INTRODUCTIONAlcoholic beverages have a colloidal structure thatdepends on primary plant raw materials or secondaryorganic compounds. Secondary organic compoundsare a product of the microbial activity. They appear asa result of various biochemical or chemical processespresupposed by the particular production technology.The combination of primary and secondary organiccompounds affects the sensory profile of the beverageand, consequently, its demand on the food market.Similarly, beer is an alcoholic drink with a complexcolloidal structure formed by organic biomolecules ofvarious molecular weights, which are interconnectedby hydrogen, covalent, disulfide, and other bonds [1, 2].Nitrogenous compounds, phenols, and carbohydratebiomolecules shape both the sensory profile of beer andits stability as a fermented drink (Fig. 1) [2]. However,flavor profile development is a versatile process. Itdepends both on the primary biomolecules that gethydrolyzed during wort production and on the secondarybiomolecules that appear as a result of biomodificationin the Krebs cycle during fermentation [3].Depending on the size and fraction, some organiccompounds develop both the sensory profile andconsumer characteristics of beer, while others areresponsible for haze.319Gribkova I. N. Foods and Raw Materials. 2022;10(2):318–328Foam stability and settling time are importantconsumer characteristics that are associated withthe quality of beer [4]. Foam quality depends onprotein fractions, bitter hop resins, pentosans, gumsubstances, and other fractions of plant materials thatproduce carbon dioxide bubbles on beer surface [5].Protein biomolecules play the key role in foamdevelopment during brewing. Some proteins possessfoaming properties, while others are responsible forstabilizing [6]. The composition of beer foam is stronglyassociated with lipid carrier proteins (LTP1). Theirmolecular weight is 9.7 kDa, and they include 91 aminoacids. Other foam-related proteins are protein Z (40 kDa)and various derivatives of hordein (10–30 kDa) [7].Beer foam has a complex composition, whichconsists not only of protein fractions but also of ligandcompounds. Ligands are formed by bitter isoformsof α-bitter acids found in hop. The carboxyl group ofthe asparagine residue in the LTP1 protein moleculeis linked by covalent bonds with the hydroxy group ofresin, flavonoids, phytosterols, etc. [8]. Foam stabilityalways correlates with the degree of malt dissolution andsometimes with another protein Z fraction [9].Protein Z is part of the fraction of hordein proteins.Good solubility of malt stimulates the release of thisprotein into the liquid fraction and causes haze [10, 11].Similarly, the intensity of haze depends on the content offractions with a molecular weight of 8–14 kDa in barleymalt and < 7 kDa in wheat malt [12].The last 40 years of beer studies have establisheda partial similarity in the composition of the proteinfractions of the foam and the body of beer. It includesthree groups of protein molecules of 40, 10, and 8 kDa(proteins and peptides), which are similar to barleynitrogenous compounds [13].Non-starch polysaccharides also affect the taste ofbeer [14]. For instance, maltodextrins and β-glucan canenhance flavor profile. The molecular weight of β-glucanin barley is 150–1937 kDa, in malt – 800–1220 kDa, andin beer – 10–10 000 kDa [15]. The content of β-glucan inthe initial barley affects that of malt, and the content ofβ-glucan in malt affects that in wort. The correlation isdifferent for different types of barley. For instance, thecorrelation coefficient was 0.9717 for barley malt and0.9998 for barley wort colloids [15].Phenolics are other important compounds of beer.Catechins, non-condensed phenolic compounds, andmonophenolic acids have a positive effect on the flavorprofile of beer, while proanthocyanidins spoil bothits taste and stability [16]. In fact, proanthocyanidinspossess an extraordinary reactivity and condenseinto large globules, dragging along proteins and otherbiomolecules [2].Thus, the effect of grain organic compounds on thefinished product is diverse and quantitatively unclear.For instance, the issue of the interrelation between grainbiomolecules and other plant materials still remainsunderstudied in the brewing industry. The researchobjective was to establish the correlation betweenthe biomolecules of beer plant