# Lysozyme



## Boatboy24 (Oct 27, 2020)

Good article by Tim Patterson in Winemaker Mag on Lysozyme. Link below. I'm also posting the text in the event the link breaks.









It's Lysozyme Time - WineMakerMag.com


Home winemakers have an important new resource in the ongoing battle against spoilage organisms — it’s called lysozyme. Discovered in the 1920s and used for decades in the pharmaceutical, dairy and cheese industries, this natural protein has come into increasing use in the wine industry in the...




winemakermag.com





*It’s Lysozyme Time*
*Written by Tim Patterson*
Home winemakers have an important new resource in the ongoing battle against spoilage organisms — it’s called lysozyme. Discovered in the 1920s and used for decades in the pharmaceutical, dairy and cheese industries, this natural protein has come into increasing use in the wine industry in the past decade. It’s now available in home-scale quantities, giving amateur winemakers a means of reducing sulfite use while still protecting the health of their wines.
Lysozyme is an enzyme found in the protective fluids (tears, saliva and mucus) of most animals. Commercial lysozyme is isolated from egg whites. Lysozyme kills certain types of bacteria by attacking their cell walls. It’s effective against most eubacteria, but not against archaebacteria or eukaryotes. (Eubacteria are “ordinary” bacteria; archaebacteria are thermal-vent bacteria and their relatives. Eukaryotes include animals, plants, protists and fungi, including yeast.)
Lysozyme works against a range of lactic-acid bacteria that can affect wine stability, including Oenococcus, Pediococcus and Lactobacillus, all of which produce off-odors in wine. Lysozyme also kills Leuconostoc, the bacteria responsible for transforming malic acid into lactic acid during malolactic fermentation. It does not interfere with yeast.
Lysozyme can “clean up” batches of wine infested with unwanted lactic bacteria. It is increasingly used in a preventive mode, to stop problems before they can start. Lysozyme is also used to delay or prevent malolactic fermentation, an important consideration for some white wines. It also aids the stability of wines that are bottled without filtration and could therefore be subject to bacterial action in the bottle.
In home winemaking, sanitation issues can be difficult to control, accurate testing may be hard to come by and sterile filtration is virtually impossible. So lysozyme has great potential. It provides a terrific alternative to sulfite additions, particularly for delaying or preventing malolactic fermentations in white wines, which always requires threading a needle between too little (leading to an unwanted malolactic fermentation) and too much (leading to the nose burn of over-sulfited wines). Within recommended ranges, lysozyme has no discernible sensory impact on wine aroma or taste, and leaves no toxic residue. All of which led one home winemaker colleague of mine to extol lysozyme as a “magic bullet.”
For the record, lysozyme is not omnipotent and it by no means eliminates the need for sulfite additions. First of all, lysozyme does nothing to prevent oxidation and browning; for this, sulfur dioxide is still required. Similarly, lysozyme is no help against many of the nasty organisms on the winemaker worry list — such as Acetobacter, the vinegar bacteria, or Brettanomyces and other rogue yeasts — that require some combination of SO2 and sterile filtration. Finally, there are even some strains of lactic acid bacteria, including certain malolactic bacteria, which are resistant to lysozyme, and can only be controlled with sulfites or sterile filtration.
Here are some guidelines and suggestions for lysozyme use.
Preparation: To prepare the enzyme, add one part lysozyme (in powdered or crystalline form) to 5–10 parts tepid water. Stir gently, as shaking or rough mixing will produce foam and “gumballs.” Allow time for the lysozyme to dissolve completely, then add to wine or must, stirring gently with a sanitized utensil.
Timing and dosage: It’s up to you to decide whether to use lysozyme as insurance at every step along the way, or simply use it for known problems or intentional stylistic decisions. The effective dosage varies with the size of the bacterial population and the tannin level of the wine, so the numbers below are given as ranges.
For translating the ranges below, adding 100 parts per million (ppm) means 0.1 gram of lysozyme for each liter of wine (or 0.013 ounce per gallon); 250 ppm means 0.25 g/L (or 0.033 ounce/gallon). Even though it mixes measuring systems, my rule of thumb is that one gram per gallon is a hair over 250 ppm.
