Strategies for combating must 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. I described the process of enzymic oxidation in my most recent post and will advance strategies to avoid/combat its effects in this one.

The chart below provides a graphical overview of the available strategies. I discuss each in the following text.

Oxygen Elimination

Reductive Winemaking

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.

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."

Ascorbic Acid

When used in combination with sulfur dioxide, ascorbic acid can be a powerful tool to utilize the dissolved oxygen available in the juice and inhibit aromatic oxidation. It can also delay the onset of browning by converting quinones back to the original phenol. The by-products of the ascorbic acid reactions are dehyroascorbic acid and hydrogen peroxide, the latter of which is itself a strong oxidant. 

Ascorbic acid converts oxygen to hydrogen peroxide 1700 times faster than sulfur dioxide alone but the sulfur dioxide is needed to quickly scavenge the hydrogen peroxide. The dehyroascorbic acid undergoes rapid degradation into a variety of species "including numerous carboxylic acids, ketones, and aldehydes."

The recommended levels of ascorbic acid addition range between 50 and 200 mg/L. The use of ascorbic acid is somewhat controversial as it tends to produce fruitier wines and is thought to increase the potential for oxidation in the longer term.

Tannins

Tannins offer some antioxidant properties in that they can (i) bind to oxygen directly, (ii) block enzymatic oxidases (prevent them from working), and (iii) inhibit the radicals that contribute to many oxidative processes.

Substrate Elimination

Hyperoxidation
The deliberate introduction of oxygen into the juice causes enzymatic oxidation of the phenols. The process entails spraying the juice in the tank with oxygen for about 30 minutes and then racking the juice from tank to tank a few times. Alternatively, oxygen can be used in lieu of nitrogen when using flotation to clarify the juice, allowing it to settle, and then racking it from the brown precipitate just prior to fermentation. This oxidation will cause browning of the juice but the phenols will have been polymerized and will precipitate out. 

Clarification is required to reduce the suspended solids to less than 1% by weight in order to remove the major part of the phenolic precipitate. This clarification must be completed before fermentation begins as the precipitate will re-dissolve in alcohol. The clarified juice will retain a brown color but this residual browning will be eliminated by the reducing conditions of alcoholic fermentation and absorption by yeasts (Schneider, Hyperoxidation: A Review, AJEV, 1998).

The brown pigment absorbed by the yeasts during alcoholic fermentation will fall to the bottom of the tank with the lees and can be removed in a post-fermentation racking. Fining and/or filtration can be utilized for additional clarification if required.This process renders the wine less susceptible to in-bottle browning (due to the elimination of the phenols) as well as reduces bitterness in the wine.

Hyperoxidation requires that SO2 additions be withheld from the must as the oxidative enzymes are inhibited in its presence. For example, tyrosinase registers a 90% decrease in activity when 50 mg/L of SO is added to the must. SO2 also reduces caftaric acid quinone and enhances the solubility of phenolic molecules. These effects will limit the extent and effectiveness of the hyperoxidation. 

Hyperoxidation, then, uses the strength of oxidation in the early stages of winemaking to neuter the substrate in the early stages of winemaking and prevent it from becoming an oxidation resource in the bottle. 

White juice that was not exposed to skin contact can be saturated once with oxygen and that should be sufficient to remove most phenolic compounds. If there has been skin contact, the juice can be left for 30 minutes after saturation and then be sparged for an additional 30 minutes.

Fining

Phenolic molecules can be removed from the must and wine by fining with gelatin, PVPP, bentonite, or activated charcoal. The latter should be handled with care as it can strip the flavor compounds from the wine if the dosage is too high.

