When researching several hypotheses were examined:

The hops pellets Hallertaur Magnum and Tettnanger varieties are of the 2019 harvest containing the following amount of oil and α-acids respectively: Magnum (1.6 – 2.6 mL.100g^-1, 12.0%) (Figure 1) and Tettnanger (0.5 – 0.9 mL.100g^-1, 3.8%) (Figure 2). The precise oil and α- acid content were determined by the producer.

All reagents and standards were of analytical grade. Quercetin, rutin, isoxantogumol, and phenolic acids standards were from Sigma-Aldrich with a purity ≥ of 99%. Potassium dihydrogen phosphate (KH₂PO₄), acetonitrile, acetic acid, orthophosphoric acid (H₃PO₄) were purchased from Galachem (Moscow, Russia).

1-butanol, hydrochloric acid (HCl), iron (II) sulfate heptahydrate (FeSO₄·7H₂O), methanol, carboxymethylcellulose, ethylenediaminetetraacetic acid (EDTA), ferric ammonium citrate ((NH₄)₅[Fe(C₆H₄O₇)₂]), ammonium hydroxide (NH₄OH) were purchased from Limited liability company “Reatorg” (Moscow, Russia).

This investigation was used centrifuge Armed 80-2 (Russia), photoelectric colorimeter Apel AP-101 (Japan), pH meter Milwaukee (USA), chromatographic equipment "Agilent Technologies 1200" ("Agilent", USA) with diode array detector. HPLC equipment was fitted column Hypersil 5 u C18 250 x 4.6 mm 5 μm (Thermo, USA) with wavelength 270 and 310 nm, and fitted column Luna 5 u C18 (2) 250 x 4.6 mm 5 μm (Phenomenex, USA) with wavelength 290 nm and column Kromasil C18 150 x 4.6 mm 5 μm (Supelco, USA) with wavelength 290 nm.

The hops selected for the research represented by T90 pellets of the Hallertaur Magnum and Tettnanger varieties purchased from Joh.Barth & Sohn Company (Freiligratstrasse, 7-9, 90482, Nuremberg, Germany).

Sample pH was determined with a pH meter. All measurements were made in triplicate.

The organoleptic analysis was done by a professional group of researchers, consisting of 10 people on a 5-point scale according to the characteristic taste descriptors selected. 5 points mean a strong descriptor shade, 4 points – a well-developed descriptor shade, 2 points – a slightly visible descriptor shade, 1 – a subtle descriptor shade. The results obtained were summarized and the average score was recorded.

The wort was produced in a pilot brewery (Bavaria, Germany) of the All-Russian Research Institute of Brewing, Non-alcoholic, and Wine-Making Industry. The original wort density produced from 100% malt was 12 °P. The wort was produced according to the conventional infusion mash procedure with temperature breaks at 52 °C, 63 °C, and 72 °C during 20 – 30 min each.

The wort was hopped with two Hallertaur Magnum and Tettnanger hop varieties to achieve a target of 12 bitterness units (BU). Hop samples were added at 15, 30 and 45 minutes after the wort boiling. The boiling time was 60 min and the post-boiling density was 1.048 (12 °P). Also, hop samples were placed to the whirlpool applying dryhopping technology. The data is shown in Table 1.

T90 pellet hops at a rate of 4 g.L^-1 (Hallertaur Magnum and Tettnanger variety) were added to the wort current pumped into the whirlpool at a 35 m.sec^-1 flow rate. The cold hopping stage lasted 25 minutes;

The boiling time for the preparation of model water hopped solutions was 15 minutes at different pH values applying the same hop doses as for the wort. The pH was adjusted by adding a 0.1% citric acid solution.

Number of samples analyzed: The number of one experiment analyzed samples in one replication was 16.

Number of repeated analyses: The analysis was done in triplicate.

Number of experiment replication: The experiment replication was done two times.

Descriptive statistics performed and values expressed as mean ± standard deviation. The Student-Fischer method was used in the studies. The obtained data reliability limit (p <0.05) was considered to assess the various factors that impact the polyphenols content in all studies; the statistical data was processed by the Statistics program (Microsoft Corporation, Redmond, WA, USA, 2006).

It is known that the finished beer organoleptic profile depends upon the hops types used in the technology and forms due to various organic compounds – bitter acids, ether compounds, polyphenolic substances (Praet, 2016; Van Opstaele et al., 2010). Sometimes the sharp shade in the hop compounds taste becomes leveled and changes also due to the presence of organic malt substances (β-glucan, starch dextrins, etc.) in the wort (Fox, 2018; Langstaff and Lewis, 1993). In our terms the boiling time of the wort with hops is of great importance.

