biomolecules

Article Chemical Composition of Sour Resulting from Supplementation the Fermentation Medium with Magnesium and Zinc Ions

Aneta Ciosek * , Katarzyna Fulara, Olga Hrabia , Paweł Satora and Aleksander Poreda

Department of Fermentation Technology and Microbiology, University of Agriculture in Krakow, 30-149 Krakow, Poland; [email protected] (K.F.); [email protected] (O.H.); [email protected] (P.S.); [email protected] (A.P.) * Correspondence: [email protected]; Tel.: +48-792-868-084

 Received: 2 October 2020; Accepted: 20 November 2020; Published: 25 November 2020 

Abstract: The bioavailability of minerals, such as zinc and magnesium, has a significant impact on the fermentation process. These metal ions are known to influence the growth and metabolic activity of , but there are few reports on their effects on bacteria (LAB) metabolism during sour . This study aimed to evaluate the influence of magnesium and zinc ions on the metabolism of Lactobacillus brevis WLP672 during the fermentation of brewers’ . We carried out lactic acid fermentations using wort with different mineral compositions: without supplementation; supplemented with magnesium at 60 mg/L and 120 mg/L; and supplemented with zinc at 0.4 mg/L and 2 mg/L. The concentration of organic acids, pH of the wort and carbohydrate use was determined during fermentation, while aroma compounds, real extract and ethanol were measured after the mixed fermentation. The addition of magnesium ions resulted in the pH of the fermenting wort decreasing more quickly, an increase in the level of L-lactic acid (after 48 h of fermentation) and increased concentrations of some volatile compounds. While zinc supplementation had a negative impact on the L. brevis strain, resulting in a decrease in the L-lactic acid content and a higher pH in the beer. We conclude that zinc supplementation is not recommended in sour beer production using L. brevis WLP672.

Keywords: sour brewing; Lactobacillus brevis; aroma compounds; carbohydrate use; magnesium; zinc; wort enrichment

1. Introduction Sour beer is characterised by a low ethanol content and a refreshing taste and is becoming more popular worldwide. In the production of sour beer, the main metabolite released by lactic acid bacteria (LAB) is lactic acid (LA), which creates the specific, crispy bouquet of the beer. The LA decreases the pH of the beer, which is the main factor inhibiting the growth of LAB cultures and LA production [1]. Peyer et al. [2] revealed that LAB metabolism was reduced below pH 4.9 and their growth was completely inhibited at pH below 3.4. Therefore, delaying or reducing the rate of pH drop is beneficial for the growth of LAB. This can be done by adding alkalising agents (e.g., calcium carbonate or potassium hydroxide) that react with LA to form lactate salts. This method can increase the production of LA up to eight-fold, but the produced LA must be recovered through separation [3]. Alternatively, the buffering capacity of the wort can be increased by, for example, changing the mash profile, prolonging the protein digestion or adding a citrate-based buffer [2]. In this way, it is possible to increase the production of LA by 30–40% compared to a standard wort while maintaining a relatively high pH (4.3) in the fermentation medium.

Biomolecules 2020, 10, 1599; doi:10.3390/biom10121599 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, 1599 2 of 14

Biomolecules 2020, 10, x 2 of 14 The nutritional requirements of LAB are complex and varied, even between strains within the same genera.The nutritional LAB require requirements sufficient of carbohydrates, LAB are comple proteins,x and varied, amino even acids, between fatty strains acids, within vitamins the and mineralssame forgenera. proper LAB growth require [ 4sufficient]. carbohydrates, proteins, amino acids, fatty acids, vitamins and minerals for proper growth [4]. The metabolic activity and fermentation efficiency of LAB depend on the availability of fructose [5], The metabolic activity and fermentation efficiency of LAB depend on the availability of fructose some amino acids (e.g., lysine, arginine, and glutamic acid) [5,6] and microelements (e.g., manganese, [5], some amino acids (e.g., lysine, arginine, and glutamic acid) [5,6] and microelements (e.g., magnesium,manganese, potassium) magnesium, [7,8 potassium)]. Scheme1 [7,8]. shows Scheme the potential 1 shows the metabolic potential pathways metabolic for pathways the generation for the of aromageneration compounds of aroma from compounds wort precursors from wort during precursors lactic acidduring fermentation. .

SchemeScheme 1. Schematic1. Schematic representation representation ofof mainmain aroma compounds compounds and and organic organic acids acids that that can canbe be generatedgenerated lactic lactic acid fermentationacid fermentation of of wort malt from wort existing from precursors.existing precursors. AAT: alcohol AAT: acyltransferase; alcohol AT:acyltransferase; aminotransferases; AT: aminotransferases; BOX: ß-oxidation; BOX: DC: ß-ox decarboxylase;idation; DC: decarboxylase; DH: dehydrogenase; DH: dehydrogenase; ES: esterase; FAD:ES: fatty esterase; acid FAD: desaturase; fatty acid H:desaturase; hydrogenase; H: hydrogenase; HPL: hydroperoxide HPL: hydroperoxide lyase; lyase; KADC: KADC: ß-ketoacyl ß- ketoacyl decarboxylase; LP: lipase; OX: oxidation; R: reductase. Based on Nsogning Dongmo et al. [9]. decarboxylase; LP: lipase; OX: oxidation; R: reductase. Based on Nsogning Dongmo et al. [9].

