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Enzyme and Tannin Applications: Potential impacts on red wine quality.

Enological tannins and enzymes are powerful winemaking tools. Proper use can improve the basic characteristics of a wine, from increasing the depth of color to imparting fuller and more supple tannins to the final wine. Other, subtler process improvements include better filtration, protection from oxidation and microbial control.

Enological enzymes

Commercial enzymes are now accepted by many as an integral part of winemaking. With many suppliers offering a vast array of enzymes under different brand names, the choice available to winemakers is staggering. Some view enzymes as commodity products to be purchased on price alone. However, significant differences in quality exist between supplier offerings. A good understanding of enzymes and their specific role in winemaking applications is necessary to make informed decisions.

The main substrate target for enological enzyme applications in red winemaking is grape pectin (see Pectin Polymer Showing Structural Components and Sites of Enzyme Activity, above). Pectin, cellulose and hemicellulose are structural polysaccharides in the middle lamella and primary cell walls of grape cells. (3) Red grape skins contain approximately 75% more cell wall tissue than grape pulp does. (10) Cell wall polysaccharide structure changes with grape ripening due to increasing activity of the grape's own pectinases.

Pectinases liberated from the grapes themselves, however, are not very active under winemaking conditions. (2) Commercial pectin ase-based enzyme preparations derived from Aspergillus species are commonly used in red winemaking to enhance color and tannin extraction, to improve yield and to aid in clarification and filtration. (3)

The predominant enzyme activity in winemaking enzyme products is pectinase, which sounds simple. The problem is that pectinase is not one enzyme; it is actually a suite of many enzyme activities. The composition and proportion of those specific enzyme activities can change the appropriate optimal enzyme application. The main enzyme activities in a commercial pectinase are pectin lyase, pectin methyl-esterase and polygalacturonase. Secondary pectinase enzyme activities also contribute to the overall activity and can determine the optimal application.

The increased enzymatic extraction of tannins in the form of grape proanthocyanidins can also contribute to the color stability of red wines over time as they react with anthocyanins to form derived pigments such as tanninanthocyanin adducts. (4,8)

Insoluble pectin, mostly in the middle lamella-the "glue" between cells-becomes more soluble as grapes mature and, as a result, its grip on the surrounding cell walls is loosened and the fruit becomes softer. The substrate of greatest concern in red winemaking is the pectin between grape cell walls and within the cell wall structure; a secondary concern is the soluble pectin released upon crushing ripe fruit. Depending upon the vintage and harvest conditions, a beta-glucanase/pectinase blend can aid in de-pectinization and breakdown of any beta-glucans present from mold pressure, which may cause filtration difficulties in later processing.

Another important factor to consider is enzyme purification, which is one of the major advances in winemaking enzymes over recent decades. During commercial enzyme production, the fungi produce a diverse suite of enzymes, including unwanted side activities including cinnamyl esterase. Cinnamyl esterase catalyzes the first reaction in the production of vinyl-phenols. This activity is always present in pectinase preparations if not removed by a specific purification step. (1)

In red winemaking where the unwanted cinnamyl esterase activity is not fully inhibited by wine tannins as previously thought, in the presence of non-purified enzymes, the concentration of vinyl-phenol precursors will increase. The danger with formation of precursors of vinyl-phenols is, if Brettanomyces spoilage occurs, they will be first decarboxylated into vinyl-phenols by the cinnamate decarboxylase activity of Brettanomyces and then reduced into ethyl-phenols by the vinyl-phenol reductase activity only present in Brettanomyces. These ethyl-phenols have a much more intense medicinal/barnyard aroma than vinyl-phenols. The use of enzymes with elevated levels of cinnamyl esterase in red winemaking can lead to increased substrate for Brettanomyces off-flavor production.

A simple one-size-fits-all approach is rarely successful in winemaking applications due to factors including varietal differences, vintage variation, differences in winemaking practices and individual stylistic goals. The differences in available enzymes and enological tannins allows for winemaker control in selection and application of enzymes and tannins. However, the fruit quality and condition, process pathway and final style goals must be taken into consideration. In order to make informed decisions, there is much to know about both enzymes and tannins; these are complex products with multiple points of differentiation.