raw materials to castlight upon the general structure of beer as a colloidalsystem. The research will make it possible to update themethodology for quality control in the brewing industry.Non-starch polysaccharides (β-glucan)PolyphenolsHop compounds (prenylchalcones,iso- α-acids)Nitrogenous compounds (LTP 1-,Z-proteins, hordein fraction of malt)AromaNon-starch di-, tri-, and mono polysaccharidesStarch carbohydrates (dextrins)Hop compounds (sesquiterpenes, ester oils)Amino acidsPolyphenols (catechins, rutins, quercetins and their homologues,phenols, phenolic acids, and aldehydes)Foam formationPalate fullnessNitrogenous compounds (LTP 1-, Z-proteins, hordein fraction of malt)Non-starch polysaccharides (β-glucan, dextrins of arabinoxylans)Mineral substances in raw materials and waterFigure 1 Colloidal structure of beer320Gribkova I. N. Foods and Raw Materials. 2022;10(2):318–328STUDY OBJECTS AND METHODSBeer samples. Samples of filtered pasteurized beerwere purchased from a retail network in Moscow andstored in the dark at temperature 15 ± 20°C and airhumidity W ≤ 75 ± 2%. The list included light beers(45 samples), dark beers (10 samples), wheat beers(10 samples), and non-alcoholic beers (5 samples), fivebottles or cans per each sort.Fractioning the organic compounds of beer. Topreserve the spatial structure of the protein fractionsof biomolecules, the protein fractioning was carriedout by two methods. High-molecular proteins andrelated organic compounds were precipitated with a 2%tannin aqueous solution. High-molecular and mediummolecularnitrogenous compounds were precipitatedusing a 50% sodium molybdate (Na2MoO4) solutionin an acid medium. The fractions of nitrogenouscompounds, polyphenols, and β-glucans in the filtratewere determined as described below.An aliquot (62 cm3) of decarbonated beer was takeninto two volumetric flasks of 100 cm3. Into the first flask,we added 35 cm3 of distilled water, followed by 2 cm3of concentrated sulfuric acid, which made it possibleto establish the acidic pH of the medium. The solutionwas stirred, mixed with a 2% tannin aqueous solution,and filtered. Into the other flask, we added 30 cm3 ofdistilled water, followed by 5 cm3 o f 5 0% Na2MoO4solution. The mix was brought to the mark with distilledwater, followed by another 5 cm3 of concentratedsulfuric acid. The resulting phosphomolybdic acid in themedium made it possible to precipitate protein nitrogenfrom beer. The initial samples of beer, post-tanninfraction, and post-molybdate fraction were tested forthe mass concentrations of soluble nitrogen, nitrogenouscompounds with unoxidized disulfide bonds, β-glucan,catechins, and polyphenolic compounds.The content of organic compounds in the highmolecular weight fraction was calculated as thedifference between the total amount of a particularcompound and its content in the post-tannin extract. Thelow molecular weight fraction was determined in thepostmolybdate filtrate. The average molecular fractionwas calculated as the difference between the totalamount of the substance and the sum of the high and lowmolecular weight fractions.Determining the nitrogenous compounds.The Kjeldahl method for determining total solublenitrogen was used according to the European BreweryConvention method No. 4.9.3 [17].Determining the total content of polyphenols.The mass concentration of polyphenols was measuredaccording to the European Brewery Convention methodNo. 9.9 [18].Determining the mass concentration of catechins.The content of catechins was determined by highperformanceliquid chromatography (HPLC). Theprocedure involved an Agilent Technologies 1200device (Agilent, USA) with a diode array detector anda Hypersil 5u C18 250×4.6 mm 5 μm column (Thermo,USA) with a wavelength of 280 nm. According tothe procedure, 0.001 cm3 of samples and all standardsolutions were injected into a reverse phase columnat 30°C. The mobile phase for HPLC was preparedas follows. Solution A included 0.1 mL of phosphoricacid dissolved in 900 cm3 of HPLC water. The volumewas brought up to 1000 cm3 with water. The solutionwas filtered through a 0.45-μm membrane filter anddegassed in an ultrasonicator for 3 min. Solution B wasacetonitrile. The mobile phase used gradient elution: at0.01 min – 11% B; 30 min – 25% B; 35–39 min – 100% B;40–50 min – 11% B. The flow rate of the mobilephase was 1.0 cm3/min, and the injection