Zinc Interactions with Brewing Yeast: Impact on Fermentation Performance

Raffaele De Nicola1 and Graeme M. Walker,2 Yeast Research Group, School of Contemporary Sciences, University of Abertay Dundee, Dundee, Scotland
ABSTRACT J. Am. Soc. Brew. Chem. 69(4):214-219, 2011 The effect of zinc on brewing yeast cells was studied in relation to zinc uptake, fermentation performance, and flavor congener formation. Experiments using malt wort with variable supplements of zinc salts were conducted in small-scale conical vessels and in pilot-plant fermentors to reproduce industrial lager beer fermentations. During small-scale fermentations, zinc was taken up completely from wort by yeast within the first 48 and 96 hr, when zinc concentrations in wort were 1.0 and 4.85 ppm, respectively. Zinc impacted fermentation performance, with wort zinc levels required for optimal fermentation ranging from 0.48 to 1.07 ppm. These initial zinc levels corresponded to final zinc yeast cell contents of 14 and 108 fg/cell, respectively. In pilot-plant fermentors, preconditioning of yeast by enriching cells with zinc prior to fermentation benefited fermentation progress when zinc-deficient wort was employed. Flavor congener profiles appeared to be affected only at high zinc levels of 10 ppm, with elevated concentrations of higher alcohols and some esters (ethyl caproate and isoamyl acetate) observed. We concluded that control of zinc bioavailability, including Zn-supplementation strategies (for both wort and yeast), plays an important role in dictating brewing yeast fermentation performance and product quality. Keywords: Brewing yeast, Pilot-plant fermentors, Saccharomyces cerevisiae, Zinc RESUMEN El efecto del zinc en las clulas de levadura cervecero se ha estudiado en relacin con la absorcin de zinc, el rendimiento de la fermentacin, y la formacin de los congneres sabor. Los experimentos que usan el mosto de malta con suplementos variables de sales de zinc se llevaron a cabo en pequea escala en tanques de forma cnica y en fermentadores en la fbrica piloto para reproducir fermentaciones industriales de cerveza lager. En fermentaciones de pequea escala, zinc fue tomada por completo del mosto por la levadura en las primeras 48 y 96 hr, cuando las concentraciones de zinc en la hierba fue de 1.0 y 4.85 ppm, respectivamente. El zinc tuvo un impacto en el rendimiento de la fermentacin, con la hierba de los niveles de zinc requerido para la fermentacin ptima que van desde 0.48 hasta 1.07 ppm. Estos niveles de zinc inicial correspondi al contenido final de zinc clula de levadura, de 14 y 108 fg/clula, respectivamente. En fermentadores de fbrica piloto, "preacondicionamiento" de la levadura, enriqueciendo las clulas de zinc antes de la fermentacin se beneficiaron el rendimiento de la fermentacin cuando un mosto con una deficiencia de zinc se utiliz. Perfiles de sabor congnere que pareca ser slo afect a los niveles de zinc de alta de 10 ppm, con elevadas concentraciones de alcoholes superiores y steres de algunos (etil acetato de isoamilo y caproato) observ. Llegamos a la conclusin de que el control de la biodisponibilidad del zinc, incluidas las estrategias de suplementacin de Zn (tanto para el mosto y la levadura), juega un papel importante en dictando el rendimiento de fermentacin de la levadura cervecero y la calidad del producto. Palabras claves: Fermentadores de fabrica piloto, Levadura cervecero, Saccharomyces cerevisiae, Zinc

Current address: DSM Nutritional Products, Wurmisweg 576, CH-4002 Basel, Switzerland. 2 Corresponding author. E-mail: g.walker@abertay.ac.uk
doi:10.1094 /ASBCJ-2011-0909-01 2011 American Society of Brewing Chemists, Inc.

