Precedent published studies dealt with composition of water and its effect on brewing. The aim of this study was to find out, whether composition of brewing water significantly influences selected analytes during beer brewing and in the beer in conditions of homebrewing.

Synthetic water was prepared according to Smith, Davison, and Hamilton-Taylor (2002) by adding selected compounds to distilled water. Thus, soft and hard model water was created.

For study of the influence of brewing water on selected analytes, beer was made from soft and hard synthetic model water in three replicates. These were the pale bottom-fermented beers also called lagers. The raw materials were pale Pilsner barley malt (Malt house Bernard, Rajhrad), Sládek hop pellets with alpha acid content of 8.08% and Saflager S-23 yeasts.

Milled barley malt was mixed with brewing water at 38 °C and then an infusion method was chosen for mashing process. Evaporated water was compensated with distilled water. The values of original gravity (OG) and pH were measured in wort after lautering. Then it was continued with wort boiling for 90 min. After this process, whirlpooling of wort was done. Also the values of OG and pH were measured. After that wort was cooled down and then it was inoculated and aerated and stored at 14 °C. The degree of attenuation was checked by measuring the OG value to confirm that yeasts completed fermentation. The OG value was in range of 7.0 – 7.3%. Values of OG and pH were measured in the samples of young beer. Then bottled beer was stored at 7 °C. The secondary fermentation and maturing last for three weeks.

The OG values were measured refractometrically (A. Krüss Optronic GmbH, Hamburg, Germany). The pH of synthetic water was measured with pH meter (WTW, Germany).

The samples of wort, hopped wort, young beer and beer were degassed by sonication (Ultrasonic Compact cleaner PS03000A 2.5 L, PowerSonic s r.o.) and then diluted with distilled water. All samples were analysed by HPLC-DAD (Agilent 1260 Infinity, Agilent Technologies, USA) to determine the quantity of B vitamins. Specifically, it was focused on riboflavin, niacin, pyridoxine and cobalamin. B vitamins were separated on Polar C18 column (150 mm x 3.0 mm; particle size 2.6 μm) with set temperature to 40 °C. The mobile phase consisted of 10 mM ammonium formate (Sigma-Aldrich Inc., USA) and 0.1% solution of formic acid (solvent A) (Sigma-Aldrich Inc., USA) and acetonitrile (Sigma-Aldrich Inc., Germany) and 0.1% solution of formic acid (solvent B) (Sigma-Aldrich Inc., USA). Gradient elution was used for the analysis. The gradient was as follows: 0 min, 100% A; 12 min, 40% A; 15.10 min, 100% A and then held for another 7 min. The samples were detected with DAD detector (260 nm, 270 nm, 271 nm, 292 nm, 360 nm). The data were collected by the Agilent 1260 Infinity chromatographic data system.

Ion chromatography (850 Professional IC, Metrohm, Switzerland) was used to determine lactic and acetic acid in the samples of wort, hopped wort, young beer and beer. Each sample was degassed by sonication (Ultrasonic Compact cleaner PS03000A 2.5 L, PowerSonic s r.o.) for 20 minutes. Then the samples were filtrated using filters with 0.45 μm pore size (Labicom, Czech Republic) and diluted with distilled water in proportion 1:1.

Organic acids were separated on Metrosep Organic Acids column (250 mm x 7.8 mm; particle size 9 μm) with temperature set to 30 °C. The mobile phase was 0.5 mmol perchloric acid (Sigma-Aldrich Inc., USA). Isocratic elution was used and the flow of mobile phase was 0.6 mL.min^-1. The analytes were detected with conductivity detector.

Data analysis and statistical evaluation was carried out by software XLSTAT (Addinsoft, USA). Influence of the brewing water on the selected parameters of beer was evaluated by Analysis of Variance (ANOVA). ANOVA was set to a confidence interval of 95%.

