Zinc

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Zinc is one mineral that is often limited in wort. Zinc is important in the yeast cell cycle (reproduction), and is a cofactor for alcohol dehydrogenase, the enzyme responsible for alcohol production. Supplementation of zinc generally has the effect of speeding up fermentation, as well as preventing stuck fermentations.[1]

Natural zinc levels in drinking water are normally less than 1 ppm, typically near 0.05 ppm. An astringent taste can be noted near 5 ppm. Zinc is a vital yeast nutrient and recommended levels in wort for optimum fermentation are 0.1-0.5 ppm. Concentrations greater than 0.5 ppm can cause over-activity and off-flavors in beer.[2]

Zinc is a trace element of great physiological importance for proteins synthesis, cell growth of the yeast, and thus fermentation. The fermentation proceeds slowly in the case of zinc deficiency because yeast propagation is retarded, which leads to incomplete reduction of diacetyl. It is there for advantageous to transfer the zinc that is present in malt to the wort as completely as possible. Only about 20% of the zinc in the malt goes into solution during mashing in. The zinc content decreases further during the course of mashing. If a threshold of 0.10 to 0.15 mg zinc per liter is not reached the aforementioned fermentation difficulties may occur.[3]

Factors that increase zinc:[3]

  • Low pH value
  • Low dough-in temperature
  • Grist to mash water ratio of 1 to 2.5

Zinc may be supplemented directly by adding a zinc salt (e.g. zinc chloride).

Zinc may also be supplemented in accordance with the "purity law" by mixing spent grain with your "sauergut" (biological acidification) at a ratio of 1:1 for at least 24 hours.[3]

Zinc is an essential trace element for the yeast and should be available in extents of about 0.1–0.2 mg/L.[4]

Zinc (Zn2+, at. wt. 65.4), if present in appreciable amounts in brewing water, usually indicates that this ion has been picked up during transfer or storage. High concentrations in ground waters are unusual. At high levels this substance can be toxic, the upper permitted concentration in potable water is 5 mg/l. High concentrations are damaging to yeasts but small amounts are essential. Not infrequently the levels of zinc in worts are insufficient to maintain good fermentations and in these cases the worts may be supplemented with additions of zinc chloride (0.15–0.2 mg/l). The recommended range in brewing liquor is 0.15–0.5 mg/litre.[5]

Zinc is the one substance needed for fermentation that may be deficient in wort. Less than 5% of the zinc present in the grist is dissolved during mashing, and the proportions dissolved can be very variable. Of the zinc present in the wort only a proportion is available to yeast, presumably because the remainder is chelated or otherwise bound to other substances. Consequently simple analyses of wort zinc contents are unreliable for predicting zinc deficiency. Probably a 12% wort should contain at least 0.08 and preferably 0.1–0.2 mg/L Zn to ensure a good fermentation. Where permitted, traces of a soluble zinc salt may be added to the wort. Where this is not permitted the use of well-modified malts and carefully acidified mashes reduce problems of zinc deficiency, as does the use of mashing equipment with metal components from which traces of zinc can dissolve.[5]

Most yeast strains require 0.1 to 0.2 mg/L of Zn2+ ions for effective fermentation.5,36 Zn2+ is essential for the structure and function of many enzymes, where it can be involved in the active site (zinc-metalloenzymes); alcohol dehydrogenase is an example.23 However, Zn2+ can inhibit yeast growth and fermentation at higher concentrations (greater than 0.6 mg/L), if wort contains less than 0.1 mg/L Mn2+ ions, but this inhibition is relieved by the addition of 0.6 mg/L of Mn2+ ions.37 Zn2+ may stimulate H2S production at levels of around 1.0 mg/L.38[6]

A lack of zinc in the pitching wort can manifest itself through poor yeast growth, slow main and secondary fermentation and an incomplete reduction in diacetyl or its precursor, 2-acetolactate. The malt contains 3–3.5 mg / 100 g dry matter: the zinc concentration is highest in the outer layers of the grain (husks, aleuron). During mashing, only 20-25% of the zinc content goes into solution during mashing. From here on, the zinc content is continuously reduced to values of 0.05–0.20 mg / l in the lauter wort. Another decimation occurs when the wort is boiled. Falling below the threshold of approx. 0.15 mg / l can cause the difficulties mentioned above.[7]

