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February 2001

Wynboer - News

The use of lysozyme

The "Organisation Internationale du Vin" (OIV) approved the use of lysozyme in wine during its 1997 General Assembly held in Argentina. In November, 2000, two presentations focused on this enzyme during the SASEV symposium in Cape Town.

Therefore, this is now the appropriate time, in preparation for the upcoming 2001 harvest, to present lysozyme, the different ways it can be used in wine, and the benefits that winemakers can expect from these different types of applications.

The man who would later discover penicillin, Alexander Fleming, first identified lysozyme in 1921 in human nasal secretion. As with many brilliant discoveries, Fleming's skills in looking beyond the obvious led to his discovery of the antimicrobial properties of these two compounds. Lysozyme has since been isolated in human tears, saliva and mother's milk, as well as viruses, bacteria, phage, plants, insects, birds, reptiles and other mammalian fluids.

Lysozyme is not just present by chance in all these organisms and their related biological fluids: lysozyme is an important part of their immune systems, and plays a key role in the defense of these organisms against bacterial infections.

Commercially, the most readily available source of lysozyme has been chicken egg white, from which it is industrially extracted. Lysozyme is likely the most scientifically studied protein of all. It was the first protein to have its structure determined by x-ray crystallography in the 1960s and is often used as a model in protein biochemistry.

Lysozyme has been used in pharmaceutical and food applications for many years, due to its lytic activity on the cell wall of gram-positive micro-organisms. These organisms are responsible for many infections of the human body as well as the spoilage of various foods.

Lysozyme is used in "over-the-counter" drugs in order to increase the natural defenses of the body against bacterial infections. The pharmaceutical use of lysozyme encompasses applications such as oto-rhino-laryngology (lozenges for the treatment of sore throats and of canker sores), and in ophthalmology (eye drops and solutions for the decontamination of contact lenses). Lysozyme is also added to infant formulae in order to make them more closely resemble human milk (cow's milk contains very low levels of a lysozyme enzyme).

Much research has been done on the use of lysozyme as a preservative in food products, particularly in the Far East and Japan. Several applications have been developed and patented, including the treatment of fresh fruits, vegetables, seafood, meat, tofu, sake and wine.

However, the most important food application of lysozyme is, in the cheese industry. Lysozyme is used to prevent a problem known as "butyric late blowing", which occurs during the ripening of certain European-type cheeses. This problem is due to the contamination of milk by a naturally occurring, spore-forming bacterium, called Clostridium tyrobutyricum. The origin of the contamination of milk by this bacterium lies in the widespread use of silage as a feed.

The research regarding the use of lysozyme in wine started in Europe in the early 1990s. The principle of using lysozyme in wine depends on the capability of this enzyme, thanks to its lytic action on gram positive bacteria, to control the growth of the lactic acid bacteria.

Lactic acid bacteria can indeed play both a positive and a negative role in wine:

  • They are responsible for the often-desirable conversion of the malic acid into the lactic acid: a reaction known as "malo-lactic fermentation".
  • They can become a problem if their growth is uncontrolled, either during the early stages of the alcoholic fermentation, or after the completion of the malo-lactic fermentation, mainly because of the production of excessive volatile acidity and biogenic amines (histamine in particular).
  • Certain spoilage lactic acid bacteria (Lactobacillus sp, Pediococcus sp) can produce particularly negative results, leading to flawed or unmarketable wines.

The traditional way of controlling the growth of the lactic acid bacteria is the use of sulphites. Although they have been used for almost as long as wine has been made, it is widely admitted nowadays that sulphites should be utilised more rationally in wine for the following reasons:

There is a general trend towards the reduction of the sulphite levels in wine due to:

  • The widespread use of sulphites in other foods (fruits, vegetables, fruit juices), and the related risk of toxicity coming from a too high cumulative intake.
  • The well-known allergenicity of sulphites.