Therefore, and at the first research stage, comparative studies of the environmental impact on the various hop polyphenolʹs dissolution (polyphenoles (POL) and anthocianogence (ANTH) in malt wort (MW) and aqueous solutions (AS) with pH = 5.2) were presented in Table 2.

Table 2 data indicates that the hop type, hopping procedure (kettle or dry-hopping, boiling time), and all together affects the polyphenolic compounds amount transferred. When comparing the data in Table 2, one can see that the hop variety affects the extraction of polyphenolʹs – use of bitter Magnum provides a lower polyphenols release comparing to the finely aromatic Tetnanger which is consistent with the quantitative polyphenolic compounds composition in the original hop varieties (Callemien and Collin, 2009). However, the data indicates that the amount of the polyphenolic compound in the boiled wort hop-free is higher than hopped with Magnum regardless of the boiling time. The polyphenol content is reduced by 33% in the thermal interaction of malt protein substances with polyphenol substances and by 40 – 60% in the thermal interaction of malt protein substances and Magnum hop polyphenolʹs as well as by 8 – 40% in the case of Tetnanger. One can note that 15 minutes less prolonged boiling with Tetnanger provides the polyphenolʹs dissolution by 78% more than their amount in the boiled wort indicating incomplete protein-polyphenol associates formation due to a short interaction time. In our terms, this may be due to the higher intensity of the association of protein and polyphenolic compounds of malt and hops in the wort (Mikyška et al., 2002). The use of fineflavored Tetnanger hops provides greater polyphenolic substances extraction both in comparison with Magnum in the hopping dynamics and with the amount in the boiled wort. Cold hopping revealed that the use of the fine-flavored hops provides 4% greater polyphenolʹs release comparing to the use of bitter Magnum.

When comparing the intensity of the polyphenol dissolution in wort (MW) and water (AS), the level of polyphenols decreased by 0.5 – 16 times when using Magnum and 2 – 6 times when using Tetnanger relatively to the content in the hopped wort under the same conditions. This indicates that the most complete hop polyphenolʹs dissolution requires not only the appropriate pH of the medium (5.2) but also other compoundsʹ presence allowing their possible release from the hop plant matrix.

As shown in Table 2, anthocyanogens pass into the wort with different intensities; they are labile and their maximum amount is observed when wort hopping with Magnum for 15 minutes and with Tetnanger for 30 minutes. A long boiling time does not provide their presence in the wort and they are more active to the malt wort protein substances responsible for colloidal haze during beer storage. Anthocyanogens amount could be determined in aqueous solutions when using Magnum hops for 15 minutes and anthocyanogens were converted and outside the detection limits in other cases. When Tetnanger hop variety hopping time was 30 minutes the maximum amount of anthocyanogens was observed in aqueous solutions. Dry-hopping conditions did not provide complete anthocyanogens release from hops in wort and aqueous solutions. The polyphenolic substance transition processes were consistent in both research objects as well as during the aqueous solution and wort hopping.

The aroma formation studies were done due to different compounds in hopped wort and aqueous extract. The data is presented in Figure 1 and Figure 2 respectively.

It ought to be remarked that a longer boiling results in deeper organoleptic aromas such as spice, rough, hop, etc. in the case of Magnum bitter hops variety (Figure 3). The herbal flavor is more present in the aroma with minimum heat treatment time while the flower and clove aromas increase when the heat treatment time is longer in the case of Tetnanger aroma hops variety hopping (Figure 4).

It ought to be remarked regarding dry-hopping that noble hop, floral and spicy tones come out first. The wort has a strongly marked hop aroma with a spicy-herbal-floral touch in the case of Tetnanger and resinous-spicy in the case of Magnum.

Researchers note that floral and citrus tones characterize the presence of linalool while floral, citrus and rose-like – geraniol, resinous – myrcene, clove – caryophile which means that they belong to terpene hydrocarbons (Kishimoto, 2008).

Dry-hopping allows enriching the aqueous solution hopped with Magnum by strong hop, resinous, spicy, and mouthpiece shades while with Tetnanger – floral-spicy aromas where the hop is at the sub-tone level. Thus, there is a fundamental difference in the manifestation of different hop varieties aromas specifications: organic compounds of malt wort (starch and non-starch polysaccharides, protein substances, etc. as well as polyphenolic compounds) responsible for secondary subtones. Aqueous solutions obtained after hopping cause more clear secondary specifications for a particular hop variety tone which is explained by a less transfer of bitter hop compounds into aqueous solutions comparing to wort and characterized by the presence of the compounds in the composition such as acids, mineral, and organic compounds, etc. which reduce the external environment polarity coefficient facilitating the transfer of bitter resins into the wort.

