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 structure of 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. Presently the most important applications of industrial enzymes in wine production involve the use of pectinase and glucanase preparations.
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.
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.
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 (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 10C. Increased enzyme dosings are recommended for red wines because partial inhibition of the activity occurs through phenolic compounds.
|Process||Untreated wine||Enzyme treated wine|
NEW ENZYME APPLICATIONS
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 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.
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. 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.
Despite considerable research having been done, bentonite is the only effective way to stabilise white wine (removal of proteins causing cloudiness). Due to current practices in the course of winemaking, resulting in increased protein extraction, higher dosages of bentonite are required, which will improve the quality of the wine. Furthermore these treatments generate bigger volumes of sediment which have to be removed and cause losses of wine during filtration. The availability of a suitable acid protease may help to solve this problem through hydrolysis of the grape proteins and thus avoid bentonite treatment altogether. The search for fungal enzymes which may degrade the wine proteins has not been successful up to now. Endogenous enzymes are involved in almost all stages of wine production. Their activities vary according to physiological and technological factors. Thanks to a better understanding of the enzymatic changes during the processing of grapes, wine makers have been able to overcome the negative results and reinforce the desired activities. As a result the use of industrial enzyme preparations has become standard practice. The most important advantages of the enzyme preparations are effectiveness, specificity and convenience. These processing aids improve the quality of the wines and ensure a more rational and economic utilisation of facilities and equipment. Further research is necessary, however, to gather information about the role of enzymes in complex processes such as extraction. Recently the study of specific areas of fruit cell wall polysaccharides resulted in the discovery of a new enzyme, a ramnogalacturonase, which is able to degrade the hairy areas of pectins. Further studies on the chemistry of plant cell wall polysaccharides and of the biochemistry and genetic regulation of the enzymes responsible for their degradation, will definitely be important to the winemaking process. With the aid of recombinant DNA technology, expectations are that an entire range of new generation enzymes will be produced for the food and liquor industries. Such enzymes will have higher specificities, purity and detailed kinetic characteristics, as well as the prospect of lower production costs. The possibility also exists to express heterologous genes coded for extracellular enzymes (e.g. pectinases, glucanases, glucosidases, proteases, etc.) in wine yeasts. These enzymes can then be produced during fermentation. This may cause the usage of purified enzyme preparations to be drastically reduced or become unnecessary altogether. This could imply significant cost savings. It would also result in a more “natural” winemaking process in the sense that external additions to must and wine will no longer be necessary. However, these developments and genetic improvement of wine yeasts will be subject to legal requirements and consumer preferences.