volume was0.001 cm3 [19].Determining the mass concentration ofnitrogenous compounds with disulfide groups. TheEllman method detected nitrogenous compounds thatcontained unoxidized sulfhydryl (thiol) groups [20].The procedure was based on the reaction of thiolwith dithiobisnitrobenzoic acid, which formed amixed disulfide and 2-nitro-5-thiobenzoic acid. Theywere quantified by anion absorption at 412 nm in aspectrophotometer. A number of reagents made itpossible to determine the concentration of thiol groups.The list included 0.1 and 0.2M phosphate bufferand Ellman’s reagent that consisted of 37 mg ofdithiobisnitrobenzoic acid dissolved in 10 cm3 of 0.1Mphosphorus buffer with pH = 7.0 and 15 mg of NaHCO3.The experiment was prepared as follows. First, 3 cm3 ofthe protein solution was poured into a test tube, followedby 2 cm3 of a 0.2M phosphate buffer solution and 5 cm3of distilled water. The aliquot (3 cm3) was poured intoanother tube, followed by 0.02 cm3 of Elman’s reagent.After 3 min, the optical density was measured at 412 nmagainst the control solution. The control solution wasprepared similarly, but 0.02 cm3 of distilled water wasadded to 3 cm3 in another test tube at the last stage.The mass concentration of thiol-containing nitrogenouscompounds (mol/dm3) was calculated by thefollowing formula:Сs-s = D·P/11,400 (1)where D is the optical density at 412 nm; Р is thedillution.Determining the mass concentration of β-glucan.The mass concentration of β-glucan was determined bythe enzymatic European Brewery Convention methodNo. 8.13.1 [21].Statistical analysis. All experiments wereperformed in five repetitions. The obtained valueswere presented as mean ± standard deviation (SD). TheStudent’s t-test was applied to test the homogeneity ofthe samples. The multivariate models in the correlationregressionanalysis were checked using the Fisher test(P ≤ 0.95). The data were processed using Statisticasoftware (StatSoft, Redmond, WA, USA, 2006).321Gribkova I. N. Foods and Raw Materials. 2022;10(2):318–328RESULTS AND DISCUSSIONRelationship between the beer quality andthe quantity of organic compounds in grain. Thefirst stage of the research was aimed at finding thequantitative characteristics of the main organiccompounds that shape the colloidal structure of beer.The list included nitrogenous compounds, polyphenols,and a non-starch carbohydrate β-glucan. Together withdivalent metal ions, hop resins, and melanoidins, thesecompounds are responsible for both haze and beerquality [22]. The dual behavior of biomolecules canbe explained by their grain origin: they originate inmalted or unmalted grain and pass into the liquid phaseduring processing. Table 1 illustrates the quantitativecharacteristics of the main organic compounds.Non-alcoholic and light beer had a similar content ofsolids in the initial wort (Table 1). As a result, they bothwere poor in β-glucan, polyphenols, and soluble nitrogen.Apparently, this fact can be explained by the technologyof removing alcohol from beer by thermal or membranemethods.Thermal de-alcoholization processes include vacuumevaporation, vacuum distillation, and centrifugation.They have a negative effect on the sensory profile ofbeer, which loses in aroma and palate fullness whileacquiring new unwanted aromas [23]. Adsorptionextraction is another de-alcoholization method. Itinvolves adsorbents, e.g., zeolites. Their surface hascharged sites that have an affinity for polar organicsubstances, which means they can adsorb them. Zeolitesoften have an affinity for Ca2+ and Mg2+ ions [24].Molecules of nitrogenous substances, polyphenols, andβ-glucan can be connected to other biomolecules viaCa2+ and Mg2+ bridges [25]. Nanofiltration can decreaseboth the level of alcohol and some polyphenoliccompounds [23].Thus, differences between the de-alcoholizationmethods can reduce the mass concentration of thesecompounds. This fact can explain the decrease in thelevel of non-starch polysaccharides, polyphenols, andsoluble nitrogen in non-alcoholic beer, compared to lightvarieties.In light beer, β-glucan, polyphenols, and solublenitrogen are proportional to the increase in the solids ofthe initial wort (Table 1).In dark beer, the content of β-glucan was 30%,and the content of soluble nitrogen was two timeshigher. This effect might have been caused by coloredmalt, which has higher dissolving