A very common question we hear is, “Malt has antioxidants?!”

Malt is a natural product, and like many natural products, it contains naturally occurring antioxidants. Here is a list of them and what they do.

In order to get an understanding of the possible pathways for Enzymatic and Non-Enzymatic consumption of Oxygen, as well as thier comparitive differences, let’s dig into some key references in scientific and technical literature.

Enzymatic Consumption of Oxygen

Bamforth et al [16] presented an estimation of the significance of thiol oxidase in the consumption of thiol groups and therefore oxygen in mashing, based on a measured specific activity for the enzyme extract at pH 5.0 of 0.015 μmol cysteine oxidized per mg of protein when measured at 30°C. At a protein concentration of approx. 4 mg/mL in a mash, the thiol oxidase potential would be of the order of 0.06 μmol/mL/min. Assuming that 65% of the activity survives mashing at 65°C and assuming that the reaction occurs two times faster for each rise in temperature of 10 degrees Celsius, then Bamforth et al [16] suggested a potential thiol oxidation rate of 0.5 μmol/mL/min in mashing. Stephenson et al [3] measured thiol levels in mashes on the order of 0.05 μmol/mL. Thus it was inferred that the enzyme potential exceeds the amount of substrate available and that it will not be the enzyme but rather the sunbstrate that will be in limiting quantities. It begs stressing that there is a decrease in the level of thiol oxidase in malt during post-kiln storage [16]. Indeed it was suggested that this event forms the scientific explanation for storing malt prior to mashing: the theory is that thiol oxidase oxidizes the amino acid cysteine (cys-SH), with the product cys-S-S-cys in turn oxidizing the sulfhydryl groups in gel proteins with the resultant cross-linking contributing to teig formation and the ensuing reduction in rates of lautering.

Kanauchi et al [15] made a similar evaluation for oxalate oxidase but concluded that this enzyme is far less relevant than thiol oxidase in scavenging oxygen from mashes, calculating that oxalate oxidase could remove all of the oxygen in 20 minutes at conversion temperature whereas the thiol oxidase would in theory eliminate oxygen instantly if there is sufficient of the other substrate. Considering ascorbate oxidase [17], we measured a level of 5 units per g malt. One unit oxidizes 1 mM substrate (ascorbate or oxygen) per minute at 25°C. This is measured in the assay at pH 7 but activity is only 40% of this level at typical mashing pHs. The enzyme (there are two isoforms) is rather heat tolerant and
50% will survive 30 minutes of mashing at 70°C.

Based on an approximation that the enzyme will work 10 x faster at conversion temperature (according to Arrhenius) and assuming a water to grist ratio of 3:1 then we can see that the ascorbate oxidase level in a mash is going to be capable of removing oxygen at a rate a little higher than 5 mM per minute. Compare this with an estimate that a mash might contain less than 0.1 mM oxygen. I.e. if there is sufficient ascorbic acid, then this enzyme alone will have a voracious appetite for oxygen. The Km for ascorbic acid of this enzyme is 0.35 mM for one isoform and 3.25 mM for the other. Taking the first value then a concentration of 5 mM ascorbic acid would saturate the enzyme and the latter would be functioning at its maximum rate.

Kaukovirta-Norja and colleagues [18] in studying lipoxygenase in model mashes showed that significant loss of oxygen occurred only if additional linoleic acid was introduced. This confirmed the prediction of Biawa and Bamforth [13] that the level of linoleic acid in a mash was insufficient to support activity of lipoxygenase.

Non-Enzymatic Consumption of Oxygen

Of course, oxygen may also enter into non-enzymic reactions in the mash. Amongst these the reactions likely to be of most relevance are of oxygen with: Metal ions, notably iron, copper and manganese -thereby setting in train the production of reactive oxygen species [ROS, 19].
Polyphenols [20]
Thiols [11]

The Comparative Significance of Enzymatic and Non-Enzymatic Consumption of Oxygen in Mashes.

The question is begged, therefore, is it enzyme catalysis or non-enzymic reactions that primarily remove oxygen from mashes? Bamforth [12] calculated that the theoretical rate of the iron catalyzed conversion of oxygen to superoxide in a mash is 3.2mM per second. Comparing this to the rate of oxygen consumption estimated for ascorbate oxidase (above) then we can see that the potential for the non-enzymic removal of oxygen is perhaps an order of magnitude greater than the enzymic approach. However by their very nature these calculations can only be an approximation. The very fact that we can demonstrate the very real benefit of ascorbate additions to a mash in terms of protecting against oxidation [17] indicates that this enzyme-catalysed reaction is capable of scavenging at least a proportion of the oxygen. The estimate for the iron catalysed consumption of oxygen was based on an iron level of 0.1 mg/L, which is likely to be vastly higher than is present in the mash. Furthermore the estimate is based on the assumption that there is unrestricted access of the iron to oxygen, which is highly unlikely to be the case, for the majority of any iron in the mash is likely to be adsorbed within particles and/ or chelated. The probability, therefore, is that the non-enzymic consumption of oxygen is actually occurring at a substantially lower rate than that theorised previously [12].

