Lysozyme
Nearly every homebrewer I know has had the disheartening experience of having a batch of beer go bad. I wish I could say that after 13 years of homebrewing (and a couple of degrees in food science) that every beer I brew is perfect, but I’d be lying. The truth is, potentially harmful microorganisms are everywhere and some of them may get into your beer despite your best efforts to keep them out.
A homebrewer’s first line of defense against spoilage microorganisms will always be cleaning and sanitizing their equipment thoroughly and taking steps to minimize opportunities for airborne contaminants to fall into their wort or beer. There is, however, one step a homebrewer can take to minimize the risk of contamination and spoilage — adding lysozyme.
Bacterial contamination can turn beer into a sensory nightmare. Bacteria cause turbidity, off odors and aromas, stuck or sluggish fermentation, acid production and even “ropiness” — the presence of gelatinous strands or blobs in beer.
Gram staining
Bacteria can be separated into two groups based on whether or not they react with Gram stain. Cells are classified as either “negative,” if the bacteria is not stained by the Gram stain, or “positive” if it is.
In brewing, the most notable member of the Gram negative bacteria is Acetobacter, which is known for its ability to produce large amounts of acetic acid from alcohol.
The most common group of Gram positive bacteria associated with beer are collectively referred to as the lactic acid bacteria. Members of this group include Lactobacillus and Pediococcus and are called lactic acid bacteria because of their ability to produce lactic acid from sugar. Some lactic acid bacteria strains can also produce large amounts of acetic acid.
In general, the lactic acid bacteria ferment wort sugars and their growth is favored by anaerobic conditions. The most common off odors associated with these bacteria are sweet, butterscotch or honey notes created by diacetyl and related vicinal diketones. They grow optimaly at a pH of 5.5 and can survive at a pH as low as pH 3.0. (The pH of wort is typically 5.2–5.6; finished beer usually has a pH of 4.0–4.4.) They can grow over a wide range of temperatures and are alcohol tolerant. Hop resins can inhibit the growth of lactic acid bacteria but, because of adaptation, lactic acid bacteria strains encountered in the brewing environment are often resistant to hops. In short, wort and beer provide an ideal growth environment for lactic acid bacteria and can contaminate beer at nearly every turn.
The presence of bacteria is not always an unwanted occurrence. Indeed, beers such as lambics and other sour beers are dependent on the action of lactic acid bacteria as well as other microorganisms to develop a distinctive flavor profile. Additionally, bacterial growth can be used to brewers’ advantage to naturally raise the acidity of the mash. If mash temperatures are held below 140 °F (60 °C), certain strains of heat tolerant lactic acid bacteria will produce lactic acid to effectively lower the mash pH. In winemaking, the growth of certain lactic acid bacteria is actually encouraged to carry out the “malolactic fermentation” which lowers wine acidity and provides microbial stability, The lactic acid bacteria are also responsible for food products as varied as sauerkraut, yogurt, cheese, salami and Thai fish sauce.
Lysozyme
Lysozyme is a well studied enzyme that can provide protection against a broad spectrum of Gram positive bacteria. It is used as a preservative in many food products including wine, tofu, cheese and sakè. The use of lysozyme in beer has only recently garnered attention and the applications in brewing, especially to the homebrewer, are numerous.
Alexander Fleming discovered lysozyme in 1922 when the scientific pioneer had a cold. He found that nasal drippings, which had dripped onto an agar plate, dissolved bacterial colonies. Fleming quickly determined that the antibacterial action was due to an enzyme effective against certain bacteria. Lysozyme is found abundantly in nature and is produced by bacteria, fungi, plants, birds, and mammals. Some viruses even contain the genetic code for lysozyme. Although lysozyme is found in high concentrations in human tears, commercial lysozyme is made from hen egg whites. (It takes over 3,000 pounds (1,360 kg) of egg whites to make 1 pound (4.5 kg) of lysozyme!)
Lysozyme is effective against Gram positive bacteria (all lactic acid bacteria) due to its ability to specifically break down the cell wall structure of those microorganisms. Once the cell wall is broken, the cell breaks open and dies. Because the lysozyme is structure specific, yeast and Gram negative
bacteria are completely unaffected by this antimicrobial.
The most appropriate places to include lysozyme in the brewing process are also the points where lactic acid bacteria have the opportunity to infect and spoil your beer.
Mashing
Certain heat-loving lactic acid bacteria associated with malt can contaminate the mash. However, the thermophilic bacteria (most often members of the genus Bacillus) are sensitive to hops and will not survive in hopped wort. All other lactic acid bacteria introduced in the mash should be killed in the boil. In a standard 60–90 minute mash in which wort separation occurs immediately after the mash, any bacterial growth will be inconsequential. However, if a mash is allowed to sit overnight and the temperature drops to around 140 °F (60 °C), the mash can become soured due to the action of lactic acid bacteria. Overnight mashers might wish to protect their mash with lysozyme. However, they would have to wait until the temperature dropped to 144 °F (62 °C) to add it, which probably would not be practical.
Wort cooling
As wort cools, it is vulnerable to contamination. For this reason, it is very important to cool wort rapidly and pitch with clean, active yeast as soon as possible. Perhaps the best time to add lysozyme is when the yeast is pitched (as pitching yeast is often the biggest source of contaminating bacteria). For this reason, it may not make sense to add lysozyme during wort cooling. If however, the wort is going to stand awhile before the yeast is pitched, lysozyme can be added to the cooling wort at a rate of 0.75 teaspoons per 5 gallons (19 L) once the temperature falls below 144 °F (62 °C).