properties duringgermination [26]. Colored malt is also responsible forthe lower total amount of polyphenols because theycontain lower amounts of such polyphenols as catechin,prodelphinidin B3, procyanidin B3, and ferulic acid [27].Wheat beer with 12–15% of solids in the initial malthad twice as much β-glucan as light barley beer. Theamount of polyphenols in these samples was higherby 30% and that of soluble nitrogen (lower limit) – by33% (Table 1). In [28], wheat beer also contained agreater amount of non-starch polysaccharides with astructure-dependent difference and a higher degree ofpolymerization, compared to light barley beer. Barleymalt has a β-glucan polymerization of 38–48, whilewheat malt has a polymerization of 38–83 [28]. Inwheat beers with 16÷20% solids, the content of nonstarchpolysaccharide was 1.5 times higher (upperlimit), polyphenols – 1.3–1.6 times higher, protein – by5.0÷32% higher than in the samples of barley-malt beer,which was probably caused by wheat malt [29].Distribution of biomolecules of grain rawmaterials by nitrogenous fractions. The content ofsoluble nitrogen in beer samples was more significant.Thus, the structure of beer was studied dependingon the ratio of different groups of biomolecules withprotein substances. The beer samples were testedfor nitrogen with thiol groups and catechins. Table 2shows the averaged data, while Fig. 2 demonstrates thequantitative distribution of biomolecules by fractions ofnitrogenous compounds.The catechin content confirmed the data obtained byMaia et al. [30]. No correlations between thiol groupsTable 1 Quantitative profile of beer compoundsBeer Solids ininitial wort, %Content* of organic substances, mg/dm3β-glucan Polyphenols Soluble nitrogenFrom To From To From ToNon-alcoholic,barley-malt, light7÷8 69.8 ± 4.9 93.0 ± 6.5 32.8 ± 3.0 65.6 ± 5.9 440.0 ± 6.6 864.0 ± 13.0Light, barley-malt 10÷11 31.0 ± 2.2 93.0 ± 6.5 70.4 ± 6.3 217.0 ± 19.5 560.0 ± 8.4 920.0 ± 13.811÷15 45.0 ± 3.2 125.0 ± 8.8 85.5 ± 7.7 225.0 ± 20.2 580.0 ± 8.7 880.0 ± 13.215÷23 78.0 ± 5.5 180.0 ± 12.6 100.0 ± 9.0 305.0 ± 27.5 850.0 ± 12.8 1350.0 ± 20.3Dark, barley-malt 10÷11 76.5 ± 5.4 125.0 ± 8.8 102.0 ± 9.2 172.0 ± 15.5 1200.0 ± 18.0 1780.0 ± 26.715÷23 120.0 ± 8.4 180.0 ± 12.6 110.0 ± 9.9 180.0 ± 16.2 1200.0 ± 18.0 1800.0 ± 27.0Light, wheat-malt 12÷15 95.0 ± 6.7 240.0 ± 16.8 110.0 ± 10.0 290.0 ± 26.0 770.0 ± 11.6 890.0 ± 13.416÷20 125.0 ± 8.8 280.0 ± 19.6 145.0 ± 13.0 290.0 ± 26.0 1150.0 ± 17.3 1380.0 ± 20.7* Each value is the mean ± standard deviation of five independent experiments322Gribkova I. N. Foods and Raw Materials. 2022;10(2):318–328were detected. However, dark beer had more catechinsbecause the malt had better dissolution and antioxidantactivities. As a result, catechins did not oxidize until thefinal stage of beer production [30].Table 2 shows a high level of nitrogen with thiolgroups in dark and light barley-malt beers with a lot ofinitial wort solids. This fact was probably associatedwith the antioxidant capacity of these samples, whichretained thiol groups in unoxidized form.Light wheat beers contained a relatively low amountof nitrogen with thiol groups (8.80–11.4 μm) comparedto barley-malt light beers (12.7–16.4 μm), as confirmedby other studies [31].The fraction distribution of organic compounds(Fig. 2a–h) depended on the type of beer.The high-molecular fraction of soluble nitrogenranged from 7 to 15% of the total amount. Its minimalamount was in dense light barley-malt beers, wherethe solids content in the initial wort was 15÷23%. Themaximal amount was in light barley-malt beer with thesolids content of 11÷15%.The average molecular fraction correlated withthe density. The biggest amount of soluble nitrogen(8÷40 kDa) was registered in the beer samples withinitial wort solids content ≥ 23%: it was 20–34% of thetotal amount of protein compounds. The low molecularfraction of soluble nitrogen was inversely related to thedensity of beer. For all samples, the higher the contentof dry matter in the initial wort, the lower the content ofprotein compounds with a molecular weight of ≤ 8 kDa.The distribution of thiol groups of nitrogenoussubstances was as follows. In light barley-malt beers, themaximal amount was in the medium molecular weightfraction (8÷40 kDa). In dark barley-malt beers, it wasin the low molecular weight fraction (≤ 8 kDa). In lightwheat-malt