Tests have shown that the following parameters can positively influence the zinc content: Mashing at 45–50 ° C. Rest 30-60 min, pH 5.45. Use a small main pour when mashing in (1:2.5), brewing to 1: 4 with hot water as the temperature increases.[7]

Cu and Zn were present in the final beer product with an average value of 0.27 ± 0.09 mg/L and 0.30 ± 0.08 mg/L respectively. The average Se content was 8 ± 4.0 μg/L. The standard error was very high because the content of these elements in the three tested brands was variable.[8]

Zn2+ is a necessary metal ion for the growth and development of yeast in the process of beer brewing, helping to improve the fermentation speed of yeast the flavor of beer (Nascentes, Fernandes, Nogueira, & Nóbrega, 2005). After the addition of Zn2+, the content of SO2 in the wort has been found to increase significantly.[9]

For metal ion content (concentration in the sparged worts compared to the first worts), a progressive reduction could be seen. Zinc and manganese showed substantial displacement from the spent grains to the sparged worts. For zinc, the extent was mainly dependent on the mash pH. On average, over all the chelator trials, the first and second sparges of the pH 5 mashes had 121% and 82% of the Zn content of the first wort, respectively. In comparison, grains mashed at pH 6 contained only 86% and 56%.[10]

Depending on the malts used, a standard wort has levels of around 100-5000 μg/L zinc.[10]