There is an oxidation of precursors originating in the grist, with unsaturated fatty acids claimed by many to be of most significance [5]. Such oxidation may be enzyme-catalysed [lipoxygenase, 6] or non-enzymic, effected through reactive oxygenspecies [7].

It is argued that the staling molecules become attached to binding agents as adducts, notably amine groups in polypeptides [8], these adducts emerging into the finished beer and progressively releasing the staling compounds overtime. Indeed, some argue that these oxidation and binding reactions occur in malting, hence the advent of lipoxygenase free malts [9].

There is a removal through oxidizing reactions of antioxidant molecules, notably polyphenols, thereby lessening the antioxidant potential of the finished beer [10].

Oxygen is consumed in certain reactions in the mash with the
production of oxidized intermediates and these are the agents
that carry the oxidizing potential into the finished beer, where they exert their effect through oxidizing precursors in the production of off flavors [11].

What Does It All Mean?

We have long said that ascorbate oxidase (also referred to as ascorbic acid oxidase) has been a key molecule for not only its strong antioxidant properties, but also primarily as a fundamental FLAVOR component in our beers. We have hypothesized that the preservation of this molecule is an essential component of lingering fresh grain flavor, or “it” if you will. We also know from the previously released blog post on hops that preserving the hop antioxidants will help you not only make the brightest and best tasting beer possible, but also help to preserve that flavor and aroma for much longer.

Malt antioxidants tend to be talked about as a staling mechanisms however, and the problem I think most people have with that concept, is that it’s assumed to be all about finished (and thus packaged) beer, much like oxidation is always assumed to only consist of Stage B (catty, cloying caramel/toffee/sweet, etc.) and Stage C, or classic stale flavors (cardboard, sherry, etc.), which we clarified here. Yet what many low oxygen brewers have experienced, and actually seen, is that it literally happens near instantaneously in the mash before our eyes. Malt aroma during mashing are these very antioxidants being expelled into the open air of the brewery, thereby changing the beer flavor and eliminating the lingering fresh grain flavors we are trying to preserve.

What Can We Do?

As with all the blog posts we put out to the general brewing public, we try and break down how, as small to medium scale brewers, we can achieve the best results possible in our brewery by utilizing direct, or indirect in some cases, adaptation of the findings from scientific and technical literature, as well as applications from larger, commercial brewing operations.

This is no different. Below are the common steps we recommend to limiting the intrusion of oxygen on the hot and cold sides of our process, which in turn helps preserve and prolong the desirable flavors we talk about on our site and forum:

• Pre-boiling strike and sparge waters, or utilizing yeast scavenging, can drastically reduce DO levels to ≤ ~0.5 ppm
• Eliminating Copper, Brass and Aluminum (if, and where, possible) from your system can reduce/eliminate the possibility of their contributions to oxidative reactions via pathways such as Fenton Reactions
• If using these metals (Copper in the form of chillers seems most likely, with Aluminum kettles a close second), use of Brewtan B or equivalent Gallotannin substances are proving themselves very useful in mitigating these possible Fenton Reactions during the hot side process
• Ensure tight hose connections
• Employ either Sodium or Potassium Metabisulfite (a.k.a. NaMeta/KMeta or SMB/PMB) or the use of a pre-packaged or DIY “Trifecta” (Metabisulfite, Ascorbic Acid and Gallotannins) as an active Oxygen scavenger
• Underlet mash liquor, if possible, into the receiving vessel to reduce dough-in DO
• Employ a mash cap
• Eliminate splashing or unnecessary aeration
• If recirculating, make sure return line is below liquid level to minimize aeration
• Consider using a lauter cap (to limit atmospheric diffusion of oxygen when lautering)
• Consider No-Sparge mashing (full volume)
• If sparging, treat sparge water as you would strike water
• Pitch enough healthy yeast to ensure fermentation starts expeditiously, as well as takes up any and all oxygen introduced
• Ensure proper keg purging
• Keep beer cold and drink fresh

Put simply, the higher the antioxidant level you have at all stages of beer production, the better the beer will be, for longer.

