The 2002 harvesting season saw some big losses due to infection by the fungi Botrytis cinerea, which can cause either grey rot or noble rot.

Depending on certain factors like weather conditions, grape cultivar etc. grapes infected with B. cinerea can either yield the sought after noble late harvest wine or must which is unsuitable to produce a good quality wine. The question often arises how to handle grapes infected with B. cinera when a non-noble late harvest wine will be made from these grapes. The aim of this article is thus not to explain the process of B. cinerea infection in the grapes, nor noble late harvest wine production, but to focus on certain aspects which must be kept in mind by the winemaker when producing a white or red table wine from grapes infected with B. cinerea.

Problems associated with rotten grapes

It is a well-known fact that rotten grapes are much more susceptible to oxidation than sound, healthy grapes. This is due to the production of laccase by the B. cinerea fungi, which are a potent oxidation enzyme. During this oxidation process phenolic compounds in the grape juice, such as caftaric acid are being oxidised to its corresponding quinone, which can form brown polymeric products. Other oxidation enzymes, like tyrosinases or ortho-diphenol-oxidases (o-DPO) are also produced in grapes, but the substrate specificity of laccase is wider (refer to table 1) and is not inhibited to the same degree by its oxidation products than o-DPO. Laccase is also more resistant to sulfur dioxide than o-DPO. The number of acetic acid bacteria can also increase drastically on Botrytis infected grapes, up to 107 cells/ml (whereas on healthy grapes their numbers are normally between 102 to 104 cells/ml). This is because of the rupture of the grape berry skin due to the infection, which allows entry for the bacteria to the juice of the berry. Normally Gluconobacter species occur on grapes, but in rotten grapes Acetobacter species can also dominate, probably due to the production of small concentrations of ethanol by wild yeasts, which Acetobacter prefer as carbon source, although most of them can also utilise sugars. Acetic acid bacteria are known to produce high levels of SO2 binding substances from glucose and fructose, like gluconic and 2-diketogluconic acid among others. Lactic acid bacterial numbers can also increase in rotten grapes. This, together with the potential oxidation problem can pose a serious problem for the winemaker.

Wynboer - December 2002 - Winemaking with rotten grapes: It can be a headache Wynboer - December 2002 - Winemaking with rotten grapes: It can be a headache Wynboer - December 2002 - Winemaking with rotten grapes: It can be a headache Wynboer - December 2002 - Winemaking with rotten grapes: It can be a headache Wynboer - December 2002 - Winemaking with rotten grapes: It can be a headache

Some experimental observations

An experiment was thus undertaken in the 2002-harvesting season to investigate how different additions prior to settling would affect the browning of a juice, together with acetic bacterial counts. Sauvignon blanc grapes that were heavily infected with grey rot were de-stemmed, crushed and pressed. The unsettled juice was than treated in duplicate with 500 mg/l PVPP, 500 mg/l bentonite, 50 mg/l sulfur dioxide, pectolytic enzyme (according to the manufacturer’s recommendation for white cultivars) and 500 mg/l activated charcoal. Fifty milligram per litre Delvocid was added to the juice to prevent fermentation during settling. After these additions the juice was transferred into plastic pipes with a length of about two metres each, to simulate a two-metre settling distance. Settling was conducted at 8C for three days. Sixteen hours after the additions as well as two days after this a sample was taken one and a half-metres from the top of the pipe to monitor acetic acid bacterial counts, which was done by plating the juice on selective media. After the settling time a hundred ml of each of the different treatments was transferred to a two hundred ml container. After half an hour the juice was filtered through a 0.45 m filter and the optical density measured at 420 nm (which measures brown colour). This can be seen in fig. 1 and the acetic acid bacterial numbers in fig. 2.

It is clear from fig 1 that the control had a deep brown colour in the control juice. This was also clearly visible with the naked eye. It is interesting to see that at these dosages the addition of activated charcoal resulted in a less brown colour compared to PVPP. Activated charcoal and PVPP are both well-known fining agents to remove phenolic compounds in wine, which are the substrates for the laccase enzyme, but the PVPP at higher dosages probably would have removed more of the brown pigment. Bentonite removes proteins in must or wine and laccase is also a protein, being an enzyme. The use of pectolytic enzymes seemed to enhance the brown colour development in this instance in the juice. This could probably be due to the release of more phenolic compounds into the juice by the use of pectolytic enzymes. SO2, however, had the most profound effect on the brown colour, with this juice being the less brown of all the treatments. SO2 can effectively de-colorise the brown juice, as well as inhibit the oxidation enzymes. SO2 are also known to prevent the formation of the brown polymer in white juice by reacting with the monomeric quinones. Levels of 15, 25 and 75 mg/L of sulphur dioxide in clarified juice were needed for a 50, 75 and 95% inhibition respectively of polyphenol oxidases. High dosages can be required in turbid juices (up to 75 mg/L), probably due to the higher activity of the oxidation enzymes with the grape pomace. Laccase are however more resistant to SO2 and at additions of up to 150 mg/L free SO2 in must, only 20% inhibition has been observed. When adding SO2 to must it is also recommended to add the SO2 all at the same time, not adding it in progressively smaller dosages, which may lead to higher O2 consumption rates.

