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A Technical Guide for Wine Producers
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Is SO2's days numbered? SO2 versus antimicrobial peptides in wine preservation
By: Heidi Schoeman, SunBio, Institute for Wine Biotechnology, Stellenbosch University
Key words: Lactic acid bacteria, bacteriocin, biopreservation.
The first use of biological methods for food preservation dates back from as far as 6000 to 1000 BC when bread, beer and wine were produced through fermentation. It may come as a surprise to today’s users of fermentation technology and the consumers of fermentation products, but the primary value of this technology to those early users and consumers was its ability to preserve food, and not the addition of one or other attractive feature (alcohol, taste) to the final product (although these aspects undoubtedly played a role in making these products popular). While fermentation technologies developed with time, it was only in 1864 that Louis Pasteur demonstrated that micro-organisms are the cause of both fermentation and food spoilage.
To control the growth of spoilage organisms, sulphur dioxide (SO2) is used to stabilise wine microbiologically. Besides being an extractive solvent, as well as an anti-oxidant, its excessive use can be harmful to the quality of the wine. A serious concern is that SO2 is causing allergic responses in sensitive people, with symptoms ranging from nausea, vomiting, gastric irritation, headaches and asthma attacks. Knowledgeable wine consumers are reacting to many of these concerns, demanding labelling that accurately reflects product contents. These demands are in line with a trend among consumers toward healthier living which is linked to consumption of healthy food and drink. Following these trends, products should also be less processed, less preserved and more natural.
The Wine Law Alerts of 2 June 2005 (www.sawit.co.za) has already announced that the labelling of all wines containing sulphites will become compulsory in both South Africa and the European Union (EU) from 25 November 2005. This will apply to all finished wines that contain more than 10 milligrams per litre of SO2, measured as total SO2. The phrases "contains sulphites" or "contains sulphur dioxide" will have to be incorporated on all labels of wines labelled from 25 November 2005. Wines labelled before this date may still be exported to the EU without the statement while stocks last. In order to respond appropriately to consumer demands, research into alternatives to the use of SO2 for chemical preservation will be important.
Uncontrolled microbial growth during and after the fermentation of wine can change the chemical composition and ultimately the quality of the end-product. Strains of lactic acid and acetic acid bacteria are usually involved in wine spoilage and can lead to ropiness, volatile acidity, acrolein formation and bitterness, tartaric acid degradation and geranium off-flavour (Du Toit and Pretorius, 2000). Certain strains of lactic acid bacteria (LAB) can also produce biogenic amines and ethyl carbamate precursors. Despite the significance of LAB in malolactic fermentation, it is important to control the presence of naturally occurring LAB in a winemaking environment.
LAB has always had a safe association with fermented food and as a result there is an increased interest in their use for the preservation of food. These bacteria produce specific proteinaceous substances, called bacteriocins, that inhibit the growth of pathogens such as Listeria, Clostridium, Staphylococcus, Bacillus spp. and Enterococcus spp. Bacteriocins also have bactericidal activity against species that are closely related to the bacteria that produce them. However, bacteriocins have no effect on Gram-negative bacteria, yeasts and moulds. Today bacteriocins are considered to be ideal food preservatives, since they are colourless, odourless and non-toxic (Abee et al., 1995).
The most famous bacteriocin is probably nisin, produced by Lactococcus lactis. It is a permitted food additive in more than 50 countries world-wide and is sold under the trade name of Nisaplin®. Nisin is used to prevent the spoilage of processed and natural cheese, canned foods, and in extending the shelf life of milk in warm countries (Vandenberg, 1993; Delves-Broughton et al., 1996). Nisin is currently the only LAB bacteriocin legally used as a food additive and its application in wine is currently under investigation.
Lysozyme is another component that has found application in food preservation. Lysozyme is an enzyme extracted from hen egg white and is highly active against certain Gram-postivie bacteria. Lysozyme is already used successfully as an antimicrobial in many foods, especially in cheese, and has had positive evaluations from international regulatory agencies such as the World Health Organization (WHO) and the Food and Drug Administration (FDA). It has recently been introduced to the wine industry and offers another means of controlling malolactic fermentation in wine. As an organoleptically neutral alternative, lysozyme can be beneficial to further reduce SO2 levels in winemaking. Since lysozyme is more active in white wines than red wines, it is believed that polyphenolic components play a role in the bacteriolytic action of lysozyme. As wine pH increases, the antimicrobial activity of lysozyme increases as well, which makes it attractive for preventing spoilage in higher pH wines (Delfini et al., 2004). Studies have shown that there are no significant changes in the chemical and organoleptic features of wine after the addition of lysozyme (Pitotti et al., 1991).