MetalMetal ions ions play play important important roles roles in in many many biologicalbiological processes in in living living organisms. organisms. They They are are crucial for to grow and perform alcoholic fermentation. For most strains of LAB, the most crucial for yeasts to grow and perform alcoholic fermentation. For most strains of LAB, the most important ions are manganese, magnesium, potassium and sodium [8,10]. Manganese and important ions are manganese, magnesium, potassium and sodium [8,10]. Manganese and magnesium magnesium are crucial for the growth and metabolism of LAB; the former is important for the are crucial for the growth and metabolism of LAB; the former is important for the functioning of functioning of numerous enzymes, such as RNA polymerase, glutamine synthetase and lactate numerousdehydrogenase enzymes, [7,8], such while as RNA the latter polymerase, catalyses glutaminereactions of synthetase cell division, and stabilises lactate dehydrogenasenucleic acids and [ 7,8], whileis theinvolved latter in catalyses protein hydrolysis reactions of[11]. cell Recently, division, potassium stabilises ions nucleic have acids been andlinked is involvedwith long-range in protein hydrolysiselectrical [11 ].signalling Recently, within potassium bacterial ions havebiofilm been communities, linked with long-rangewith potassium electrical ions signalling triggering within bacterialdepolarisation biofilm communities,across the bacterial with ecosystem potassium [12]. ions LAB triggering show no spec depolarisationific iron requirements across the and bacterial the ecosystemsupplementation [12]. LAB of show growth no media specific with iron iron requirements does not stimulate and the lactobacilli supplementation growth [11]. of growth media with ironMalt does wort not has stimulate been shown lactobacilli to be a very growth good [11 source]. of nutrients for yeast [1]. The microelements contentMalt wort is usually has been sufficient shown tofor be microorganisms, a very good source but of some nutrients of them foryeast (mainly [1]. Thezinc) microelements are often contentreplenished. is usually Some suffi studiescient for have microorganisms, shown the role but of somemagnesium of them and (mainly zinc ions zinc) in arethe oftenmetabolism replenished. of 2+ Someyeast studies cells have [13,14] shown and thebacteria role of [8,15]. magnesium In yeast, and Mg zinc ionsaffects in the metabolismpermeability of of yeast cytoplasmic cells [13 ,14] membranes, participates in the construction of the most important cellular structures (i.e., ribosomes, and bacteria [8,15]. In yeast, Mg2+ affects the permeability of cytoplasmic membranes, participates DNA and RNA) [8,15] and activates over 300 enzymes, including synthetase, phosphofructokinase, in the construction of the most important cellular structures (i.e., ribosomes, DNA and RNA) [8,15] keratin kinases and membrane ATPase [16]. While zinc stimulates the protein synthesis during andfermentation activates over and 300 is one enzymes, of the key including glycolytic synthetase,enzymes (e.g., phosphofructokinase, ethanol dehydrogenase keratin and aldolase). kinases So and membranethat Zn2+ ATPase are crucial [16]. to While perform zinc a stimulates successful thefermentation. protein synthesis Their deficiency during fermentation may result in and a stuck is one of the key glycolytic enzymes (e.g., ethanol dehydrogenase and aldolase). So that Zn2+ are crucial to perform a successful fermentation. Their deficiency may result in a stuck fermentation [17]. In the past, Biomolecules 2020, 10, 1599 3 of 14 research has revealed the requirements for metal ions by brewers’ yeast, showing the change in the uptake and release of metal ions during the fermentation process, and also how it changes with successive reuse of yeast biomass in the fermentation process [18]. It has also been revealed that ionic composition of wort has significant influence on the resistance of yeast in the stressful environment (high osmotic pressure, ethanol concentration, etc.) [17]. Previous studies on the metal ion requirements of LAB were mostly performed using milk or MRS (De Man, Rogosa and Sharpe) medium (the model growth medium) and focused on the growth of the microorganisms. In the case of LAB, the addition of magnesium ions to the culture medium was shown to reduce the growth of the bacteria [13]. However, it has been proved that the growth conditions during the propagation of Lactobacillus in malt wort are significantly different to those in MRS [19]. Therefore, the novelty of the work is based on the approach of evaluating the influence of metal ions on the LAB in the brewers’ wort as the culture medium. Therefore, this study aims to evaluate the influence of the addition of magnesium and zinc ions on the metabolism of Lactobacillus brevis WLP672 during fermentation of brewers’ wort. The rate of fermentation, pH decrease, metal ion content and the concentration of organic acids. Key volatile compounds resulting from the mixed fermentation of lactic acid bacteria and brewers’ yeast were also evaluated in the sour beer.

2. Materials and Methods

2.1. Wort Wort (12% w/w extract content) was prepared using pale (EBC < 9), un-hopped powdered malt extract (SLADOVNA, SPOL. S ro., Bruntal, Czech Republic) and divided into five lots: two were supplemented with magnesium (Mg.1 and Mg.2) and two with zinc (Zn.1 and Zn.2), while the remaining one was used as the reference sample (REF). Magnesium sulphate (MgSO 8H O) was added at two 4· 2 different concentrations (60 mg/L and 120 mg/L), as was zinc chloride (ZnCl 4H O; 0.4 mg/L and 2· 2 2 mg/L). All samples were sterilised in an autoclave (121 ◦C for 30 min) and then cooled in cold water to room temperature. Atomic absorption spectrometry was applied to check the initial concentrations of zinc and magnesium after the sterilisation (and potential precipitation). The initial magnesium and zinc ion contents of the different wort samples are shown in Table1. The wort (50 mL) was poured into 100 mL sterile bottles and inoculated with microorganisms to perform the fermentation. Each treatment had three repeats.

Table 1. Initial magnesium and zinc ion contents [mg/L] in the different wort samples before fermenting.

REF Mg.1 Mg.2 Zn.1 Zn.2 Mg2+ 115.0 6.0 173.7 2.0 242.7 11.6 113.4 0.2 114.4 2.2 ± ± ± ± ± Zn2+ 1.7 0.1 1.6 0.1 1.6 0.0 2.1 0.2 3.9 0.5 ± ± ± ± ±

2.2. Microorganisms and Fermentation A culture of Lactobacillus brevis WLP672 (White Labs Inc, Old Sarum, United Kingdom) was propagated according to a procedure described in previous research [19] (12% wort extract, temp. 25 C, 72 h). The cultured bacteria (0.1 mL contained 6.5 107 cells) was then added to the wort ◦ × to start LA fermentation. Fermentations continued for 3 days at 25 ◦C under anaerobic conditions. After 72 h, the alcoholic fermentation was initiated by the addition of dry yeast (Fermentis Safale US-05, Lesaffre, France) rehydrated in water. An aliquot of rehydrated yeast slurry (0.17 mL; 5.8 108 cells/mL) × was added to each bottle to obtain an initial yeast content of 1.2 107 cells/mL (automatic yeast × counter Oculyze BB 2.0, BIOTECON Diagnostics, Potsdam, Germany). The samples were closed with a fermentation tube and then transferred into a thermostatic cabinet at 20 ◦C for alcoholic fermentation. At the end of fermentation, the produced sour beer samples were subjected to physicochemical analysis (ethanol content, real extract, pH, metal ions, volatile compounds, organic acids, and carbohydrates profiles). Biomolecules 2020, 10, 1599 4 of 14

2.3. Analytical Methods

2.3.1. Physiochemical Analysis A DMA 4500 M (Anton Paar, Warsaw, Poland) beer analyser was used to determine the ethanol content and real extract. Before measurement, the samples were degassed and filtered with diatomaceous earth to clarify the beer. The pH of the beer was measured daily using a CP-411 pH meter (Elmetron, Zabrze, Poland), which was calibrated using standard buffers before each measurement was taken.

2.3.2. Analysis of Metal Ions Samples of wort and beer (3 mL) were placed in sealed pressure vessels, with the addition of nitric acid (5 mL, 68%), and subjected to wet mineralisation in a Mars Xpress microwave oven (1200 W, 170 ◦C, 15 min) (CEM Corp., Matthews, NC, USA). The samples were then diluted with deionised water and their absorbance at 202.6 nm for Mg2+ and 213.9 nm for Zn2+ was determined by atomic absorption spectrometry with a flame atomization technique (Varian AA240FS), using an automatic dispensing sample system (SIPS-20, Agilent, Santa Clara, CA, USA). Gas flow was 3.5 dm3/min (acetylene) and air was 14 dm3/min. Standard solutions (Mg2+, Zn2+–respectively 100 and 5.0 mg/L) were prepared from 1000 mg/L standard solution (Merck, Bilerica, MA, USA).

2.3.3. Organic Acid and Carbohydrate Profile Analysis Specific enzymatic kits (Megazyme, Wicklow, Ireland) were used to determine the concentrations of L-lactic acid (kit K-LATE 06/18), acetic acid (kit K-ACET 04/18), L -malic acid (kit K-LMALQR 03/18), succinic acid (kit K-SUCC 06/18) and carbohydrates such as D-glucose, D-fructose, maltose and sucrose (K-FRGLQR 02/17, K-MASUG 11/16) according to the manufacturer’s instructions. LA content was determined daily during lactic fermentation and all acids were measured at the end of the process. The carbohydrate concentration was measured before (in the wort) and after fermentation (in beer). The concentrations of D-glucose and D-fructose were also measured after LA fermentation.