Commercial enzyme product differentiation is produced by the use of specific genus and species of fermentation organism and altering the fermentation conditions and substrates that determine the proportion of individual enzyme activities produced by the fermentation organism. Further product differentiation is achieved through proportional blending of enzyme preparations that have been produced with differing ratios of specific activities. Even though regulations limit the source organisms for enological enzyme production, different strains of the source organisms can have genes for isozymes that have the same activity but slightly different properties, such as pH and temperature optimums.

Use of an enzyme product with good extraction properties while not being overly aggressive and releasing too many soluble solids into the must or attacking the seed coat is of primary concern. A proper enological extraction enzyme should have a high PG (enzyme polygalaturonase) to PL (enzyme pectin lyase) ratio and be low in side activities of cellulase and hemi-cellulase which can release unwanted seed tannins. Enzymes such as Laffort's HE Grand Cru and Lafase Fruit are optimized for extraction of quality parameters such as color, tannins and aroma precursors. Lafase HE Grand Cru also contains high levels of a specific enzyme activity, Rhamnogalacturonase-II (RGII-ase), which selectively releases the pectin structure RGII, which aids in stabilizing color and structure in red wines. (7)

Enological tannins

Enological tannins are often used in red winemaking to achieve the same objective as enzymes in terms of color and structure improvement. (9) The action of enzymes, however, is to extract more from the grape, whereas the action of tannins is to protect what is already extracted.

However, not all enological tannins are the same in terms of structure, composition or intended purpose. Commercially available tannin products fall into three categories: fermentation tannins, aging tannins and finishing tannins. The source material for the tannins and the extraction method and composition of the final blended product all contribute to the differentiation of the product, which, in turn, determines its intended application and impact.

Tannins are a structurally diverse group of molecules with quite significant differences, but from a simplified viewpoint we can separate enological tannins into two categories: hydrolysable tannins and condensed tannins. Within each category there are main points of difference, and the diversity of structure and composition as well as polymer diversity make tannins very heterogeneous.

Even though tannins as a molecular class are very heterogeneous, there are some characteristic properties that tannins exhibit, including protein binding (sacrificial enological application); color stabilization depending upon reactivity; anti-oxidation effects; balancing body; and enhancing wine structure, as well as the potential of masking green pyrazine character. While the basic structure that makes tannins what they are provides for all of these activities, these properties can be exhibited to varying degrees based upon the source, extraction processes and final blending formulation. This differentiation of tannin products determines their optimal application and impact.

The two main classifications of enological tannins are hydrolysable tannins and condensed tannins. Hydrolysable tannins consist of a glucose molecule, either in ring (gallotannin) or linear (ellagitannin) conformation with gallic acid residues attached to the hydroxyl groups on each carbon. Condensed tannins are basic flavan-3-ol structures, which have substitutions off of the three-ring structure including gallic acid. These flavan-3-ol three-member rings can polymerize in many conformations and form the polymers that form the phenolic structure desired in fine wines. The association of other molecules in wine, including proteins, polysaccharides and organic molecules, along with hydrolysable and condensed tannin interactions, create the backbone of desired wine structure.

This discussion is mainly focused on fermentation tannin composition and application in red winemaking, hence the tannins should be easy to handle and prepare whether in liquid or powder form, while providing protein-binding/sacrificial effects to help preserve the early extracted grape skin tannins as well as providing good structure-building potential.

Ellagic tannins, mostly derived from oak or other wood extraction, provide good protein-binding capacity with some aging potential. Tannin reactivity with anthocyanins provides for color stabilization and preservation. Reactive catechins form an ethanal (acetaldehyde) bridge with anthocyanins beginning the polymerization process, which stabilizes color.

Enological fermentation/aging tannin products produced from grape skin tannins can be somewhat reactive with anthocyanins but are quite expensive. Laffort has patented a process to produce highly reactive catechin tannins. (6) The resulting commercial product, Tanin VR Color, has an approximately 10-fold higher reactivity than grape skin tannin products and an approximately 100-fold higher reactivity than standard fermentation tannin products, greatly facilitating stabilization of color in red wines.

The use of enological tannins is also recommended in the case of botrytis infection to minimize the oxidative browning caused by laccase (for more information, please review the website: awri.com.au.). Tannins bind to protein surfaces and can inhibit enzymatic activity, including that of laccase at concentrations less than it takes to actually facilitate precipitation and removal.