Since polyphenolʹs are labile compounds capable of exhibiting not only antioxidant properties but also oxidation and transformation, the pH impact on the organoleptic parameters state and the polyphenol content in aqueous extracts of aromatic hop varieties were researched; furthermore, due to the studies (Table 2), the aromatic hops precisely contains more polyphenolic compounds. The data contains in Table 3 and Figure 5.

Studies done with malt wort polyphenolʹs have indicated that lowering the pH changed the chemical structure of some malt polyphenols imparting them more light absorption at 275 nm and potentially more bitterness (Jurić et al., 2015) unlike hop polyphenolʹs as Table data 4 shows. The medium pH is of great importance for the polyphenolʹs and related substances extraction since the hop’s aroma is formed due to the hops ether compounds – terpenes, terpenoids, oxygenated sesquiterpenes, sulfur compounds, and glycosylated species (Almaguer et al., 2014; Dresel, Dunkel and Hoffman, 2015) – Figure 5.

pH values up to 5.2 cause an increase in the release of polyphenolic compounds. More alkaline extraction conditions cause a conversion of polyphenolʹs and anthocyanogens negatively affecting the extracts aroma. Accordingly, the spatial polyphenols configuration with associated ester compounds should be in a more acidic environment.

What is since polyphenolʹs and anthocyanidins color particularly are pH-dependent (Тsao, 2010) being chemically stable in acidic solutions which are confirmed by experiments results (Table 3). Besides, it is well known that anthocyanidins are unstable and decompose at high temperatures and neutral pH (Dangles and IFenger, 2018). When the medium reacts alkaline at pH 7 or higher, a reduction reaction occurs in the medium. Besides, the composition OH groups responsible for the polyphenolʹs stability are oxidized and the polyphenolʹs lose their stability and due to the detachment of the carbohydrate residue as well.

On the other hand, it is known that flavanols and proanthocyanidins can react to associating with other polyphenolʹs and forming coloration up to brown (Shoji, 2007).

Figure 5 indicates that polyphenolʹs critically affect aroma at a pH close to neutral when the aroma shades specifications of the ester compounds disappear and only the phenolic-pharmaceutical aroma is visualized inherent in the hop polyphenols group of hop polyphenols.

We have researched the possible use of amino acids and monosaccharides in a model solution to stabilize various polyphenolʹs as well as to examine the various compoundsʹ impact on changes in the polyphenol structure and state.

Itʹs clear, that polyphenolʹs are synergistic with several organic substances such as organic acids, amino acids, carbohydrates, etc. (Baxter et al., 1997; Codorniu‐ Hernández et al., 2005; Gauche, da Silva Malagoli and Bordignon Luiz, 2010; Guerra and Yaylyan, 2014; Renard, Watrelot and Le Bourvellec, 2017).

For example, experiments with glycine indicated that (+) – catechin forms various adducts with this amino acid through a dehydration reaction in the ring as well as with the Schiff bases formation at the attachment point to the oxidized b-ring (Guerra and Yaylyan, 2014).

Malt wort contains several amino acids in minor concentrations (Otter and Taylor, 1976), as well as reducing substances represented by low-molecular dextrins, non-staining with iodine, maltose, glucose, fructose (Floridi et al., 2001).

It was of interest to study the polyphenol system stabilization at pH = 7; for this purpose, the amino acids present in malt wort were used – glycine (Gly), leucine (Leu), alanine (Ala), argenine (Arg), aspartic acid (Asp.), and monosugar (glucose (Gl) and fructose (Fr)). The data are presented in Table 4 and Table 5.

According to data in Table 4 and Table 5, glycine, leucine, arginine, aspartic acid and fructose monosaccharide significantly affect polyphenol stability. Alanine and glucose use led to anthocyanogen degradation. Therefore, compounds were selected – amino acids stabilizing polyphenolʹs better than others such as arginine, glycine, and aspartic acid. This fact is explained by the hydrophilic nature and ability to interact with polyphenols easier than other amino acids in the case of arginine and aspartic acid (Higgs et al., 2008). There is a carboxyl group binding to the polyphenolic compoundsʹ active sites on the surface in the main chains of these amino acids.