2.3.4. Analysis of Aroma Compounds by Solid-Phase Microextraction–Gas Chromatography/ Mass Spectrometry A 1 mL aliquot of saturated saline with an internal standard solution (5 mg/L 4-methyl-2-pentanol and 0.05 mg/L ethyl nonanoate, Sigma-Aldrich, St. Louis, MO, USA) and a 1 mL sample of beer were added into a 10 mL vial. The SPME device (Supelco Inc., Bellefonte, PA, USA) containing PDMS (100 µm) fibres was first conditioned by placing it in the GC injector port at 250 ◦C for 1 h. For sampling, the fibre was inserted into the headspace under stirring (300 rpm) for 30 min at 40 ◦C. Subsequently, the SPME device was introduced into the injector port of an Agilent Technologies 7890B chromatograph system equipped with a LECO Pegasus high-throughput time-of-flight mass spectrometry and kept in the inlet for 3 min. The SPME process was automated using a Gerstel MultiPurpose Sampler (MPS). The tested components were separated using a Restek™ Rxi-1ms Capillary Column (Crossbond 100% dimethyl polysiloxane, 30 m 0.53 mm 0.5 µm). The column was heated using the following × × temperature program: 40 ◦C increasing to 230 ◦C, over 3 min at a rate of 8 ◦C/min, then held at a constant temperature of 230 ◦C for 9 min. Carrier gas: helium at 1.0 mL/min constant flow. Electron ionisation mass spectrometry energy: 70 eV; ion source temperature and connection parts: 250 ◦C. Analyte transfer was performed in splitless mode; the mass spectrometry detector was set to scan mode from m/z = 40 to m/z = 400. Compounds were identified using mass spectral libraries and linear retention indices, calculated from a series of n-alkanes from C6 to C30. The qualitative and quantitative identification of the aroma compounds (Ethyl Acetate; 1-Propanol, 2-methyl-; 1-Butanol, 3-methyl-; 1-Butanol, 2-methyl-; Butanoic acid, ethyl ester; 2,3-Butanediol; 1-Hexanol; 1-Butanol, 3-methyl-, acetate; 2-Furanmethanol, Butyrolactone; Isomaltol; Hexanoic acid; Hexanoic acid, ethyl ester; 2-Ethyl-1-hexanol; Furaneol; Biomolecules 2020, 10, 1599 5 of 14

Biomolecules 2020, 10, x 5 of 14 1-Octanol; Phenylethyl Alcohol; Octanoic acid; α-Terpineol; Octanoic acid, ethyl ester; Benzothiazole; OctanoicButyrolactone; acid, Isomaltol; 2-methyl-, Hexanoic ethyl ester;acid; Hexanoic Acetic acid,acid, ethyl 2-phenylethyl ester; 2-Ethy ester;l-1-hexanol; Geraniol; Furaneol; 1-Decanol, 1- γ-Nonalactone;Octanol; Phenylethyl n-Decanoic Alcohol; acid; Octanoic 1-Undecanol; acid; αβ-Terpineol;-Damascenone; Octanoic Decanoic acid, ethyl acid, ester; ethyl Benzothiazole; ester; Dodecanoic acid;Octanoic Dodecanoic acid, 2-methyl-, acid, ethyl ethyl ester; ester; Benzophenone, Acetic acid, Benzyl2-phenylethyl benzoate; ester; Tetradecanoic Geraniol; 1-Decanol, acid, ethyl γ ester;- HexadecanoicNonalactone; n-Decanoic acid, ethyl acid; ester; 1-Undecanol; Sigma-Aldrich, β-Damascenone; St. Louis, MO, Decanoic USA) acid, was ethyl based ester; on theDodecanoic comparison ofacid; retention Dodecanoic times acid, and peakethyl ester; areas Benzophenone, from the sample Benzyl and benzoate; standard Tetradecanoic chromatograms. acid, Otherethyl ester; detected Hexadecanoic acid, ethyl ester; Sigma-Aldrich, St. Louis, MO, USA) was based on the comparison of components were determined semi-quantitatively by comparing the relative peak area of each retention times and peak areas from the sample and standard chromatograms. Other detected identified compound, according to the National Institute of Standards and Technology Database components were determined semi-quantitatively by comparing the relative peak area of each (http://webbook.nist.gov/chemistry/), to that of the internal standard. identified compound, according to the National Institute of Standards and Technology Database 2.4.(http://webbook.nist.gov/chemistry/), Statistical Analysis to that of the internal standard. 2.4.Values Statistical are Analysis given as mean standard deviation. Data were analysed using a two-way analysis ± of varianceValues (ANOVA) are given as to mean test the ± standard significance deviation. level ofData each were factor’s analysed influence. using a two-way The differences analysis in of each parametervariance (ANOVA) were analysed to test separately the significance using level Duncan’s of each Multiple factor’s Rangeinfluence. Test The (DMRT) differences (Statistica in each v. 10, StatSoftparameter Inc., were Krakow, analysed Poland). separately For both using ANOVA Duncan’s and DMRT,Multiple values Range< Test0.05 (DMRT) were considered (Statistica statistically v. 10, significantStatSoft Inc., (p < Krakow,0.05). Poland). For both ANOVA and DMRT, values <0.05 were considered statistically significant (p <0.05). 3. Results and Discussions 3. Results and Discussions 3.1. Organic Acid Production and pH 3.1.The Organic LA concentrationAcid Production and and pH pH are the main characteristics used to classify a sour beer. According to the literature,The LA concentration sour should and pH contain are the LAmain in characte the rangeristics of 3–6used g /toL classify and have a sour a pH beer. of 3.3–3.9According [1,2 ,20] butto somethe literature, sour beers sour containing beers should lower contain levels LAof in LAthe (2.1–3.4range of g3–6/L) g/L have and also have been a pH reported of 3.3–3.9 [21 [1,2,20]]. butFigure some sour1 shows beers the containing LA concentration lower levels and of LA pH (2.1–3.4 respectively, g/L) have measured also been every reported 24 h [21]. during the LA fermentationFigure 1 and shows in thethe producedLA concentration beer. The and LA pH content respectively, increased measured during every LA 24 fermentation h during the to LA reach thefermentation maximum and level in of the 0.82–0.90 produced g /beer.L in allThe samples LA content (Figure increased1a, 72 during h LAF). LA After fermentation 24 h, there to reach were no significantthe maximum differences level of between0.82–0.90the g/L reference in all samples and the(Figure supplemented 1a, 72 h LAF). samples After 24 (the h, lacticthere acidwerecontent no wassignificant ca. 0.4 gdifferences/L). After between 48 h, the the LA reference content and had th increasede supplemented to 0.68 samples g/L in the(the referencelactic acid samplecontent and 0.75–0.82was ca. 0.4 g/ Lg/L). in theAfter supplemented 48 h, the LA content samples. had incr Thus,eased the to total 0.68 amountg/L in the of reference lactic acid sample produced and 0.75– by the bacteria0.82 g/L was in the the supplemented same in all trials, samples. but supplementationThus, the total amount with eitherof lactic magnesium acid produced or zinc by the ions bacteria shortened was the same in all trials, but supplementation with either magnesium or zinc ions shortened the the time required (48 h vs. 72 h). time required (48 h vs. 72 h).