First, the pH of prepared synthetic model water was measured. The average pH value of soft water was 7.68 ±0.23, the average pH value of hard water was 8.47 ±0.08. Compared to theoretical pH, the measured values were lower. The theoretical model of soft water pH was 7.83 and 8.49 hard water (Smith, Davison and Hamilton-Taylor, 2002).

Figure 1 shows difference between pH of soft and hard water during mashing. During mashing, the pH decreases due to malt enzymes activity, which releases phosphates from nucleic acids. In the process of wort boiling, the wort acidity is increased by precipitation of phosphates in the presence of calcium and magnesium ions. Hop bitter acids and Maillard reaction products further contribute to pH reduction. When fermenting, the pH declines by the activity of yeasts that consume amino acids and produce organic acids. The pH also changes because of presence of the carbon dioxide, which dissolves in the solution (Basařová et al., 2010).

OG (original wort extract) and ABV (Alcohol by Volume) were selected as the basic quantitative indicators for beer brewing. The extract of the original wort was used to express the total carbohydrate content presented in the medium. Differences in beers brewed from different kind of synthetic water were observed. Both, original gravity and concentration of alcohol were statistically different. The mean value of wort OG prepared by using soft water was 12.6 ±0.1%, while wort OG of hard water was 13.05 ±0.05% (see Figure 2). Higher yields in hard water are probably due to higher amounts of Mg²⁺ and Ca²⁺ ions, which stabilize α-amylases and increase its activity (Karbassi and Saboury, 2000; Saboury, Ghasemi and Umar Dahot, 2005). After wort boiling, the OG slightly increased, probably due to the extraction of chemical compounds in hop. A large decline occurred during fermentation when yeasts utilized carbohydrates. A small drop occurred during secondary fermentation and maturing in bottles because yeasts largely depleted the substrate and was no longer as vital as at the beginning. The process of fermentation of all samples is shown in Figure 3. As the yeast assimilated the substrate, the OG also declined and the ethanol concentration in the medium grew as expected. Complete fermentation of experimental beers took 70 hours.

Determination of B2 (riboflavin), B3 (niacin), B6 (pyridoxine), B12 (cyanocobalamin) vitamins were done to assess the influence of the two different kind of experimental brewing waters on content of B vitamins. The concentration of the last two mentioned vitamins was below the detection limit in all samples. The results of the analysis of the determined B vitamins are shown in Table 1 and Table 2.

Statistically significant differences between hard and soft water were found in wort (p = 0.0007, F = 92.214). Hard water seemed to be a better extraction agent due to different pH and ionic strength. Content of B3 vitamin changed during the brewing. In the presence of yeast, the level of B3 vitamin declined rapidly as yeast used vitamin in the wort in biochemical processes and nicotinic acid synthesis did not occur (Basařová et al., 2010). Relatively small, but significant difference in concentration of B3 vitamin was found in the final product (p = 0.0448, F = 8.3243). A slightly higher average concentration of vitamin B3 was determined in beer from hard water. This phenomenon is probably caused by releasing cell content during yeast autolysis.

Usual content of niacin in beer is around 5 mg.L^-1 (Basařová et al., 2010). The measured values are higher in comparison with the literature, approximately 8 – 12 mg.L^-1. High vitamin content can be caused by no further treatment after beer bottling, unlike commercial beers, which are filtered and often pasteurized.

Very similar trend was observed for B2 vitamin. Statistically significant differences were found in wort (p = 0.0385, F = 9.2210) and young beer (p = 0.0008, F = 83.2461) (shown in Table 2). Riboflavin was better extracted from barley malt during mashing in hard brewing water than in soft. This could be due to different pH of the water. Riboflavin is a thermostable vitamin, there were observed no loss of the vitamin in the process of wort boiling. During fermentation there was a certain decrease in vitamin content because riboflavin participates in many biochemical processes (Hucker, Wakeling, Vriesekoop, 2016). The greatest difference was observed in soft water. In hard water, to thereby prevent high losses probably because riboflavin form stable complexes with Zn²⁺, Ni²⁺, Co²⁺, Cu²⁺, Ca²⁺, Mg²⁺ (Sheraz et al., 2014). In beer, the vitamin concentration was probably increased by the release of yeast autolysis (Hucker, Wakeling, Vriesekoop, 2011).

