1, which was retained after 3 months of storage and the alcohol content fluctuated between 0.19% (vol.) and 0.22% (vol.). In fortified apple juice the sugar value dropped from the initial 156.54 g.L^-1 to 147.65 g.L^-1, and its alcohol content also reduced from 0.12% (vol.) to 0.09% (vol.). Conversely, for a white grape alcohol drink, which reached an average of 11.74% (vol.) alcohol content, it contained only 9.37 g.L^-1 to 10.44 g.L^-1 of sugars.

Fortified apple juice contained less alcohol from 9.02 – 9.37% (vol.), the sugar content ranged from 25.78 – 22.97 g.L^-1. Pickering et al. (1998) determined the effect of ethanol concentration (range 0-14% vol.) on perception of the "fullness" of beverages. It has been found that ethanol concentrations were highly correlated with perceived intensity, physical viscosity and density measurements (Nurgle and Pickering, 2005).

Volatile and total acids were also determined. Volatile acids were determined in very small amounts in all analyzed samples. Most of them were determined for fortified apple juice with an alcohol of 0.52 – 0.54 g.L^-1, on the other hand, the least amount in the same sample without alcohol, and in the range of 0.01 – 0.03 g.L^-1 during the three month storage. Total acids were at highest as measured for fortified apple juice without alcohol in the range of 10.45 – 10.47 g.L^-1. For all other beverages, the total acid value was approximately the same as 9 g.L^-1.

Asymilatable nitrogen were considered to be the initiator of fermentation, affecting its kinetics, aromatic content, acetic acid and others. Table 1 – 4 shows the results of determining the amount of assimilable nitrogen in natural beverages. Highest values of assimilated nitrogen were determined for non-alcoholic beverage variants. The highest was determined for a drink from white grapes wine 177.04 mg.L^-1 and dropped to 134.45 mg.L^-1. The fortified apple juice was 156.42 mg.L^-1 and dropped to 124.37 mg.L^-1 during 3 month storage. Conversely, for alcoholic beverages, the values were lower. Table 2 shows a decreasing value from 50.15 mg.L^-1 to 40.18 mg.L^-1. Table 4 shows a high drop from 99.45 mg.L^-1 to 76.34 mg.L^-1.

This confirmed (Steidl, 2002) that the more ammonium ions are in the environment, the more yeast consume them.

It is essential to select and evaluate sources to prepare raw material base for the production of natural beverages without and with alcoholic fermentation using exclusively natural starting values. During production it is necessary to assess natural values and quality of these basic rescources.

The high content of biologically active substances from grape vines and their incorporation into all musts is an integral part of the entire technological, production process and in combination with the corresponding fruit sources it appears to be a positive step towards enriching food with natural additives.