Sulphites are not an ideal technological tool to control the growth of lactic acid bacteria:

  • Sulphites have a non-specific antimicrobial activity: besides inhibiting lactic acid bacteria, they will also inhibit the yeasts responsible for the alcoholic fermentation.
  • Sulphites are mostly active at low pH, which makes them relatively inefficient on wines having a low acidity.
  • When used to stabilise the wine after malo-lactic fermentation, sulphites have a detrimental impact on the organoleptic qualities of the wine ("bleaching effect" on the colour and off-flavours).

All these drawbacks have led enological research to look for alternatives to sulphites. The work done with lysozyme has shown that this enzyme can be a good alternative/ complement to the use of sulphites:

  • Lysozyme has a very specific activity: it is active only on lactic acid bacteria and does not interfere with the alcoholic fermentation.
  • Contrary to the sulphites, the lysozyme activity increases with the pH.
  • Lysozyme is a safe product which has been positively evaluated by several regulatory agencies (WHO, FDA, Scientific Food Committee of the EU), and which has a safe track record during many years of use in the pharmaceutical and food industries.

The research done on the use of lysozyme in wine has resulted in the identification of three major types of applications:

1. The preventive control of the onset of the malo-lactic fermentation:

An early addition of 100 to 150 ppm of lysozyme (10 to 15 grams/ hectoliter) to the must will momentarily inhibit the development of the lactic acid bacteria, particularly in musts having a high pH, with the following benefits:

  • significant reduction of the risk of interference between the malo-lactic fermentation and the alcoholic fermentation, both fermentations competing on the same substrates: because of this type of interference, an early start of the malo-lactic fermentation in the must is often responsible for a stuck or sluggish alcoholic fermentation.
  • inhibition of many of the spoilage organisms which may be present when the grapes arrive at the winery (i e Lactobacillus).
  • control of the volatile acidity development.
  • start of the malo-lactic fermentation, spontaneously or through normal inoculation, and its subsequent normal development, once the alcoholic fermentation is completed.
  • a total inhibition of malo-lactic fermentation can even be achieved for white wines, in which it is not desired, by raising the quantities of lysozyme to be used up to 500 ppm (50 g/hl).

2. The protection of the wine during sluggish or stuck alcoholic fermentation accidents:

The addition of 250 to 350 ppm (25 to 35 g/hl) will inhibit the growth of the lactic acid bacteria during the time necessary to re-start the alcoholic fermentation, thus allowing considerable savings on expensive treatments designed to remove the excess of volatile acidity in the wine (like the reverse osmosis). In this case, it is important to add the lysozyme as soon as the problem is identified - if the bacterial load is above 106 cfu/ml, a dosage of 500 ppm (50g/hl) is recommended.

Note: Lysozyme cannot inhibit Acetobacter (a gram negative bacteria) in the same manner - its efficacy lies in inhibiting gram-positive bacteria.

3. The microbiological stabilisation of wine after malo-lactic fermentation:

When sulphites are used at this stage, they present the most detrimental effect on the organoleptic properties of the wine (bleaching effect, sulphur taste). An addition of 250 to 300 ppm (25 to 30 g/hl) of lysozyme will allow for a dramatic reduction in the quantity of sulphites necessary to stabilise the wine, down to the minimum level required to ensure the protection against oxidation and the control of the acetic acid bacteria (not inhibited by lysozyme).

These three types of applications have been validated by more than three years of experiments carried out by research centres (in particular by ITV in France), and by wineries in different parts of the world. Lysozyme is therefore a very flexible tool that can be used at different stages and at different concentrations, depending on the objectives contemplated by the winemakers.

These experiments have led to the approval of the use of lysozyme in wine (up to 500 ppm) by the OIV at the end of 1997. Final approvals are underway in the European Union and in the US where large experiments can be carried out with a simple declaration of intent (with no labeling requirement).

Inovatech Lysozyme will be used in large quantities for the first time in South Africa this season. Inovatech Lysozyme is available ex-stock from Columbit (Pty) Ltd. For enquiries, please contact:

  • Johan Engelbrecht: 083 270 5416
  • Pat Gerstner/Timothy Njana: 593 3161
  • Joubert Parsons: ?083 628 7855
  • Philip du Toit: 083 449 7288
  • Frank Lima: 082 330 7179
  • Dirk Wiese: 082 785 0199.