Other antimicrobial enzymes (e.g. chitinases, endoglucanases, etc.) and peptides (zymocins and bacteriocins other than nisin) are currently being investigated and considered as possible biopreservatives. Purified antimicrobial enzymes and bacteriocins will however be very expensive to use. In wine, this problem might be solved through the expression of effective antimicrobial enzymes and peptides in wine yeast starter culture strains. The hen egg white lysozyme gene (HEL1), the Pediococcus acidilactici pediocin gene (PED1) and the Leuconostoc carnosum leucocin gene (LCA1) have already been expressed in yeast at the Institute for Wine Biotechnology. Pediocin and leucocin were already tested for their efficiency as biopreservatives in wine. It was found that these bacteriocins had no effect on yeasts and acetic acid bacteria normally associated with wine and were stable for a sufficient period of time in a simulated wine environment (Du Toit et al., 2002). In future, bacteriocins may provide winemakers with a valuable, additional and controllable tool as an alternative to SO2. Since SO2 is still necessary in the winemaking process to prevent oxidation, bacteriocins can possibly be used in combination with SO2 and other physical, chemical and microbial preservatives to inhibit the growth of spoilage bacteria.
In order to address the wine consumers’ call for healthier wines of higher quality, SunBio, a commercialisation initiative of the Institute for Wine Biotechnology was launched earlier this year. SunBio is fully funded by Cape Biotech, a Biotechnology Regional Innovation Centre (BRIC) established by the Department of Science and Technology. The venture aims to establish a sustainable product development process that will combine the research output and intellectual property generated by the Institute for Wine Biotechnology with sound commercialisation practices. Various projects are under way through the SunBio venture, all targeted toward developing yeast starter cultures with defined market advantages. One of these projects is geared toward the production of bacteriocins in commercial wine yeast strains.
Several important issues (e.g. scientific, safety, regulatory, legal and ethical) will certainly play a significant role in the eventual commercialisation of transgenic yeasts. Addressing many of the legal, safety, scientific and ethical concerns of the public will form part of a strategy to illustrate the potential benefits of transgenic yeasts. One can however be optimistic that in the long term these yeasts will become important tools to the fermentation-based industries of the future.
Contact person: Heidi Schoeman, tel: (021) 808-4867, fax: (021) 808-3771, e-mail: hs2@sun.ac.za
References
Abee, T., Rombouts, F.M., Hugenholtz, J., Guihard, G. & Letellier, L. (1994) Mode of action of nisin Z against Listeria monocytogenes Scott A grown at high and low temperatures. Applied and Environmental Microbiology 60, 1962-1968.
Delfini, C., Cersosimo, M., Del Prete, V., Strano, M., Gaetano, G., Pagliara, A. & Ambrò, S. (2004) Resistance screening essay of wine lactic acid bacteria on lysozyme: efficacy of lysozyme in unclarified grape musts. Journal of Agricultural and Food Chemistry 52, 1861-1866.
Du Toit, M. & Pretorius, I.S. (2000) Microbial spoilage and preservation of wine: Using weapons from nature’s own arsenal - a review. South African Journal of Enology and Viticulture 21, 74-96.
Du Toit, M., Du Toit, C., Krieling, S.J. & Pretorius, I.S. (2002) Biopreservation of wine with antimicrobial peptides. Bulletin of the Office International de la Vigne et du Vin 75, 855-856, pp. 284-302.
Pitotti, A., Zironi, R., Dal Bo, A. & Boschelle, O. (1991) Assay of lysozyme by its lytic action on Leuconostoc oenos: a suitable substrate at acidic pH. Journal of Food Biochemistry 15, 393-403.
Delves-Broughton, J., Blackburn, P., Evans, R.J. & Hugenholtz, J. (1996) Applications of the bacteriocin, nisin. Antonie van Leeuwenhoek 70, 193-202.
Vandenberg, P.A. (1993) Lactic acid bacteria, their metabolic products and interference with microbial growth. FEMS Microbiology Reviews 12, 221-238.
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