In wine the activity of oxidation enzymes generally decreases, due to precipitation during fermentation and the inhibitory effect of ethanol. The activity of laccase is however higher in wine than other oxidation enzymes. Prolonged maceration times during red wine making are therefore not recommended when wine is being made from Botrytis infected grapes. Care should also be taken during subsequent running off of the wine from the skins and pressing to avoid oxygen pick-up. It is sometimes recommended to add SO2 to these wines at running off at levels as high as 50 mg/L. This would protect the wine to a large degree to oxidation caused by laccase, but the destruction of laccase in wines containing 20 to 30 mg/L of free SO2 may take several days. The addition of SO2 at this stage of the wine making process could, however, influence the subsequent malolactic fermentation.

The addition of SO2 at different levels to a red wine with subsequent aeration and the laccase activity and development of brown (measured at 420 nm) and red colour (measured at 520 nm) is shown in fig. 3 and 4. It is clear that the laccase activity declined faster with higher SO2 additions. It is however still high after 120 hours in the wine which did not receive any SO2. The brown and red colour increased in this wine after aeration, but decreased drastically with precipitation in the latter stages. In the wine that received a total of 36 mg/L SO2 the laccase activity decreased, but the enzyme was still active after a few days and all the free SO2 disappeared at this stage, allowing the enzyme to reduce the red colour in the latter stages. Only in the wine receiving 55 mg/L total SO2 was the laccase activity destroyed, which allowed for good development of the red colour especially.

The settling during the 2002 experiment did decrease acetic acid bacteria numbers, but to a lesser extent than expected (fig 2). The different treatments did not have a dramatic influence on acetic acid bacterial numbers, except for SO2, which did however decrease acetic acid bacterial numbers. At a low settling temperature of 8C an increase in acetic acid bacteria is, however, unlikely.

To summarise a few important steps that a winemaker can take when having to make wine with Botrytis infected grapes:

1.By selecting the healthiest bunches in the vineyard the quantity of rotten grapes being brought into the cellar can be reduced.
2.The addition of SO2 and an inert gas in the vineyard can be considered.
3.The addition of high dosages of SO2 (up to 80 mg/L) at crushing.
4.To minimise damage to the grapes during crushing and pressing to prevent excessively high phenolic content in the must which serves as a substrate for laccase.
5.Avoid skin contact and the use of enzymes which can liberate phenolic compounds.
6.The addition of acid to lower the pH of the must. At lower pH values more SO2 is in the free form, which is active towards laccase and bacteria.
7.Efficient and quick cooling of the pressed must for effective settling.
8.The addition of a fining agent during settling, such as bentonite to remove part of the laccase or other fining agent to remove phenolic compounds.
9.To inoculate with a strong fermenting yeast strain with the addition of sufficient diammoniumphosphate. Botrytis is known to utilise a large quantity of the nitrogenous compounds in the grapes. This fungus also produces polyosides, like botrytidial, which have fungistatic activity. This can also lead to fermentation problems.
10.Avoid excessive skin contact after fermentation.
11.The addition of SO2 after fermentation. During this time the wine must be protected from air, to allow the SO2 to destroy remaining laccase activity.

The occurrence of rotten grapes can be a headache for the winemaker, but some sound winemaking practices can assist the winemaker to tackle these problems successfully.

References

Boulton, R.B., Singleton, V.L., Bisson, L.F., Kunkee, R.E. (1995). Principles and Practices of Winemaking. New York: Chapman & Hall.

Du Toit, W.J., Pretorius, I.S. (2002). The occurrence, control and esoteric effect of acetic acid bacteria in winemaking. Annals of Microbiology, 52, 155-179.

Ribereau-Gayon, P., Glories, Y., Maujean, A., Dubourdieu D. (2000). Handbook of Enology, Volume 2, The chemistry of wine and stabilization and treatments, John Wiley & Sons Ltd., Chichester: England.

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