It has long been known that zinc has a crucial role in wort fermentation and Zn is the most commonly utilized supplement in the brewing industry. Brewery wort can contain between 0.1 and 5.0 ppm Zn and concentrations are dependent on a number of factors including the malt used97, wort preparation and composition94,95,111,120. Wort Zn concentration represents only a fraction of the Zn present in malt and most Zn is retained with the used grains11 or is lost through protein precipitation and wort clarification94,120. In wort, Zn may be rendered unavailable for biological use through its association with amino acids94. Even exogenously supplied Zn may have limited availability depending on when it is added to wort, with addition at time of pitching appearing to be optimal203. Consequently, worts are commonly Zn-deficient, a factor which in the past may have been ameliorated by the presence of galvanised components within the brewery vessels and pipes. The predominance of stainless steel in the modern brewery and ‘dilution’ of all-malt worts with sugar adjuncts results in an increased requirement for Zn supplementation. Zn has multiple roles to play in yeast biology, acting as a structural or catalytic co-factor for a number of proteins, including enzymes associated with glycolysis and alcohol synthesis56,130. Zn deficiency results in the differential regulation of tens of genes in both shake flasks85,130 and chemostat cultures54 and approximately 3% of the yeast proteome requires Zn to function normally65. Interestingly, De Nicola and co-workers54 found that the genes whose transcription were regulated by Zn availability had varied biological roles, including the metabolism of storage carbohydrates and the biogenesis of mitochondria, as well as flavour development through the regulation of branched chain amino acid synthesising genes. Zn also has an important role in processes involving Zn-finger DNA-binding proteins25 such as Msn2p and Msn4p, which regulate the general stress response135. Furthermore, Zn appears to play a role in maintaining the redox balance within cells and its limitation can result in overproduction of reactive oxygen species (ROS), potentially resulting in DNA damage, necessitating an antioxidant response in the yeast cell229. The importance of Zn during brewery fermentation is reflected in the relatively rapid uptake by yeast cells in the first hours of fermentation31,53,55,122,165. Zn is first adsorbed to the cell wall and subsequently imported into the cell, where it accumulates in the vacuole55. Wort Zn levels are variable but a value of 0.2 ppm Zn results in optimum fermentation time in standard gravity (ca. 12°P) worts31,84,93, and values lower than 0.1 ppm typically result in sub-optimal fermentation. Zn supplementation increases yeast growth132 and improves fermentation rates31,93,39,189,214. A number of factors are, however, known to influence the requirement of yeast for Zn during fermentation. Helin and Slaughter84 found, for example, that the requirement for Zn was influenced by the concentration of Mn ions in the wort, with 0.6 ppm Zn having an inhibitory effect on fermentation when wort Mn levels were low (<0.01 ppm). Brewing yeast performance is, however, known to be unaffected by Zn concentrations as high as 500 ppm71. Similarly high levels of Zn tolerance have been observed in other studies and have also been associated with Mn availability102,173. Rees and Stewart173 reported that viability of yeast strains was in many cases not decreased in the presence of 65.5 ppm wort Zn, and a concentration of 327.5 ppm did not affect viability significantly in some strains. The same authors noted that increasing the Zn content of the fermentation wort led to reduced attenuation time, increased uptake of fermentable trisaccharides and increased ethanol production by lager strains173. An improvement in fermentation performance was generally seen up to 65 ppm, though in some cases a Zn concentration as high as 1,300 ppm was found to have a positive effect on fermentation. Generally, lager strains were better able to cope with excessive levels of Zn than were ale strains, though strain-specific differences were also observed within the lager strain group. Interestingly, the toxic effects of excess Zn were less pronounced at very high gravity (20°P), possibly due to a lower level of Zn in the original wort or the availability of Mn173. Ion-ion interactions are not however limited to Mn and other metal ions including those of calcium, magnesium, potassium and sodium have also been found to influence the effect of Zn on yeast fermentation performance39,214. These effects may be related to the ions’ competition for binding sites on chelating molecules such as wort amino acids and peptides. While Zn can have a toxic effect on yeast, the concentrations found naturally in worts are unlikely to be high enough for this to occur. Rather, wort Zn may act as a stress protectant. Improvement in ethanol production with increasing Zn supplementation39,173 may suggest that Zn has a role in protecting cells against ethanol toxicity. Addition of Zn to growth media has been shown to protect yeast cells against subsequent ethanol shock in the form of a 30-minute exposure to an 18% (v/v) ethanol solution, with a value of 0.02 g/L Zn sulphate (8 ppm) being optimal230. This result was confirmed in further studies that showed that ethanol tolerance and synthesis were improved in Zn-supplemented media and that the greater resistance to ethanol was associated with increased production of trehalose and ergosterol234, which are known to protect membranes against ethanol-induced damage118,134. The fact that the ethanol-resistant cultures were also resistant to heat shock supports the idea that resistance was brought about through