Usual content of riboflavin in beer is approximately 0.25 mg.L^-1 (Olšovská, Jurková and Čejka, 2012). When comparing the values and literature the B2 vitamin concentration is several times higher – 0.6 to 1.2 mg.L^-1. Again, it is probably the reason for omitting post fermentation adjustments.

Determination of organic acids was done to assess the influence of soft and hard experimental brewing waters. Retention time of lactic acid was 11.17 min, acetic acid 14.22 min. The results are shown in Table 3 and Table 4.

The results have shown that there are no statistically significant differences in content of acetic acid in beer made from soft and hard brewing water (Table 3).

The content of acetic acid increases rapidly during fermentation (South, 1996). The greatest changes between samples made from soft and hard brewing water were found in young beer, though Analysis of Variance did not prove statistically significant differences. This difference was probably due to a divergent ratio of minerals in hard and soft brewing water, which can either positively or negatively affects the level of organic acid synthesis.

Concentration of acetic acid in beer brewed from soft and hard brewing water varied from 106 to 144 mg.L^-1, whereas Vontrobová et al. (2017); Coote and Kirsop (1974); Walker (1998); Zhang, Jia and Zhang, (2012) indicate values ranging from 30 to 200 mg.L^-1.

Lactic acid consists of two isomers. D-lactate, which comes from malt and the content may vary during mashing, and L-lactate, which indicates bacterial activity. The D and L isomers of this acid have been reported in wort and beer on a number of occasions (Coote and Kirsop, 1974). Lactate was excreted throughout the period of sugar utilization (Coote and Kirsop, 1974; Whiting, 1976).

The results of determination of lactic acid are shown in Table 4. The difference in composition of brewing water had no influence on content of lactic acid. During wort boiling, it was observed no decline of acid, concentration slightly increased in hard brewing water. It could be possibly related with extraction of organic acids from hops. High production of acid occurred during fermentation. Higher amounts of lactic acid were obtained in young beer made from hard brewing water (p = 0.098 8, F = 4.589 4). On the contrary, higher concentration was determined in beer prepared from soft water (p = 0.102 0, F = 4.469 0), while the concentration in beer made from hard water remained the same.

Experimental beers content high amounts of lactic acid, the concentration was ranging between 289 – 305 mg.L^-1. (Coote and Kirsop, 1974; Whiting, 1976) present big range of lactic acid concentration in beer. Specifically, this span varies from 37 to 233 mg.L^-1 in lagers, ales 44 – 276 mg.L^-1 and dark strong beers 276 – 292 mg.L^-1.

Lactic acid bacteria (LAB) may be the reason of high concentrations of lactic acid. They may proliferate in wort, hopped wort and even during fermentation. They survive in pH 4 – 7. Optimal growth temperature is 20 – 30 °C, but psychrophilic strains occur, which may grow in conditions of low temperatures during fermentation and maturing of beer (Basařová et al., 2010).

The results of iso-α-acids analysis are in Table 5, Table 6 and in Table 7. The retention time of isocohumulone was 7.481 min, isohumulone 9.610 min and isoadhumulone 10.256 min.

Impact of brewing water composition was studied on the most represented analogues of iso-α-acids – isohumulone, isocohumulone and isoadhumulone. Analysis of variance did not prove statistically significant differences neither in soft nor hard brewing water. Isomers of α-acids are unstable and during beer production its concentration declined rapidly. Of the total amount of bitter compounds 30% at most will end in beer, whereas approximately 20% remains in the spent hops, 20 – 30% is captured in waste and 20 – 30% is captured in the head of foam on the top of the fermentation. Isomerization is also affected by time of wort boiling and OG value (Basařová et al., 2010).