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June 2018 WINES&VINES 35 PRACTICAL WINERY & VINEYARD WINEMAKING The increased enzymatic extraction of tan- nins in the form of grape proanthocyanidins can also contribute to the color stability of red wines over time as they react with anthocya- nins to form derived pigments such as tannin- anthocyanin adducts. 4,8 Insoluble pectin, mostly in the middle la- mella–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 condi- tions, 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 produc- tion, the fungi produce a diverse suite of en- zymes, including unwanted side activities including cinnamyl esterase. Cinnamyl ester- ase catalyzes the first reaction in the produc- tion of vinyl-phenols. This activity is always present in pectinase preparations if not re- moved 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 concen- tration of vinyl-phenol precursors will increase. The danger with formation of precursors of vinyl-phenols is, if Brettanomyces spoilage oc- curs, they will be first decarboxylated into vi- nyl-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 me- dicinal/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 path- way and final style goals must be taken into consideration. In order to make informed deci- sions, there is much to know about both en- zymes 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 fermenta- tion 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 extrac- tion properties while not being overly aggres- sive and releasing too many soluble solids into the must or attacking the seed coat is of pri- mary 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 un- wanted 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 spe- cific enzyme activity, Rhamnogalacturonase-II (RGII-ase), which selectively releases the pec- tin structure RGII, which aids in stabilizing color and structure in red wines. 7 Enological tannins Enological tannins are often used in red wine- making to achieve the same objective as en- zymes 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 in- tended 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 differen- tiation of the product, which, in turn, deter- mines its intended application and impact. Tannins are a structurally diverse group of molecules with quite significant differences, but from a simplified viewpoint we can sepa- rate 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 char- acteristic properties that tannins exhibit, in- cluding protein binding (sacrificial enological application); color stabilization depending upon reactivity; anti-oxidation effects; balanc- ing 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, extrac- tion processes and final blending formulation. This differentiation of tannin products deter- mines their optimal application and impact. The two main classifications of enological tannins are hydrolysable tannins and con- densed tannins. Hydrolysable tannins consist of a glucose molecule, either in ring (gallotan- nin) 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 sub- stitutions off of the three-ring structure includ- ing 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 inter- actions, create the backbone of desired wine structure. This discussion is mainly focused on fermen- tation 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-bind- ing/sacrificial effects to help preserve the early extracted grape skin tannins as well as provid- ing 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. Reac- tive catechins form an ethanal (acetaldehyde) bridge with anthocyanins beginning the po- lymerization process, which stabilizes color. Enological fermentation/aging tannin prod- ucts produced from grape skin tannins can be 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.