Karien O’Kennedy1, Céline Fauveau2 and Patrice Pellerin2
1Anchor Yeast, Epping Industria, South Africa (www.anchorwineyeast.com)
2 DSM Food Specialties Oenology, Montpellier, France (www.dsm-oenology.com)

This is a review on the different types of enzyme preparations available and their applications. The enzyme production methods and compositions discussed in this article are based on the enzyme technology of DSM Food Specialties and may differ from those of other enzyme producing companies.  

To produce the enzymes used in winemaking, selected strains are cultivated in fermentors under aerobic conditions: Aspergillus niger for the production of pectinases and b-glycosidases, Trichoderma harzianum in the case of b-glucanases and Lactobacillus fermentum in the case of urease.

A well-defined composition of the growth medium induces optimal production of the desired enzymatic activities. For example, a growth medium rich in pectin induces the microorganisms to secrete pectinases into the media. After fermentation, the pectinases and enzymatic side activities are isolated by centrifugation, ultra filtration and concentration. During these stages microorganisms are completely eliminated from the end product.  

Hen egg-white (HEW) lysozyme is a well-known protein, which has been used in pharmaceutical compositions and food since the 1950s. The product is separated from hen egg white with an ion exchange resin, then purified and dehydrated.

Pectinases

The main activities in winemaking enzyme formulations are derived from the pectinase family. They include pectin lyase (PL), pectin methyl-esterase (PME) and polygalacturonase (PG). PL-type activity, known as de-polymerization, cleaves the pectin chain between two methylated galacturonic acids, while the PG prefers a non-methylated substrate. PME activity does not depolymerise the pectin chain but releases a methanol molecule from galacturonic-esterified acids. It eases the action of PG.
Pectinases, by breaking down pectin, do provide a number of obvious technical advantages, such as accelerating the pre-fermentation stages, increasing free run juice yield, enhancing clarification and pressing leading to an overall improvement in the grape must quality, with increased aroma and polyphenol concentrations.

Today, each enzyme producer uses their own pectinase activity measuring method and unit. These measures do not provide an indication of the enzyme efficiency in winemaking. The efficiency of an enzymatic preparation in winemaking is strongly related to the presence of side activities. The main pectinase activity alone does not ensure all benefits. For this reason, enzyme efficiency in winemaking ought to be checked through application trials.  It is not possible to compare products from different origins based on the activity level provided by the supplier on the product label or specification sheet.

Such enzymes are of importance for grape varieties that contain aroma precursors linked to sugar moieties (terpenes, C13-norisoprenoid derivatives). Aroma bound with sugars is not volatile. When the sugar is removed the aroma becomes volatile and thus aromatic. In Vitis vinifera there are mainly di-glycosides, which means that the monoterpenes are bound to glucose and another carbohydrate residue such as arabinose, rhamnose or apiose.  Grapes contain glycosidases capable of releasing aromatic terpenols from their non-aromatic precursors.  However under winemaking conditions these enzymes are not very efficient. Fungal glycosidases are effective at wine pH. Glycosidase is the common word given to these activities. Glucosidase on its own is ineffective in releasing the aromatic components from the di-glycosylated precursors, as sugar breakdown is sequential and the other sugars must be removed first before glucose can be removed.

β-Glucanases are produced by Trichoderma harzianium.  Traditionally β-glucanases have been used to improve filtration of wines obtained from grapes infected with Botrytis cinerea. Glucans are secreted by Botrytis into the juice during infection and can cause filter clogging. As β-glucans are the main component of yeast cell walls, a more recent alternative use of these enzymes is to enhance yeast autolysis. Natural yeast autolysis is a long-term process that occurs for more than 12 months after fermentation. Commercial glucanase-containing enzymes facilitate improved yeast autolysis through reduction in time as well as increased quantity of released yeast cell wall compounds into the wine.

The nature and complex composition of the growth substrate used to produce enzymes induces the production of a wide pool of enzymatic activities. The main (principal) activity in the obtained enzymatic product is accompanied by numerous secondary (side) activities that play roles of varying degrees of importance, some being essential, other neutral or detrimental in specific winemaking applications. By using Aspergillus niger strains specifically selected for enological purposes, DSM Food Specialties enzyme preparations are formulated to naturally maintain any unwanted activities to a negligible level. Some side activities might, in certain types of wines, have an undesirable effect and a positive effect in other. Although these secondary activities are tolerated under the legislation, these enzymes can in certain cases spoil wine quality.

These side activities are present in varying amounts in pectinase preparations. They are wanted in red grape maceration in order to extract the maximum skin cells content. They are unwanted in white grapes maceration to limit over extraction.