FigureFigure 1. 1.L-Lactic L-Lactic acid acid concentration concentration ( a(a)) and and pH pH ( b(b)) of of fermenting fermenting wort wort measured measured every every 24 24 h h during during lactic acidlactic fermentation acid fermentation (LAF)and (LAF) in theand beer; in the REF: beer; reference REF: reference sample (withoutsample (without supplementation); supplementation); Mg.1, Mg.2: wortMg.1, supplemented Mg.2: wort withsupplemented magnesium with at two magnesium different levels;at two Zn.1, different Zn.2: wortlevels; supplemented Zn.1, Zn.2: withwort zinc atsupplemented two different with levels. zinc Bars at two show different the average levels. Bars of three show replicates the average standardof three replicates deviation. ± standard*, ** and *** ± indicatedeviation. homogeneous *, ** and *** indicate groups homogeneous (p < 0.05) within groups the ( samplesp < 0.05) within analysed the samples at a certain analysed timepoint. at a certain timepoint. Biomolecules 2020, 10, 1599 6 of 14

Biomolecules 2020, 10, x 6 of 14 A further increase in the lactic acid content was observed after ethanol fermentation. The highest levelsA were further obtained increase in in the the reference lactic acid sample content andwas theobserved samples after supplemented ethanol fermentation. with magnesium The highest ions levels were obtained in the reference sample and the samples supplemented with magnesium ions (1.55–1.65 g/L), while the lowest levels were observed in the samples supplemented with zinc ions (1.55–1.65 g/L), while the lowest levels were observed in the samples supplemented with zinc ions (1.40–1.50 g/L). As expected, zinc ions enhanced the fermentation performed by yeast but did not (1.40–1.50 g/L). As expected, zinc ions enhanced the fermentation performed by yeast but did not improve lactic acid production. McLeod and Snell [10] showed that zinc can be toxic to some LAB if improve lactic acid production. McLeod and Snell [10] showed that zinc can be toxic to some LAB if the growth media does not contain sufficient manganese. The requirement for zinc is around 10 times the growth media does not contain sufficient manganese. The requirement for zinc is around 10 times lowerlower in in the the case case of of LAB LAB as as compared compared to to yeast yeast [11 [11]]. Therefore,. Therefore, the the addition addition of of zinc zinc did did not not enhance enhance LAB fermentationLAB fermentation but improved but improved ethanol ethanol production production by yeast. by yeast. According According to the to literature, the literature, the L-LAthe L-LA content incontent green beerin green reaches beer ca.reaches 1 g/L ca. when 1 g/LL. when brevis L.is brevis used is [ 1used,5,22 [1,5,22],], which which was concordant was concordant with with our results.our results.Considering that the rate of LA production was enhanced in the wort samples supplemented with eitherConsidering magnesium that the or zincrate of ions, LA butproduction zinc supplementation was enhanced inhibitedin the wort the samples production supplemented of LA during thewith mixed either fermentation magnesium or performed zinc ions, bybut bacteria zinc supplementa and yeast,tion it is inhibited concluded the that production zinc supplementation of LA during is notthe recommended mixed fermentation in sour performed beer production. by bacteria and yeast, it is concluded that zinc supplementation is notThe recommended addition of magnesiumin sour beer production. ions to the wort had a positive influence in the performance of the LAB asThe shown addition by the of magnesium rate of pH decrease ions to the of thewort wort had (Figure a positive1b). influence Supplementation in the performance with Mg 2 +of(at the both 60LAB and as 120 shown mg/ L)by resultedthe rate of in pH a lower decrease wort of pHthe measuredwort (Figure 72 1b). h after Supplementation addition of LAB with (pH Mg 3.902+ (at andboth 3.85, respectively)60 and 120 mg/L) as compared resulted in to a thelower reference wort pH wort measured (pH 3.95). 72 h after Consequently, addition of LAB the beers(pH 3.90 produced and 3.85, with Mgrespectively)2+ supplementation as compared (after to the alcoholic reference fermentation) wort (pH 3.95). had Consequently, lower pH (< 3.4)the beers than theproduced reference with beer 2+ (>Mg3.4). supplementation On the contrary, (after the beers alcoholic produced fermentation) from the had wort lower supplemented pH (<3.4) withthan zincthe reference ions had beer slightly higher(>3.4). pHOn (3.45the contrary, and 3.5) the than beers the produced reference from beer. the wort supplemented with zinc ions had slightly higher pH (3.45 and 3.5) than the reference beer. In addition to LA (the main metabolite), L. brevis also produced other organic acids. In addition to LA (the main metabolite), L. brevis also produced other organic acids. Their Their concentrations are shown in Figure2. concentrations are shown in Figure 2.

FigureFigure 2. 2. AceticAcetic acid concentration (a (a) )after after 72 72 h hof of lactic lactic acid acid (LAF) (LAF) and and ethanol ethanol fermentation fermentation (beer), (beer), andand succinic succinic andand malic acid ( (bb)) in in beer; beer; REF: REF: reference reference sample sample (without (without supplementation); supplementation); Mg.1, Mg.1, Mg.2: Mg.2: wortwort supplemented supplemented withwith magnesium at at two two different different levels; levels; Zn.1, Zn.1, Zn.2: Zn.2: wort wort supplemented supplemented with with zinczinc atat two two di differentfferent levels.levels. Bars show the the average average of of thre threee replicates replicates ± standardstandard deviation. deviation. * and * and ** indicate ** indicate homogeneous groups within the same compound (p < 0.05). ± homogeneous groups within the same compound (p < 0.05).

AccordingAccording to to the the literature, literature,L. L. brevisbrevisis is ableable toto produceproduce 0.2–0.40.2–0.4 gg/L/L ofof aceticacetic acidacid [[1,22–24],1,22–24], while the detectabilitythe detectability threshold threshold in beerin beer is is 0.2 0.2 g /g/LL[25 [25].]. InIn the current current work work significantly significantly higher higher acetic acetic acid acid concentrations 0.6–0.7 g/L were obtained (Figure 2a). L. brevis produces CO2 and ethanol or acetic concentrations 0.6–0.7 g/L were obtained (Figure2a). L. brevis produces CO2 and ethanol or acetic acid in addition to LA. The proportions of these metabolites depend on the composition of the acid in addition to LA. The proportions of these metabolites depend on the composition of the medium: if it is rich in glucose, LA is the main metabolite, whereas if the medium is low in glucose medium: if it is rich in glucose, LA is the main metabolite, whereas if the medium is low in glucose or if maltose/galactose dominate, the amounts of acetic acid/ethanol and CO2 produced increase, or if maltose/galactose dominate, the amounts of acetic acid/ethanol and CO produced increase, while LA biosynthesis decreases [23]. This phenomenon was confirmed in our research.2 The main whilecarbohydrate LA biosynthesis in malt wort decreases is maltose [23 ].(approximately This phenomenon 50 g/L), was while confirmed glucose accounts in our research. for only 12% The of main carbohydratethe carbohydrates in malt (7–8 wort g/L), is which maltose is why (approximately higher concentrations 50 g/L), while of acetic glucose acid were accounts obtained for onlythan 12%the of thevalues carbohydrates reported in (7–8 the gliterature./L), which After is why alcoholic higher fermentation, concentrations the ofamount acetic of acid acetic were acid obtained did not than Biomolecules 2020, 10, 1599 7 of 14 the values reported in the literature. After alcoholic fermentation, the amount of acetic acid did not change significantly in any of the samples. On the contrary, the l-LA content more than doubled after alcoholic fermentation (Figure1a). Succinic and malic acids are also produced in the course of fermentation. According to the literature, LAB can produce 0.2–0.8 g/L of succinic acid but this is very strain-dependent [5,22]. Figure2b shows the succinic and malic acid contents in the beer. In the samples supplemented with zinc ions (Zn.1 and Zn.2), the succinic acid content was almost double that in the reference (0.36 and 0.77 g/L, respectively). Tyrell [26] studied various parameters affecting the formation of succinic acid in beer and concluded that aeration, extract content in the wort, pH and fermentation temperature are the major ones. Our research proves that zinc ions can be also taken into account as a factor contributing to the succinic acid concentration in beer. Nevertheless, a high concentration of succinic acid in beer is not desirable because it stimulates the secretion of stomach acids, causing indigestion and lowering the drinkability of the beer [27]. Supplementation of the wort with magnesium ions did not lead to a significant difference from the reference values (0.28–0.43 g/L). Malic acid is produced during the malting process by endogenous microorganisms [28]. In malt wort, malic acid is present at low levels of around 0.4 g/L, decreasing to around 0.1 g/L after lactic fermentation [5]. Bacteria transform malic acid as a result of malolactic fermentation, and the intensity of this process depends on the bacterial strain [6]. In the present study, there were no significant differences in malic acid content across all samples (0.07–0.1 g/L).