Enzyme and tannin application criteria and potential benefits

Enological enzymes and tannins are not necessarily meant to be used in a formulaic or recipe fashion. There are specific circumstances where their use is most beneficial, as well as some stylistic goals that may necessitate consistent use.

The first consideration is the quality and condition of the fruit. Enzymes can certainly aid in extraction of fruit that is picked before optimal ripeness due to vineyard conditions, while mold pressure may indicate early tannin application with minimal pre-fermentation extraction either by maceration or enzyme application. When fruit conditions do not indicate an early enzyme application, an addition of a pectinase/beta-glucanase enzyme blend after pressing off the skins will help with wine clarity and filterability and will lower the microbial load in the resulting wine.

When using gentle cap-management techniques, an enzyme application can help the wine reach optimal color and tannin potential. In challenged vineyards or difficult vintages, where color and/or tannin is underdeveloped, enzyme extraction and tannin stabilization can be used to maximize these wine quality parameters. Selective extraction of wine quality components such as color, soft grape skin tannins and aroma precursors can be achieved by the proper enzyme selection and application.

Grape varieties that are high in soluble proteins when crushed are particularly good candidates for a sacrificial tannin addition at the crusher. Soluble proteins as well as cell-surface-bound proteins can bind and sequester the valuable grape skin tannins that extract early in the aqueous phase upon grape crushing. The application of enological tannins strong in protein-binding can help in preservation of the native grape skin tannins by occupying the protein surface tannin-binding sites present in the must.

Enological tannin addition can also help to fill in the structure and body of a wine. An early addition of tannin can allow for beneficial integration of the added tannin into the desirable oligomeric polyphenolic structures. In fruit that may be low in tannin, supplementation can fill in gaps in the structure that would otherwise compromise wine quality. As another tool in balancing the structure of challenged vintages, the availability of selective fining agents can work with enological tannin additions to achieve the balance sought.

Use of enological tannins is also indicated when fruit is compromised by mold or mildew. Tannins can react with mold proteins that give a musty character to wines and remove them, as well as neutralizing a portion of the laccase activity that many molds produce, causing rapid and excessive oxidation of phenolics and resulting in color degradation and loss. High ellagic tannin products are generally good protein-binding options.

The benefits of enological enzyme and tannin application are plentiful. Proper selection and addition can maximize stable color and tannin, aid in building structure and assist in bringing flavor and aroma consistency across variable vintages. The addition of enological enzymes and tannins can be integrated into almost any existing maceration, fermentation and style differentiation or aging strategy.

Regarding the addition of enological enzymes and tannins with existing flash detente technology: The rapid extraction possible with flash technology almost necessitates the use of exogenous agents to balance and stabilize the resulting must and wine. Consult with a flash technology provider and enological product supplier for optimal product offerings and applications.

Case study: Optimization of color and tannin in Merlot

In this collaboration between Delicato Family Vineyards, Fresno State University and Laffort USA, Merlot fruit from the San Bernabe Vineyard in King City was donated by Delicato to Fresno State for use in a student-based trial of enological enzyme and tannin applications for the purpose of improving color and tannin extraction and stabilization in the finished wine. Laffort USA provided all enological products and assisted the Fresno State Winery in technical guidance of the student winemakers.

The trial was set up with three lots: a control of standard practice with no enzyme or tannin additions; one treatment of standard practice with a 40 grams per ton dose of Laffort HE Grand Cru enzyme; and a second treatment of standard practice with a 40 grams per ton dose of Laffort HE Grand Cru and a fermentation tannin addition of 200 ppm Laffort Tanin VR Supra at the crusher and a 300 ppm dose of Laffort Tanin VR Color at 5[degrees] to 7[degrees] Brix depletion during fermentation.

All other additions, conditions and treatments were kept as similar as possible. After primary fermentation in half-ton picking bins, the wines were pressed off and placed in neutral barrels for malolactic fermentation and aging. Samples were taken after primary fermentation, at six months post-fermentation and 12 months post-fermentation. After 12 months in neutral barrels, the wines were bottled under cork and placed in cellar conditions.

Trial measurements were standard wine chemistry panels done at Fresno State, HPLC phenolic measurement done at ETS Laboratories in St. Helena, Calif., and CIE-LAB and A520 spectrophotometric analysis also done at ETS Laboratories. Sensory evaluation was done in a blind tasting at Fresno State with a 19-member untrained panel consisting of Fresno State winemaking students, faculty and staff and Laffort personnel.