Yeast growth and metabolism are influenced by trace elements, particularly by zinc. Zinc is a cofactor in several of the six enzymes classes (30), including alcohol dehydrogenase (ADH), the terminal enzyme of the fermentation pathway. Consequently, zinc plays an essential role in alcohol production (15). This metal also governs protein synthesis (18) and the phospholipid composition of membranes (11) in yeasts, and these processes are strongly diminished at low levels of zinc, resulting in impaired yeast cell growth and fermentation performance. In brewing, the risk of encountering low critical concentrations of zinc in malt wort is very high due to zinc binding and coprecipitation with proteins during the malt wort boiling process (12). Optimal zinc requirements are yeast-strain dependent: in the range of 0.25 to 0.50 g/mL for cell growth and 1 to 2 g/mL for glycolysis (14). Zinc concentrations lower than 0.1 g/mL (ppm) are generally considered too low and may lead to sluggish or stuck fermentations (2,9,13). To prevent such problems, brewers may supplement wort with additional zinc salts during malt boiling (31) or yeast storage. However, information is limited concerning the minimal critical content below which zinc supplementation is necessary. Yeast cells accumulate zinc first by chemically binding it with specific sites on the cell wall and second by active membrane transport. At the transcriptional level, three or more uptake systems are known to control zinc uptake. Under zinc-limited conditions, the protein Zap1 induces expression of the genes ZRT1, ZRT2, and FET4 (8). Subsequently, zinc becomes localized inside the yeast cell vacuole (17), where it is stored together with other metal cations (19,23). De Nicola and Walker (7) described a mechanism of distribution of zinc within a yeast population whereby daughter cells receive part of the zinc previously accumulated (in the vacuole) by mother cells during budding. Consequently, intracellular zinc content is related to the zinc taken up by the yeast cells from the medium and the zinc inherited by the cells during cell division. Therefore, measurement of intracellular zinc content in pitching yeast could be a more meaningful way to determine optimum zinc requirements for fermentation. Few studies have been conducted on the effect of zinc on production of flavor congeners by yeast in potable alcohol fermentations. Skanks et al (26) reported that higher alcohols and esters were elevated but acetaldehyde levels reduced with increasing zinc concentrations. However, zinc supplementation may lead to an increase in the concentration of medium-chain fatty acids such as caproic, caprylic, capric, and lauric acids (33). De Nicola et al (5) found that distillates obtained from fermented malt wort produced by Zn-preconditioned ale and distilling yeast strains had altered ester and higher alcohols profiles. For brewers, it would be useful to define, for each yeast strain employed, the optimal zinc cellular content for best fermentation performance and for producing final beers with the desired aroma and flavor profiles. This study evaluated the impact of variable zinc concentrations in malt wort on zinc accumulation by brewers yeast, fermentation performance, and beer flavor congeners. Experiments were carried out in both laboratory and pilot-plant fermentors. Additionally, cellular zinc levels were measured in a lager yeast strain to determine the zinc contents that resulted in optimal fermentation performance. 214

Zinc and Yeast EXPERIMENTAL Yeast Strains and Growth Media The lager beer yeast strain Saccharomyces pastorianus (carlsbergensis) AL-1 (from the University of Abertay Dundee yeast culture collection) was employed in this study. In small-scale conical vessel fermentations, yeast cells were grown in malt wort prepared with a 25% (wt/vol) suspension of barley malt grist (variety Optique provided by Baird Malt) mashed for 1 hr in preheated deionized water at 65C. After filtration, the malt wort (original gravity [OG] 1.055) was autoclaved at 120C for 20 min, and proteins were separated by aseptic centrifugation. Zinc was removed from the malt wort through bio-chelation as follows: yeast cells were inoculated in the medium at an initial cell density of 5 106 cells/mL and kept in suspension for 1 hr at 25C. This treatment has previously been shown to remove all zinc from the medium (7). Cells were removed after centrifugation at 1,100 g for 5 min 5C, and the supernatant was sterile-filtered (0.45-m cellulose acetate; Whatman) into a sterile and deionized Buchner flask. Such a medium was assumed to be normal in all aspects except measured zinc levels, which were below the detection limit after centrifugation. Prior to yeast pitching, Zn-free wort was pre-aerated for 2 hr at 14C with sterile air. Seed cultures were prepared in malt wort with zinc concentrations of 0.10 ppm. Yeast cells were grown in shake flasks at 25C and 180 rpm for 48 hr and transferred at 14C for 8 hr prior to the onset of experimental fermentations to preadapt the cell population to the fermentation temperature. In pilot-plant fermentation experiments, hopped malt wort with an OG of 1.057 (14P) was aerated prior to inoculation. The dissolved oxygen measured at inoculation was 8 ppm. The measured zinc concentration was below the limit of detection of the atomic absorption spectrophotometer (metal ion analyses are described below) used for zinc determinations (0.10 ppm). The medium without added zinc will be referred to as zinc-free. Yeast cells used as seed cultures were collected from a yeast storage vessel after one fermentation in malt wort with a zinc concentration of 0.5 ppm. Growth Conditions and Sampling Yeast cell seed cultures were inoculated from cryovials and grown in an orbital incubator at 200 rpm and 25C for 24 hr to reach a final cell density of 1.9 108 cells/mL. Cell numbers were determined by bright-field microscopy using a hemacytometer (Neubauer improved type), and yeast viabilities were assessed using methylene violet staining in accordance with the method of Smart et al (27). Mean yeast cell volumes were measured cell counter (Coulter Counter Multisizer, Beckman-Coulter Electronics). Yeast dry weight was assessed according to the following modified method of Postma et al (20). Using a calibrated pipette, 10 mL of culture sample was poured into a preweighed membrane filter (0.45-m cellulose acetate; Whatman) on a Buchner flask with a filtering device connected to a vacuum pump. The preweighted filter was washed once with 10 mL of deionized water and placed in microwave at 360 W for 20 min. The filter was dried in a desiccator for 30 min and weighed. Cell biomass was calculated by difference and measured in grams per liter. Each sample was analyzed in duplicate. Small-scale fermentation experiments were performed in polycarbonate conical Imhoff vessels (Nalgene) with a 1-L volume and 74 cone angle. Fermentation locks were used to minimize the ingress of oxygen. Yeast cells were inoculated into malt wort at 5 106 cells/mL and thoroughly mixed at the onset of fermentation. Experiments were carried out for 264 hr at 14C under static conditions. Samples were taken from the middle of the conical Imhoff vessel (500 mL) without homogenization and using a sterile syringe. Analyses included the number of cells in suspension, size of the

