Lysozyme in Food and Pharmaceutical Applications

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

Pharmaceutical Applications of Lysozyme

Hen egg white lysozyme is used in over-the-counter drugs to increase the natural defenses of the body against bacterial infections. Since lysozyme forms part of the human immune system, it has been proposed that supplementation with chicken lysozyme may have benefits. The pharmaceutical use encompasses applications such as otorhinolaryngology(lozenges for the treatment of sore throats and canker sores) and ophthalmology (eye drops and solutions for disinfecting contact lenses). Lysozyme can be added to infant formulas to make them more closely resemble human milk (cow’s milk contains very low levels of lysozyme).

Over the past decade there has been interest in the antiviral properties of lysozyme, with some research demonstrating anti-HIV activity of both human and chicken lysozyme in vitro. It is possible that these findings may lead to new means of treatment for this infection.

Food Applications of Lysozyme

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

The most important food application of lysozyme is the prevention of the problem known as ‘butyric late blowing,which occurs during the ripening of certain European-type cheeses. This problem is due to contamination of milk by spores of Clostridium tyrobutyricum. The origin of this contamination lies in the widespread use of silage as a feed. The spores of C. tyrobutyricum are present in the soil and are incorporated,together with soil particles, into the corn or hay used to make silage. The spores will proliferate in the silage if a rapid acidification does not take place. When the cows are fed the contaminated silage, the spores are excreted into the manure and, if the milking is not carried out under strict hygienic conditions (e.g., thorough washing of the udder, elimination of the first drops of milk), the spores can subsequently contaminate the milk. It has been demonstrated that a very small amount of manure (less than 1 g) is enough to contaminate a tank containing several thousand gallons of milk.

If cheese is made with milk contaminated with spores of C.tyrobutyricum, the majority of the spores are retained in the curd. Here, the conditions (absence of oxygen and presence of large amounts of lactic acid) are favorable for the germination and development of vegetative forms during the ripening of the cheese. Lactic acid can be metabolized as the primary source of carbon by C. tyrobutyricum to produce butyric acid and a combination of two gases: hydrogen and carbon dioxide. The accumulation of butyric acid is responsible for organoleptic defects in the cheese due to the characteristic off-flavor caused by this short-chain fatty acid. The production of large volumes of hydrogen (totally insoluble in the water phase of the cheese curd) and carbon dioxide (partly soluble), leads to an increase in the internal pressure of the cheese and, subsequently, to the formation of slits and cracks in the cheese during the ripening process. This has a dramatic and detrimental impact on the quality of the cheese and, consequently, on its commercial value. Cheese with a late-blowing problem usually has to be downgraded or, in severe cases, cannot be sold.

Before the use of lysozyme, cheesemakers developed a number of techniques to try to prevent butyric late blowing.Two commonly implemented techniques are (a) a physical process to eliminate the spores by centrifugation (known as ‘bactofugation’) and (b) the use of chemical inhibitors of C. tyrobutyricum, such as nitrates. Neither method can guarantee a complete solution to the problem.

Research begun in the late 1960s and 1970s, followed by cheese trials carried out in Europe in the early 1980s, demonstrated the efficacy of lysozyme to prevent late blowing in different types of cheese. The principle of lysozyme action is based on its capacity to be retained in the cheese curd, through electrostatic attraction with the casein, and on the stability of its enzymatic activity throughout the ripening process. Lysozyme is active on the vegetative cells of C. tyrobutyricum, which appear during the ripening process. The usage level is usually 25 ppm in the cheese milk. At this concentration, most of the lactic cultures used in the production of cheese, although Gram-positive bacteria, are not sensitive to the lytic action of lysozyme.

Lysozyme has been approved by the European Union and has now been used with success for more than 25 years in several European countries (e.g., France, Italy, Spain, Portugal,Germany, Denmark, the Netherlands). Its use has been successful in different types of cheeses, such as hard cheeses(Parmesan, Swiss), semihard cheeses (Gouda, Manchego), and soft cheeses (Brie). Lysozyme received Generally Recognized as Safe status from the U.S. Food and Drug Administration in 1998 and is raising a lot of interest in North America for its application in specialty cheeses.

Another food application of lysozyme of growing importance is in alcoholic beverages like wine or beer. In wine-making, the Organisation International du Vin (OIV) has permitted its use since 1997 at a level not exceeding 500 ppm.Some of the types of applications are as follows:

1.Delaying malolactic fermentation

2.Stabilizing wines after malolactic fermentation

3.Preventing spoilage by lactic acid bacteria that cause stuck fermentations

The industrial use of lysozyme in winemaking has become common worldwide for these noted applications. Beer is an unfavorable medium for many microorganisms due to the presence of ethanol and the hop bitter compounds, the high content of carbon dioxide, the low content of oxygen, the low pH, and the lack of nutritive substances. So-called beer spoilage microorganisms, however, still manage to grow in beer. Most breweries now commonly filter sterilize or pasteurize their beers to prevent bacterial spoilage during storage of the beer before consumption. Beer spoilage bacteria are mostly lactic acid bacteria. For some specialty beers, like the top-fermenting beers with refermentation (e.g., cask-conditioned beer and bottle-conditioned beer), these treatments are not possible because the viable microorganisms present are part of the production process of those beers. Lysozyme has proven to be a suitable antibacterial agent for brewing purposes, and it is effective in inhibiting lactic acid bacteria added to finished beers. Therefore, it is used to preserve beers that will not receive either pasteurization or sterile filtration.

The high specificity and effectiveness of lysozyme against only certain Gram-positive organisms are what allows it to be used in the applications discussed. For example, in the wine-making examples, it inhibits spoilage organisms (mainly lactic acid bacteria), while at the same time not interfering with the yeast that are essential to making the wine. This specificity is what makes lysozyme unsuccessful as a broadly effective antimicrobial. Some efforts have been made to increase lysozyme’s range of food applications by combining it with other natural antimicrobials. For example, work in the 1980s on combinations of lysozyme with the antimicrobial peptide nisin led to patents for its use against Listeria monocytogenes in meat products. To date, industrial use of lysozyme in processed meat preservation has not been as significant as its use in cheese and wine.