membrane protection, as both parameters are known to affect membranes in a similar manner and membrane integrity is a main determinant of heat and ethanol stress resistance78,164. The mechanism by which Zn protects cells from ethanol toxicity is not fully understood, but could conceivably involve direct interaction with cellular membranes21 and alteration of membrane fluidity (i.e., rigidification of the membrane to offset the fluidifying effect of ethanol). The antioxidant properties of Zn are well-known, particularly in relation to oxidative stress in animal cells, with possible antioxidant mechanisms including protection of sulfhydryl groups from oxidation, prevention of hydroxyl radical generation through antagonistic effects on Fe and Cu ions and support of proper functioning of the antioxidant enzyme Cu/Zn superoxide dismutase166,216. Zn deficiency in yeast cells is also known to result in oxidative stress through the intracellular production of ROS and Wu et al.229 have shown that the TSA1 gene, encoding cytosolic peroxidase, is activated under Zn-deficient conditions. Zn supplementation was also required to repair growth defects observed in a Δtsa1 mutant strain229. The antioxidative properties of Zn may be particularly important during higher gravity fermentation, as ethanol is known to elicit an antioxidant response in yeast4,215 and yeast strains with a compromised antioxidant potential are known to be hypersensitive to ethanol stress45,46,160. The importance of antioxidants in protecting cells against ethanol toxicity is also shown by the fact that Mn superoxide dismutase has an important role in protecting cells against ethanol toxicity, most likely due to the location of this antioxidant enzyme in mitochondria, the main source of ROS in cells80. ROS production in response to ethanol has even been observed under hypoxic conditions115, suggesting the possibility of similar reactions occurring during brewery fermentation. While in vitro studies have identified effects of Zn on yeast flocculation properties, it is unlikely that these effects will be apparent during wort fermentation. Taylor and Orton207 have, for example, noted that 0.1 M Zn chloride (6,540 ppm Zn) inhibits flocculation in a buffer solution at pH 7.6. Raspor and co-workers170 have observed enhanced flocculation up to a concentration of 2.6 ppm, with reduced flocculation occurring thereafter, though this was only observed with Saccharomyces cerevisiae and S. diastaticus; no effect whatsoever was observed with a lager strain. Conversely, Wackerbauer et al.220 found that propagation of yeast with Zn supplements subsequently led to enhanced flocculation during fermentation, most probably as a result of increased growth rate and more rapid depletion of sugars from the system. Given the relatively low concentrations of naturally-occurring Zn in wort and its low level of redox activity compared with other metal ions such as Cu and Fe236, it is unlikely that Zn ions will have significant direct effects on beer flavour. The effect of Zn supplementation (up to 0.5 ppm) on yeast metabolism does however lead to significant changes in the synthesis of higher alcohols and esters126,168,187,189. Zn supplementation has been found to result in increased synthesis of isobutanol126,187 and amyl alcohols126,168, including isopentanol187 and 3-methyl-1- butanol189. Ester synthesis is also stimulated by exposure to increased Zn concentrations, with increased concentrations of ethyl butyrate, isobutyl acetate, isopentyl acetate, ethyl hexanoate187, ethyl acetate and isoamyl acetate189,168 observed in fermented wort. Seaton et al.187 attributed increased ester synthesis to changes in carboxylation, while Skands et al.189 suggested that increased higher alcohol synthesis (and hence increased ester synthesis) was related to higher alcohol dehydrogenase activity, a result which would also explain the reduced levels of acetaldehyde observed in that study. Similar results have been observed when cells have been pre-conditioned to have higher concentrations of cellular Zn53. In a study to determine the effect of Zn pre-conditioning on flavour attributes of distillates produced from fermented distillery malt worts, De Nicola et al.53 found a general increase in higher alcohols, though ethanol concentration was reduced by up to 1.8%. It is not clear why increased cellular Zn levels do not promote ethanol production by the Zndependent alcohol dehydrogenase. In the same study total ester levels were decreased by 25 or 38%, depending on yeast strain used. Considering the relationship between amino acids and ester synthesis, it is also possible that the reduced amino acid uptake associated with Zn deficiency126 will also result in altered flavour profiles. If extra Zn supplementation is to be carried out in an effort to improve yeast ethanol tolerance or productivity during higher gravity fermentations, the potential effects on ester synthesis and overall flavour profile of the finished product must naturally be considered. The available evidence suggests that the influence of Zn on foam properties is, like the influence on flavour, indirect. Evans and Sheehan68 reported an unpublished study by Evans and Stewart, which showed a positive correlation between the concentrations of various metal ions (including Zn) in malt and foam stability. It was suggested that the effect may be due to an alteration of malt protein content or an increase in wort Zn content (and yeast vitality) with a subsequent reduction in the release of foamnegative molecules such as proteases. The effect of metal ions on foam stability was found to be related to the malt ion content and not the beer ion content in that study, suggesting that ions were not stabilising foam through crosslinking with iso-α-acids89.[11]

