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Enzymes in Winemaking


Karien Lourens1 (pictured) & Patrice Pellerin2
1 Anchor Yeast, Epping Industria (www.newworldwinemaker.com)
2 DSM Food Specialties Oenology, Montpellier, France (www.dsm-oenology.com)

Introduction

Today it is commonplace to use commercial enzymes during winemaking. This is a short review on the different types of commercial enzymes available and their applications. The enzyme production methods and compositions discussed in this article are based on the enzyme technology of DSM and may vary from other enzyme producing companies.



The main enzymes used during winemaking are pectinases. Pectinases occur naturally in all fruit - including grapes - and are partly responsible for the ripening process. Grape pectinases are however inactive under the pH and SO2 conditions associated with winemaking. Fungal pectinases are resistant to these winemaking conditions. The method used to produce wine enzymes for use in the European Union is regulated by the OIV. The OIV has established that only Aspergillus niger and Trichoderma can be used for enzyme production. Producers that export wine to the EU - if they use enzymes during production - are obliged to use enzymes that comply with these prerequisites.

Commercial enzyme types

The most widely used enzymes available for commercial use are: pectinases, hemicellulases, glucanases and glycosidases. The latter three types are generally sold as blends with pectinases. With the exception of glucanase all the enzymes are produced by Aspergillus niger, whereas glucanase is produced by Trichoderma harzianium.

Enzyme Production

For Aspergillus to produce pectinases it must grow on a medium of pectin as a carbon source. The fungi must therefore be stimulated to produce the desired enzymes and their side activities and will produce very little if grown on a normal sugar source like molasses. Only genetically modified fungi can grow on other substrates and produce the desired enzymes as they are genetically manipulated to continually produce the enzyme. They do not require a specific substrate for enzyme production to be stimulated. It is important to note that the enzyme structure produced in this manner does not differ in any way from the enzyme structure produced by the un-manipulated organism. The enzyme is not modified - only the organism used in the production is, and the organism is removed from the final product to the consumer. There is however a very noticeable decrease in side activities if this type of enzyme preparation is used in its pure form and not blended, since only the enzyme from the gene that is manipulated will be produced. This form of production holds no danger to the consumer and is legal in many industries.


White wine production enzymes

After crushing, negatively charged pectin molecules 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 are the most basic commercial enzymes with regards to their composition and mode of action. They have three main activities: Pectin lyase (PL), pectin methylesterase (PME) and polygalacturonase (PG). 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. The polygalacturonic acid units that make up pectin are bound to a methyl group. Pectin lyase recognizes this structure and is able to cut between these units to break up the pectin structure. During ripening the PME content of grapes increase and this enzyme cleaves the methyl units off the polygalacturonic acid chain and pectin becomes pectate. When the methyl groups are removed pectin lyase is unable to recognize its substrate. PG recognizes galacturonic acid units without the methyl unit (pectate). As a result very ripe grapes require settling enzymes with higher concentrations of PG compared to normal settling enzymes. When settling problems take place with very ripe grapes it is suggested that skin contact enzymes be used as they contain a higher PG content.


White skin contact enzymes

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 (Rapidase X-Press) are more concentrated and contain more side activities compared to normal settling enzymes (Rapidase Vino-Super). Skin contact is done on white grapes for two reasons, namely juice and flavour 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 to break this physical barrier and therefore increase the yield per ton of grapes obtained.

Most grape flavours such as norisoprenoids, pentanones (Sauvignon blanc) and terpenols (Muscats) are more concentrated in the grape skins and with skin contact the levels are increased in the must. Skin contact also increases the nitrogen content in the must, which is generally a good feature, however it can also cause an increase in the concentration of heat unstable proteins and polyphenols. This explains why higher dosages of bentonite are often needed to protein stabilise wines that have had skin contact. An important concern with skin contact enzymes is over-maceration. This happens if the skins are exposed to enzyme for too long. This can cause a settling problem that is characterized by clear juice in the top of the tank, compact lees in the bottom of the tank and a meter or two of "fluff" above the lees. This "fluff" consists of the very fine pieces of the grape skins that stay in suspension. Skin contact protocols should be meticulously followed.

Red Skin Contact Enzymes

There are two main differences between the red skin contact enzyme (Rapidase Ex-Color) and the white skin contact enzyme (Rapidase X-Press) from DSM. The red skin contact enzyme contains more hemicellulase than the white skin enzyme for improved maceration. The red skin contact enzyme also has very low concentration of anthocyanase activity. Anthocyanases are able to break off sugar units from more complex molecules. Grape anthocyanins are stabilised by covalent linkage with one glucose unit. They become unstable and become colourless when these linkages are broken. It is important that commercial red wine enzymes do not contain this activity. During industrial fermentation Aspergillus produces a whole range of enzymatic activities including glucosidase activity. Rapidase Ex-Color is produced by fungal strains that naturally produce glucosidase activity far below the concentration that becomes harmful to red wine colour.

If winemakers use enzymes for red skin contact containing high anthocyanase activity, colour stability problems may occur over time. Anthocyanase activity in white wine enzymes is harmless. The use of liquid pectinases suited for the clarification of white musts and wines has to be avoided on red grapes unless the producer can guarantee the absence of anthocyanase activity.