In white wines this activity contributes to the hydrolysis of hydroxycinnamyl-tartrate esters, releasing coumaric and ferulic acids, which, after decarboxylation by a POF+ (phenyl off flavour) yeast strain, lead to the formation of vinyl-4-phenol and vinyl-4-guaiacol. These compounds give unpleasant poster paint and nail polish smells. In red wines vinyl phenols react with polyphenols to form colour-stabilising compounds. It is therefore important to use white wine enzymes that contain negligible levels of this side activity in order to limit the formation of volatile phenols, thereby maintaining their concentration under the perception threshold.  

This activity could cause a colour loss in red wines as it releases anthocyanins from their bound sugar. This hydrolysis results in an unstable anthocyanidin form.  

Enological enzymes can be formulated in liquid or micro granulated form.

Micro granulate enzymes
These enzyme preparations offer good storage stability; their activity level is stable when stored under the recommended conditions of humidity and temperature. At room temperature the shelf life ranges from 24 to 36 months. Granulated enzymes have no risk of being contaminated after opening, even though no preservatives are added.

These enzyme preparations should be stored at cold temperature. The shelf life of these products when stored under recommended conditions ranges between 12 to 24 months. Their microbiological stability is more difficult to guarantee and their formulation often requires the use of preservatives. For example, sorbate salts and potassium chloride are authorised preserving agents used in liquid enzyme preparations. Another stabilising agent commonly used in liquid enzymes is glycerol.

White winemaking enzymes

Settling enzymes

After crushing, negatively charged pectins form a protective layer around positively charged grape solid particles.  This keeps the grape solid particles in suspension.  Pectinase enzymes break the pectin molecules into smaller components, thereby exposing some of the positively charged grape solid particles underneath this protective layer.  These positive charges bind to the negative charges of the pectin protected grape solids and bigger particles form.  When particles become too big, they settle out.

Settling enzymes work mainly on the soluble pectins (mainly homogalacturonans) of the pulp of grapes. The skins of grapes contain more insoluble pectin (protopectin) with more “hairy regions” (side chains).  Skin contact enzymes therefore - in addition to the basic settling enzyme components - contain more side activities that specifically work on the hairy parts of the pectin. Like all fruits the pectin structure changes during ripening and the grapes become softer. Very ripe grapes require settling enzymes with higher concentrations of PG. When settling problems take place with very ripe grapes it is suggested that skin contact enzymes be used as they contain more PG.

As mentioned previously the structure of insoluble pectin in grape skin cell walls is more complex than pulp soluble pectins. It is for this reason that skin contact enzymes are highly concentrated and contain essential side activities.  Skin contact is performed for two reasons, namely juice and aroma extraction. Grape cell walls form a physical barrier between the juice in the vacuole of berry cells and the outside medium.  Since grape cell walls contain +/- 30% of pectin, pectinases help break this physical barrier and therefore increase juice yield.

Most grape aroma and their precursors such as norisoprenoids, pentanones, thiols  (Sauvignon blanc) and terpenols (Muscats) are located in the grape skins. Skin contact increases their concentration in the must. An adapted white skin contact enzymatic formulation contains reduced levels of cellulases and hemicellulases to avoid over maceration.

Red winemaking enzymes

Red maceration enzymes can contain hemicellulase for improved maceration.  Red enzyme formulations should also contain very low levels of anthocyanase activity, which break off sugar units from more complex molecules. Grape anthocyanins are stabilised by covalent linkage with one glucose unit. They become unstable when these linkages are broken.

Maceration increases the anthocyanin content, however the more important action of enzymes used on red grapes is the increase in colour stability. Recent studies on complex phenolic compound chemistry, allows for a new approach to increase and stabilise red wine colour. This consists of the synergy between an enzyme preparation Rapidase Maxifruit and an oenological yeast strain Fermicru XL that both present specific characteristics that enhance the formation of stable pigments.

To achieve true yeast autolysis within three to eight months a commercial glucanase-containing enzyme should be used. Autolysis has many advantages for the wine quality such as mouth feel that is acquired from the polysaccharides that are released into the wine. Certain mannoprotein fractions improve protein stability whilst others improve tartrate stability. Other components released into the wine during autolysis have an impact on wine flavour and complexity.  Autolysis releases many amino acids and nucleotides into the wine that are a source of nutrition for organisms such as bacteria and Brettanomyces. In the case of malo-lactic fermentation this can be advantageous; on the other hand if you suspect Brettanomyces contamination in the cellar then it should not be used.  The danger of feeding Brettanomyces is greater when the enzyme is used on a red wine.

References

1. Pellerin, P. DSM Food Specialties Oenology. Personnel communication.
2. Ribéreau-Gayon, P., et al. Handbook of Enology Vol. 2.
3. Van Rensburg, P. and Pretorius, I.S. Enzymes in Winemaking: harnessing natural catalysts for efficient bio-transformations. South African Journal of Enology and Viticulture Vol. 21, Special Issue 2000.
4. www.dsm-oenology.com