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yeast cone, mean cell volume, viability, zinc concentration in medium and cells, specific gravity, and ethanol concentration. Sampling times were 0, 1, 6, 24, 48, 96, 144, 192, and 264 hr. For brewery pilot-plant experimental fermentations, 200-L stainless-steel fermentors (cone angle between 60 and 70) were used. A system of tubes directly connected to the malt wort preparation vessel facilitated introduction of pre-aerated hopped malt wort into the fermentors. Cells were pitched at approx. 10 106 cells/mL. Inoculation was performed from the top of the fermentor under sterile conditions using an open flame during the malt wort filling process to allow initial homogenous mixing of the yeast cell culture. Fermentation was carried out for 186 hr at 11C and stopped when the specific gravity was stable at 2.5P (OG 1.010) for 48 hr. Samples were taken at 0, 18, 41, 65, 87, 138, 161, and 186 hr. Analyses included cell counts, dry weight, Zn cell content, ethanol, specific gravity, turbidity, and pH. Maintenance of Zinc-free Conditions and Zinc Supplementation Metal cations, including zinc, were removed from glassware, flasks, and conical vessels according to the following washing procedure: overnight soaking in 2% nitric acid, two washes with deionized and distilled water (ddH2O), one wash with 0.1M EDTA, four washes with ddH2O, and final drying (6). Zinc concentrations in conical vessels were altered by adding calculated volumes of a sterile 1,000 ppm zinc acetate stock solution to attain Zn concentrations of 0, 0.05, 0.48, 1.07, 4.85, and 10.8 ppm. A solution of zinc sulfate (1,000 ppm) was used to adjust the zinc concentration in the medium used in the pilot-plant studies. Zinc concentrations were altered to 0.5, 1, 5, and 10 ppm. Previous studies showed that zinc acetate and zinc sulfate did not impact zinc uptake rates by yeast cells (6). Therefore, pilot-plant fermentations were conducted with zinc sulfate as a cost-effective source of zinc for scale-up studies and further process implementation. Metal Ion Analyses Yeast cells were separated from the supernatant and washed three times in deionized water before being hydrolyzed with concentrated nitric acid (69% AnaLar grade; Fisher Scientific) at 90C for 1 hr. Nitric acid was added to malt wort supernatants (dilution 1:1) without high-temperature treatment to hydrolyze malt wort protein-binding metal ions. Metal ions in acid-treated and diluted yeast and wort hydrolysates were analyzed using an atomic absorption spectrophotometer (1100B, Perkin Elmer). Lanthanum chloride was added to a final concentration of 0.2% in samples analyzed for calcium. This addition was necessary to avoid matrix interference due to the presence of phosphates. Triplicate analyses were conducted for each sample. Ethanol Analysis Ethanol production during fermentation was analyzed using a gas chromatograph mass spectrometer (GCMS-QP2010, Shimadzu) fitted with an HP blood alcohol capillary column (0.32 mm i.d., 7.5 m long, and 20-m film; Agilent Technologies). Program conditions were as follows: column temperature: 125C; injector temperature: 250C; split ratio: 20:1; linear velocity: 200 cm/sec; detector temperature: 250C; temperature program: 125C; rate: 15 degrees Celsius/min; and final temperature: 150C. Statistical Analyses All experiments were performed in duplicate, and analyses of experimental samples were carried out in either duplicate or triplicate depending on the experimental conditions. To verify the consistency of zinc concentration during fermentation, zinc levels were