In recent years there has been a move towards development of yeast foods to correct specific wort deficiencies. Zn deficiency has, for example, been considered in the formulation of a supplement consisting of Zn-enriched intact yeast cells. These enriched cells can enhance fermentation performance over and above the effects seen with Zn salts alone69 or other Zn-containing yeast foods217. This approach allows for Zn-supplementation of worts where legislation restricts the use of all but the basic ingredients in the production of beer.[11]

Zinc is extremely important for yeast growth, metabolism and fermentation. However, it is only present at low concentrations in malt wort, usually at sub-ppm levels. Only part of the Zn present in grains is transferred to wort during brewing. The majority remains in the spent grains, precipitates with proteins and is removed during wort clarification. As a result, wort is frequently deficient in zinc (De Nicola & Walker, 2009; Gibson, 2011). Zinc is a cofactor for almost 300 enzymes, including those involved in glycolysis and alcohol synthesis (Tenge, 2009; Udeh et al., 2014). Zinc is also a stress protectant against ethanol toxicity and heat shock (Zhao et al., 2009). Zinc has been shown to affect processes associated with Zn-finger DNA-binding proteins, which regulate the stress response (Zhao & Bai, 2012). Moreover, zinc has a role in maintaining the redox balance within yeast cells (Gibson, 2011). Brewing wort is often supplemented with zinc. In a study by Bromberg et al. (1997), an increase in zinc concentration from 0.02 to 0.05 mg/L allowed to shorten beer fermentation time by more than 5 days. According to these authors, zinc concentrations in wort between 0.1 and 0.15 mg/L give maximum fermentation performance. Jacobsen et al. (1981) observed a steady growth in fermentation rate for wort with zinc content in the range of 0.05–0.3 mg/L. An elevated level of zinc in wort (0.6 mg/L) can be toxic to yeast when a manganese content is low (<0.01 mg/L) (Helin & Slaughter, 1977). However, a too high concentration of manganese is not desired since manganese catalyses oxidative staling reactions and has a detrimental effect on beer flavour stability.[12]

Wort zinc concentration can ber very low (e.g. 0.01 mg/L). the minimum level of zinc needed to obtain maximum fermentation performance was between 0.1 and 0.15 mg/L of Zn ++ (Fig. 2). Levels considerably less than 0.1 mg/L gave excessively long fermentations. when repitching yeast from a low level zinc wort to a higher level zinc wort, or vice versa, the full effect is not seen immediately. There is evidence that there are interactions between the trace metals, and that a requirement for another trace metal in the poor quality malt could be offset by additional zinc (10). This may be expressed as a requirement for more zinc in a wort made with poor quality malt to obtain the same fermentation performance as with a better quality malt. There is no correlation between zinc concentration and cell growth. Thus, zinc increases the specific fermentation rate of each cell.[13]

zinc is an essential micronutrient for yeast and occasionally brewer’s wort may be Zn-deficient, resulting in impaired fermentation performance (Densky et al, 1966; Desmartez, 1993; Bromberg et al, 1997; Stehlik-Thomas et al, 1997; Rees and Stewart, 1998). This phenomenon, which can lead to slow, or socalled “sluggish”, fermentations in breweries, is yeast strain-dependent but may encountered when wort zinc levels are generally below 0.1ppm. Zinc plays a major role in yeast fermentative metabolism not only because it is essential for ethanol dehydrogenase activity (the terminal Zn-metalloenzyme in alcoholic fermentation – see Magonet et al, 1992), but also because it can stimulate uptake of maltose and maltotriose into brewing yeast cells, thereby augmenting fermentation rates. Furthermore, elucidation of possible effects of zinc on cell membrane stability and dynamics may define further fermentation improvements. In some instances zinc bioavailability may be limiting due to decrease of zinc levels during mashing, lautering and boiling through complexing in precipitated trub.[14]

Results shown in Fig 2 indicate that zinc availability significantly affected fermentation performance. Additionally, we found that even at very high Zn levels studied (23ppm) zinc was not toxic to the yeast cells. The lack of toxicity may have been due to presence of manganese ions, although Mn levels in our wort (0.20ppm) were half of that previously reported to be required for yeast to tolerate levels of zinc above 2 ppm (Jones and Greenfield 1984). This level may be strain dependent and the lager strain used in this experiment may have high zinc tolerance.[14]

Zinc accumulation was very rapid and zinc in the media became depleted within the first hour or so of fermentation. From this data, it appears that yeast demand for zinc is very high and immediate during the initial stages of fermentation.[14] At higher concentrations, zinc accumulation was slower and 1-2 hours were required to uptake all zinc from wort. Uptake is slower at lower temperatures. Eventually, all zinc is taken up, irrespective of fermentation temperature.