3.2. Carbohydrate Use The four fermentable carbohydrates and their initial concentrations in wort were: maltose (49.5 1.3 g/L), glucose (7.7 1.6 g/L) fructose (3.0 0.6 g/L) and saccharose (1.8 0.3 g/L). Maltose is ± ± ± ± the most abundant carbon source in malt wort, and it is fully used by yeast during fermentation [29]. However, this is not the case for Lactobacillus-according to some studies, only about 4% of maltose contained in the wort is used by Lactobacillus [1,5]. The crucial carbohydrates for LAB are glucose and fructose, with their order of uptake depending mainly on the strain e.g., L. amylolyticus and L. plantarum show a preference towards glucose, while L. brevis mainly ferments fructose [5,24]. Therefore, we focused on the use of those carbohydrates (Table2).

Table 2. Concentration of carbohydrates [g/L] in malt wort before fermentation, after 72 h of lactic acid fermentation and after alcoholic fermentation depending on the initial concentration of magnesium and zinc ions: REF: reference sample (without supplementation); Mg.1, Mg.2: wort supplemented with magnesium at two different levels; Zn.1, Zn.2: wort supplemented with zinc at two different levels.

Wort After Lactic Acid Fermentation After Ethanol Fermentation Carbohydrate REF Mg.1 Mg.2 Zn.1 Zn.2 REF Mg.1 Mg.2 Zn.1 Zn.2 REF Mg.1 Mg.2 Zn.1 Zn.2 [g/L] 6.7 7.1 6.7 7.1 8.1 0.1 0.1 0.1 0.1 0.1 Glucose 7.7 1.6 ± ± ± ± ± ± ± ± ± ± ± 0.5 0.8 0.2 0.1 0.3 0.0 0.0 0.0 0.0 0.0 * * * * ** * * * * * Fructose 3.0 0.6 0.0 0.0 0.0 0.0 ± ± ± Sucrose 1.8 0.3 0.0 0.0 ± ± 3.3 3.6 3.4 3.6 3.6 Maltose 49.5 1.3 ± ± ± ± ± ± 0.5 0.4 0.4 0.4 0.6 * * * * * Results are presented as the mean of three replicates standard deviation, * indicates that the sample is not significantly different from the reference and ** indicates± that the sample is significantly different from the reference sample (p < 0.05).

After 72 h of lactic fermentation, fructose was fully used, regardless of the supplementation variant. There have been no reports to date proving that fructose is an essential carbohydrate for LAB Biomolecules 2020, 10, 1599 8 of 14 growth, but it can be supposed that its higher concentration in the medium could contribute to an increase in the bacteria’s metabolic activity. The use of fructose as an electron acceptor gives the LAB an additional ATP gain without ATP expenditure when converting acetyl-P to acetate [30]. Therefore, it is suggested that the production of fructose-rich wort (e.g., with the use of unmalted raw materials) may be a good solution for the production of sour beers, especially when Lactobacillus brevis is used. Glucose is the main carbohydrate fermented by LAB according to many studies [1,23,24]. In work presented by Nsogning et al. [5], LAB used 20–50% of the glucose present in the wort. The strain L. amylolyticus La.TL3 has the highest preference for glucose (using 6 g/L), while the lowest preference is expressed by L. brevis Lb.986 (2 g/L). In the current work, glucose was only slightly processed at about 1 g/L. Interestingly, an increase in glucose content was noted in samples supplemented with zinc after lactic fermentation (8.06 g/L in Z.2 vs. 6.68 g/L in REF), which could be associated with the action of enzymes released from the cytoplasm after cell death that may have broken down sucrose into glucose and fructose. A similar relationship was observed in research by Peyer [1], where he noted an increase in glucose content in the medium during L. brevis R2∆ fermentation while hydrolysing sucrose. Nsogning et al. [5] noted an increase in fructose content during LAB fermentation, with a high percentage of dead cells (60–80%). Sucrose is not considered as an essential carbohydrate for LAB fermentation, especially for heterofermentative strains [5], so we did not monitor its concentration beyond the 72 h of lactic fermentation. However, after alcoholic fermentation, all the monosaccharides present in the wort had been completely consumed (Table2), while around 93% of the maltose was used, regardless of the experiment variant (p = 0.9). Assume that, supplementation with Mg2+ and Zn2+ had no effect on carbohydrate use by bacteria and yeast, but slightly affected the ethanol content and the real extract in beer (Table3). The addition of zinc positively influenced alcoholic fermentation, resulting in a slightly higher ethanol concentration compared to the reference.

Table 3. Ethanol and real extract content in beer depending on the initial concentrations of magnesium (Mg.1 Mg.2) and zinc (Zn.1 and Zn.2) ions in wort. REF-beer obtained from wort without supplementation.

REF Mg.1 Mg.2 Zn.1 Zn.1 Ethanol [% v/v] 3.9 0.0 * 4.0 0.0 ** 3.9 0.0 * 4.0 0.0 ** 4.2 0.0 ** ± ± ± ± ± Real extract [% w/w] 6.2 0.0 * 6.5 0.1 ** 6.4 0.0 ** 6.3 0.0 * 6.6 0.0 ** ± ± ± ± ± Results are presented as the mean of three replicates standard deviation, * indicates that the sample is not significantly different from the reference and ** indicates± that the sample is significantly different from the reference sample (p < 0.05).

3.3. Aroma Compounds According to the literature, the main volatile compounds produced by LAB in malt-based fermented beverages are principally associated with carbohydrate metabolism (esters, aldehydes and ketones) and amino acid metabolism (higher alcohols and aldehydes) [31,32]. They may arise as a result of malt wort fermentation or the conversion of precursors derived from malt wort by LAB enzymes [32]. Both LAB and yeast produce many aroma compounds during the fermentation of malt wort. Yeast produces mainly higher alcohols, esters, aldehydes and fatty acids, while LAB produce organic acids and aldehydes [1,33]. It was proved that the initial concentration of metal ions in wort influences the concentration of some aroma compounds in beer. Zinc ions have a significant impact on the biosynthesis of higher alcohols, carboxylic acids and terpenes; their content was higher by more than double in samples supplemented with zinc ions, as compared to the reference and to the magnesium supplemented samples (Table4). Biomolecules 2020, 10, x 9 of 14