beer, it was in the high molecular weightfraction (40÷100 kDa).The β-glucan dextrins differed in distribution. Inlight barley-malt beer, 58–68% of the total content ofnon-starch polysaccharide fractions accounted for theprotein fraction with a molecular weight of 8÷40 kDa.In dark barley-malt beer, 59–63% of β-glucan moleculeswere concentrated in the fraction of nitrogenoussubstances of ≤ 8 kDa, and 73–79% of its totalcontent was distributed in nitrogenous substances of40÷100 kDa.Catechins did not depend on the type andcomposition of beer: 45–74% of the total contentaccumulated in the high molecular weight fractionof soluble nitrogen. However, the total content ofpolyphenols showed strong correlation with the type ofbeer.Table 3 shows the correlation between the totalpolyphenol content and the catechin content.Table 3 revealed a strong correlation between thetotal polyphenols and catechins and the type of beer.According to the determination coefficient, the totalpolyphenols depended on the content of catechins whenthe latter was 50 99%. Therefore, some unknown factorsaffected the total polyphenols in different beer samples.The lowest determination coefficient was registeredin light barley-malt beers 15÷23%, dark beers 15÷23%,and wheat-malt beers 16÷20%. When the solids in theinitial wort was high, the composition of polyphenoliccompounds experienced a stronger impact fromanthocyanogens, phenolic acids, aldehydes, hop resins,and prenylflavanoids. Apparently, strong beer requires agreater proportion of hops, which, like grain, is a sourceof polyphenolic compounds [32]. On the other hand,the stability of phenolic compounds depends on manyfactors, e.g., temperature, pH, coactivating substances,polar solvents, etc., which makes the amount of alcohol amore significant factor for strong beer sorts [33].Table 4 illustrates the dependence of the distributionof thiol groups and catechins.Table 4 shows that the change in β-glucan was 50%,while the content of thiol groups and catechins changedby 80%, which depended on the parameters of the plantmaterial, i.e., barley or wheat malt. On the one hand, thisfact can be traced back to grain varieties. On the otherhand, non-starch polysaccharides can develop colloidalsuspensions and links with other beer compounds,which leads to product losses and affects the content ofβ-glucan [15, 34].Table 2 Thiol nitrogen-containing compounds and catechins in beer samplesBeer type Solids in initial wort, % Content in beerProtein with thiol groups, μmoL/dm3 Catechins, mg/dm3Non-alcoholic barley malt 7÷8 5.61 ± 0.56 2.25 ± 0.23Light, barley malt 10÷11 12.7 ± 1.26 6.33 ± 0.6511÷15 16.4 ± 1.55 8.14 ± 0.8015÷23 36.7 ± 3.60 14.4 ± 1.40Dark barley malt 10÷11 28.0 ± 2.80 14.9 ± 1.5015÷23 35.5 ± 3.50 18.0 ± 1.80Light, wheat malt 12÷15 11.4 ± 1.00 1.98 ± 0.2016÷20 8.80 ± 0.90 6.90 ± 0.70Each value is the mean ± standard deviation of five independent experiments323Gribkova I. N. Foods and Raw Materials. 2022;10(2):318–328Figure 2 Distribution of compounds by fractions of soluble nitrogen: (a) non-alcoholic barley-malt beer; (b) light barley-malt beerwith 11÷12% initial wort solids; (c) light barley-malt beer with 12÷15% initial wort solids; (d) light barley-malt beer with 15÷ 23%initial wort solids; (e) dark barley-malt beer with 10÷11% initial wort solids; (f) dark barley-malt beer with 15÷23% initial wortsolids; (g) light wheat-malt beer with 12÷15% initial wort solids; (h) light wheat-malt beer with 16 ÷ 20% initial wort solidsFigures 3 and 4 illustrate the analysis of correlationsand regressions, which registered the presence anddegree of the relationship between the content of solublenitrogen and other parameters. The analysis establisheda close and logical relationship between the amount ofraw materials (solids in the initial wort) and the contentof alcohol and polyphenols, which was confirmed byprevious studies [32, 33]. Fermentation and the content0 20 40 60 80 100soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 80 100soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa 80 100Content, %40÷100 kDa 8÷40 kDa ˂ 8 kDaa b0 20 40 60 80 100soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 80 100soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa20 100 120soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 80 100 120soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 