An interesting effect was observed in the case of adding a monosaccharide, fructose, to the medium (data in Table 5). Considering the amount increase of anthocyanogens (proanthocyanidins, prodelphinidins, etc.) comparing with the control it prevented their oxidation binding to them in other words. This is confirmed by the literature proving the same (Andrade et al., 2017). Researchers attribute interactions with carbohydrates due to van der Waals interactions providing a degree of polymerization, molecular flexibility, number of external hydroxyl groups, or number of terminal galloyl groups (Bordenave, Hamaker and Ferruzzi, 2014).

In our opinion, it is interesting to note that the anthocyanogens presence affects the aroma and color change of the aqueous extract desired while the sugar adding caused no impact on the total polyphenols content.

However, a significant effect, judging by Table 4 and Table 5, we did not observe. Therefore, it was decided to examine hydrophilic amino acids at a concentration equal to the total sum of amino acids contained in malt wort – 150 mg.100mL^-1 (Otter and Taylor, 1976), data are shown in Table 5.

According to Table 5, a distinct medium acidification effect was achieved by the aspartic acid adding since the medium pH became 5.2 which is bound to increased polyphenols and anthocyanogens content. The pH changes effect on the anthocyanogens color change has been examined indicating that the acidic to neutral medium acidity changes caused complete but reversible discoloration of the anthocyanidin molecule due to the colorless isoforms formation (Basílio and Pina, 2016).

The results obtained using aspartic acid exceed the control sample obtained by boiling hops in water with a pH of 5.2, provided by the citric acid adding (Table 5). In this case, the anthocyanogens amount tripled indicating their great resistance to critical conditions of extraction from hops at the temperature of 100 °C. Terpene hydrocarbons and alcohols are the main aromatics in hops. Hydrocarbons are isomerized by the ionic mechanism in case of acidic pH values with the amino acids adding (Bejblová, Žilková and Čejka, 2008). Isomers are reversibly formed, enhancing the characteristic aroma. The isomerization transformations of alcohols (linalool, etc.) are more diverse than the transformations of hydrocarbons, due to the presence of a hydroxyl group (Basílio and Pina, 2016). Unsaturated terpenoids undergo irreversible polymerization when a pH close to neutral and alkaline causing an odor loss (Bejblová, Žilková and Čejka, 2008), thus, when a pH close to neutral, oxidized hop tones are present; pH at 5.2 shows unoxidized hop tones and the anthocyanogens and other polyphenolʹs color are lighter indicating a creep of the structure of the unoxidized compound (Basílio and Pina, 2016). After all the anthocyanins color change is bound to an intramolecular binding of p-coumaric residues to the anthocyanin chromophore (Mori, Kondo and Yoshida, 2009).

Table 5 data indicates the change in the polyphenol accumulation in different aqueous extracts groups under different experimental conditions. According to Table 5 data the isoxanthohumol extraction is consistent with the pH medium: the closer to a neutral and alkaline medium the higher the prenylflavanoid extractability which is consistent with the literature data (Moens et al., 2020). Most of all isoxanthohumol is extracted during boiling, rather than with vigorous stirring. The polar amino acid addition affects the isoxanthohumol extraction intensity since the addition of the amino acids changes the water solution polarity and increases its dissolution (Radzicka and Wolfenden, 1988). When comparing the boiling method with the cold hopping vigorous stirring at pH 7.0, the isoxanthohumol goes least of all into solution with stirring without exposure to high temperatures. This can be caused by its isomerization (Magalhães et al., 2008).

The quercetin extraction did not depend on external conditions (temperature, pH, stirring), and a stabilizer was added remaining at the same level. The rutin extraction was maximum under the acidic pH value conditions provided by organic or amino acid. However, the temperature factor (t = 99 °С) allowed extracting double more rutin comparing with the extraction at t = 25 °С along with stirring during the same time.

We can say that pH values have a more significant effect comparing with other factors (thermal, the presence of sugar, stirring) regarding the recoverable low molecular weight phenolic compounds (LMPS) amount. The LMPS are most intensively extracted in the citric acid 10% solution state at pH 5.2 during boiling. The LMPS amount is somewhat lower at the same pH value achieved by adding aspartic acid.

It is also necessary to pay attention to the variability of the hopped water aroma depending on the amino acid used (Table 5). Which could hypothetically contribute to the aroma and flavor development in hopped wort made from a variety of grains. There is a wide variety of amino acids containing in the wort qualitatively and quantitatively in this case (Zhuang et al., 2017). Thus, one can testify that the beer taste and aroma formation begins at the hopping stage, when amino acids and mono sugars extracted from various grain raw materials in different percentages react with the hop anthocyanogens but not at the fermentation stage as was stated before so far (He et al., 2014).