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De Nicola, R., and Walker, G. M. 14.13 fg/cell (0.48 ppm), 108 fg/cell (1.07 ppm), 120.73 fg/cell (4.85 ppm), and 214 fg/cell (10.83 ppm) in cells collected from the yeast cone. It is conceivable that during cell division zinc was shared between mother and daughter cells (7) and that the latter continued to accumulate further zinc from the medium during growth. The time required for complete zinc uptake in small-scale brewing fermentation experiments was longer when compared with shake-flask experiments in which complete removal of zinc occurred within the first hour (7). This may have been due to the lower temperature employed in simulated lager beer fermentations (14C) and heterogeneity in the medium due to the absence of agitation of the conical vessels. Oxygen levels in the medium at the initial stages were high due to oxygenation of the malt wort prior to yeast pitching. Only when cells actively fermented did the generated carbon dioxide contribute to natural mixing of the yeast cell population throughout the growth medium. The resulting cell population heterogeneity may also explain why cells in the yeast cone of zinc-free medium, at the end of fermentation, had a zinc cell content that was higher than at the start of fermentation. Assuming that bigger (and older) cells sediment before younger cells, it is conceivable that their vacuoles contained higher amounts of zinc compared with smaller (and younger) cells. This seems to be consistent with the heterogeneity in cellular age found in various portions of the yeast cone by Powell et al (21). In similar experiments in 2-L static fermentors, Mochaba et al (16) described different patterns that occurred during zinc uptake in malt wort. During the first 4 hr of fermentation, with zinc at 0.75 ppm, all zinc was taken up from the medium, and levels fluctuated during fermentation, reaching maximum intracellular zinc levels at the end of fermentation. After repitching in fresh malt wort, Mochaba et al (16) found that yeast cells released zinc ions back to the medium, followed by subsequent reaccumulation. Different zinc uptake kinetics and high sensitivity to zinc ions probably were due to the yeast strain employed in the experiments described by Mochaba et al (16). In the current study, high zinc content was not accompanied by loss of viability, which remained above 95% in all experimental fermentations in conical vessels (data not shown). The presence of manganese ions may influence the yeast response to variable zinc levels. Initial manganese levels in malt wort were 0.20 ppm, which was half the level of Mn (0.40 ppm) reported by Jones and Greenfield (14) to be necessary to support levels of zinc above 2 ppm. The lager yeast strain used in this experiment may have a high zinc tolerance regardless of available man-

analyzed in cells and supernatant, and a zinc balance was calculated. When comparing the data for different experimental conditions, appropriate statistical tests (e.g., Students t test and ANOVA) were applied. In the figures presented, error bars are provided, when available, to indicate the statistical significance of our observations. RESULTS AND DISCUSSION Studies on Zinc Influence on Brewing Fermentation (Small-Scale Conical Vessels) In simulated small-scale brewing fermentors, lager yeast cells (strain AL-1) were able to take up zinc completely from their growth media, as previously described in shake-flask experiments with the same yeast strain (7). The time required for the complete removal of zinc from the medium depended on the initial zinc concentration. For example, with extracellular zinc at 0.48 ppm, all zinc was taken up within the first 24 hr (Fig. 1). At higher initial zinc concentrations, removal was gradually delayed: removal was complete after 96 hr when the initial zinc was 4.85 ppm and only after 144 hr when the initial zinc was 10.83 ppm (with residual zinc concentrations of 0.3 ppm after 96 hr) (Fig. 1). As a result, the mean zinc content changed from the initial value of 1.22 fg/cell in the inoculated (pitching yeast) cells to final contents of 3.9 fg/cell (0.05 ppm),

Fig. 1. Zinc levels in wort during fermentations with varied initial zinc concentrations. Yeast cells (lager strain AL-1) were pitched in conical Imhoff vessels containing malt worts with altered zinc levels. Fermentations were carried out for 264 hr. Zn residual levels in supernatants were analyzed (P < 0.05) during fermentation with initial wort Zn concentrations ranging from 0 to 10.83 ppm.

