Enzymic oxidation of wine must

In my most recent post, I initiated a series on wine faults with a discourse on sulfur taint in wine production. I continue in that vein with a discussion of must oxidation.

Wine oxidizes when exposed to air via two primary mechanisms: enzymic and non-enzymic oxidation. Enzymic oxidation primarily afflicts wine must and requires the presence of the enzyme Tyrosinase (or Lacasse, in the case of botrytized must), phenolic compounds (flavonols, anthocyanins, tannins, etc.), oxygen, and metallic co-factors (iron, copper, etc.). Non-enzymic oxidation, also known as chemical oxidation, occurs in two steps: (i) Oxygen in the air reacts with wine phenols to create hydrogen peroxide and (ii) hydrogen peroxide reacts with ethanol to form acetaldehyde. The effects of oxidation on wine are browning, loss of fruity aromas, and aldehydic aromas. Because of these characteristics, oxidization is widely viewed as a wine fault.

Enzymic oxidation (which primarily afflicts wine must) requires the presence of the enzyme Tyrosinase* (or Laccase**, in the case of botrytized must), phenolic compounds (hydroxycinnamic acids, with the main player being caftaric acid but others — including coumaroyl, tartaric acid, and catechin — as alternates) to perform the role of substrate, oxygen, and metallic co-factors (iron, copper, etc.). These enzymes interact with the substrates to form caftaric acid quinone which, in turn, reacts with glutathione (normally a powerful anti-oxidant) in the must to form Grape Reduction Product (GRP). The conversion of the oxidized quinone to GRP limits the browning of the juice to some extent (duToit and Kritzinger). Once the glutathione is depleted, the remaining caftaric acid quinone reacts with other must constituents to form caftaric acid and begins the oxidation process anew. Browning occurs when the flavanols oxidized by caftaric acid quinones polymerize and precipitate out. Unlike the case of wines, these brown pigments are insoluble in must.

Because tyrosinase is associated with grape solids, its enzymic activity is significantly diminished once the solids have been removed from the equation. Laccase is difficult to eliminate from grape juice.

The activity of these enzymes will be impacted by (Boulton, et al, Principles and Practices of Winemaking):

• The concentration of major phenols
• Competition between substrates for binding and reaction
• The caftaric and glutathione content of cultivar (the state at which glutathione is depleted will determine the level of potential browning)
• The ascorbic acid content
• Temperature
•  Wine pH.

Both tyrosinase and laccase use catechin, anthocyanin, flavanols, and flavanone as substrates but, as indicated in Table 2.1 of Boulton et al., laccase acts on a far wider range of substrates than does tyrosinase. UCDavis pegs the added scope of laccase as encompassing anthocyanin pigments and ascorbic acid, the latter of which is itself used as an antioxidant

A number of suggestions have been advanced to impede/control the oxidative activities of the enzymes:

• Boulton has reported that the addition of sulfur dioxide at levels between 25 and 75 mg/L will reduce tyrosinase activity between 75 and 97%. This inhibition may be the result of a binding of a sulfhydril group on the enzyme or a bisulfite inhibition resulting from the interaction of sulfites and intermediate quinones. (Sulfur dioxide does not significantly impact the operation of laccase (addition of 150 mg/L sulphur dioxide yields only a 20% reduction in oxidative activity); as a matter of fact, it can serve as a substrate. The only way to completely stop laccase is to use HTST to denature it.) Sulfur dioxide can reverse the effects of oxidized quinones to colorless phenols. Sulfites react slowly with oxygen, but react with important intermediates (hydrogen peroxide, acetaldehyde, quinones)
• Judicious harvesting, crushing, pressing to minimize enzyme reaction subtrates and limit oxygen exposure
• Bentonite fining to remove the enzyme
• Hyperoxidation to reduce phenols
• Use healthy grapes
• Keep wine containers full
• Use of inert gas to limit oxygen contact

According to Boulton et al., ascorbic acid can be used as an antioxidant either alone or in cooperation with sulfur dioxide. It does not have any direct effect on the enzyme but it reduces the potential for browning by consuming the available oxygen. It can also delay the onset of browning by converting quinones back to the original phenol. The recommended levels of ascorbic acid addition ranges between 50 and 200 mg/L. Ascorbic acid will react with oxygen to form dehydrascorbic acid and, upon the consumption of the former, browning may be initiated. Conversely, unused ascorbic in finished wines can act like a phenol, leading to the production of acetaldehyde where oxygen is introduced.

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*Tyrosinase is located in the chloroplasts of the grape berry cell membrane and, in the must, is predominantly attached to grape particles. Its concentration in the must is dependent on the variety and the maturity of the fruit. Warmer temperature and higher pH increases the activity of this enzyme.

**Laccase is generally associated with Botrytis and can cause bunch rot and grey mold in grapes. Moisture on the berry surface can cause infection. Farming and harvesting practices can impact the levels of rotten fruit brought into the cellar and, potentially, included in the must. This enzyme is considered more dangerous than tyrosinase because it oxidizes a wider range of phenolic compounds and is resistant to sulphur dioxide. As is the case for tyrosinase, this enzyme is more active at higher temperatures and pH.

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