ENZYMES INVOLVED IN WINE PRODUCTION (Article 3)
P van Rensburg and IS Pretorius
P van Rensburg and IS Pretorius - Institute for Wine Biotechnology and Department of Viticulture and Oenology, University of Stellenbosch
In this third article about enzymes involved in wine production we look
more specifically at the industrial enzyme preparations as well as possible new enzyme applications.
INDUSTRIAL ENZYME PREPARATIONS
As mentioned, the endogenous enzyme activities of grapes and
micro-organisms are often not effective or sufficient under winemaking
conditions. Presently enzymes from exogenous sources are considered
valuable means of processing, as they can reinforce or replace the
enzymatic preparations in the grape. The use of enzymatic preparations in
the food and liquor industry was investigated and specific applications
made for the wine industry. Extensive knowledge of the nature and structureof the macro molecules occurring in must and wine offer new possibilities for the use of this enzyme in wine production. Changes which are therefore obtained by means of enzymatic treatment will not only influence the clarification and filtration, but also the extraction and stabilising processes in both white and red wine production (Fig. 1.). Presently the most important applications of industrial enzymes in wine production
involve the use of pectinase and glucanase preparations.
Pectinases
Most industrial pectinase preparations are isolated from cultures of Aspergillus, especially Aspergillus niger, a fungus species with GRAS (Generally Regarded As Safe) status and accepted by the Office
International de la Vigne et du Vin (OIV).
The technology used for the production of industrial enzymes was
investigated. After fermentation the desired enzyme activities are
recovered and purified. Standardised enzyme preparations undergo various
quality control tests before being sold in liquid or granulated form.
Except for the main pectolytic activity, industrial pectinase preparations
also contain hemicellulolytic and cellulolytic activities. Additional
glycosidase and protease activities may also be present. The relationship
between the most important activities and the so-called secondary
activities defines the value of an enzyme preparation for a specific
application. Some of the applications are discussed below.
Pulp treatment for juice extraction
The pulp of a number of grape varieties is rich in pectic compounds. The
incomplete hydrolysis of these molecules by the endogenous enzymes may
therefore cause problems during processing. Tests on laboratory and
industrial scale have demonstrated the advantages of using exogenous
pectolytic enzymes for the processing of grape juice. This technology is
commonly used for the treatment of "smooth" grapes, which are otherwise difficult to press.
Experiments were conducted on various grape varieties and showed that
enzyme treatment of the pulp considerably increased the juice recoveries
and, compared to the control, the wines were of equal or better quality.
Furthermore the tests indicated that clarification by centrifugation may be
improved significantly as a result of changes in the physical qualities of
the flocculating material. More recent tests with Muscat Gardo Blanco have
confirmed the considerable improvement in juice recovery from enzyme
treated samples.
Juice clarification and wine filtration
After pressing the grape juice is cloudy and has a certain viscosity due to the presence of pectins. The pectin content is dependent on the grape
variety, the degree of ripeness and the effect of technological factors
(mechanical harvest, pressing conditions). These parameters all play a role in the time required for good clarification.
During the production of white wine the viscosity of the must is quickly
reduced by the hydrolysis of pectins with pectinases. This allows for
quicker and easier stabilisation of the juice and causes aggregates to
become finer and eventually disappear.
The result of enzymatic clarification appears later, after the filtration
stage, where the remaining pectins from the control lot are responsible for the rapid increase in filter pressure and consequently also clogging of the filter. Improved filtration is therefore obtained with enzyme treatment, followed by the use of less filter material and less wine loss. Other experiments also showed that the addition of a suitable protease to the must after pasteurisation may also improve the clarification and filtration of the wine as well as reduce or prevent the formation of foam. With red wines obtained by traditional pulping, the use of enzyme preparations after pressing is also favourable (clarification tank) for clarification and filtration as indicated in Table 1.
