The roles of Tyrosinase and Laccase in enzymic 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 Laccase, in the case of botrytized must), phenolic compounds (flavonols, anthocyanins, tannins, etc.), oxygen, and metallic co-factors (iron, copper, etc.). 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. In this post I will focus on enzymatic oxidation.

Polyphenols

According to Jackson (Wine Science), "In contrast to red wines, the limited antioxidant character of white wines (ed: tannins and anthocyanins provide substantive antioxidant capability in red wines with red wine polyphenol content ranging between 300 and 5000 mg/L while white phenolics range between 60 and 200 mg/L) make them more susceptible to oxidative browning." Hydroxycinnamic acid is the most important of the non-flavonoids, comprising 80% of non-skin-contact-white-wine phenolics. It is the first white wine polyphenol to be oxidized.

Further, grape varieties differ markedly in the amount of phenolics released during crushing or extracted during maceration (an extremely important consideration given that phenolics are the main substrate for oxidation activity). The table below shows the levels of flavonoid accumulation during crushing or maceration of selected white varieties.

Table 1. Phenolics released/extracted during crushing/maceration

Variety	Flavonoid Accumulation
Palomino	Low
Sauvignon Blanc	Low
Riesling	Moderate
Semillon	Moderate
Chardonnay	Moderate
Muscat Gordo	Extensive
Colombard	Extensive
Trebbiano	Extensive
Pedro Ximinez	Extensive

Tyrosinase

Polyphenol oxidases (PPOs) are a widespread group of enzymes found in plants, fungi, bacteria, and animals. In plants, they are located in the plastid of the chloroplast and in the mitochondrion, separate from the unsuspecting polyphenols (which are located in the vacuole). 

It is thought that these PPOs contribute to the defense of the plant against predators such as herbivores and insects. 

Enzymic oxidation (which primarily afflicts wine must) requires the presence of the PPO 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.). 

Once the grape berry integrity has been compromised, the enzyme and phenols are exposed to each other and to atmospheric oxygen. Juice or wine that is saturated with oxygen contains about 7 - 8 mg/L (depending on the temperature).

As shown in the figure above, the enzyme has an active site where its interaction with the substrate will eventually occur. The copper contained in the enzyme is located at this active site.

In the presence of oxygen, this copper-containing enzyme oxidizes the phenolic groups to reactivate oxygen molecules known as quinones. The active site of tyrosinase undergoes transitions among mel-, oxy-, and deoxy-forms in a cyclic manner. In each cycle, two molecules of catechol are oxidized and one molecule of oxygen is reduced to water, resulting in the formation of two quinone products.

These quinones, in turn, continue reacting with each other, and other cellular factors, to form brown spots known as melanin.

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

Glutathione

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). While my intent was to discuss anti-oxidants in a totally separate post, tight integration of the naturally occurring glutathione into the enzymatic oxidation process dictates that it be an integral part of this discussion. 

Glutathione is a grape- and yeast-produced tripeptide which contains three amino acids: glutamate, cystine, and glycone. It is generally found in must, yeast and wine in its reduced (GSH) or oxidized (GSST) forms, the latter of which contains two molecules of glutathione linked by a sulfide bridge.

Glutathione is important in the wine space, firstly, because of its ability to scavenge ortho-quinones, the main culprit in oxidative browning. The compound plays a critical role in preventing the oxidation of phenols in must by reacting with caftaric ortho-quinones to generate Grape Reduction Product, a stable, colorless compound. 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. The process is illustrated graphically in the figure above.

The GSH form of glutathione can also compete with several aromatic compounds for ortho-quinones, thus protecting and preserving certain wine aromas.

Minimizing the loss of glutathione is one of the keys in white winemaking as there is a strong correlation between the concentration of glutathione in the must and the concentration in the young wine and a positive correlation between glutathione in the wine and the wines freshness and longevity.

The glutathione-to-caftaric-acid ratio can give an indication of the oxidation sensitivity of certain cultivars.

Laccase

Laccase is produced by the phytopathogenic fungus Botrytis cinerea and enters the must with contaminated grape berries. Laccase resides in the glycan sheath surrounding the hyphae of Botrytis. High levels of Botrytis is often correlated with high levels of laccase. 

