Until recently, the use of lysozyme was mostly recommended to delay or block malolactic fermentation, prevent unwanted bacterial growth during storage or ageing, and inhibit further bacterial spoilage in the case of stuck or sluggish fermentation.

However, a new study conducted by Chr. Hansen, together with Canadian Inovatech, has focused on the preventive use of lysozyme where uncontrolled bacterial populations pose a higher risk during alcoholic fermentation. The studies were carried out with Chr. Hansen's lysozyme product, which is available under the brand name Lactizyme.

As expected, the results demonstrated that lysozyme, besides being a natural alternative to high levels of SO2, can ensure better control over undesirable bacterial growth during alcoholic fermentation without compromising the development of malolactic fermentation,

A Benefit to Producersof High pH Wine

While wines of high pH represent a very suitable environment for lactic acid bacteria to grow, they have an inverse effect on the antimicrobial properties of SO2, which are significantly reduced at those conditions. In addition, some Lactobacilli strains are quite resistant to SO2.

Lactobacillus species are believed to be one of the causes of stuck or sluggish fermentation, although more research needs to be done in this area.

Stuck or sluggish fermentation will leave room for lactic acid bacteria to compete against yeast for the sugar and nutrients present in the wine. As a result, a considerable delay in the fermentation processes or worse, the formation of volatile acidity and off-flavors may spoil a whole production.

"By adding lysozyme when the fermentation problem is detected, it is still possible to stop or slow down the deterioration of the wine. Lysozyme will, at least, ensure that you only have one problem--stuck or sluggish fermentation-- not several, like bacterial spoilage. In some cases, adding lysozyme will revert the fermentation process to normal conditions; i.e., inhibit further bacterial growth and regain the activity of the yeast. However, the enzyme is not able to "fix" the damages in terms of flavor and acidity already caused. Sometimes these damages are irreversible and not even the most modern--and expensive-- technique can fix them," explains Lars Petersen, Chr. Hansen's Application Manager for Wine Bacteria.

"This is why we strongly recommend the preventive use of lysozyme in combination with SO2, specially in wines of high pH."

How does lysozyme work?

Lysozyme cleaves the peptidoglycan in the cell wall, leading to fractures in the cell membrane.

Water enters, the cell swells and eventually bursts, a process called lysis.

When Lysozyme has fractured the cells, a fine sediment will occur.

The sediment consisting of cell debri will resemble the texture of fine lees.

Impact of lysozyme on yeast and LAB Lysozyme attacks the cell walls of Gram-positive bacteria and causes the cell to rupture and die. Lysozyme has no effect towards yeast or Gram-negative bacteria.

A Natural Alternative

Besides being susceptible to elevated pH values and not effective against some Lactobacilli strains, the use of SO2 has other implications that restrain winemakers from adding larger amounts to ensure protection.

"For consumers, a high level of sul fite in wine is considered a health concern. Cases of severe allergic reactions have been related to SO2. In production, high concentrations may inhibit the yeast fermentation and affect the sensory qualities of the wine, besides severely interfering with the activity of malolactic cultures," says Petersen. Lysozyme is a natural alternative to the use of high levels of SO2. Even in higher concentrations, lysozyme has no effect on the yeast fermentation and, since its activity gradually decreases through the course of the alcoholic fermentation, it can have a lower or insignificant impact on malolactic cultures depending on the selected strains, thus enabling the malolactic fermentation to complete.

Lysozyme can therefore supplement SO2 for protection of must and wine against lactic acid bacteria spoilage, but cannot completely replace it. "SO2 is a "necessary evil" as it is so far the only antimicrobial agent effective against acetic acid bacteria and some undesirable yeasts, besides being an antioxidant," adds Petersen.

Excellent Results Under Real Winemaking Conditions

The study entitled "Prophylactic Use Of Lysozyme To Control Indigenous Lactic Acid Bacteria During Alcoholic Fermentation" had three main objectives: to investigate the effect of lysozyme on (1) the indigenous lactic acid bacteria (LAB) before alcoholic fermentation (AF), (2) the subsequent inducement of malolactic fermentation (MLF), and (3) different strains of Oenococcus oeni.