Enzyme Elimination

Sulfur Dioxide
Sulfur dioxide is the most important and widely used chemical in the battle against oxidative browning. It is a colorless, non-flammable gas which exists in wine both in the dissolved molecular state as well as ionized bisulfate and sulfite. It is a normal wine constituent -- a by-product of yeast metabolism -- levels of which (depending, primarily, on yeast strain and fermentation conditions) can range between 10 and 100 mg/L, but is usually around 30 mg/L. Because of toxicity at high levels of use, the levels in wine are regulated with the US stipulating levels at or below 350 mg/L and the French 150 mg/L for red wines and 200 mg/L for whites.

Sulfur dioxide can be found in juice and wine in both free and bound forms. In juice, the molecule binds to sugars, grape skins, grape solids, and laccase (catalyzing enzyme found in botrytized grapes) compounds while, in wine, it binds to acetaldehyde, keto acids, glucose, quinones, and monomeric anthocyanins. The total sulfur dioxide in a wine is the sum of free plus bound fractions with the bound component being unavailable for "beneficial" activities.The distribution and characteristics of free sulfur dioxide in wine is presented in Table 1 below. 

Free Sulfur Dioxide State	Characteristics
Molecular (SO₂)	- Exists as gas or single molecules dissolved in wine - Most important antimicrobial form of SO₂ - Can act indirectly as an antioxidant - Sensorially detectable
Bisulfite (HSO₃)	- Predominant form of free SO₂at wine and juice pH values (3 - 4) - Binds with many wine components - Prevents enzymatic oxidation - Insignificant as an antimicrobial
Sulfite (SO₃)	- Small quantities - Not a very effective antioxidant in wine

Table 1. Distribution and characteristics of free sulfur dioxide in wine (Source: Compiled from UCDavis material).

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 can reverse the effects of oxidized quinones to colorless phenols. Sulfites react slowly with oxygen, but react with important intermediates (hydrogen peroxide, acetaldehyde, quinones).

Pasteurization

Laccase comes into the winemaking environment on the back of Botrytis-cinerea-infected fruit. Sulfur dioxide does not significantly impact the operation of laccase. For example, the addition of 150 mg/L sulphur dioxide yields only a 20% reduction in laccase oxidative activity; as a matter of fact, sulfur dioxide can serve as a substrate in the oxidation process.

The Australian Wine Research Institute (AWRI) recommends testing for laccase activity as early as possible and, if detected, heat treatment (pasteurization) should be considered to deactivate the enzyme prior to fermentation. The application of 70℃ heat for 30 seconds is sufficient to destroy laccase activity. 

Laccase requires oxygen to cause oxidative damage therefore the winemaker should employ techniques which reduce must, juice, and wine contact with oxygen. Recommended strategies for handling white grapes include (AWRI):

• Whole-bunch press with a CO₂ cover
• Add pectic enzyme at the higher end of the recommended range and cold soak at low temperature to achieve rapid settling
• Rack and discard the heavy lees
• Trial bentonite additives (starting rate of 0.5 - 1 g/L) to remove moldy characters and settle for 24 hours.

For red grape handling (AWRI):

• Minimize the time between crushing and yeast inoculation and avoid cold soaking
• Laccase is an enzyme and, as such, a protein, which can be fined out using tannin. Use 200 - 500 mg/L of enological tannin at crushing to bind the laccase.

Reducing Agent

Glutathione

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). 

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.

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The next post in this series will explore the non-enzymatic (chemical) oxidation of wine.

©Wine -- Mise en abyme

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.

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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

Oxygen management: The phenols that are the substrates in enzymic oxidation

In the winemaking-process chart below, our first encounter with oxygen is the concern with enzymic oxidation in the must stage of wine production.

The figure below graphically illustrates the concept of enzymatic oxidation. In the figure, an enzyme binds with a substrate (in the presence of O₂), with the substrate broken into two products as a result of this coupling. In chemical terms, oxygen attracts electrons from other molecules, changing their chemical nature. 

Source (sites.google.com)

In the case of wine, this oxidation can cause binding of thiol (aroma) compounds and a browning of the color. In the case of wine, the substrate are phenolic compounds. The phenols in grapes are discussed in greater detail below.