As in the case with tyrosinase, laccase oxidizes phenolic compounds into quinones which polymerize in the presence of oxygen. Polymerized quinones form pigmented compounds "associated with laccase-induced browning and discoloration."

Both tyrosinase and laccase use catechin, anthocyanin, flavanols, and flavanone as substrates but 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. Laccase can oxidize the GRP to the corresponding quinone which can, in turn react with glutathione to form GRP2, GRP3, etc.

***********************************************************************************************************

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.

Allowing the juice to brown prior to fermentation may be beneficial to some non-aromatic varieties as oxidized phenolics will not contribute to post-fermentation astringency.

In my next post I will discuss how anti-oxidants can be deployed to counteract the effects of oxidation.

©Wine -- Mise en abyme

An architecture for the production of reductive white wines

Traditionally, wine has been made in an oxidative style. But, beginning in the 1950s and 1960s, grape growers and winemakers began to employ new tools to attain specific "stylistic and qualitative ends."

Based on Clark Smith's interpretation of the history of that period, the "tools of 20th century winemaking" were stainless steel, inert gas, refrigeration, and sterile filtration (a product of nuclear energy) and this "modern winemaking revolution exploded out of Germany" in the form of Rieslings that were fresh, sterile-filtered, and completely without oxidative characters. According to Smith: "the idea of a light, sweet, fresh, fruity wine like Blue Nun was as world changing as color television." 

These tools and techniques were adopted by Emile Peynaud and other scientists in France and, from there, migrated to the US. According to Smith, prior to the 1960s, 95% of California wines were either port or sherry styles. With the introduction of Blue Nunn, and the adoption of the associated technologies in Bordeaux, US winemakers followed suit such that, by 1970, the majority of California wine contained less than 14% alcohol. These tools and techniques allowed the introduction and use of a reductive style of winemaking.

Hydrogen sulfide is the result of a severe case of reduction in wine but, lower down on the scale, Benzene thiol and furfural thiol contribute bread crust, smoke, and struck flint aromas. Fruity thiols provide notes of passion fruit, citrus zest/cat pee, and grapefruit, aromas associated with Sauvignon Blanc (Remy Charest, Fashionable Chemistry ..., nomacorc.com; Jackson, Wine Science). But it is preservation of freshness and fruit aromas and flavor that the winemaker pursues when he/she decides to employ a reductive winemaking style.

The essence of reductive winemaking is the production of wine without the presence of oxygen. Grapes are harvested from cool regions and the juice is fermented cold in closed stainless steel tanks. Juice is protected as is the wine through maturation and bottling. This method is particularly beneficial for grape varieties such as Sauvignon Blanc, Petit Manseng, Chenin Blanc, and Gewurtztraminer that are rich in varietal aromas that can be placed at risk in the face of oxidizing effects.

The winemaker's plight in producing a reductive white wine
(Underlying picture source: http://cdn.pcwallart.com/)

Before I get into the elements of the architecture, I would like to highlight a Remy Charest report wherein he illustrated a shift to reductive winemaking among Burgundy producers. According to Remy, Jancis Robinson had written about in a shift in Burgundy from buttery, rich, toasty Chardonnays to Chardonnays that exhibited:

• High acidity
• No trace of toastiness or obvious oak
• Leanness on the palate
• The flinty smell of recently struck match.

In her discussions with Jean-Marc Roulot (Domaine Roulot), she was told that this result was largely due to a more reductive style of winemaking, itself a reaction to the premox crisis that rocked the region's white wines in the 1990s.

Remy described the reductive program as:

• Long, slow, delicate pressing
• Protection from oxygen through vinification and aging
• Finishing the aging in tank

Some additional (and widely accepted, though not necessarily reductive) characteristics of these wines include:

• Relatively high SO2 addition (Probably related to the battle against oxidation but may also be linked to the appearance of the struck-match character in some of the wines).
• Fresher, crisper wines resulting from earlier pick dates and moving vineyards to cooler sites.

Reductive white wines are all the rage today but, as Lance Cutler (Achieving Balance in Reductive Winemaking, Wine Business) points out, "Keeping wine away from oxygen can create some vibrantly fruity wines, but this same lack of oxygen might encourage the development of reduced sulfur compounds."