At the crush: Add 50–150 ppm lysozyme to red grape must or white grape juice. The lysozyme addition should be made after any addition of SO2, to allow the SO2 to diffuse and bind. Used this way, lysozyme will inhibit lactic acid bacteria and delay the onset of malolactic fermentation, thereby reducing any competition with the yeast for nutrients during the alcoholic fermentation.
Stuck fermentations: For red wines, add 150–400 ppm lysozyme. For whites, add 300–500 ppm, then restart fermentation with a new yeast culture. Stuck fermentations with rapid lactic acid bacteria buildup are great breeding grounds for volatile acidity. Fighting the lactic acid bacteria with heavy sulfite additions can also inhibit yeast fermentation activity, keeping it stuck. In contrast, lysozyme targets the bacteria and lets the yeast do its thing.
Preventing malolactic: Once primary fermentation is complete, malolactic activity can be blocked with an addition of 250–500 ppm of lysozyme. This dosage will also stop a malolactic fermentation in progress, resulting in a partial malolactic conversion. For wines that received a small lysozyme treatment at the crusher, inoculation with a malolactic starter should work normally after the alcoholic fermentation has run to completion.
Aging stability: For wines that go through a malolactic fermentation, wait until this fermentation is complete, then add 250–500 ppm of lysozyme to prevent lactic acid bacterial activity during aging.
Bottling: For unfiltered or lightly filtered wines that have not already been treated with lysozyme, an addition of 250–500 ppm can be done a few weeks before bottling for greater stability. In the bottle, free SO2 becomes bound and less effective over time. Lysozyme’s effectiveness decreases at a slower rate.
In all these situations, lysozyme does its work in about a week or ten days, at which point testing or further treatment can be done.
Blending: Blending different wines means that lysozyme treatment has to be reconsidered, or perhaps redone. For example, if a malolactic white wine is blended with another non-malolactic wine, the resulting blend will need a lysozyme addition to remain stable. If the stylistic goal is a partially malolactic-fermented wine, mixing batches in this way is probably the most practical method. It offers more control than starting a malolactic fermentation, then stopping it, in a single batch. The same is true if a treated wine receives a significant amount of topping with a wine untreated with lysozyme.
Testing: In a commercial setting, lysozyme treatments are accompanied by several rounds of testing: to determine which problem bacteria are present, and in what numbers; to determine if any resistant strains are present, requiring different treatment; to make sure the killing action worked and the bacteria are eliminated; and to determine if a malolactic fermentation happened according to plan.
For home wineries, testing for malolactic activity should at least be done. (In my book, bottling anything without knowing its malolactic status is a serious leap of faith.)
If testing indicates that a lysozyme addition has not prevented malolactic activity, the problem may be a resistant bacterial strain and sulfite control may be the only option. With stuck fermentations, testing could determine the lactic bacterial load and therefore indicate the appropriate lysozyme addition. However, if a fermentation is truly stuck, and testing isn’t available, don’t be afraid of lysozyme additions at the upper end of the ranges above.
Clarification and fining: Because lysozyme is a protein, it can contribute to protein haze in some wines when they are stored at warmer temperatures. Its killing action can also result in some precipitation of the lees. Precipitation, if it does appear, normally shows up in a few days and the wine can simply be racked for clarification. Be cautious and allow at least a couple weeks between a final lysozyme addition and bottling.
Fining: Bentonite neutralizes enzymatic activity, including that of lysozyme. The same is true for carbon fining agents, oak chips and high tannin levels. Yet for good heat stability, and the prevention of cloudiness, bentonite may need to be part of your winemaking routine.
For non-malolactic wines, treat with lysozyme first to kill bacteria, then fine with bentonite later — but remember that the killing power of the lysozyme has been greatly reduced. A light second addition of lysozyme can serve as insurance. For reds and whites that do go through malolactic, fine with bentonite after fermentation is complete, then treat with lysozyme later for barrel and bottle stability.
The same logic applies to oak chip additions. Let the lysozyme have two weeks to kill malolactic bacteria; test to make sure; add oak chips for whatever duration is appropriate; rack the wine off the chips and then protect the wine with a second treatment.