In the first experiment, 800 L of a Merlot must prepared at a commercial winery in South Africa (pH 3.7) was divided into two lots; one lot had 45 mg/l SO2 , added at crush, while the other lot had 30 mg/l SO2 and 150 mg/l lysozyme added at crush. After completion of alcoholic fermentation, MLF was induced with Chr. Hansen's Viniflora® oenos.

"Based on our experiments, the use of lysozyme not only allows winemakers to lower the dosage of SO2 ,but in some cases can also be more effective at killing indigenous lactic acid bacteria. On top of this, it can make malolactic fermentation run faster, " says Petersen.

Another experiment evaluated the sensitivities of four commercially avail able strains of O. oeni to remaining lysozyme activity after alcoholic fermentation. In the control wine all four strains completed MLF in 14 days. When adding 250 ppm of lysozyme at crush to the same wine, they behaved completely differently. Two of the strains needed more than 32 days to complete MLF.

Efficiency and Control at the Right Times

Preventing the growth of undesirable bacteria during alcoholic fermentation is more effective than inhibiting their activity later on, and avoids further damages to the wine.

The prophylactic use of lysozyme is especially recommended in wines of high pHs; wines with a history of stuck or sluggish fermentation or a history of high levels of volatile acidity and off-flavors, as well as wines or wineries that have frequent contamination problems with Lactobacilli or Pediococci", says Petersen.

"For an isolated occurrence of stuck or sluggish fermentation, lysozyme should, at least, be added to interrupt or avoid further spoilage of LAB." The addition of lysozyme at crush is also beneficial to wines where malolactic fermentation is desired. "But it is fundamental that the selected bacteria strain is compatible to this application of lysozyme," Petersen emphasizes.

Managing the development of malolactic fermentation by inoculating selected strains of malolactic cultures also ensures better technical and risk control. "The formation of biogenic amines--that may cause allergic reactions to consumers--is one of the risks of letting malolactic fermentation be performed by the indigenous bacteria of the wine" Petersen adds.

Controlling both fermentations at the right times can drastically reduce the chances of bacterial contaminations during storage or ageing. In any case, lysozyme can also be added at these stages.

Freedom to Develop the Desired Characteristics of the Wine

Preventing bacterial contamination problems from the very beginning of the production process gives winemakers more freedom to focus on what consumers most appreciate: the sensory characteristics of the wines.

"The pre-alcoholic maceration, for example, is a technique believed to enhance flavor and color in red wine," comments Petersen. "Nevertheless, the high risk of bacterial contamination has discouraged many producers to run this step of winemaking.

Enabling better control during pre-alcoholic maceration is just one more advantage winemakers can obtain when adding lysozyme at crush," concludes Petersen. wbm

FAQs About Lysozyme

Can lysozyme replace the use of SO2?

SO2 cannot be completely replaced by lysozyme because its antimicrobial effect covers also Gram-negative bacteria and undesirable yeasts. In addition, SO2 is also used as an antioxidant.

Lysozyme has a lytic effect only against Gram-positive bacteria and has no antioxidative effect. Therefore, lysozyme should be seen as a natural strong supplement to SO2 so that its dosage can be reduced.

Will the addition of lysozyme at crush protect my wine so that contamination will not happen at later stages?

When adding lysozyme at crush, its activity gradually decreases during the alcoholic fermentation. This makes it possible to inoculate with a MLF starter. To ensure protection later in the winemaking process, you can add more lysozyme or use SO2.

How much lysozyme should I add at crush to get these benefits?

We normally recommend around 150 ppm (mg/l) added at crush, if malolactic fermentation is desired, but our recommendation varies depending on the following conditions:

• If the pH is very high (more than 3.7), add another 50 ppm.

• If you want to do several days of pre-alcoholic maceration, add another 50 ppm.

• If you use lysozyme in white wine, lower the addition by 50 ppm.

• If you know you have high levels of LAB at crush, add another 50 ppm.

Which MLF starter should I use for this application?

The starters have to be compatible with the use of lysozyme. Both Viniflora Oenos and Viniflora CH-35 have shown good compatibility with this application.

If I have a stuck or sluggish fermentation, how much lysozyme should I add?

If there is higher risk of getting a problem with LAB, we recommend increasing the dosage to 200 ppm. If you see climbing volatile acidity or detect large bacteria populations in the microscope, increase to 300 ppm. In this case, we normally recommend waiting a minimum of one week before inoculating a malolactic starter. wbm