The main considerations in producing a reductive white wine are as follows:

1. Healthy fruit from a cool vineyard. The climate in the vineyard will help to preserve freshness and crispness of aromas while healthy fruit will have an adequate supply of the vitamins and minerals to ensure a successful fermentation. The viticultural factors affecting the supply of yeast assimilable nitrogen include: cultivars, rot incidence, block, vineyard mulch, crop load, moisture stress, and grape maturity level (Zoecklein)
2. Minimize the use of sulfur in the vineyard and ensure adequate time spacing between application and harvest
3. Harvest at night to preserve freshness and flavors
4. Application of inert gases during harvest (mainly CO₂ in dry-ice form)
5. Provide antioxidant treatment to the free-run juice. Most normally ascorbic acid and sulfur dioxide but there is some concern that ascorbic acid switches from protection to oxidative mode over the long term and, as such, is not suited for wines destined for aging. In many cases inert gases such as CO₂ and N₂ are used to protect the juice from oxygen.
6. Measure fermentable nitrogen as too high, or too low, concentrations can result in the formation of undesirable sulfur compounds during fermentation. According to Zoecklein these measurements can be carried out with either Formol titration or a NOPA test.
7. Turbidity should be adjusted such that stylistic goals and aromatic finesse of the wine is achieved. Juice clarity should be measured in Nephol units and should fall between 100 and 150 (Zoecklein)
8. Non-soluble solids concentration should be monitored as both high and low concentrations can result in the production of undesirable sulfur compounds
9. The appropriate yeasts should be selected for the effort as strains differ in their capacity to transform the non-volatile grape derived precursors to odor-active volatiles (Zoecklein)
10. Keep yeast cells suspended in the tank during fermentation in order to allow an even distribution of fermentation as well as to allow full access to distributed nutrients
11. Rack gently under a carbon dioxide or nitrogen blanket. Use an in-line sparging device to sparge the wine with CO₂ or N₂. Add SO₂ to the wine as it is being racked to prevent oxidation
12. No malolactic fermentation in order to preserve varietal character, flavors, and freshness
13. No oak aging in order to continue to continue to deny oxygen access to the wine
14. Select an appropriate closure. According to Remy Charest, "Precise control of oxygen in the bottle, for example by selecting a closure allowing small and consistent amounts of oxygen, can prevent extreme reduction without compromising the more interesting flinty and fruity aromas."

In the cases where the odors are manifested in the wine, remedies include (i) blowing it off through volatility; (ii) inert gas sparging; (iii) precipitating with copper additions; and (iv) fining.

©Wine -- Mise en abyme

Walking the tightrope between oxidation and reduction in white wine production: Reduction

Reduction is the other side of the oxidation/reduction coin. When elemental oxygen combines with wine compounds, it can take a pair of electrons from the compound. The compound losing the electrons is said to have been "oxidized" while the oxygen, which has gained two electrons, is said to be "reduced."

Reduction is of principal importance to the winemaker as it relates to sulfur compounds. According to Jackson (Wine Science):

When present, elemental sulfur can be assimilated and used in the synthesis of sulfur-containing amino acids and coenzymes. It also may be oxidized to sulfate and sulfur dioxide or reduced to hydrogen sulfide. The reduction of sulfide to hydrogen sulfide may be a means, albeit aromatically unpleasant, of maintaining a favorable redox balance in yeast cells under anaerobic conditions.

According to Zoecklin (Enology Notes #96, 12/20/2004, vtwines.info):

Since wine is fermented by yeast through an anaerobic process (without oxygen), a number of reduced compounds are produced. Reduced sulfur and and nitrogen compounds, in the form of hydrogen sulfide and mercaptans (ammonia and amines), are known particularly for the negative characters they impart to wines. Thus, it is possible to have a wine with an unpleasant and undesirable reduced character.

The sulfur compounds associated with sulfur taint, and the population of odors associated therewith, are illustrated in the figure below.

Sulfur taint has its origins in either the vineyard, the cellar, or both. In the vineyard, elemental sulfur is sprayed on the vines to combat the potential effects of powdery mildew. If this spraying is conducted too close to harvest, portions of the sulfur will remain on the grapes and make its way into the fermentation process. An example of sulfur-like off odors created in the cellar is the case of hydrogen sulfide production by the yeast to synthesize the sulfur-containing amino acids methionine and cysteine. This process is facilitated by the reduction of sulfates via the sulfur-reduction pathway. A lack of intracellular nitrogen will not curtail the process and the excess hydrogen thus created cannot be incorporated into the amino acid. Rather, it is secreted into the medium (Kennedy and Reid, Yeast nutrient management in winemaking, The Australian and New Zealand Grapegrower and Winemaker, 537, October 2008).