The duration of red skin contact is much longer compared to white skin contact. Tannins can bind to enzymes and thereby inactivate them. This explains why higher concentrations of enzymes are needed for red grapes compared to white grapes. Red skin contact increases the anthocyanin content, however the more important action of enzymes used on red grapes is the increase in colour stability. Many grape varieties possess ample colour and a number of winemakers feel it is unnecessary to add enzymes. Enzymes however increase tannin extraction that is vital for colour stabilisation.

Glycosidases

As already mentioned these enzymes remove the sugar moieties from more complex molecules. Such enzymes are of commercial importance for grape varieties that contain flavour groups attached to sugar moieties (or residues). Examples of these flavour groups are monoterpenes and C13-norisoprenoid derivatives. Aroma precursors in a bound form with sugars are not volatile. When these sugars are removed, the flavour becomes volatile and thus aromatic and then contributes to wine aroma. In vitis vinifera there are mainly di-glycosides, which means 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 mainly because their optimum pH is at five and wine pH is between three and four. Clarification of the must also removes glycosidase activity. Certain wine yeasts also have glycosidase activity but the optimum activity is at pH five, rendering it not very effective at wine pH. Fungal glycosidases are effective at wine pH and must ideally contain all four enzyme activities namely, glucosidase, arabinosidase, rhamnosidase and apiosidase. 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. Fungal glycosidases are effective when used on grape varieties containing such precursors such as Muscat, Gewürztraminer and (Weisser) Riesling. They can be added to a finished wine or a wine with a residual sugar of 50 g/L or less. This is because fungal ß-glucosidases are repressed by glucose. It is suggested that an enzyme like AR 2000 is only used on part of a final blend because it is not desired that all the bound flavours are released into the volatile form. Normally monoterpenes are fairly stable molecules and are hydrolyzed over time, releasing a floral aroma over a long period of ageing. The enzyme will release a lot of flavour all at once and by treating only a part of a blend the rest of the blend will supply the flavours to enhance the longevity of the wine. Certain grape varieties like Sauvignon blanc and Chardonnay contain monoterpenes in addition to their specific varietal character. However it is not always desirable for these grape varieties to have a terpene background aroma so glycosidase enzymes should be used carefully on these varieties. The enzyme action must be stopped after one to four months depending on the desired effect that is required. The enzymes have to be removed with 5 - 10 g/hL bentonite.

Beta-Glucanases

ß-Glucanases are produced by Trichoderma harzianium. Beta-glucans are the main component of cell walls of all fungi, including the yeast Saccharomyces cerevisiae. Traditionally ß-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 blockages during filtration. A more recent alternative use of these enzymes is to enhance yeast autolysis. The yeast cell wall is composed of glucan chains and mannoproteins. Natural yeast autolysis is a longterm process which occurs for more than 12 months after fermentation. During and after fermentation yeasts are able to release various components that are small enough to move out through the cell membranes and cell walls. True autolysis is where the cells break open and usually takes much longer. To achieve true yeast autolysis within three to eight months - which is the normal lees contact time - a commercial glucanase-containing enzyme like Rapidase Filtration 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, it should not be used. The danger of feeding Brettanomyces is greater when the enzyme is used on a red wine.

Cinnamyl esterase free enzyme preparations

Cinnamyl esterase (CE), an enzyme activity present in certain pectinase enzyme preparations, together with cinnamyl decarboxylase produced by certain yeast strains (Pof+, phenyl off flavour), can be responsible for the production of volatile vinyl-phenols. These volatile phenols can cause off-flavours in white wine, which are described as phenolic or medicinal. The production of these volatile phenols is thus a two-step process requiring both enzyme activities. Cinnamyl esterase is sometimes referred to as "depsidase". The name corresponds to the first description of the enzyme but is incorrect and the word depsidase should not be used.

The first enzymatic reaction is the cleavage by cinnamyl esterase of the ester linkage between tartaric acid and hydroxycinnamic acid, neither products of which are volatile. These esters are the major group of polyphenols in white grape musts and are also abundant in reds. The second reaction is the transformation of the hydroxycinnamic acids by the wine yeast decarboxylase, into vinyl-phenols, which are volatile and have an impact on wine quality. When present in low concentrations, vinyl-phenols can have a very pleasant clove-like smell in red wines. However when present in higher concentrations the effect on wine aroma is negative.

Brettanomyces also contains the cinnamate decarboxylase enzyme activity and can therefore also decarboxylase hydroxycinnamic acids to vinyl-phenols. However in addition to the decarboxylase, Brettanomyces also contains the enzyme vinyl-phenol reductase. This enzyme converts vinyl-phenols to ethyl-phenols and this is unique to Brettanomyces. The result is similar, though much stronger phenolic off-flavours. The effect of vinyl-phenols is much less in red wines as they react with tannins, forming covalent linkages, making them non-volatile. Thus phenolic off-flavours in red wine are usually the result of ethyl-phenols and thus of Brettanomyces contamination.

Non-Brettanomyces phenolic off-flavours are therefore really only an issue in white wines. Only when the industrial enzyme preparation contains significant levels of cinnamyl esterase together with the use of a Pof+ yeast strain will these off-flavours form. Both Anchor VIN 13 and Anchor NT 116 are Pof-.

The DSM range of enzymes have naturally low levels of cinnamyl esterase - lower than purified enzymes - and their use would thus not contribute significantly to the production of volatile phenols, even if Pof+ yeast strains are used for fermentation.

References

Pellerin, P. DSM Food Specialties Oenology. Personal communication.

Ribéreau-Gayon, P., et al. Handbook of Enology Vol. 2.

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.

www.dsm-oenology.com.

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