60 80 100 120soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 80 100 120soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 100 120soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 80 100 120soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 80 100 120soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 80 100 120soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 80 100soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 80 100soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDac d0 20 40 80 100 120soluble nitrogenthiol groupsβ-glucanpolyphenolscatechins%40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 80 100 120soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 80 100 120soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 80 100 120soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 80 100 120soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 80 100 120soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 80 100 120soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 80 100 120soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 80 100soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 80 100soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDae f0 20 40 60 80 100soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 80 100soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 80 100soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 80 100soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 80 100soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDa0 20 40 60 80 100soluble nitrogenthiol groupsβ-glucanpolyphenolscatechinsContent, %40÷100 kDa 8÷40 kDa ˂ 8 kDag h324Gribkova I. N. Foods and Raw Materials. 2022;10(2):318–328Table 3 Analysis of correlation and regression between components and beer parametersBeer type Solids in initialwort, %Correlationcoefficient (r)Equation of dependance Correlation according toChaddock scaleDeterminationcoefficientNon-alcoholicbarley malt7÷8 0.744 y = 48.2 – 2.56 x Direct, high 0.553Light, barley malt 10÷11 0.713 y = 77.4 + 6.22 x Direct, high 0.50811÷15 0.975 y = 54.9 + 12.2 x Direct, high 0.95215÷23 0.517 y = 81.9 + 15.3 x Direct, moderate 0.267Dark barley malt 10÷11 0.999 y = –133 + 19.4 x Functional 0.99915÷23 0.556 y = 294.7 – 8.9 x Direct, moderate 0.310Light, wheat malt 12÷15 0.959 y = 19.8 + 38.7 x Direct, high 0.91916÷20 0.557 y = 225.5 – 2.1 x Direct, moderate 0.310Significance level ≤ 0.05x – type of beer; y – total polyphenols and catechinsTable 4 Analysis of correlation and regression between beer parameters and raw materialComponentCorrelationcoefficient (r)Equation of dependance Correlation according toChaddock scaleDetermination coefficientThiol-containing Proteins 0.920 y = –9.8 + 1.9 x Direct, high 0.846Catechins 0.896 y = –4.0 + 0.8 x Direct, high 0.803β-glucan 0.708 y = 19.5 + 9.8 x Direct, high 0.501*Significance level ≤ 0.05x – solids in initial wort; y – component amounof polyphenols in the finished product also provedclosely interconnected. This fact has been described indifferent publications [32].The content of soluble nitrogen proved to dependon the color (type) of beer. This result was quitepredictable since a greater degree of dissolution ofcolored malt means a greater effect of low molecularweight nitrogenous compounds on colored compounds.Similar conclusions were obtained by Castro et al. andFilipowska et al. [26, 35]. Partial correlation coefficients(Fig. 4) were based on the changes in the pair correlationof the corresponding features (Y and Xi), provided theyexperienced no effect from other factors (Xj). Thisaspect demonstrated much deeper dependencies ofthe analyzed indicators. The change in the content ofsoluble nitrogen was confirmed by the conclusion aboutthe correlation with the color (type) of beer, as well ascorrelation coefficients YX3, YX5, and X5. The experimentconfirmed the hypothesis about the relationship ofnitrogenous fractions of nitrogenous substances withpolyphenolic and non-starch compounds. X2, X4,and Y also appeared to correlate, which means thatpolyphenolic compounds affected soluble nitrogenfraction. Polyphenols transformed when the parametersof young beer changed during fermentation while pHbecame more acidic, oxygen dissolved, carbon dioxideaccumulated, etc.The calculations represented in Figs. 3 and 