|
A Technical Guide for Wine Producers
|
|
RECENT ARTICLES | WYNBOER HOME
Enzymes in winemaking - Method of production, the enzymatic reactions and their effect on the transformation of grapes into wine
DSM Food Specialties, a Dutch company specializing in chemicals and biotechnology, is the world leader in the agro-food market for ingredients and technological auxiliaries. Among its highly varied product range are yeast and yeast by-products, as well as enzymes including a specific winemaking range. Frequently asked questions, both on the means of production and on the use of the enzymes in vinification, have led DSM's technical and application service to write this article. Inducing the desired enzymatic activity To produce the enzymes used in winemaking DSM Food Specialties cultivates selected micro organisms, Aspergillus niger or Trichoderma, in fermenters under aerobic conditions. The composition of the vegetal-origin growing medium induces optimal reproduction of the desired enzyme. For example, a growing medium rich in pectin induces the micro organisms to secrete pectinase (pectolytic enzymes) into the media. The enzymes are then isolated by centrifuge, ultra filtration and concentration. During these stages micro organisms are completely eliminated and an enzymatic product is obtained whose main activity is accompanied by numerous secondary (side) activities that play different roles of varying degrees of importance, some being of great value, in winemaking. Reminder of the structure and composition of the grape To understand the importance of enzymes in winemaking, their action must be understood. Enzymes play an important role in breaking down grape pulp and skin cells. Although the composition of the grape depends on its variety, the soil and the climatic conditions, there is little variation in the actual cell structure of the plant. During vinification, the grape skin plays a vital role. It represents 6 to 9% of the berry mass. Within the skin cells are found anthocyanins, tannins and aromas or aroma precursors. The thick pecto-cellulosidic wall of the skin cells provides rigidity while also preventing the intracellular components from being diffused into the must during vinification. The pulp represents 75 to 85% of the berry at maturity. It comprises large cells with fine pecto-cellulosidic walls offering little mechanical resistance to the grape transformation. These cells are a source of pectic polysaccharides in the must; inside their vacuole is a concentrated solution of organic acids and fermentable sugars (glucose and fructose at between 150 and 300 grams per litre). The pectin is located in the primary wall and the sheath between the skin cells and the pulp. The pecto-cellulosidic wall is a complex structure. It is comprised of cellulose microfibrilles, linked together by a matrix of xyloglucan, mannan, xylan (generally known as hemi-cellulose) and pectin, all consolidated by a secondary protein network. Some neutral sugars (galactose and arabinose) make up part of the structure of the lateral pectin chains, and form macromolecules with the proteins that hinder clarification of the must. As the grape ripens, the grape berry endogenous pectinase activities slowly solubilize the pectin. This partial hydrolysis contributes to the gradual softening of the berry. In its soluble form the pectin passes into the must during pressing. The grape's pectin content varies in relation to the grape variety. See Table I.
Pectin - a complex molecule Pectin is probably one of the most complex macromolecules found in nature. It has three main components (2, 3):
There are several schools of thought on how these three constituents found in the cell walls are organized. It has, however, been shown that they are combined by covalent acidic links (Figure 2) to form pectin. Crossed links between different pectin chains (ionic, electrostatic, and diester borate bridges) determine the porosity of the cell wall. The high level of viscosity of pectin solubilized after crushing, hinder juice extraction, clarification and filtration. Soluble pectin is a major constituent of the cell walls preventing the diffusion of phenolic compounds and aromas into the must during the pre-fermentation and fermentation stages.
Enzyme activity permitted in winemaking The activities authorised in winemaking are covered by the European Union article 1493/1999. The OIV resolution, oeno 11-18/2004, recognizes the importance in the vinification process of the following enzymatic activities: pectin lyase, pectin methyl-esterase, polygalacturonase, glycosidase, hemicellulase, cellulase and b-glucanases. In this article we will focus mainly on the effect of enzymes on grape berry components. The main activities currently used in winemaking preparations are derived from the pectinase family. They include pectin lyase (PL), pectin methyl-esterase (PME) and polygalacturonase (PG). PL-type activity, known as depolymerising, cuts the pectin chain between two galacturonic methylated acids, while the PG prefers a non methylated substrate. PME activity does not depolymerise the pectin chain but releases the methylic group from galacturonic esterified acids. It eases the PG action.
The enological formulations produced by DSM are the result of work conducted jointly with research institutes such as INRA (the French National Institute for Agricultural Research). For over twenty years the DSM plant at Seclin has been producing Aspergillus Niger pectinase suitable for many different winemaking applications. These enzyme preparations are produced from classic strains (non GMO), within an HACCP framework with ISO 9002 (4) certification. The uses and advantages of enzymes in winemaking Yields from free run juice and pressing By weakening the cell walls of the pulp and hydrolyzing the soluble pectin, the enzymes in white grapes maceration facilitate juice release and thus increase the free run juice yield, which avoids excessively harsh pressing. The enzyme activities required for this application are: pectinase (PG, PL, PME) as well as some activities that promote hydrolysis of the pectin "hairy regions".