Fig. 2. Yeast cone sizes in brewing fermentations with varied zinc concentrations. The size of the yeast cone formed by sedimented (flocculated) cells (lager strain AL-1) in 1-L conical Imhoff vessels was determined at regular intervals during fermentation (P < 0.05). In zinc-free malt wort, yeast cells did not flocculate. This may have been due to the high levels of carbon dioxide still present after 264 hr as a result of the slow fermentation of sugars. For clarity, standard deviations are not reported in the graph.

Fig. 3. Brewing yeast growth during fermentations with varied zinc concentrations. Yeast cells (lager strain AL-1) were sampled from the middle of 1-L conical Imhoff vessels and counted using a hemacytometer to determine, together with yeast cone size analysis, the level of cell growth and flocculation (P < 0.05). Suspended yeast cell numbers in the fermentation with a very low zinc concentration was consistently higher compared with higher zinc concentrations tested. This was likely due to slow fermentation in the zinc-depleted medium.

Zinc and Yeast ganese. Other metals in malt wort may also influence yeast cell zinc tolerance. For example, Bromberg et al (2) found that zinc requirements for brewing fermentations were higher in poor quality malt wort, indicating a possible interaction between trace metals and zinc. Cell growth was largely unaffected by various zinc levels (Figs. 2 and 3). This finding agreed with the work of Jones and Greenfield (14) and Bromberg et al (2). After 264 hr, the number of cells in suspension ranged between 0.77 106 and 1.37 107 at zinc concentrations higher then 0.05 ppm, and the number of cells was 2.25 107 in the fermentation with zinc at 0.05 ppm (Fig. 3). At the same time, yeast cells flocculated at the bottom of the conical vessels, forming a yeast cone. The size of the cone was 15 mL at all zinc concentrations studied, with the exception of Zn at 0.05 ppm, for which the yeast cone was 10 mL (Fig. 2). This was most likely due to turbulence produced by carbon dioxide (still present after 264 hr at very low zinc concentrations) when sugars were still not completely utilized (Fig. 4A). Zinc-free wort did not suppress yeast cell growth when Zn cell content was 3.9 fg/cell, indicating there was sufficient intracellular zinc reserves to support cell division. The various zinc concentration levels tested in this study did not greatly influence yeast flocculation. The uptake of other divalent cations may play a role in this phenomenonnotably, calcium ions. Throughout fermentation, residual concentrations of calcium, magnesium, and manganese were not affected by altering the initial wort zinc concentration (data not shown), which demonstrated the absence of interactions among these cations in binding to the cell surface sites involved in flocculation. Rapid metal ion uptake during fermentation appeared to be specific to zinc. De Nicola (3) previously showed no influence of calcium on zinc uptake by four different industrial yeast strains in malt wort. Taylor and Orton (29)