Phenols

Phenols are highly reactive chemical compounds of primary importance in the quality of red wines. Phenol, the basic building block, is an aromatic organic compound (formula C6H5OH) where the phenyl group (C6H5, where six bonded carbon atoms with alternating double bonds are connected to five hydrogen atoms) is bonded to a hydroxyl group (OH, where the oxygen atom is covalently bonded to an hydrogen atom). A graphical representation of a phenol is provided below.

Phenolic compounds are:

• Responsible for the color of red grapes and wine
• Involved in the oxidative browning of white wines
• Contributors to taste and astringency through interactions with salivary proteins
• Another measure of wine quality.

The two major classes of wine phenolic compounds are flavonoids (defined by a C6-C3-C6 skeleton consisting of two phenolic rings joined by a central, oxygen-containing ring -- Jackson) and nonflavonoids (possessing a C6-C1 or C6-C3 skeleton; all numbers following "C" are subscripts). The sources and roles of the phenolic compounds falling into these two classes are illustrated in the figure below and the relative concentrations of selected classes are provided in the table following.

Table 1. Generalized concentration of various phenolic compounds
present in wine.

Phenolic	White Wine (mg/L)	Light Red Wine (mg/L)	Full Red Wine (mg/L)
Volatile	Trace	10	40
Hydroxycinnamic acids	150	200	200
Other nonflavonoids	25	40	60
Anthocyanins	0	200	400
Catechins	25	150	200
Polymeric catechins	0	600	900
Totals	200	1200	1800

Source: Kennedy, et al., Grape and wine phenolics: History and perspective,
AJEV, 57(3), September 2006.

Sources of Phenolics: Skins, Seeds, and Stems

The figure below illustrates the flavonoid phenolic compounds in wines and their sources. 

Sources of Phenols (Source: mdpi.com)

Skin

The berry skin consists of an outer layer with a wax-like coating (cuticle) and 6 to 10 layers of thick-walled cells (hypodermis) which accumulate phenolic compounds in fairly high concentrations as the berry matures (Dharmadhikari, McGlynn). The main components of the skin are phenols, aromatic substances, potassium, and other minerals.

Skin contact increases the amount of hydroxycinnamates, gallic acids, and flavonoids in a wine. Flavonoids increase slightly with contact time but strongly with temperature. These compounds are of concern because they contribute to bitterness and astringency and also serve as substrates for oxidation in white wines. While there are elevated levels of astringency in skin-contact white wines, they are nowhere near as high as in red wines. First, even though tannin is extracted from the skin of the white grape, the lack of anthocyanins means that only tannin-tannin bonds are formed, a combination that is less soluble in alcohol. Second, during fermentation, most of the tannin will precipitate out, thus limiting its ability to negatively impact the wine's sensory characteristics.

Aromatic substances are located in the skin and layers of cells immediately below it. Examples of these compounds include (Dharmadhikari):

• 2-methoxy-3-isobutyl pyrazine -- imparts bell pepper odors to Cabernet Sauvignon and Sauvignon Blanc
• 4-vinylguaiacol and 4-vinylphenol -- spicy, clove-like, and medicinal odors in some Gewurtztraminers
• Terpenes -- can be found in Muscats and Rieslings.

Seeds
Grape seeds are comprised of an outer seed coat, an endosperm, and an embryo, with the seed coat containing relatively large concentrations of tannin. Jackson stipulates that the predominant phenolics in seeds are the flavan-3-ols catechin, epicatechin, and procyanidin polymers (the latter a condensed tannin).  The tannins in the seed walls are more polymerized, and contain a higher proportion of epicatechin gallate, than those in the inner portions. Phenol levels in the seed are higher than in the skin or stems (60% versus 20% each) but they are seldom extracted to their "full potential" during wine production due to the lipid coating which retards tannin extraction until alcohol content becomes a facilitator (Jackson).