4 resultedin the following multiple regression equation (2):Y = 117.2991 – 33.1413 · X1 + 15.1575 · X2 ++ 34.8177 · X3 + 2.6063 · X4 + 7.7755 · X5 (2)Color or type of beer (X3) was the most significantparameter in the regression equation. This resultconfirmed our previous conclusion that the fractiondistribution of biomolecules depended on the typeof beer (Fig. 2). The overall coefficient of multiplecorrelation R equaled 0.9073, while the multipledetermination coefficient R2 equaled 0.82. Thedifference indicates that the change in the contentof soluble nitrogen depended the abovementionedparameters by 82%.The study of the protein fractionation could beused to determine the accompanying groups of organicmolecules. The acidic extraction regime of biomoleculeswas quite sparing. Different conditions, e.g., alkaline pH,organic polar solvents, etc., disrupt the equilibriumof nitrogenous substances, polyphenols, and othercompounds. As they oxidize, their amount in equilibriumsystems cannot be determined [36, 37].The behavior of organic compounds in the colloidalsystem of beer revealed a strong correlation between thetechnological conditions and the low amount of β-glucan,polyphenols, and soluble nitrogen. In particular,thermal or adsorption de-alcoholization had a greatimpact on the abovementioned substances, whichis consistent with data obtained Muller et al. andYassue-Cordeiro et al. [23, 24].325Gribkova I. N. Foods and Raw Materials. 2022;10(2):318–328Figure 3 Correlation coefficients of beer parametersFigure 4 Pair correlation coefficients of beer parametersThe distribution of biomolecules by types of beeralso revealed an obvious connection between thetype of beer and the biochemical composition of theraw materials (barley or wheat malt), productiontechnology, and the amount of mashed grain(Table 1). These results are consistent with otherpublications [26–30].The quantitative assessment of organic compoundsand their biochemical properties resulted in thehypothesis about the structural character of nitrogenoussubstances in the colloidal system of beer. Thisexperiment also made it possible to trace the changesin polyphenols, carbohydrates, and other compoundsrelative to the fraction distribution of nitrogenouscompounds [38].The results of nitrogenous fractionation (Fig. 2)showed its obvious correlation with the beer type. Thehigh molecular weight fraction of soluble nitrogen(40÷100 kDa) varied in the range of 7÷15%, dependingon the solids in the initial wort. The higher was thesolids content, the lower was the amount of the highmolecular weight fraction of nitrogenous compounds.High-molecular fractions of nitrogenous substancesare associated with the palate fullness, which is mostSoluble nitrogencontent, mg/L(Y)Raw materialscontent, %(X1)Alcoholcontent, %(X2)Beer type(color, EBC)(X3)Polyphenolscontent, mg/L(X4)β-glucancontent, mg/L(X5)Y 1 0.33** 0.35 0.74 0.49 0.57X1 – 1 0.956*** 0.02* 0.79 0.42X2 – – 1 0.13 0.864 0.30X3 – – – 1 0.42 0.10X4 – – – – 1 0.12X5 – – – – – 1* – weak bond strength; ** – moderate connection; *** – strong bondYX1 YX2 YX3 YX4 YX5Y – – – – –X1 – 0.156* 0.777*** 0.395 0.501X2 –0.0502 – 0.748 0.381 0.518X3 0.462 0.383 – 0.287 0.740X4 –0.112 –0.148 0.677** – 0.587X5 0.117 0.239 0.836 0.512 –X1X2 X1X3 X1X4 X1X5 X2X3Y 0.952 –0.348 0.769 0.302 –0.204X1 – – – – 0.395X2 – –0.376 –0.229 0.500 –X3 0.963 – 0.865 0.420 –X4 0.885 –0.566 – 0.540 –0.500X5 0.961 –0.0229 0.825 – 0.111X2X4 X2X5 X3X4 X3X5 X4X5Y 0.847 0.121 0.102 –0.583 –0.218X1 0.594 –0.410 0.663 0.098 –0.390X2 – – 0.609 0.0612 –0.282X3 0.898 0.285 – – 0.085X4 – 0.384 – 0.053 –X5 0.874 – 0.413 – –* – weak bond strength; ** – moderate connection; *** – strong bondSoluble nitrogencontent, mg/L(Y)Raw materialscontent, %(X1)Alcoholcontent, %(X2)Beer type(color, EBC)(X3)Polyphenolscontent, mg/L(X4)β-glucancontent, mg/L(X5)Y 1 0.33** 0.35 0.74 0.49 0.57X1 – 1 0.956*** 0.02* 0.79 0.42X2 – – 1 0.13 0.864 0.30X3 – – – 1 0.42 0.10X4 – – – – 1 0.12X5 – – – – – 1* – weak bond strength; ** – moderate connection; *** – strong bondSoluble nitrogencontent, mg/L(Y)Raw materialscontent, %(X1)Alcoholcontent, %(X2)Beer type(color, EBC)(X3)Polyphenolscontent, mg/L(X4)β-glucancontent, mg/L(X5)Y 1 0.33** 0.35 0.74 0.49 0.57X1 – 1 0.956*** 0.02* 0.79 0.42X2 – – 1 0.13 0.864 0.30X3 – – – 1 0.42 0.10X4 – – – – 1 0.12X5 – – – – – 1* – weak bond strength; ** – moderate connection; *** – strong bond326Gribkova I. N. Foods and Raw Materials. 