Colour extraction
The extraction of phenolic compounds usually occurs during the pulping of
the mixture in the course of alcoholic fermentation. The efficiency of this extraction is dependent on the variety and quality of the grapes, as well as technological parameters such as pressing, addition of SO2, pulping time, temperature, racking, etc. Although trial runs on colour extraction are difficult to conduct, due to the heterogeneous nature of the mixture and the influence of processing factors, good results were obtained with enzyme treatment on an industrial level. Improved anthocyanin extraction was obtained with enzyme addition to the grapes at the beginning of pulping. Racking provides good enzyme distribution in the mixture after filling the tank. The improved colour of the wine remained stable during maturation and it was found that enzyme treatment had a positive effect on the organoleptic parameters such as fruitiness and structure. However, basic research on colour extraction and stabilisation is still required for a full understanding of the mechanisms involved in these processes.
GLUCANASES
Glucanase preparations for wine production were developed and tested in the
1980's. The only specific industrial enzyme preparation available was
prepared from a culture of a selected strain of a Trichoderma species. It
was developed to solve the problems with clarification and filtration in
juices extracted from grapes infected with Botrytis cinerea. Processing
problems occur due to the high molecular mass of polysaccharides secreted
by this fungus. The structure of these macro molecules was identified as a
ß-(1,3-1,6)-glucane. During the processing of grapes the Botrytis cinerea
glucane is released in the juice and then later found in the wine. As it is
impossible to remove this polysaccharide through hydrolysis with endogenous
enzymes or by conventional treatments (centrifugation, clarification
agents), the degradation thereof by exogenous enzymes is a unique solution.
Various trial runs have demonstrated the efficiency of such treatments as
well as the impact thereof on the efficieny of filtration. The addition of
enzymes is preferable in young wines just after completion of the alcohol
fermentation. Contact time with the enzyme is 7 - 10 days and the
temperature should not be below 10?C. Increased enzyme dosings are
recommended for red wines because partial inhibition of the activity occurs
through phenolic compounds.
Table 1:
Result of enzyme treatment on the clarification of pressed red
wines. Influence on fining and physical processes.
| Process |
Untreated wine |
Enzyme treated wine |
| None |
60 |
38 |
| Fining |
55 |
8 |
| Centrifugation |
20 |
12 |
| Filtration |
2 |
0.3 |
Fining &
centrifugation |
13 |
2.3 |
NEW ENZYME APPLICATIONS
Aroma release
Since the enzymatic mechanisms for the hydrolysis of terpinylglycosides
have been determined, studies have been targeted at searching fungal
enzymes which may improve the aroma of wine. Tests conducted with an
experimental ß-glucosidase preparation from Aspergillus species have
demonstrated that positive results may be obtained under winemaking
conditions. This new enzyme can be added to fermented wines as soon as the
glucose has been used by the yeast, or it may be added to young wines.
However, further tests have to be conducted on different grape varieties.
This technology can be used to reinforce the varietal aroma and the bouquet
of certain wines.
Low alcohol wines
Low alcohol wines may be produced by various physical treatments involving
the use of expensive equipment such as retro-osmosis. An enzymatic method
based on the use of glucose-oxidase and catalase has been suggested.
Through the enzymatic treatment of the juice, glucose is converted to
gluconic acid, which cannot be metabolised by yeast. Wine produced in this
way has a reduced alcohol content and higher acid. This technology can
therefore also be used for the production of a reserve sour must or wines
for blending purposes.
Colour stabilisation
The removal of phenolic compounds to prevent oxidation, cloudiness, colour
changes (browning) and taste changes, is usually done with clarifying
agents (gelatine, poly-vinylpolypyrolidone). Treatments with enzymes such
as laccase, tannase and peroxidase were also tested for their ability to
impart stability in juice and wine. The effect of the fungal laccase was
thoroughly investigated since it has the ability to react with a wide range
of phenolic compounds. It was found that laccase treatment may improve the
effect of conventional clarification (Fig. 2). In other words, laccase is
added before clarification, so that the reaction products of laccase and
the phenolic compounds may be removed during the clarification stage. In
this way, phenols are being eliminated in advance.
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