Seed tannins weigh, on average, 3.5 - 5 mg per berry while skin tannins weigh in between 0.5 and 0.9 mg. Seed tannin polymers are shorter than skin tannin polymers (the longer the tannin chain the higher the astringency) yet seed tannins are perceived as harsher, greener, and more astringent due to a greater degree of galloylation.

Grape tannins accumulate during the first period of berry growth with skin tannin synthesis beginning earlier than seed tannin synthesis and then ending with the conclusion of the first phase of growth. Seed tannin synthesis continues into the early period of berry ripening before concluding. Both skin and seed tannins continue to mature during the berry ripening phase.

Skin tannins release early and easily but then plateau. Seed tannin release is slow, steady, and long and requires alcohol as a solvent. Tannin extraction will continue throughout fermentation with the ratio tilting in favor of seed tannin sometime during the process.

Stems
Grape stems are comprised of cellulose (approximately 30%), hemicellulose (21%), lignin(17%), tannin (16%), proteins (6%), and other constituents. As was the case for seeds, stem flavan-3-ols occur primarily as catechin, epicatechin, epigallocatechin, epicatechin-gallate, and condensed tannins (procyanidin oligomers and polymers).

In a study of wines made with varying levels of stem inclusion, Suriano, et al., came to the following conclusions:

• Wines vinified in the presence of stems were higher in tannins, flavans, vanillin, and proanthocyanidins
• Stems conferred more structure and flavor to the wines
• Stems facilitated must aeration thus promoting synthesis of higher alcohols and ethyl esters by the yeast.

In addition to the foregoing, it should be mentioned that the stems reduce the compactness of the cap thus providing pathways for carbon dioxide escape during fermentation and wine flow-through during pumpovers. On the minus side, stems in the cap increase the difficulty of manual punchdowns. 

Stem condensed tannins are also considered to be very bitter and astringent and fall between skin and seed tannins in size. Green stems should be avoided as it will take years for the wine in which it is included to mellow out (Pambianchi).

Summary

To summarize (Gil, et al.):

• Seeds and stems are major sources of phenolic compounds that condition the final composition of the wine
• Seeds release significant amounts of flavan-3-ol monomers as well as proanthocyanidins with relatively low mean degree of polymerization and a high percentage of galloylation
• Seeds also increase astringency and bitterness and generate a slight but significant decrease in ethanol content, probably through the release of potassium and water
• Stems also release flavan-3-ol monomers and proanthocyanidins but their composition differs from those of the seeds
• (+)-gallocatechin replaces (-)-epicatechin
• Procyanidins had a higher mean degree of polymerization than those from seeds and a higher percentage of prodelphinidins
• Stems significantly increased the pH and decreased the titratable acidity and ethanol content (probably through the solubilization of potassium and water) of the finished wine.

Phenol Characteristics: Tannins

Key tannin properties are:

• Astringency
• Bitterness
• Reaction with ferric chloride
• The ability to bind with protein.

As shown below, flavan-3-ols are the primary tannins in the flavonoid class of phenolics and are derived from the skin, seed and stem while hydrolizable tannins are primarily oak derivatives. There is some small amount of hydrolized tannin in the flavonoid group that is derived from the fleshy part of the fruit but they are bound with other non-phenolic compounds and play no part in tannin-tannin or tannin-anthocyanin interaction. Hence, flavonoid hydrolyzable tannins are not included in the following discussion.

Flavan-3-ols
Grape-derived tannins are primarily monomers and increase in quantity from fruit set through veraison. It is thought that the primary purpose of these compounds in the plant is as a defense against bacteria, viruses, and higher herbivores. The naturally occuring flavan-3-ol compounds are catechin and epicatechin which register at between 10 and 50 mg/L in white wines and 200 mg/L in reds. Catechin and epicatechin are characterized by a single OH group at position 3 of the C ring (shown below). The formation of the compounds gallocatechin and epigallocatechin is signaled by the presence of three OH groups in the B ring. We can also have a gallic acid acylated at position 3 of the C ring to form catechin-3-o-gallate or epicatechin-3-o-gallate.