Example with Rapidase©X-PRESS (5) The respective yields were 132 kg of grapes per hl juice (76% of juice extracted) for the reference pectinase, compared with 124 kg per hl (80.5% of juice extracted) for the DSM specific preparation. This increase was particularly noticeable in respect of pressed juice (+15%). Figure 3 summarizes the results obtained. Racking and clarification Several phenomenon induce clarification:
It is important that the enzyme used for settling has a sufficiently high PG/PL ratio to promote rapid hydrolysis of soluble pectin.
Example with Rapidase©CB Rapidase© CB was used at 1 g per hl. After 24 hours contact time (see Photo 2) turbidity was of 208 NTU for the control and 7 NTU for the enzyme treated batch. It should be noted that when settling a high sugar content must, such as a Muscat intended for a naturally sweet wine, the sedimentation phase is always slow. This is due to the viscosity of the must and the abundance of sugar. Colour extraction and stabilization As was explained above, the grape skin forms a physical barrier to diffusion of anthocyanins, tannins and aromas contained in the skin cells. Thus, to release the cell content, the polysaccharides found in the pecto-cellulosic cell wall and the middle sheath of the berry must be hydrolyzed. When making red wines, these components are extracted during alcoholic fermentation with the effects of temperature, alcohol and mechanical operations such as pumping over, punch downs and delestage. Some physical methods such as thermo-vinification or flash expansion increase these extraction processes. Commercial enzymatic preparations for red grapes extraction strengthen and promote these physical methods. In order to weaken the cell walls and facilitate the diffusion of the vacuole content, secondary hemi-cellulosic activities are needed in addition to the pectolytic activities required. Recent studies (6, 7) show that tannin diffusion is achieved in a selective way. Only the free tannins from the vacuole and those included in the cell wall are released by the previously mentioned enzymatic action. This affects the tannin structure in enzyme-treated wines; these tannins are generally more stable and supple. Example with Rapidase© ExColor Comparative enzyme trial at the University of Talca (Chile) on Cabernet Sauvignon: enzyme dose 3 g per hl, maceration 15 days at 21°C. The results are shown on figures 4 & 5 below: Release of aroma precursors Odourless aromatic precursors are present in a form linked to sugars (8) in the grape skin. Their composition and content varies according to the grape variety. Among these components, terpenols comprise a major part of the varietal aromas in white grapes, in particular in Muscat-type varieties. In varieties such as Muscat or Riesling, the linalol, nerol and geraniol glycosylated precursors are the most abundant, their "sugar" part comprising ramnose-glucose for rutinosides, arabinose-glucose for arabinosides and apiose-glucose for apiosides. Sequential hydrolysis of these sugars by glycosidase enzymes releases the highly odorous terpenols. Firstly, terminal sugars are cut by a rhamnosidase, an arabinosidase or a apiosidase. Next, the b-glucosidase releases the terpenol. Thus the aroma releasing enzyme process cannot be reduced to just b-glucosidase since this acts only after the di-glucoside has been hydrolyzed. These enzymes thus have the effect of releasing the aromatic components from their glycosylated precursors, not only the terpenols but also some esters and norisoprenoids (C13 components). Example with Rapidase© AR2000 Rapidase© AR2000 is a pectinase produced by a strain of Aspergillus niger. This product naturally contains secondary glycosidase-type activities: b-glucosidase, arabinosidase, rhamnosidase and apiosidase. These activities are present in optimum proportions for releasing aromas. The enzyme produced by DSM is a unique enzyme preparation, the only one that contains these four enzymatic activities at effective concentration levels. INRA and DSM hold a joint patent for its application. A Riesling must was treated with 3 g per hl Rapidase© AR2000. Table II shows the aromatic compounds content in an enzyme treated wine by comparison with a control without any enzyme treatment. Conclusion In conclusion, the pectinases used in winemaking give the winemaker many advantages such as
By using Aspergillus niger strains specifically selected for enological purposes, DSM enzyme preparations are formulated to naturally maintain any unwanted activities (anthocyannases, cinnamyl esterases) to a negligible level. Analysis shows that the level of cinnamyl-esterase activity in DSM enzymes is naturally lower than that of purified enzymes. Using DSM enzymes in vinification limits the formation of volatile phenols maintaining their concentration under the perception threshold. References J. Mourgues, 1983, Doctoral-Engineering Thesis, University Paul Sabatier, Toulouse France. F. Voragen and al. (eds.), Advances in Pectin and Pectinase research, 47 - 59. S. Vidal, P. Williams, M.A. O'Neil, P. Pellerin, Carbohydrate Polymers 45 (2001) 315 - 323. P. Pellerin, C. Bajard-Sparrow, C. Fauveau and F. Strozyck, Revue Française d'Oenologie, 2006, nø216. O. Fernandez, C. Bajard-Sparrow, C. Fauveau and P. Pellerin, Revue des Oenologues, nø116, Juillet 2005. D. Lefebvre, VITI nø316, Mars 2006. J. Ducruet, 2000, Doctoral Thesis Oenologie - Ampélologie, University V. Segalen, Bordeaux France. Z. Gunata, C. Bayonove, R. Baumes and R. Cordonnier, 1985, J. of Sci.Food Agric., 36, 9, 857. K. Lourens & P. Pellerin, WineLand November 2004.
http://www.oiv.org/fr/accueil/index.php.
|
|
Wynboer is incorporated in WineLand, magazine of the SA wine producers.
Visit our sister sites:
|
| UP |
COPYRIGHT (C) 2000 WineLand |