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## cmason1957 (Oct 27, 2020)

I wonder if you could add potassium sorbate to a wine that underwent MLF after adding the proper dosage of lysozome? Of course, why someone would make a sweeter red wine is beyond my comprehension, but we always hear of this coming up.


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## BernardSmith (Oct 27, 2020)

Does anyone know the source of this enzyme? Is it vegetable or animal ?


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## stickman (Oct 27, 2020)

Chicken Eggs.


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## BernardSmith (Oct 27, 2020)

Fertilized eggs or simply the eggs themselves?


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## cmason1957 (Oct 27, 2020)

The words of the article above says egg whites, I would imagine unfertilized.


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## BernardSmith (Oct 27, 2020)

Yup, sounds like it's the protein in the albumen. I ask because I am a vegetarian but I eat eggs - though they cannot be fertilized.


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## TurkeyHollow (Oct 28, 2020)

How does it affect the amount of SO2 to be added? It seems you would use this so less sulfites would be required. Is there any formula bringing lysozyme into the equation (along with pH and SO2)?


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## Johnd (Oct 28, 2020)

TurkeyHollow said:


> How does it affect the amount of SO2 to be added? It seems you would use this so less sulfites would be required. Is there any formula bringing lysozyme into the equation (along with pH and SO2)?


No, use your sulfite as you normally would, you still need the protection from oxidation and all of the other things lysozyme doesn’t inhibit. If you read the article,it clearly states: 

“For the record, lysozyme is not omnipotent and it by no means eliminates the need for sulfite additions. First of all, lysozyme does nothing to prevent oxidation and browning; for this, sulfur dioxide is still required. Similarly, lysozyme is no help against many of the nasty organisms on the winemaker worry list — such as Acetobacter, the vinegar bacteria, or Brettanomyces and other rogue yeasts — that require some combination of SO2 and sterile filtration. Finally, there are even some strains of lactic acid bacteria, including certain malolactic bacteria, which are resistant to lysozyme, and can only be controlled with sulfites or sterile filtration.”


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## Boatboy24 (Oct 28, 2020)

To build on John's response, I think the only impact it has on required sulfites is if you were trying to use sulfite alone to inhibit MLF. And that's not an approach I think I'd want to take.


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## sjjan (Oct 29, 2020)

I use lysozyme to inhibit MLF after primary fermentation and this allows me to use less SO2 as I can go with the 5 versus 8 ppm SO2 addition regime as I don’t have to add extra SO2 anymore to inhibit the start or ongoing of MLF.


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## Johnd (Oct 29, 2020)

sjjan said:


> I use lysozyme to inhibit MLF after primary fermentation and this allows me to use less SO2 as I can go with the 5 versus 8 ppm SO2 addition regime as I don’t have to add extra SO2 anymore to inhibit the start or ongoing of MLF.


You’re not applying SO2 practices properly if you’re using 5 ppm vs 8 ppm additions, neither 5 nor 8 ppm is enough to protect a wine. I believe you are referring to the molecular levels of .5 and .8. The use of these molecular level additions is based upon the COLOR of your wine and it’s pH, reds use .5 molecular levels, whites use .8 molecular levels See the chart below, which gives the recommended dosage, in ppm, for both reds and whites, based upon their respective pH.

For instance, using the chart below, if you have a white wine with pH of 3.5, you’d use the blue curve (.8 molecular level) to determine that 40 ppm is the proper level of free SO2 for your wine. Similarly, a red wine with a pH of 3.5, using the red / pink curve (.5 molecular level) would yield a free SO2 level of 25 ppm.

The only wines in which 5 ppm or 8 ppm free sulfite would be appropriate, would have a pH value below 3.0, in which case, you wouldn’t even need Lysozyme to inhibit MLF, the pH would prevent it. 

If you’re shorting your SO2 additions because you’ve added Lysozyme, you’re playing with fire. I will again cite the article that started this discussion: “For the record, lysozyme is not omnipotent and it by no means eliminates the need for sulfite additions. First of all, lysozyme does nothing to prevent oxidation and browning; for this, sulfur dioxide is still required. Similarly, lysozyme is no help against many of the nasty organisms on the winemaker worry list — such as Acetobacter, the vinegar bacteria, or Brettanomyces and other rogue yeasts — that require some combination of SO2 and sterile filtration. Finally, there are even some strains of lactic acid bacteria, including certain malolactic bacteria, which are resistant to lysozyme, and can only be controlled with sulfites or sterile filtration.”