2022;10(2):318–328typical for light beers with low density [14, 39]. In thesamples where the content of extractive substancesof the initial wort was 15÷23%, the palate fullnessdepended not only on the raw materials but also on thesecondary products of yeast metabolism, i.e., secondaryalcohols, aldehydes, ketones, ethers, and other carbonylcompounds. Our results were quite similar. The mediummolecular fraction (8÷40kDa), which is responsible forfoam structure, correlated with the density of beer or theproportion of grain products in it, which is consistentwith some previously obtained data [40]. In all samples,the low molecular weight fraction of soluble nitrogen(≤ 8 kDa) developed inversely to the density of beer,which is consistent with other studies on sensoryperception of beer body [14, 39]. In other words, thelow molecular weight fraction of protein compoundsdepended on the yeast metabolism, i.e., the enzymesystems of the strain.Thiol groups of nitrogenous substances areresponsible for foam and palate fullness. Theirdistribution proved to depend on the grain raw material –barley or wheat malt. Thus, light barley-malt beercontained the maximum of thiol groups in the mediummolecular weight fraction, dark barley-malt beer – inthe low molecular weight fraction, and wheat-malt beer –in the high molecular weight fraction. This findingindicates a great effect of the type of grain on beerquality.The fraction distribution of non-starch β-glucandepended on the type of malt. In light beers, thisnon-starch polyaccharide was mostly represented inhigh- and medium-molecular fractions of nitrogenoussubstances (Fig. 2). In dark beers, up to 63% of β-glucanmolecules concentrated in low molecular weightfractions of nitrogenous compounds, which meansthey linked to peptides through hydrogen bonds [12].Probably, this fact can be explained by the competitivedistribution of catechins and their bonding withnitrogenous biomolecules in high and medium molecularweight fractions of dark beer (Fig. 2).The correlation analysis revealed a close and logicalrelationship between catechins and total polyphenols(Table 3) in different types of beer. The amount ofpolyphenols depended on the density of the initial wort,as well as on the increase in the alcohol content, whichstabilized polyphenolic compounds [33].The analysis of correlation and regression(Figs. 3 and 4) showed the strong impact of the rawmaterial factor (light, dark barley, and wheat malt) onthe content of alcohol and polyphenols. This finding wasconsistent with the previously obtained research results(Tables 1 and 2) [32, 33].The statistical analysis revealed a correlationbetween the color (type) of beer and the amountof nitrogenous compounds in terms of colloidalstructure (Fig. 3). This correlation is associated withthe technology of coloring malts and the degree ofdissolution of malt endosperm during the hydrolysis thatoccurs during barley germination [33].Therefore, the experimental part of the researchconfirmed the hypothesis that fractionation of nitrogenouscompounds can be conducted by the methodspecified in Study Objects and Methods. Fractionsof soluble nitrogen and polyphenolic compoundsdemonstrated a close correlation under various beerproduction technologies. This relation can be illustratedby a multiple correlation equation (2), in which the color(type) of beer is the most significant parameter.CONCLUSIONThe present research featured the fractionation oforganic compounds in various beers. It established thedependences and factors affecting the distribution ofnitrogenous compounds in the colloidal system of beer,as well as the relationship between polyphenolic andnon-starch biomolecules. The study also revealed therelationship between the fractional composition of beerand such parameters as contents of solids in the initialwort, raw materials, alcohol, color, etc.CONTRIBUTIONI.N. Gribkova designed the research, collected,analyzed, and interpreted the data. M.N. Eliseevdesigned the article, developed the concept, andinterpreted the data. M.A. Zakharov and V.A. Zakharovacollected and analyzed the data. O.A. Kosareva editedand proofread the manuscript.CONFLICT OF INTERESTSThe authors declare that there is no conflict ofinterests regarding the publication of this article.