Delvozyme® Lysozyme

Enzyme preparation based on lysozyme

Avoidance of bacterial spoilage during alcoholic fermentation. Adding Delvozyme® at a dose of 100 to 150 mg/l on putting into tanks assists in avoiding and limiting the proliferation of spoilage bacteria. This facilitates the sound progression of alcoholic fermentation and reduces the risks of increased volatile acidity, production of unpleasant taste and nutritional competition.

Oenobrands Delvozyme lysozyme is an enzyme (protein) isolated from egg whites with a bacteriolytic function.  It is able to lyse gram-positive bacteria and thereby inhibiting their growth.  Lactic acid bacteria (LAB) – Oenococci, Lactobacilli and Pediococci - are gram-positive and can therefore be killed by lysozyme.  Acetic acid bacteria, the other group of bacteria associated with winemaking, are gram-negative and are un-affected by lysozyme.  Lysozyme also has no influence on wine yeasts and fungi.

Lactic acid bacteria can play both positive and negative roles in winemaking.  Most LAB found naturally on grape skins and in resulting musts / wines are heterofermentative.  That means they can have different metabolic end products depending on the substrate they grow on.  When LAB utilize malic acid as a carbon source – mainly lactic acid is formed.  However, when certain LAB utilize sugar or citric acid as a carbon source, then acetic acid is often one of the end products.  This second outcome is unwanted and causes an organoleptic defect in the wine.  Lactic acid bacteria can utilize sugar in must at two stages:  during the lag phase of the wine yeast before the onset of active fermentation or if cold soaking is applied; or when fermentation becomes sluggish towards the end due to high alcohol concentration and other factors affecting yeast viability.

This last scenario usually has a snowball effect. With the decrease in fermentation activity by the yeast, the opportunistic LAB - oppressed by the fast metabolism of the yeast up until now - start growing and can utilize the residual sugar or citric acid rather than - or in addition to - the malic acid.  The result is the production of acetic acid.  According to literature as well as Oenobrands practical experience, yeast fermentation activity rapidly decreases when volatile acidity levels exceed 0.7 g/L (tartaric).  The acetic acid formed by the LAB can thus be the cause of a stuck fermentation.

If LAB growth and thus acetic acid formation can be inhibited – it is possible that sluggish fermentations can ferment to lower residual sugar concentrations or even finish fermentation.   Depending of course on how severe the factors were that caused the sluggish fermentation in the first place. 

Lysozyme can be used at various stages during the winemaking process:

• Controlling the start of MLF – when it is important to minimize the risk of MLF starting before the end of alcoholic fermentation: vinification of whole grape clusters, Beaujolais vinification or maceration with high pH.  The addition of 100 mg / L Delvozyme before fermentation will delay MLF by ±10 days.
• Preventing lactic disease at the end of a sluggish alcoholic fermentation – the addition of 250 mg/L Delvozyme at the start of fermentation will prevent LAB growth and VA formation in lagging fermentations.  Commercial freeze dried malo-lactic cultures must be inoculated if MLF is desired after the completion of the alcoholic fermentation since the natural population concentration will then be eliminated by Delvozyme treatment.
• Lagging or stuck fermentations – the addition of 250 – 350 mg/L lysozyme at the first sign of a sluggish fermentation could prevent the proliferation of the LAB if the cell counts are not already too high.
• Complete inhibition of MLF in white and rosé wines – the addition of 300 - 500 mg/L Delvozyme after settling will prevent any MLF.
• Microbiological stabilization of wine after MLF – the addition of 150 - 250 mg/L Delvozyme will enable LAB population to be controlled and thus avoid unwanted organoleptic defects. 
• Protection against LAB spoilage during barrel ageing – the addition of 100-150 mg/L Delvozyme at beginning of ageing in barrel will kill all LAB present and thus prevent possible volatile acidity increase during ageing.

Theoretically the work of lysozyme should be completed within two days. However, various factors can have an influence on lysozyme activity.  While lysozyme is un-effected by alcohol and SO₂ concentrations present in wine, pH, temperature and bacterial cell counts do have an effect.  At a cell count of 1x10⁵ cells / ml lysozyme will not be able to eliminate the whole population, not even at a dosage of 500mg/l.  Activity is also dependent on the concentration of enzyme added as well as timing of addition.  It is therefore advisable to add lysozyme to fermentations before LAB cell counts start to increase.

Lysozyme activity in wine is unstable.  It decreases faster in red wine than in white wine due to the presence of polyphenols.  If Delvozyme is added at the start of a red wine fermentation there will be no activity after 7 – 14 days.  If Delvozyme is added after fermentation then residual activity can be present as long as a few months especially in white wine.

Lysozyme can have an influence on red wine color since it is able to bind polyphenols.  The effect can differ from wine to wine and is dependent on the tannin concentration and the dosage lysozyme used (100 – 200 mg/l has no effect).  It is therefore necessary to conduct laboratory trials first before the addition of Delvozyme to a finished wine.   When Delvozyme is used during fermentation then the effect on color can be ignored.

Since lysozyme is a protein it cannot be used in the presence of bentonite.  The use of lysozyme can thus also increase a wine’s protein instability.  The following components will bind to lysozyme and precipitate it:  tannins and polyphenols, wood chips, activated carbon and silica sol.  Lysozyme is thus not a permanent preservative and cannot replace SO₂ since it has no anti-oxidative function.  It is merely an aid to control LAB in situations where SO₂ alone is not effective.

The use of lysozyme in commercial winemaking is approved by the OIV. 

References:

Gerbeaux, V. et al. Use of lysozyme in enology. Bulletin de l’OIV. 1999.