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## stickman (Oct 29, 2020)

I remember seeing some manufacturer marketing information on Lysozyme a number of years ago, and they did indicate a lower use of SO2 was possible, but they didn't provide any numbers associated with the concept. For big red wines with a pH in the 3.7 to 3.9 range, 20 to 30 ppm free SO2 is adequate to prevent oxidation, but wouldn't suppress all of the microbial activity, so I'm speculating here, that the marketing people were targeting winemakers that, for various reasons, would prefer not to maintain adequate molecular SO2. 

As the article above points out, not many things are perfect, it really comes down to how much risk the winemaker is willing to accept to achieve the particular style of wine.


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## sjjan (Oct 29, 2020)

I was referring to 0.5 ppm molecular vs 0.8 ppm molecular. There are a few conversion charts around (all more or less giving the same info) that can help to calculate how much SO2 or PMBS (converted) to add. For example, at a pH of 3.10 you would end up with 10 ppm SO2 as target level when looking down the 0.5 ppm molecular column vs 16 ppm [email protected] when looking down the 0.8 ppm molecular column which would translate (in the case of 10 ppm SO2 to 1.85 grams of PMBS per hL (hecto liter) or e.g. 1.97 gram per 30 Gal.

The idea is that you use the 0.8 ppm molecular schema if you have to protect the wine as well as to prevent MLF from happening, but use the 0.5 ppm molecular schema if you are past the MLF phase and do not have to prohibit MLF anymore. In that case, the idea is to go with the 0.5 ppm molecular schema. In my case, I am using lysozyme at a dosis that completely knocks out the MLF possibility, so I would assume that I could then add SO2 according to the 0.5 ppm molecular schema. In any case, I will test for free SO2 levels soon.

I am just starting out making wine so am absolutely not sure if this is correct. Any tips or suggestions are appreciated.


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## montanarick (Oct 29, 2020)

Agree with Johnd


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## sjjan (Oct 30, 2020)

montanarick said:


> Agree with Johnd


I am here to learn. 

Most of the information I have been reading refers to the 2 targets of 0.5 and 0.8 ppm molecular SO2 in relation to stopping MLF or not and do not differentiate between red and white wine in this matter. I by now found some articles that do mention the use of the different targets in relation to red and white wine. For example, there is this calculator at Winemaking Magazine that differentiates the target molecular SO2 based on red vs white wine. It thus all depends on which literature you read and what the reasoning is behind it. What would the reasoning behind it be one way or the other?

Most wine yeasts (S. cerevisiae) are inhibited at 0.8 ppm molecular SO2. Bacteria, wild yeast and Brett are inhibited at 0.4 ppm molecular SO2. Lysozyme at a high enough dosis kills off most of the bacteria. After primary fermentation, the yeast is dead at the bottom of the tank and MLF is based on bacteria and can be inhibited by lysozyme. Lysozyme cannot replace sulfur dioxide (SO2) because it will not prevent infections by other spoilage organisms such as Acetobacter or Brettanomyces.

I just don’t know  and am glad for the feedback received.


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## Ajmassa (Oct 30, 2020)

sjjan said:


> I am here to learn.
> 
> Most of the information I have been reading refers to the 2 targets of 0.5 and 0.8 ppm molecular SO2 in relation to stopping MLF or not and do not differentiate between red and white wine in this matter. I by now found some articles that do mention the use of the different targets in relation to red and white wine. For example, there is this calculator at Winemaking Magazine that differentiates the target molecular SO2 based on red vs white wine. It thus all depends on which literature you read and what the reasoning is behind it. What would the reasoning behind it be one way or the other?
> 
> ...