A listing of the sources of sulfur-like off odors is presented in Table 1.

Table 1: Sources of sulfur taint in wine production.

Environment	Source	Action	Impact
Vineyard	Elemental sulfur used as fungicide	Reduction during fermentation
	Sulfur-containing pesticides	do.
	Excess of metal ions
	Vine stress
	Unsound fruit
Cellar	Cold soaking	Growth of yeasts such as Kloeckera	Depletion of amino acids and micronutrients
	Native Yeasts	High hydrogen sulfide production	Compete against other yeasts for dominance of fermentation
	Excess hydrogen sulfide from sulfate reduction	Hydrogen sulfide used to synthesize	Absence of nitrogen causes produced hydrogen sulfide  to be secreted into the medium
	High levels of sulphur dioxide added to must at crush	Allows sulphur dioxide to bypass the sulfate reduction system	Sulfur dioxide enters the yeast cell directly
	Vitamin shortage in high YAN musts
	Nitrogen limitation	Produces sulfur-like off odors	Production begins 30 minutes after ammonia starvation initiates

Source: Compiled from Lansing and Kennedy and Reid)

The timing of the production of sulfur-like off odors is shown in Table 2 below.

Table 2. Production timing of sulfur taint by sulfur class.

Sulfur Class	Production Timing	Source
Hydrogen Sulfide	Early in fermentation (2 - 4 days)	Nitrogen/vitamin deficiency
	Fermentation end	Degradation of sulfur-containing compounds
	Sur lie aging	Autolysis
	In bottle	Generally under screw cap
Higher Sulfides	Late in fermentation/Sur lie aging	Release of compounds by metabolically active yeasts Degradation of sulfur-containing amino acids Degradation of cell compounds during autolysis

Source: Compiled from Lansing

In the cases where the odors are manifested in the wine, remedies include (i) blowing it off through volatility; (ii) inert gas sparging; (iii) precipitating with copper additions; and (iv) fining.

Oxidation and reduction are twin evils in the world of (especially) white wine production but there are aspects of both that are beneficial to the final product. Making wines which call on these qualities is called oxidative and reductive winemaking, respectively, and I will cover those styles in upcoming posts.

©Wine -- Mise en abyme

Walking the tightrope between oxidation and reduction in white wine production: Oxidation

In this series I will be examining the winemaker's challenge in navigating between the twin evils of reduction and oxidation, both faults but both having potentially desirable characteristics close to the center of the continuum. I begin with this post on oxidation.

According to Lukacs' research (Inventing Wine), modern wine did not arise until the advent of the relevant scientific and technological advances of the Enlightenment. Prior to that period, wine drinkers consumed oxidized, sour wines which were "fortified" with all manner of additives designed to either slow its decay or make it more "palatable." Lukacs points out that winemaking in the first half of the 20th century was a reprise of thousands of years past -- "a process of letting nature run its course."

When Emile Peynaud (famed Bourdeaux enologist) began his work in the early 1950s, growers were harvesting early and, as a result, the wines were "excessively green or vegetal." He observed that there was a further striking uniformity about the wines: they were all oxidized.

Wine oxidizes when exposed to air via two primary mechanisms: enzymic and non-enzymic oxidation.  The effects of oxidation on white wine are browning, loss of fruity aromas, and aldehydic aromas. Because of these characteristics, oxidization is widely viewed as a wine fault.

Enzymic Oxidation

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

Non-Enzymic Oxidation

Non-enzymic oxidation, also known as chemical oxidation, occurs in fermented wine. In this case, oxygen does not react directly with phenolic compounds. Rather, it functions through a reaction catalyzed by Cu+ or Fe+ that converts oxygen into a highly reactive radical capable of oxidizing organic compounds.

Non-enzymic oxidation in white wines can result in premature aging, browning, and pinking, all resulting in wine deterioration and loss of quality. Strategies for combating this fault include removing metals -- oxidation catalysts -- and reducing the concentration of phenolic compounds -- oxidation precursors --in wines.

©Wine -- Mise en abyme