BiomoleculesTable2020 4. The, 10, 1599amounts of aroma compounds (higher alcohols, esters, fatty acids and terpenes) in sour9 of 14 beer produced during subsequent lactic acid and alcoholic fermentation of brewers’ wort depending on the initial concentrations of magnesium (Mg.1 Mg.2) and zinc (Zn.1 and Zn.2) ions. REF-beer Table 4. The amounts of aroma compounds (higher alcohols, esters, fatty acids and terpenes) in sour obtained from wort without supplementation. beer produced during subsequent lactic acid and alcoholic fermentation of brewers’ wort depending on Sample [µg/L] the initial concentrations of magnesium (Mg.1 Mg.2) and zinc (Zn.1 and Zn.2) ions. REF-beer obtained from wort without supplementation. LRI 2 REF Mg.1 Mg.2 Zn.1 Zn.2 Sig.1 Esters Ethyl acetate 614 2716 a 3224Sample a [µ2740g/L] a 4099 b 2733 a * 3 b b c a a Butanoic acid, ethyl ester LRI 2 789 REF0.3 Mg.10.4 Mg.20.5 Zn.10.0 Zn.2 0.0 Sig. 1 *** 1-Butanol, 3-methyl-, acetate 872 16.7 d 9.5 c 6.6 b 3.7 a 2.7 a *** Esters Hexanoic acid, ethyl ester 986 2.8 b 2.5 b 2.1 ab 1.6 a 1.9 ab * Ethyl acetate 614 2716 a 3224 a 2740 a 4099 b 2733 a * Octanoic acid, ethyl ester 1180 34.6 a 53.6 b 61.9 b 54.6 b 57.9 b * Butanoic acid, ethyl ester 3 789 0.3 b 0.4 b 0.5 c 0.0 a 0.0 a *** Octanoic acid, 2-methyl-, ethyl ester 3 1209 69.3 b 33.4 a 26.3 a 30.6 a 30.1 a *** 1-Butanol, 3-methyl-, acetate 872 16.7 d 9.5 c 6.6 b 3.7 a 2.7 a *** c c b ab a AceticHexanoic acid, acid, 2-phenylethyl ethyl ester ester 986 1228 2.8 b 64.2 2.5 b 63.2 2.1 ab 46.8 1.6 a 37.4 1.9 ab 30.5 * *** DecanoicOctanoic acid, acid, ethyl ethyl ester ester 1180 139734.6 a11.8 b 53.6 b 11.4 b 61.9 b 11.7 b 54.6 b 7.9 ab 57.9 b 6.9 a * * DodecanoicOctanoic acid, acid, 2-methyl-, ethyl ester ethyl ester 3 1209 158169.3 b 6.3 a 33.4 a 8.1 b 26.3 a 6.2 a 30.6 a 6.0 a 30.1 a 4.7 a *** * BenzylAcetic Benzoate acid, 2-phenylethyl ester 1228 175064.2 c 1.3 a 63.2 c 3.8 b 46.8 b 3.8 b 37.4 ab 10.7 c 30.5 a 13.2 d *** *** TetradecanoicDecanoic acid, acid, ethyl ethyl ester ester 3 1397 179011.8 b 0.8 a 11.4 b 2.6 ab 11.7 b 3.5 b 7.9 ab 12.6 c 6.9 a 14.7 d * *** HexadecanoicDodecanoic acid, acid, ethyl ethyl ester ester 3 1581 1990 6.3 a 0.6 a 8.1 b 2.0 a 6.2 a 2.6 a 6.0 a 12.2 b 4.7 a 14.7 c * *** a b b c d HigherBenzyl alcohols Benzoate 1750 1.3 3.8 3.8 10.7 13.2 *** 3 a ab b c d 1-Propanol,Tetradecanoic 2-methyl- acid, ethyl ester 1790 617 0.8 433.6 a 2.6 699.2 b 3.5 725.5 b 12.6 977.8 c14.7 1046.8 c*** *** 3 1990 0.6 a 2.0 a 2.6 a b 14.7 c *** 1-Butanol,Hexadecanoic 3-methyl- acid, ethyl ester 723 13,787 a 21,007 bc 19,298 b12.2 24,456 c 29,083 d *** Higher alcohols 1-Butanol, 2-methyl- 740 4984 a 6818 b 5173 a 6093 ab 7235 b *** 1-Propanol, 2-methyl- 617 433.6 a 699.2 b 725.5 b 977.8 c 1046.8 c *** 2,3-Butanediol 768 1663 bc 1861 c 1322 b 838 a 553 a *** 1-Butanol, 3-methyl- 723 13,787 a 21,007 bc 19,298 b 24,456 c 29,083 d *** a b a a a 1-Hexanol1-Butanol, 2-methyl- 740 865 4984 a23.7 6818 b 36.0 5173 a 25.8 6093 ab 24.9 7235 b 23.8 *** * 3 a c ab b a b 2-Furanmethanol2,3-Butanediol 768 8801663 bc48.8 1861 c130.1 1322 b73.1 838 a 75.4 553 a 73.3 *** *** Isomaltol1-Hexanol 3 865 975 23.7 a 48.5 a 36.0 b104.4 c 25.8 a 72.1 ab 24.9 a 77.3 b 23.8 a 146.0 d * *** 2-Ethyl-1-hexanol2-Furanmethanol 33 880 102048.8 a 99.9 b 130.1 c97.3 b 73.1 ab 78.1 a 75.4 b 73.2 a73.3 a b 64.5 a *** *** FuraneolIsomaltol 3 3 975 105748.5 a 35.8 104.4 c 43.3 72.1 ab 41.2 77.3 b 40.1 146.0 d 42.3 *** ns 1-Octanol2-Ethyl-1-hexanol 3 3 1020 107099.9 b 19.9 97.3 b 21.5 78.1 a 16.9 73.2 a 14.7 64.5 a 13.6 *** ns 3 PhenylethylFuraneol Alcohol 1057 1084 35.83320.3 a 43.34966.0 b 41.2 3233.5 a 40.1 3193.6 a 42.35582.0 b ns *** 3 1-Decanol1-Octanol 3 1070 1272 19.9 192.3 a 21.5 179.6 a 16.9 312.3 c 14.7 244.2 b 13.6159.3 a ns *** a b a a b 1-UndecanolPhenylethyl 3 Alcohol 1084 13743320.3 44.2 4966.0 59.0 3233.5 56.3 3193.6 57.55582.0 73.3 *** ns 1-Decanol 3 1272 192.3 a 179.6 a 312.3 c 244.2 b 159.3 a *** Fatty acids 1-Undecanol 3 1374 44.2 59.0 56.3 57.5 73.3 ns Hexanoic acid 982 2.7 a 7.2 b 6.9 b 19.7 c 27.6 d *** Fatty acids a a a b c OctanoicHexanoic acid acid 982 1160 2.7 a 62.2 7.2 b 173.5 6.9 b 184.2 19.7 c 584.3 27.6 d 1102.2 *** *** a a a b b n-DecanoicOctanoic acid acid 1160 136862.2 a 3.9 173.5 a 9.5 184.2 a 11.2 584.3 b 30.7 1102.2 c 36.8 *** *** Dodecanoicn-Decanoic acid acid 1368 1568 3.9 a 0.6 a 9.5 a 2.0 b 11.2 a 1.4 ab 30.7 b 4.3 c 36.8 b 6.8 d *** *** TerpenesDodecanoic acid 1568 0.6 a 2.0 b 1.4 ab 4.3 c 6.8 d *** α-TerpineolTerpenes 1171 13.0 a 28.6 ab 50.3 b 96.8 c 159.1 d *** Geraniolα-Terpineol 1171 125713.0 a 27.6 a 28.6 ab59.2 ab 50.3 b 91.5 b 96.8 c 201.1 159.1c d248.0 d*** *** a ab b c d β-DamascenoneGeraniol 3 1257 138627.6 29.2 59.2 30.6 91.5 32.6 201.1 31.6 248.0 30.5 *** ns 3 Lactonesβ-Damascenone 1386 29.2 30.6 32.6 31.6 30.5 ns Lactones Butyrolactone 3 916 79.7 72.0 80.9 73.3 42.9 ns Butyrolactone 3 916 79.7 72.0 80.9 73.3 42.9 ns γ-Nonalactone 3 1360 64.4 75.2 62.2 54.2 57.4 ns γ-Nonalactone 3 1360 64.4 75.2 62.2 54.2 57.4 ns OtherOther compounds compounds 3 a b c c d BenzothiazoleBenzothiazole 3 1186 1186247.8 a247.8 437.2 b437.2 592.8 c592.8 586.1 c586.1772.4 d772.4 *** *** BenzophenoneBenzophenone 3 3 1610 1610 5.2 5.2 6.5 6.5 4.9 4.9 4.8 4.8 5.05.0 ns ns