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demonstrated that yeast flocculation occurred only at zinc concentrations higher than 6,500 ppmwell above the concentrations normally found in malt wort. Raspor et al (24) showed that only some ale strains may flocculate in vitro with zinc at 2.6 ppm. Lager yeast flocculation appeared unaffected by varied zinc concentrations, and this phenomenon may be due to differences in the structural characteristics of cell surfaces compared with ale brewing strains. Zinc concentrations ranging from 0.48 to 1.07 ppm appeared to be optimal in terms of yeast fermentation performance, and this was supported by data for both sugar utilization and ethanol production (Fig. 4). A lesser impact on fermentation rate was shown at higher Zn concentrations (5 and 10 ppm), but after 264 hr, the final ethanol concentration was the same as for zinc concentrations ranging from 0.48 to 1.07 ppm. Yeast cells used as seed cultures in this experiment were grown in low-zinc medium (0.10 ppm) to lower the initial zinc cell content and to better observe the effect of various zinc wort concentrations on fermentation. Cells preconditioned in a medium with more zinc, and thus with a higher initial zinc cell content, performed better in zinc-free medium, as was observed in pilot-plant fermentations (discussed below). Results indicated that final zinc cell contents ranging from 14 to 108 fg/cell were optimal for fermentation performance, corresponding to initial zinc concentrations of 0.48 and 1.07 ppm in malt wort. Initial zinc concentrations between 1.5 and 2.5 ppm were optimal for two wine strains of S. cerevisiae in a similar study (4) with grape juice fermentation medium. This indicates that optimal zinc intracellular contents for industrial fermentations are both strain and medium dependent. Studies on Influence of Zinc on Brewing Pilot-Plant Fermentation In brewing pilot-plant experimental fermentations, yeast cells accumulated higher levels of zinc when it was present in wort at higher initial concentrations, similar to observations in laboratoryscale fermentations. Zinc cell contents at the beginning of fermentations were 8.18 fg/cell and, at the end of fermentation, ranged between 7 and 220 fg/cell from the lowest (0.05 ppm) to highest (10 ppm) initial zinc concentrations tested (data not shown). The high initial zinc content of the propagation culture (8.18 fg/ cell) was approximately seven times higher than in experiments with brewing in small-scale conical vessels (1.22 fg/cell). This level of zinc cellular content was sufficient to sustain good fermentation at all zinc concentrations examined in the pilot-plant study. Fermentation performance remained unchanged, as shown by the analyses (P < 0.05) related to the decline of specific gravity and the concentration of ethanol produced (Fig. 5). It is noteworthy that supple-

Fig. 4. Performance of brewing yeast in fermentations with varied zinc concentrations. Fermentation performance was evaluated in 1-L conical Imhoff vessels by analyzing specific gravity (A) and ethanol (B) at regular intervals. Data were statistically significant (P < 0.05). For clarity, the zinc concentrations of 0.48 and 4.85 ppm were omitted from the graphs.

Fig. 5. Influence of zinc on fermentation performance of brewing yeast in 200-L pilot-plant fermentors. Specific gravity and ethanol production were measured during fermentation to determine the fermentation rate at various initial wort zinc levels. Open circles and squares refer to ethanol concentration. Filled circles and squares refer to wort gravity. Data were statistically significant (P < 0.05).

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De Nicola, R., and Walker, G. M.

TABLE I Influence of Initial Wort Zinc Concentration on Green Beer Congenersa

Compound Acetaldehyde (ethanal) (mg/L) Dimethylsulfide (DMS) (g/L) Acetone (propanone) (mg/L) Ethylformiate (mg/L) Ethylacetate (mg/L) Methanol (mg/L) Ethyl propionate (mg/L) n-Propylalcohol (1-propanol) (mg/L) Iso-butylalcohol (iso-butanol) (mg/L) Iso-amylacetate (mg/L) Amyl alcohols (mg/L) Ethyl caproate (ethylhexanoate) (mg/L) Total higher alcohols (mg/L) Diacetyl (2,3-butanedione) (g/L) 2,3-Pentanedione (g/L)
a

0.05 ppm 7.25 31.90 0.20 0.10 31.00 2.00 0.15 13.86 19.50 4.09 81.00 0.20 114.35 94.20 128.70

0.5 ppm 10.70 32.15 0.20 0.10 27.30 2.01 0.10 12.65 19.35 3.72 77.65 0.20 109.70 90.70 132.70

Initial Wort Zinc Concentration 1 ppm 5 ppm 8.70 32.30 0.21 0.10 30.85 2.18 0.10 12.79 19.60 4.54 79.55 0.20 111.95 100.65 126.60 9.20 32.40 0.20 0.10 29.05 2.10 0.10 12.63 20.20 4.27 79.50 0.20 112.35 99.75 127.75

10 ppm 8.05 32.70 0.19 0.10 32.50 2.01 0.10 13.67 21.20 5.10 83.65 0.25 118.50 114.65 122.15

Data were obtained from experimental pilot-plant fermentations after 192 hr.