Sources:

1. O’Rourke, T,; Brown, J. W,; Theaker, P. D. and Archibald, H.W.: The effect of oxidation during wort production on the processing and flavour of beer, Proceedings of the Convention of the Institute Brewing(Australia New Zealand Section), 1992, pp. 52-58.
2. Narziss, L.: Technological factors of flavor stability, Journal of the
   Institute of Brewing, 92 (1986), pp. 346-353.
3. Stephenson, W.H,; Biawa, J.-P.; Miracle, R.E. and Bamforth, C.W.:
   Laboratory-scale studies of the impact of oxygen on mashing, Journal
   of the Institute of Brewing, 109 (2003), pp. 273- 283.
4. Narziss, L.; Reicheneder, E. and Lustig, S.: Oxygen optimization in
   wort preparation. Brauwelt International, 3 (1989), pp. 238- 250.
5. Kobayashi, N.; Kaneda, H.; Kano, Y. and Koshino. S., The production
   of linoleic and linolenic acid hydroperoxides during mashing, Journal
   of Fermentation and Bioengineering, 76 (1993), pp. 371-375.
6. Walker, M.D.; Hughes, P.S. and Simpson, W.J. Use of Chemilumine-
   scence HPLC for Measurement of Positional Isomers of Hydroperoxy Fatty Acids in Malting and the Protein Rest Stage of Mashing, Journal of the Science of Food and Agriculture, 70 (1996), pp. 341-346.
7. Bamforth, C.W. and Parsons, R.: New procedures to improve the flavor
   stability of beer. Journal of the American Society of Brewing Chemists,
   43 (1985) pp. 197-202.
8. Noel, S. and Collin, S.: Trans-2-nonenal degradation products during
   mashing, Proceedings of the European Brewery Convention Congress,
   Brussels, 1995, pp. 483-490.
9. Hirota, N.; Kuroda, H.; Takoi, K.; Kaneko, T.; Kaneda, H.; Yoshida, I.;
   Takashio, M.; Ito, K. and Takeda, K.: Brewing performance of malted
   lipoxygenase-1 null barley and effect on the flavour stability of beer.
   Cereal Chemistry, 83 (2006), pp. 250-254.
10. Bamforth, C.W.: The science and understanding of the flavour stability
    of beer: a critical assessment. Brauwelt International, 17 (1999)
    pp. 98-110.
11. Wu, M.J.; Rogers, P.J. and Clarke, F.M.: The role of proteins in beer
    redox stability, Journal of the Institute of Brewing, 118 (2012), pp. 1-11.
12. Bamforth, C.W.: Enzymic and non-enzymic oxidation in the brewhouse;
    a theoretical consideration. Journal of the Institute of Brewing, 105
    (1999) pp. 237-242.
13. Biawa, J.-P. and Bamforth, C.W.: A two-substrate kinetic analysis of
    lipoxygenase in malt. Journal of Cereal Science, 35 (2002), pp. 95-98.
14. Baxter, E.D.: Lipoxidases in malting and mashing. Journal of the Institute
    of Brewing, 88 (1982), pp. 390-396.
15. Kanauchi, M.; Milet, J. and Bamforth, C.W.: Oxalate and Oxalate
    Oxidase in Malt. Journal of the Institute of Brewing, 115 (2009), pp.
    232-237.
16. Bamforth, C.W.; Roza, J.R and Kanauchi, M.: Storage of malt, thiol
    oxidase and brewhouse performance. Journal of the American Society
    of Brewing Chemists, 67 (2009) pp. 89-94.
17. Kanauchi, M.; Simon, K.J. and Bamforth, C.W.: Ascorbic acid oxidase
    in barley and malt and its possible role during mashing. Journal of the
    American Society of Brewing Chemists, 72 (2014), pp. 30-35.
18. Kaukovirta-Norja, A.; Laakso, S.; Reinikainen, P. and Olkku, J.: The
    effect of kilning on the ability of malt to oxidise lipids, Journal of the
    Institute of Brewing, 104 (1998), pp. 327-332.
19. Bamforth, C.W.: Oxido-reduction processes and active forms of oxygen
    in aqueous systems. Cerevisiae Biotechnology, 26 (2001), pp. 149-154.
20. Bamforth, C.W.; Muller, R.E. and Walker, M.D.: Oxygen and oxygen
    radicals in malting and brewing: a review. Journal of the American
    Society of Brewing Chemists, 51 (1993) pp. 79-88.
21. Bamforth, C.W. and Lentini, A.: The flavor instability of beer, pp. 85-109,
    in Beer: A Quality Perspective, Academic Press, San Diego, 2009
22. Bamforth, C.W.: A critical control point analysis for flavor stability of
    beer, Technical Quarterly of the Master Brewers Association of the
    Americas, 41 (2004), pp. 97-103.