The colours colours (from (from light light to dark) to dark) indicate indicate increasing increasi valuesng within values the within same volatile the same compound. volatile1 Sig.:compound. significance; 1 Sig.: *significance; and *** indicate * significanceand *** indicate at the 5, significance 1 and 0.5% levels at the by least 5, 1 significant and 0.5% di fflevelserence; by ns: least not significant. significant Values difference; with different superscript roman letters (a–d) in the same row are significantly different according to the Duncan test a–d (ns:p < 0.05)not 2significant.LRI: Linear retentionValues indexwith 3 diDeterminedfferent superscript semi-quantitatively roman by letters measuring ( )the inrelative the same peak arearow of are eachsignificantly identified compound,different according to theto NISTthe Duncan database, andtest comparing(p < 0.05) it to2 thatLRI: of theLinear internal retention standard. index 3 FattyDetermined acids havesemi-quantitatively a beneficial eff ectby onmeasuring yeast growth the re duringlative peak fermentation area of each as wellidentified as a negative compound, effect on theaccording foam and to the sensory NIST stabilitydatabase, ofand beer comparing [34]. In it general, to that of long-chain the internal fatty standard. acids originate from raw materials, whereas short-chain acids are produced by yeast during fermentation [35]. The high levels of Fatty acids have a beneficial effect on yeast growth during fermentation as well as a negative fatty acids seen in the samples supplemented with zinc ions (Table4) may be caused by increased yeast effect on the foam and sensory stability of beer [34]. In general, long-chain fatty acids originate from metabolism (as in the case of higher alcohols) or a LAB response to stress factors (oxidative, osmotic, rawacid ormaterials, heat) during whereas fermentation short-chain [36 ].acids Usually, are produc high levelsed by of yeast hexanoic, during octanoic fermentation and n-decanoic [35]. The acid high levelsin beer of are fatty not desirableacids seen because in the theysamples cause supplemen an unpleasant,ted with rancid zinc smell ions and (Table taste 4) [34 may]. be caused by increased yeast metabolism (as in the case of higher alcohols) or a LAB response to stress factors Biomolecules 2020, 10, 1599 10 of 14

Biomolecules 2020, 10, x 10 of 14 Geraniol, α-terpineol and β-damascenone (Table4) are always associated with hop oils but they have(oxidative, also been osmotic, reported acid to beor presentheat) during in un-hopped fermentation wort [36]. [37 Usually,]. They high may levels be formed of hexanoic, during octanoic malolactic fermentationand n-decanoic by glycosidase acid in beer and are arabinosidasenot desirable becaus frome precursors, they cause an or unpleasant, thermal cleavage rancid smell by heat and treatmenttaste [34]. under acidic conditions [32]. In the trials supplemented with zinc ions, we observed 7–12 times higher Geraniol, α-terpineol and β-damascenone (Table 4) are always associated with hop oils but they concentrations of geraniol and α-terpineol compared to the reference. It is not entirely clear why the have also been reported to be present in un-hopped wort [37]. They may be formed during malolactic increasedfermentation zinc supplementation by glycosidase resultedand arabinosidase in higher amountsfrom precursors, of these twoor thermal terpenes. cleavage Metal ions,by heat such as manganese,treatment have under been acidic reported conditions to increase [32]. In the the tria beta-glucosidasels supplemented activitywith zinc by ions, as muchwe observed as twofold 7–12 [38]; 2+ perhapstimes Zn higheralso concentrations plays an important of geraniol role and inα-terpineol the activity compared of glucosidase, to the reference. which It isis responsiblenot entirely for releasingclear terpenoidswhy the increased from the zinc bound supplementation form [24]. Nevertheless, resulted in higher the values amounts are below of these the two flavour terpenes. threshold of 1 mgMetal/L[ ions,39]. such as manganese, have been reported to increase the beta-glucosidase activity by as muchFurthermore, as twofold the [38]; contents perhaps of Zn higher2+ alsoalcohols, plays an important such as 1-Propanol, role in the activity 2-methyl-; of glucosidase, 1-Butanol, which 3-methyl-; 1-Butanol,is responsible 2-methyl- for releasing and Phenylethyl terpenoids from Alcohol the bo wereund form significantly [24]. Nevertheless, higher inthe thevalues trials are withbelow Zn2+ supplementationthe flavour threshol as comparedd of 1 mg/L to [39]. the reference sample (Table4). Similar results were obtained by Furthermore, the contents of higher alcohols, such as 1-Propanol, 2-methyl-; 1-Butanol, 3- Gutierrez [40], who revealed lower production of 1-Propanol, 2-methyl and 1-Butanol, 3-methyl- with methyl-; 1-Butanol, 2-methyl- and Phenylethyl Alcohol were significantly higher in the trials with zinc deficiency in the fermentation medium. This can be explained by the essential role played by Zn2+ supplementation as compared to the reference sample (Table 4). Similar results were obtained zincby ions Gutierrez in the operation[40], who revealed of several lower enzymes, production such of as 1-Propanol, alcohol dehydrogenase, 2-methyl and 1-Butanol, aldolase 3-methyl- and alkaline phosphatasewith zinc [11deficiency]. Higher in alcoholsthe fermentation originate medium. from the This metabolism can be explained of amino by acids. the essential In yeast, role the played synthesis of branched-chainby zinc ions in aminothe operation acids isof regulatedseveral enzymes, by the such expression as alcohol of thedehydrogenase, genes ILV2 ,aldolaseILV3 and andBAT2 . Thesealkaline genes phosphatase were found [11]. to be Higher downregulated alcohols originate in zinc-limited from the yeast metabolism growth of in amino the presence acids. In oryeast, absence of oxygenthe synthesis [41]. of branched-chain amino acids is regulated by the expression of the genes ILV2, ILV3 andIn work, BAT2. the These ester genes concentration were found wasto be not downregulated influenced by in zinc-limited the initial zinc yeast and growth magnesium in the presence ion content. Thisor may absence be because of oxygen ionic [41]. supplementation of wort does not affect the production of amino acids or fatty acids,In work, which the are ester precursors concentration of esters, was as not the influenced ions are notby cofactorsthe initial forzinc enzymes and magnesium involved ion in the content. This may be because ionic supplementation of wort does not affect the production of amino production of esters (i.e., esterase, alcohol acetyltransferase) [42]. Biosynthesis of esters by LAB is acids or fatty acids, which are precursors of esters, as the ions are not cofactors for enzymes involved based on the transfer of a fatty acyl group from acylglycerol and acyl-CoA to an alcohol (Scheme1). in the production of esters (i.e., esterase, alcohol acetyltransferase) [42]. Biosynthesis of esters by LAB However,is based as on a result the transfer of the of low a fatty ethanol acyl production group fromby acylglycerol LAB (<0.5% andv acyl-CoA/v)[2], lactic to an fermentation alcohol (Scheme does not favourably1). However, affect as esterification, a result of the which low ethanol is generally producti desirableon by LAB because (<0.5% esters v/v) [2], give lactic a sweet, fermentation fruity taste. LA anddoes acetic not favourably acid (the mainaffect organicesterification, acids producedwhich is generally during lactic desirable acid because fermentation) esters give may a be sweet, involved in thefruity formation taste. LA of theand estersacetic ethylacid (the lactate main and organic ethyl acids acetate, produced respectively during [lactic32]. acid fermentation) may be involved in the formation of the esters ethyl lactate and ethyl acetate, respectively [32]. 3.4. Metal Ion Release by LAB and Yeast 3.4. Metal Ion Release by LAB and Yeast Figure3 shows the concentration of magnesium (a) and zinc (b) ions in the wort during the mixed fermentationFigure depending 3 shows the on concentration the initial content of magnesium of this ions (a) in and the zinc wort. (b) Theions lactic in the acid wort fermentation during the did not causemixed any fermentation changes in depending the magnesium on the content initial inconten the wort,t of this which ions indicates in the wort. that the The LAB lactic did acid not take up orfermentation release magnesium did not cause during any thechanges 72 h in of the the magnesium process. On content the contrary, in the wort, yeast which released indicates magnesium, that the LAB did not take up or release magnesium during the 72 h of the process. On the contrary, yeast which resulted in a slight increase in the magnesium content. released magnesium, which resulted in a slight increase in the magnesium content.