In industry, zinc supplementations (if required) may be more conveniently applied at beginning of fermentation, when sources of energy are available for active zinc translocation across yeast membrane.[14] Physiologically speaking, the best time proposed for any zinc supplementations would be at pitching, where bioavailability would be higher.

We have also found that dead yeast cells (killed by treatment at 65°C for 1 hour) were unable to actively uptake zinc.[14]

the practice of removal of part of the yeast cone may deplete yeast biomass of an important reservoir of zinc.[14]

Zinc is found in trace amounts in wort. It is of great physiological importance for protein synthesis and yeast cell growth and thus for fermentation. If zinc is deficient, yeast growth is retarded, fermentation proceeds slowly and there is incomplete reduction of diacetyl. During the brewing process a higher zinc content can be favored by low pH, low mashing temperature, and a grist:liquor ratio of 1:2.5 (Kunze, 2004). However, only about 20% of the zinc in the malt goes into solution on mashing and the zinc content decreases further during mashing. If zinc levels of 0.10–0.15 mg/l are not maintained, the above-mentioned fermentation difficulties may occur because zinc is bound by a number of enzymes, like alcohol, dehydrogenase, and regulatory proteins (Bromber et al., 1997; Kunze, 2004). To compensate for zinc deficiency, zinc chloride, or zinc sulfate can be added to the wort, but this is not permitted in all countries. Zinc is present in beer at a concentration of 0.02–4.5 ppm.[15]

Literature to review:

References[edit]

  1. ↑ White C. Yeast nutrients make fermentations better. White Labs. Accessed 2020.
  2. ↑ water ch3
  3. ↑ a b c Kunze p. 233
  4. ↑ Pahl R, Meyer B, Biurrun R. Wort and Wort Quality Parameters. In: Bamforth CW, ed. Brewing Materials and Processes: A Practical Approach to Beer Excellence. Academic Press; 2016.
  5. ↑ a b Briggs DE, Boulton CA, Brookes PA, Stevens R. Brewing Science and Practice. Woodhead Publishing Limited and CRC Press LLC; 2004.
  6. ↑ Taylor DG. Water. In: Stewart GG, Russell I, Anstruther A, eds. Handbook of Brewing. 3rd ed. CRC Press; 2017.
  7. ↑ a b Narziss L, Back W, Gastl M, Zarnkow M. Abriss der Bierbrauerei. 8th ed. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA; 2017.
  8. ↑ Fantozzi P, Montanari L, Mancini F, et al. In vitro antioxidant capacity from wort to beer. LWT - Food Sci Technol. 1998;31(3):221–227.
  9. ↑ Yang D, Gao X. Research progress on the antioxidant biological activity of beer and strategy for applications. Trends Food Sci Technol. 2021;110:754-764.
  10. ↑ a b Mertens T, Kunz T, Wietstock PC, Methner FJ. Complexation of transition metals by chelators added during mashing and impact on beer stability. J Inst Brew. 2021;127(4):345–357.
  11. ↑ a b Gibson BR. 125th anniversary review: improvement of higher gravity brewery fermentation via wort enrichment and supplementation. J Inst Brew. 2011;117(3):268–284.
  12. ↑ Kordialik‐Bogacka E, Bogdan P, Ciosek A. Effects of quinoa and amaranth on zinc, magnesium and calcium content in beer wort. Int J Food Sci Technol. 2019;54(5):1706–1712.
  13. ↑ Bromberg SK, Bower PA, Duncombe GR, et al. Requirements for zinc, manganese, calcium, and magnesium in wort. J Am Soc Brew Chem. 1997;55(3):123–128.
  14. ↑ a b c d e f Walker G, De Nicola R, Anthony S, Learmonth R. Yeast-metal interactions: impact on brewing and distilling fermentations. In: Proceedings of the Institute of Brewing & Distilling Asia Pacific Section 2006 Convention. 2006.
  15. ↑ Montanari L, Mayer H, Marconi O, Fantozzi P. Chapter 34: Minerals in beer. In: Preedy VR, ed. Beer in Health and Disease Prevention. Academic Press; 2009:359–365.
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