Source: ergogenics.de

Tannins have the ability to associate (form long chains; also called polymerization) and grape tannin polymers are called proanthocyanidins or condensed tannins. These condensed tannins are unstable and, in the acidic wine environment, are subject to polymerization, hydrolysis, and depolymerization. A limited degree of polymerization occurs during fruit maturation.

If a tannin is hydrolyzed under the acidic conditions in wine, it can break up into shorter lengths, producing one electron-neutral and one positively charged tannin. The positively charged tannin thus released will react with another tannin or with an anthocyanin. In the case of tannin-tannin interaction, a longer, non-colored polymer is formed. This tannin polymerization continues until the chain is end-capped by an anthocyanin molecule.

Increasing polymerization brings increased polymer size which is quantified by a measure called degree of polymerization (DP). DP increases with wine age, yielding greater wine suppleness and a reduction in astringency. Tannin quality is generally considered to be a function of the degree of polymerization and the level of association with other molecules.

Hydrolizyable Tannins
According to Puech, et al., hydrolizyable tannins contain a polyhydric alcohol (more than one hydroxyl group) as the basic structural unit of which the hydroxyl group has been esterified by gallic and hexahydroxydiphenic (HHDP) acid. The bonds between these units can be easily broken -- through enzymatic action or contact with an acid or base -- to produce free gallic acid and HHDP acid, the latter of which spontaneously converts into the lactone ellagic acid by internal condensation. Oak-sourced tannins are classified as gallotannins or ellagitannins depending on the type of acid formed.

Ellagitannins may comprise up to 10% of heartwood. In the plant, ellagitannins are toxic to micro-organisms and provide the oakwood with a defense against fungal degradation. It differs from lignin by its ability to bind with, and precipitate, alkaloids, gelatins, and other proteins. Ellagitannins may be monomeric (one glucose core) or oligomeric with differences based on the position of the couplings. The most frequent ellagitannin monomers extracted from oak are vescalagin and castalagin while the most important oligomers are roburin A and E, both vescalagin or castalagin dimers, or granidin. Fully 50% of the total ellagitannin in heartwood is unextractable.

Ellagitannins influence the structure of phenolic compounds and red wines by speeding up the condensation of procyanidins while limiting their oxidative and precipitative degradation.

Phenol Characteristics: Anthocyanins

The most important source of color in red wines is anthocyanin, a class of phenolic compound resident in grape skins. As is the case for all phenolic compounds, anthocyanin is synthesized from the amino acid phenylalanin through the phenylpropanol and flavonoid pathways.

Anthocyanins are synthesized directly from anthocyanidins by glycosylation (adding of a sugar to a protein) at the 3 and 5 positions of the C ring and are accumulated in the berry skins from veraison until full maturity. After synthesis in the cytosol (fluid portion of the cell cytoplasm), anthocyanins are transported into the vacuole (cell organelle responsible for a number of functions including nutrient storage) where they are stored as colored coalescences called anthocyanic vascular inclusions.

Environmental effects can influence anthocyanin content in the fruit but its composition is most closely associated with grape variety. In the fruit, anthocyanin has the following functions (Flamini, et al.):

• Protection against solar exposure and and UV radiation
• Protection against pathogen attacks
• Protection against oxidation attacks
• Protection against attacks by free radicals
• Attracting animals for seed dispersal after the fruit has attained maturity

Each anthocyanin has a particular hue ranging from red to blue (shown below) and the combination of quantities and profiles will determine the color intensity of fruit and wine.

Grape anthocyanins. Source: gopixpic.com

As discussed previously, anthocyanins exist in grapes as glycosides (a bonding of the flavonoid component -- called an anthcyanidin -- with a sugar), a situation which increases both the chemical stability and solubility of the anthocyanidin.