Pitching
Pitching yeast, in terms of the number of contaminants introduced into the process, is the most significant reservoir of contamination. So, it stands to reason that this is one of the most opportune times to add lysozyme.
Yeast contamination is especially critical when using active dried yeast. Even though there have been huge advances in dried yeast technology, dried yeast is not produced under
sterile conditions and there is great potential for introducing lactic acid bacteria into the fermentation via the yeast preparation.
A clean, active yeast slurry is also an excellent way to minimize spoilage. Many, perhaps most, homebrewers rely on using new cultures of liquid yeast with every brew. The yeast from liquid yeast producers should be free from contamination and safe to pitch. With appropriate care taken with cleaning and sanitation, homebrewers can make a healthy yeast starter with a minimum of unwanted microbial growth. Many homebrewers even report success in reusing yeast used for one or more fermentation cycles.
If yeast to be used (or reused) is suspect, lysozyme can be added to kill any lactic acid bacteria that are present. Lysozyme can be added to either active dried yeast or fresh yeast cultures. By mixing powdered lysozyme with active dried yeast, contaminating lactic acid bacteria can be inhibited upon yeast rehydration. Additions to both dried and fresh yeast will result in the presence of residual lysozyme activity in the fermenting wort. Just “a pinch” of lysozyme in a 2 qt. (~2 L) yeast starter or 1 cup yeast sample should do the trick.
There is one other way to clean up pitching yeast — acid washing. Acid washing is a technique used by some professional brewers to clean up yeast prior to pitching. It is a process whereby phosphoric acid is added to the yeast slurry to drop the pH. The intent is to kill off contaminating bacteria and weak yeast cells leaving only the strong viable yeast for pitching. However, acid washing can sometimes harm the yeast culture and fail to kill all the bacterial cells. This technique merely reduces the bacteria in number and should not be considered a cure. The practice of acid-washing yeast is probably out of reach for most amateur brewers. (And, for what it’s worth, many professional brewers really dislike yeast washing).
Lysozyme addition to fresh yeast cultures can be used in place of acid-washing to “clean-up” the preparation. Keep in mind however, that — unlike acid washing — lysozyme only targets certain types of bacteria.
During fermentation
If microbial contamination is suspected, use lysozyme to prevent further growth of lactic acid bacteria. If lactic acid bacteria are present in fermenting wort, they probably originated in the pitching yeast rather than in the sweet wort. However, if open-fermentation vessels are used or equipment is not thoroughly cleaned, contamination at this stage can occur.
Also, if the primary fermentation stops or slows prematurely, use lysozyme to stabilize the wort while the fermentation is being restarted. Use 0.75–1.0 teaspoons of powder per 5 gallons (19 L) of wort.
In finished beer
Lysozyme can be added to the final product. Research has shown that a lysozyme addition of 150 ppm is high enough to inhibit sensitive bacteria and achieves good maintenance of activity over time with very little physical changes to the beer. The addition rate here is around 0.75 teaspoons of powder per 5 gallons (19 L) of beer.
In the prevention of unwanted microbial growth in wort and beer, nothing replaces brewhouse cleanliness and sanitation. With proper techniques, it is possible to brew clean, unspoiled beer batch after batch. However, microorganisms are everywhere and homebrewers frequently worry about the possibility of contamination. Lysozyme is another tool for the control of microorganisms previously unavailable to the homebrewer.
Lysozyme: Frequently Asked Questions
How long does it take for lysozyme to work?
The rate of activity depends on many factors including temperature, pH, bacterial load and bacterial resistance. Even though lysozyme starts working immediately, it doesn’t necessarily kill all the bacteria immediately.
At what temperature is lysozyme most active?
Lysozyme has optimal activity between 104–113 ºF (40–45 °C), but will remain active up to 144 ºF (62 °C). If the lysozyme is added at lower temperatures, the rate of activity will be dramatically slowed (but not stopped).
At what pH is lysozyme most active?
Lysozyme activity is highest from pH 3.5 to 7.0, although lysozyme is active over a pH range of 2.0–10.0.
Is lysozyme effective against all lactic acid bacteria?
No. A few lactic acid bacteria strains have shown resistance to lysozyme.
How long does lysozyme remain in beer?
The amount of residual lysozyme activity depends on the type of beer, timing of addition, initial bacterial population and the addition rate. Activity loss is greatest in darker beers, which contain more polyphenols. Research has shown that lysozyme is relatively stable in beer and will maintain at least 50% of its activity over a six month period when added to the final bottled product.
Does lysozyme affect the properties of beer?
According to research conducted by Dr. Mark Daeschel at Oregon State University, lysozyme has minimal effects on the physical and sensory properties of beer. Daeschel has shown that using lysozyme at a maximum rate of 200 ppm did not cause chill haze and did not affect foam stability. Lysozyme also has been shown to have no impact on the flavor or aroma of beer. Two sensory studies (also at OSU) were performed by both a regular consumer panel and a brewing industry professional panel. Niether panel could detect a difference in lysozyme treated beers from untreated beers.
Which fining agents react with lysozyme?
Lysozyme is a protein and will be removed by any fining agent that removes protein.
If I add the lysozyme before the boil will it still be active in the fermenting wort or in the final package?
No. Lysozyme loses all activity at 144 ºF (62 °C) and so will be inactivated during the boil.
What is the shelf life of lysozyme?
When stored in a closed container at room temperature in a dry environment, the powder form of lysozyme retains 95% activity after five years. A rehydrated 22% stock solution lysozyme kept in the refrigerator at 39–41 ºF (3.8–5.0 °C) has been shown to retain 90% activity after 12 months.
