214

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. Experi-ments using malt wort with variable supplements of zinc salts were con-ducted in small-scale conical vessels and in pilot-plant fermentors to repro-duce 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, respec-tively. 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 fermen-tation 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 bioavail-ability, including Zn-supplementation strategies (for both wort and yeast), plays an important role in dictating brewing yeast fermentation perform-ance and product quality.

Keywords: Brewing yeast, Pilot-plant fermentors, Saccharomyces cer-evisiae, 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 pe-quea escala en tanques de forma cnica y en fermentadores en la fbrica piloto para reproducir fermentaciones industriales de cerveza lager. En fer-mentaciones 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 fermen-tadores de fbrica piloto, "preacondicionamiento" de la levadura, enrique-ciendo las clulas de zinc antes de la fermentacin se beneficiaron el rendi-miento de la fermentacin cuando un mosto con una deficiencia de zinc se utiliz. Perfiles de sabor congnere que pareca ser slo afect a los ni-veles de zinc de alta de 10 ppm, con elevadas concentraciones de alcoholes superiores y steres de algunos (etil acetato de isoamilo y caproato) obser-v. 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 fer-mentacin de la levadura cervecero y la calidad del producto.

Palabras claves: Fermentadores de fabrica piloto, Levadura cervecero, Saccharomyces cerevisiae, Zinc

Yeast growth and metabolism are influenced by trace elements, particularly by zinc. Zinc is a cofactor in several of the six en-zymes 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 di-minished 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 copre-cipitation 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 glyco-lysis (14). Zinc concentrations lower than 0.1 g/mL (ppm) are generally considered too low and may lead to sluggish or stuck fer-mentations (2,9,13). To prevent such problems, brewers may sup-plement wort with additional zinc salts during malt boiling (31) or yeast storage. However, information is limited concerning the mini-mal 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 trans-port. 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 ca-tions (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 vacu-ole) 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 pro-duction of flavor congeners by yeast in potable alcohol fermenta-tions. 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 in-crease 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. Ad-ditionally, cellular zinc levels were measured in a lager yeast strain to determine the zinc contents that resulted in optimal fermentation performance.

1 Current address: DSM Nutritional Products, Wurmisweg 576, CH-4002 Basel, Swit-zerland.

2 Corresponding author. E-mail: g.walker@abertay.ac.uk

doi:10.1094 /ASBCJ-2011-0909-01 2011 American Society of Brewing Chemists, Inc.

Zinc and Yeast / 215

EXPERIMENTAL

Yeast Strains and Growth Media The lager beer yeast strain Saccharomyces pastorianus (carls-

bergensis) AL-1 (from the University of Abertay Dundee yeast cul-ture 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 Op-tique provided by Baird Malt) mashed for 1 hr in preheated deion-ized 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-fil-tered (0.45-m cellulose acetate; Whatman) into a sterile and de-ionized 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 concentra-tions 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 popula-tion 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 dis-solved 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 be-low) used for zinc determinations (0.10 ppm). The medium with-out 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 deter-mined by bright-field microscopy using a hemacytometer (Neu-bauer 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 meth-od 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 de-vice connected to a vacuum pump. The preweighted filter was washed once with 10 mL of deionized water and placed in micro-wave 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 poly-carbonate conical Imhoff vessels (Nalgene) with a 1-L volume and 74 cone angle. Fermentation locks were used to minimize the in-gress 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 con-ditions.

Samples were taken from the middle of the conical Imhoff ves-sel (500 mL) without homogenization and using a sterile syringe. Analyses included the number of cells in suspension, size of the

yeast cone, mean cell volume, viability, zinc concentration in me-dium and cells, specific gravity, and ethanol concentration. Sam-pling times were 0, 1, 6, 24, 48, 96, 144, 192, and 264 hr.

For brewery pilot-plant experimental fermentations, 200-L stain-less-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 cul-ture. 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. Analy-ses included cell counts, dry weight, Zn cell content, ethanol, spe-cific 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 pro-cedure: overnight soaking in 2% nitric acid, two washes with de-ionized 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 solu-tion 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 ad-just 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 implemen-tation.

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) with-out high-temperature treatment to hydrolyze malt wort protein-bind-ing metal ions.

Metal ions in acid-treated and diluted yeast and wort hydroly-sates 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 ad-dition was necessary to avoid matrix interference due to the pres-ence 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) fit-ted 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 tem-perature: 250C; temperature program: 125C; rate: 15 degrees Cel-sius/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 trip-licate depending on the experimental conditions. To verify the con-sistency of zinc concentration during fermentation, zinc levels were

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

analyzed in cells and supernatant, and a zinc balance was calcu-lated. When comparing the data for different experimental condi-tions, 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 concentra-tions, 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 concentra-tions 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 inocu-lated (pitching yeast) cells to final contents of 3.9 fg/cell (0.05 ppm),

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 oc-curred 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 agi-tation 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 gen-erated 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 fermen-tation, had a zinc cell content that was higher than at the start of fermentation. Assuming that bigger (and older) cells sediment be-fore 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 fluc-tuated 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. Dif-ferent zinc uptake kinetics and high sensitivity to zinc ions prob-ably were due to the yeast strain employed in the experiments de-scribed 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 ex-periment may have a high zinc tolerance regardless of available man-

Fig. 1. Zinc levels in wort during fermentations with varied initial zinc con-centrations. Yeast cells (lager strain AL-1) were pitched in conical Imhoffvessels containing malt worts with altered zinc levels. Fermentations were carried out for 264 hr. Zn residual levels in supernatants were analyzed (P