I believe the difference from .5 and .8 is more than just
Protection from o2, bacteria
vs
Protection from o2, bacteria, & Mlf prevention

though that’s probably part of it too I’d guess. But it also has to do with white wines generally requiring a higher level of protection in general. White wines are more fragile where lesser amounts of o2 have ability to affect the wine more than reds—- quicker then reds to see color affected I think. And typically abv is lower in whites which naturally means less protection—in spite of typically stronger acid protection.
<— this has been the way I viewed the reason whites are recommended for .8 while reds .5. And figured Mlf prevention seemed more of byproduct of the higher protection needs- not the reasoning for higher so2.


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## sjjan (Nov 1, 2020)

Ajmassa said:


> I believe the difference from .5 and .8 is more than just
> Protection from o2, bacteria
> vs
> Protection from o2, bacteria, & Mlf prevention
> ...


I have by now adopted my procedure and use the .8 for white wine. That is thanks to the input from WMT.


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## TD Fermenting (Dec 14, 2020)

Great and helpful thread. 
I have 5 gallons of sauv blanc made from frozen juice from WGD. Supplier numbers were TA of 0.70, pH 3.37, Brix 24.0. I measured pH at 3.5, brix at 23.5, SpGr of 1.100. Supplier included 10 gm Tartrate which I added before primary. Immediately before primary pH was 3.6 (went the wrong way!) I used bentonite in primary fermentation which took 10 days to finish to a SpGr of 0.992. I racked it with 1/4 tsp KMeta. Post primary fermentation pH is 3.65. It has been in a carboy for 4 days and is slowly clearing. 
I don’t want MLF and am considering lysozyme, but am wondering about dropping the pH with some tartrate. I’m intending to leave on fine lees with some battonage for a few months. 
This is my first non-kit wine, I am getting a TA kit and am slightly intimidated by measuring free SO2, so haven’t attempted.
Any thoughts, advice or comments much appreciated.


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## montanarick (Dec 15, 2020)

use of lysozyme should not have adverse impact on your wine and is a sure fire way to prevent MLF


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## Ajmassa (Dec 15, 2020)

TD Fermenting said:


> Great and helpful thread.
> I have 5 gallons of sauv blanc made from frozen juice from WGD. Supplier numbers were TA of 0.70, pH 3.37, Brix 24.0. I measured pH at 3.5, brix at 23.5, SpGr of 1.100. Supplier included 10 gm Tartrate which I added before primary. Immediately before primary pH was 3.6 (went the wrong way!) I used bentonite in primary fermentation which took 10 days to finish to a SpGr of 0.992. I racked it with 1/4 tsp KMeta. Post primary fermentation pH is 3.65. It has been in a carboy for 4 days and is slowly clearing.
> I don’t want MLF and am considering lysozyme, but am wondering about dropping the pH with some tartrate. I’m intending to leave on fine lees with some battonage for a few months.
> This is my first non-kit wine, I am getting a TA kit and am slightly intimidated by measuring free SO2, so haven’t attempted.
> Any thoughts, advice or comments much appreciated.



Congrats on the first non kit wine!
I Just recently learned the major affect freezing temps have on ph readings. your initial readings were likely skewed by temps I bet. And If WGD supplied a bucket with known levels and gave you a specific amount of tartaric acid to dial in the ph- chances are that they won’t steer you wrong.

And even now at 3.65– I wouldn’t lower the ph just for the sake of it. It’s already fermented and it’s at a healthy range. Any adjusting at this point can just do by taste after a few months. TA testing isn’t necessary. Could just take a few small samples for bench trial- like 50mL or something. Figure the math and add .25g/L, .50g/L, .75g/L and 1.0g/L 1.5g/L etc. Pick your favorite (or none) and dose the batch. Plus this way ensures you don’t overdo it.

TA testing not required for it. And also ya don’t need a kit. Kits go by color change and are a pain. You can just use your ph meter, a bottle of NaOH, and a lab syringe and you’re good to go. 

As far as so2, i used the titrets for a few years and they were awesome. Definitely accurate enough for my needs. About $20 for a 10pack. Cheaply testing so2


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## hounddawg (Dec 15, 2020)

cmason1957 said:


> I wonder if you could add potassium sorbate to a wine that underwent MLF after adding the proper dosage of lysozome? Of course, why someone would make a sweeter red wine is beyond my comprehension, but we always hear of this coming up.


easy  
Dawg


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