Figure 3. Magnesium (a) and zinc (b) concentration in the wort before fermentation, after lactic acid fermentation and in the produced beer (after lactic acid and alcoholic fermentation) depending on the Biomolecules 2020, 10, 1599 11 of 14

initial concentrations of magnesium and zinc ions: REF: reference sample (without supplementation); Mg.1, Mg.2: supplemented with magnesium at two different levels; Zn.1, Zn.2: supplemented with zinc at two different levels. Results are the means of three replicates and the error bars are SD, *, ** and *** ± indicate homogeneous groups within the same trial (p < 0.05).

In all of the trials supplemented with metal ions (either magnesium or zinc), the yeast released magnesium into the medium at the end of the process. In the samples with magnesium supplementation, the release was less pronounced than in the case of the zinc supplementation. In the former one (trial Mg.1, 60 mg/L supplementation) the increase in the Mg2+ content was ca. 10% (from 165 to 190 mg/L), while in the case of trial Mg.2 (120 mg/L supplementation) it was ca. 20% (from 225 to 280 mg/L). In the latter case (zinc supplemented samples), the increase in the magnesium ion in the beer after fermentation was much higher, reaching 50% (from 120 to 175 mg/L in both the 0.4 and 2 mg/L Zn2+ supplementation trials). Similar trends were observed in study Poreda et al. [43], which proved that increased content of zinc ions in wort can block the uptake of magnesium ions by the yeast. The current results show that in the case of L. brevis strain the uptake and release of magnesium ions is not affected by the zinc ion content. The pattern of release of zinc ions during subsequent LA and alcoholic fermentation was different from that observed for magnesium (Figure3b). First of all, there was already an increase in the zinc ion concentration in the wort after performing the lactic acid fermentation (in all treatments except for the one with the highest level of Zn2+ supplementation). It is worth noting that the highest release of zinc ions (Zn2+ increased from 2 to 6 mg/L in the wort) was observed in the trial with the highest supplementation of magnesium ions (120 mg/L). In the reference sample and the sample with 60 mg/L magnesium supplementation, the zinc ion concentration in the wort increased from around 2.5 mg/L up to ca. 4.5–5 mg/L. In the trial with Zn2+ supplementation at 2 mg/L, neither the LAB nor the yeast cells released zinc ions into the wort. It seems that the concentration in the wort was high enough to block any leakage of Zn2+ ions out of the cells. Based on the results two conclusions can be drawn. First of all, the release of zinc ions from both LAB and yeast cells proceeds via passive transport dependent on the Zn2+ concentration gradient. The higher the content of zinc ions in the wort, the fewer ions were released by the microbes. Secondly, the rate of zinc ion release from the yeast cells depends not only on the zinc ion content in wort but also on the magnesium ion content. The more magnesium that was present in the wort (Mg.2), the more zinc ions were released out of the cell. A similar trend was observed in earlier studies, which proved that at levels above 150 mg/L, magnesium ions effectively limit zinc bioaccumulation in yeast biomass [43].

4. Conclusions The objective of this study was to determine how metal ion (magnesium and zinc) supplementation affects the metabolism of Lactobacillus brevis WLP672 in brewers’ wort, the production of organic acids and volatile compounds, and the rate of sour beer fermentation. Supplementation of wort with magnesium resulted in a slightly faster decrease in the pH during the fermentation and an overall the lowest pH of the sour beer. The differences, though statistically significant, may be negligible at the industrial production, so more work is necessary in this field to further increase the rate of pH decrease during LAB fermentation. Additionally, the concentration of lactic acid after 48 h of LAB fermentation was higher in the samples with Mg2+ supplementation, compared to the reference sample. On the other hand, zinc ions supplementation resulted in a higher pH and lower content of lactic acid in the sour beer. It is an important finding, as zinc supplementation is a standard procedure in many , and it is worth considering reducing or abandoning Zn2+ supplementation when producing wort for sour beer. We also found almost twofold higher concentrations of succinic acid in the Zn2+ supplemented samples than in the reference sample or the Mg2+ supplemented samples. High concentration of succinic acid in beer is not desirable, as it results in beer with lower drinkability. Regarding aroma compounds, supplemental magnesium increased the Biomolecules 2020, 10, 1599 12 of 14 concentrations of 1-Propanol, 2-methyl-; 1-Butanol, 3-methyl-; Hexanoic acid, Octanoic acid, ethyl ester; Benzothiazole and Benzyl Benzoate, while zinc most of all enhanced the synthesis of carboxylic acids and terpenes. Supplementation with magnesium and zinc had no effect on carbohydrate use but slightly affected the ethanol content and the real extract in beer. The addition of zinc positively influenced alcoholic fermentation, resulting in a slightly higher alcohol concentration compared to the reference. This work will contribute to the brewing industry by providing new knowledge about the impact of magnesium or zinc supplementation on the lactic acid and alcoholic fermentation. It will also help to understand the relation between ionic content of wort and the aroma profile of beer, which can lead to improvement of sour beer quality. Further work is needed to evaluate how changes in other wort characteristics (carbohydrates profile, free amino nitrogen content) influence the lactic acid production and sensory quality of sour beers.

Author Contributions: Conceptualization, A.P. and A.C.; methodology, A.C.; validation, A.P., P.S.; formal analysis, A.C., A.P. and P.S.; investigation, A.C. and K.F.; resources, A.P. and P.S.; data curation, A.C. and A.P.; writing—original draft preparation, A.C.; writing—review and editing, A.C., A.P., P.S., O.H.; visualization, A.C.; supervision, A.P.; funding acquisition, A.P. All authors have read and agreed to the published version of the manuscript. Funding: The research was financed in part by the Ministry of Science and Higher Education of Poland. Conflicts of Interest: The authors declare no conflict of interest

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