The figure at the top shows the anthocyanidins in native form while the bottom shows a glucosylated Malvidin, Malvidin-3-glucoside.

The anthocyanin can be further complexed through the bonding of the sugar with either acetic, coumaric, or caffeic acid (Jackson).

Malvidin-3O-coumaroylglucoside

In wine, spectral color is a function of anthocyanin concentration, the concentration of co-factors which bind with anthocyanin to cause co-pigmentation, and the number and quality of polymeric pigments (Enology Note #120).

In young red wines, anthocyanins occur predominantly as a dynamic equilibrium between one molecular state bonded to sulfur dioxide and four free-form states. Most of these forms are colorless within the range of of wine pH. The exception is a small portion that exists in the flavylium state (2-phenylchromenylium ion), that portion being dependent on pH and free-sulfur dioxide content. According to Jackson, low pH increases the concentration of the flavylium (thus increasing redness) while increases in pH will decrease the color density as the proportion of anthocyanin in the flavylium declines.

When sulfites bind to anthocyanins, the anthocyanins go from a colored to a colorless form (Practical Winery). Because of its effectiveness as an anthocyanin bleacher, sulfur dioxide is the most significant factor affecting the color density of young red wines (Jackson).

The free form of anthocyanin is rendered stable by a number of short- and long-term factors. The short-term factor that is most impactful is co-pigmentation, a process whereby anthocyanin complexes are held together by hydrophobic interaction with non-anthocyanin compounds (anthocyanin-anthocyanin aggregates combined similarly are referred to as self-associations). According to Jackson, the stacking of molecules in these complexes "physically limits water access to (and hydration of) red flavylium and bluish quinoidal base states to colorless carbinol forms." In the case of these co-pigmentation complexes "covalent bonds form between acyl groups of anthocyanin and co-pigments."

The principal compounds which act as co-pigments are epicatechin, procyanidins, cinnamic acids, and hydroxycinnamoyl esters but a wide array of compounds may be involved in this role (Jackson).

Co-pigmentation has the following effects on young red wines (Jackson; Boulton):

• It significantly increases color density and may affect color tint
• It accounts for over half of the observed color of young red wines
• It is important to the purplish coloration of young wines
• Color enhancement has been found to be between two and ten times that expected from the pigment alone
• It also adds temporary stability towards oxidation and SO₂ bleaching.

During the color extraction phase of wine production, co-pigmentation co-factors seem to attain a maximum extraction after which the ability to absorb additional pigments is exhausted. The general consensus seems to be that extraction seems to peak after approximately six days.

Phenols Levels in Grapes

The phenol levels in grapes can vary based on a number of factors (Angelosante, Guildsomm):

• The grape variety
• Yields -- excessive yields can result in lower levels of color and tannin
• Growing environment
• Higher elevations mean more sunlight and higher polyphenol levels
• Cooler temperatures prolong the ripening period -- tannins and color ripen at lower sugar levels
• Hillsiide vineyards have less water and higher polyphenol production
• Hydric stress
• Seems to induce the plants to produce more skin tannins
• Smaller berries so higher skin-to-juice ratio
• Excess nitrogen causes the vine to cease production of polyphenols

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I will cover the enzymic oxidation process in my next post.

©Wine -- Mise en abyme

An overarching view of oxygen management considerations during winemaking

Bodegas Torres has identified four key factors that are determinative of wine quality:

• The plant's environment
• Species and varieties
• Viticultural practices
• Enological practices.

In the area of enological practices, Torres focuses on oxygen management: "Keeping in mind that oxygen oxidizes wines, its proper management is crucial during vinification and aging in order to attain the desired result."