mentation with zinc salts during malt wort preparation is not always effective. For example, the practice of adding zinc at the end of wort boiling defeats the purpose, because most of the zinc precipitates with protein and polyphenols during the hot break (31). Therefore, preconditioning of pitching yeast cells with elevated zinc levels during propagation, or even in yeast storage vessels, may circumvent the need to supplement zinc salts at wort collection or in fermentation medium after yeast cells have been inoculated. In a similar vein, Walker et al (34) and Smith and Walker (28) previously showed that preconditioning brewing yeast cells with magnesium enhanced fermentation performance in terms of rate and yield of ethanol produced. pH and turbidity (data not shown) patterns were the same for all tested zinc concentrations, confirming no particular differences in fermentation performance. Similarly, yeast growth did not differ appreciably with the different zinc concentrations studied, and the yeast cone was the same size. Cell viability was 9095% at the end of fermentation (both small-scale and pilot-plant), even at relatively high zinc concentrations, which was similar to results reported by Jones and Greenfield (14) and Bromberg et al (2). With regard to the influence of zinc on green beer flavor congeners, only the wort zinc concentration of 10 ppm resulted in discernable differences in ethyl caproate, isoamyl acetate, iso-butanol, amyl alcohol, diacetyl, and total higher alcohol concentrations (Table I). Ethyl caproate and isoamyl acetate are two esters responsible for apple sour and fruity banana aromas, respectively. Ester synthesis is known to be strain dependent (lager strains more than ale strains) and is influenced by several parameters, such as wort specific gravity, wort nitrogen content, oxygen, wort lipid content, fermentation temperature, activity of several enzymes, and concentration of acetyl-CoA and higher (fusel) alcohols (32). Isoamyl acetate is produced by the esterification of amyl alcohol with acetyl-CoA, and Quilter et al (22) previously observed a continuing increase in amyl alcohol content (approx. 25%), as well as isoamyl acetate, in brewing fermentations when zinc was increased from 0.015 to 0.12 ppm. In contrast, De Nicola et al (5) did not find elevated levels of isoamyl acetate following preconditioning of ale and distilling yeasts with zinc. Zinc can stimulate the production of higher alcohols and, hence, indirectly the production of esters (5,10,26). Primary fermentation of sugars and biosynthesis and catabolism of amino acids are responsible for higher alcohol formation. Therefore, either the higher concentration of amino acids in malt wort or the amino acid uptake rate could indirectly impact levels of higher alcohols in beer. Higher alcohols can also be produced from ketoacids after breakdown in the presence of zinc (25). Iso-bu-

tanol and diacetyl production were stimulated by zinc and are known to impart a desirable warming character and a strong butterscotch or toffee aroma and flavor to beer, respectively (1). Iso-butanol is derived from the branched-chain amino acid valine, the synthesis of which is regulated by the expression of the gene ILV3, which is down regulated when zinc is limited in both aerobic and anaerobic fermentations (6). In addition, when an ale brewing strain of yeast was preconditioned with Zn, De Nicola el al (5) found that iso-butanol production was elevated. Because reduction of acetaldehyde to ethanol by ADH is zinc dependent, zinc deficiencies in wort may lead to excess acetaldehyde production, imparting a grassy, green-apple flavor to beer. However, acetaldehyde levels were unaffected by low wort zinc levels and it appears that yeast zinc cellular contents of 7 fg/cell are sufficient to impart desirable flavor profiles during fermentation. Overall, zinc supplementation during propagation and from previous fermentations was satisfactory for the progress of the fermentations with zinc-free or zinc-supplemented media described in this study. This suggests yeast cells can accumulate zinc to satisfactory levels and then utilize zinc reserves to successfully complete brewing fermentations. CONCLUSIONS In conclusion, this study has highlighted the importance of zinc in brewing yeast physiology and fermentation performance. In simulated brewing fermentors (1-L and 200-L scales), zinc was taken up completely by yeast cells. Wort zinc concentrations ranging from 0.48 to 1.07 ppm appeared best in terms of fermentation performance. Only when wort was supplemented with higher zinc levels did key beer flavor congeners (higher alcohols and some esters) vary appreciably. As an alternative to wort zinc supplementation, preconditioning of pitching yeast with Zn may provide benefits in subsequent fermentations, which may prove of practical value to the brewing industry.
LITERATURE CITED 1. Briggs, D. E., Boulton, C. A., Brookes, P. A., and Stevens. R. Brewing Science and Practice. Woodhead Publishing Limited, Cambridge, 2004. 2. Bromberg, S. K., Bower, P. A., Duncombe, G. R., Fehring, J., Gerber, L., Lau, V. K., and Tata, M. Requirements for Zn, Mn, Ca, and Mg in wort. J. Am. Soc. Brew. Chem. 55:123-128, 1997. 3. De Nicola, R. Interaction of zinc with the yeast Saccharomyces cerevisiae. Ph.D. thesis. University of Abertay Dundee, Dundee, Scotland, 2006.

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