Previous studies of enological tannin application compared to oak chip use in fermentation have shown that lasting differentiation occurs after six months, with consistent characteristics developed at one year post-fermentation. The sampling times of six and 12 months were based upon previous results.

The differences between control and treatments can be seen in Stabilization and Retention of Color and Tannin, on page 36, where the 12-month time-point amount of color in the enzyme-tannin treatment is 41% greater than in the control and the amount of tannin is 56% greater. As in all red wine aging, color and tannin naturally decrease over time. The differences between the six-and 12-month points are due to the reduced amount of color and tannin disappearance. The decrease in the enzymetannin treatment is significantly less than in the control and, along with the additional color and tannin extracted and stabilized early in fermentation, accounts for the improvement in these two important wine quality characteristics.

Looking at some specifics, the basic wine chemistries between control and treatments were not differentiated. The A520 data tracked along with the CIE-LAB data, with the differences reported in the color graph in Stabilization and Retention of Color and Tannin. The phenolics panel revealed significant differentiation, with the control wine analysis set as 100% for all parameters measured (see Enzyme-Tannin Application Effects on Red Wine Phenolics, above). The HE Grand Cru treatment shows good extraction of both tannins and anthocyanins, while the enzyme-tannin treatment has an increase in total tannin and catechin due to the 300 ppm addition of Laffort

Tanin VR Color (a reactive catechin with extremely low astringency characteristics), as well as a dramatic shift in the amount and distribution of monomeric and polymeric anthocyanins. The enzyme-tannin treatment reveals a large shift of monomeric anthocyanins into the pool of polymeric anthocyanins, a much more stable form of the colored compounds in red wines.

While the chemistry of the trial wines is quite interesting, quantitative evaluations of wine can only get you so far. Sensory evaluation of the wines is critical to understanding the impact of the quality components and characteristics that a trial seeks to impact and measure. The sensory panel determined in a blind tasting that the enzyme-tannin treated wine was preferred by a very wide margin of 79%, compared to 16% for enzyme alone and 5% for the control.

There were many other presentations of these trial wines beyond the Fresno State sensory panel, and in less formal settings, the 80% preference for the enzyme-tannin treated wine held up consistently (data not shown). Comments suggested that the color differentiation was so noticeable that the naked eye could detect the difference, and the depth of body and intensity of flavors were also noted as differentiating factors.

Powerful tools for your winemaking toolbox

This specific Merlot trial from the Central Coast may not create a paradigm in wine production, yet it does serve as an example where enological enzyme and tannin application can act to increase measurable and detectable quality parameters in a wine.

The doors left to be opened include the impact these treatments can have on the most important quality parameters of other varieties, as well as dose response differentiation under various variety and vintage conditions. The possibility of altering standard winemaking practices such as length of cold soaks, post-fermentation macerations, process applications and even yeast selection in response to integrating enzyme-tannin treatments into existing winemaking practices also brings the potential of additional impacts on specific wine quality characteristics.

KEY POINTS

Enological enzyme products have many specific catalytic activities.

Enological tannins are complex and diverse tools.

Extraction and stabilization of color and tannins can be achieved through enzyme/tannin synergy.

Peter Salamone, an Independent consultant, received his Ph.D. from Washington State University and has spent 18 years in both the biotechnology and wine production industries. Shaun Richardson, Laffort USA general manager, has spent 25 years in the wine industry, receiving his undergraduate degree in Oenology from the University of Adelaide, and MBA in Wine Business from Sonoma State University.

Caption: PECTIN POLYMER SHOWING STRUCTURAL COMPONENTS AND SITES OF ENZYME ACTIVITY

Caption: STABILIZATION AND RETENTION OF COLOR AND TANNIN

Caption: ENZYME-TANNIN APPLICATION EFFECTS ON RED WINE PHENOLICS

Please Note: Illustration(s) are not available due to copyright restrictions.
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Title Annotation:WINEMAKING: PRACTICAL WINERY & VINEYARD
Comment:Enzyme and Tannin Applications: Potential impacts on red wine quality.(WINEMAKING: PRACTICAL WINERY & VINEYARD)
Author:Salamone, Peter; Richardson, Shaun
Publication:Wines & Vines
Date:Jun 1, 2018
Words:3086
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