Oxygen primarily enters the wine through air of which it comprises approximately 20%. Oxygen affects the juice/wine by attracting electrons from other molecules, thus changing their chemical nature. This electron loss can affect aspects of the wine to include color, aroma, flavor development, and tannic stability. But the role of oxygen is not all deleterious; there are areas of what I call beneficial instances of oxygen application that can redound to the benefit of the winemaker.

The scope of oxygen impact includes:

• Acting as a substrate of enzymatic reactions
• Involvement in direct chemical reactions
• Causing phenol polymerization
• Affecting the growth and metabolite production of wine micro-organisms.

I will be developing an oxygen management needs proposal and an associated solution architecture over the course of a number of forthcoming posts. The framework within which this work will be conducted is shown in the chart below.

My first post in the series will be a deep dive into enzymic oxidation.

©Wine -- Mise en abyme

Piemonte dry white wines: The instances of Favorita and the Malvirà example

My recent experiences with Piemonte dry whites have piqued my curiosity to the extent that I will be exploring the category in greater detail. I explore Favorita-based wines in this post.

Favorita has been shown to be the exact same variety as Vermentino, which is, itself, primarily grown in Sardegna (68%) and Tuscany (14%). Fully 80% of known Favorita plantings are found in Piemonte. The main characteristics of the variety are shown in the chart below.

Favorita wines are made in Piemonte according to the requirements shown in the following chart.

It should be noted that, in addition to Favorita, Colli Tortonesi DOC hosts high-quality white wines from the Timorassa and Cortese varieties.

I secured a bottle of the 2015 Langhe Favorita from Malvirà in order to evaluate this instance of Piemontese white.

Malvirà, located in Canale in Roero, was established in the 1950s by Giuseppe Damante. He subsequently passed the business over to his sons Roberto and Massimo in 1974. The sons transformed the business from a primarily bulk-wine supplier to one focused on quality. Today they plant 42 ha across six cru vineyards. The vineyards are farmed organically with all of the wines being sourced from estate fruit.

The Malvirà Favorita is sourced from three Canale vineyards: Saglieto (south to southeast exposure at 200 m elevation), Trinita (southwest exposure), and Bossola. The soils in these vineyards are calcareous clay and sand and the vines are between 30 and 40 years of age. Yields are at 50 hl/ha.

The wine is made from 100% Favorita grapes. The grapes are fermented in stainless steel tanks for 7 10 days and then aged in tanks for 4 - 8 months.

Floral, initially, with green herbs accompanying rich, intense fruit notes and spice. Full, round fruit intensity on the palate. Bright. Ripe lime with a slate minerality that gives way to a chalky minerality. Persistence of acidity through all phases. Excites the salivary glands in a low-key fashion. Loooong finish.

After a respite, I went back to the wine. I was now getting sweet white fruit, limestone minerality, salinity, and red pepper. On the palate, sweet fruit along with acidity and salinity. Great weight and lengthy finish. 

This wine continues the string of excitement on which I have been tugging in this exploration of Piemonte dry whites. I loved it. 

In terms of pairings, the literature sees this class of wine as perfect as an aperitif or as an accompaniment to fish, a typical Piemonte starter, or Risotto. I can't wait to try examples from the other identified regions.

©Wine -- Mise en abyme

Benanti Winery: Its "whole-Etna" product strategy and distribution relationship with Wilson Daniels positions it well for the future

From its founding in 1988 as Tenuta di Castiglione, Benanti has exhibited a proclivity for experimentation, innovation, and strategic property acquisition/de-acquisition. And the entity continues in this vein to this day, as illustrated graphically by the timeline below.

Giuseppe Benanti, once he hit on the idea of making quality wine on the mountain, saw experimentation as the key to determining the best varieties and soil that should be utilized in the effort. To aid in the endeavor, he enlisted the assistance of Professor Rocco di Stefano (Experimental Institute for Enology, Asti), Professor Jean Siegrist (Institut National de la Recherch Agronomique, Beaune, Burgundy), Langhe winemakers Gian Domenico Negro and Marco Monchiero, and local winemaker Salvo Foti.

The team conducted over 150 micro-vinifications in the initial trials and, after two experimental harvests, designated the 1990 vintages of Pietra Marina Etna Bianco Superiore and Rovitello Etna Rosso for bottling.

Vinous has, on a number of occasions, paid homage to Benanti and its role in the development of winemaking on Etna. In a December 2016 note Vinous mentioned Benanti as the "... first to believe and insist upon Etna's native grapes at a time when everyone on Sicily was rushing to plant Chardonnay, Cabernet Sauvignon, and Merlot." In another mention, Vinous stated: "Credit must go to Benanti for having created the I Monovitigni series of wines, which showcased to great effect the characteristics and high quality of the likes of monovariety Nerello Cappucchio, Nerello Mascales, and even Minella Bianco, at a time when little was known of these cultivars."

Benanti has shifted its territorial holdings from time to time to comport with its evolving business strategy. For example, Benanti initiated the company in Castiglione di Sicilia, even though the family owned property in Viagrande; property which had been used to grow grapes since the 1800s. When Giusepe felt that a broader Sicilia portfolio was in order, he procured property in Noto and Pantelleria. When he handed management of the business over to his sons Antonio and Salvino, they opted to narrow the focus to selected sites on Etna and sold the Noto and Pantelleria properties, along with some under-performing Etna properties, in order to effect their vision.

And that strategic repositioning continues today as Benanti deploys a "whole-Etna" strategy and streamlines its product portfolio to reflect that direction.

In an InstagramLive Chat with Anotnio earlier this year, I queried him about widespread deployment of Contrada-based winemaking on the mountain. He was emphatic that they were nowhere near knowing, with any degree of confidence, what effects individual contrada had on the wines; but they had that information for the slopes (what I call sub-regions). Shortly after this meeting with Antonio I convened a session with Benjamin North Spencer (The New Wines of Mt. Etna) and he laid out the sub-zone architecture that I subsequently captured in the below figure.

The figure below shows the Benanti property holdings/grape sources at the time of my 2016 visit. 

The figure below shows the current Benanti vineyard architecture and two things should be noted vis a vis the 2017 map: (i) grape sources have declined from six locations to four and (ii) the current locations map closely to the sub-zone architecture which I depicted above.

The product architecture is built around the "whole-Etna" core with the Contrada series of wines but further leverages those sites to provide higher-value Riserva products to the customer base. At the other end of the spectrum, the Traditional wines provide a seamless entry point into these higher-order offerings. 

It should be reiterated here that the Contrada series wines are not intended to showcase/compare contrada-specific qualities. If Benanti wanted highlight contrada differences, that would have been best accomplished by featuring intra-slope, rather than cross-slope, contrade.

The Etna terroir-based framework provides a flexible architecture for future Benanti growth. The northeast slope of the mountain is currently the only gap in the Benanti terroir wall. Beyond that, the company has shown that it can expand its product base by elevating portions of the existing stock. For example, it introduced two Riservas by so designating the upper portions of existing vineyards (Serra della Contessa and Rovitello) and devoting the remaining vines to the production of Contrada wines. 

The company introduced the Rosata (successfully) a little over two years ago; is introducing a new sparkling wine in the near term (and bringing all sparkling wine production in-house); and is introducing the Contrado Rinazzo Etna Bianco Superiore to the market in 2020 (the 2018 vintage).

The true success of this effort will be reflected in how well the products are received on the market. And Benanti has opened a new front in this battle by collaborating with Wilson Daniels to have that organization import and represent its products in the New York tri-state area. Wilson Daniels represents producers such as DRC in that market and numbers the top restaurants and collectors among its clients. This partnership will add cachet to the Benanti line and, for current customers, may signal rising costs for these products over time.

In the short-to-midterm, however, we are all hostage to the whims of Covid-19.

©Wine -- Mise en abyme