Lysozyme Questions

[HoCo Al]

I'm planning to add Lysozyme to several of my white wines before bottling, since I plan to back-sweeten them and use potassium sorbate, as an insurance policy to prevent MLF from starting. Other than a final fining, racking and back-sweetening, the wines are ready to bottle. I've just adjusted the sulfite levels, and my plan is to add the lysozyme and leave it in just long enough to make sure it destroys any lactic acid bacteria before fining, racking, back-sweetening and bottling.

Questions:

1. What is the minimum wait time after putting the Lysozyme in before fining and racking? Daniel Pambianchi's Modern Winemaking book recommends waiting two weeks after adding the lysozyme, but is that really necessary? I would really like to bottle at least some of this wine as soon as possible -- preferably no longer than a 3 or 4 days or at most one week after adding the Lysozyme.

2. I normally use Super-Kleer (Chitosan + kieselsol) to fine my white wines. Will that also take care of the protein stabilization required after the use of Lysozyme?

3. Is there a low temperature limit below which Lysozyme will not be effective in destroying LAB? One research study I read determined that the optimal temperature for Lysozyme action, a.k.a. "lytic activity", is 22 °C. (Note: The researchers used several different pH buffer solutions from pH 4.5 to 9, not wine, as the media. And they used it against a bacterium called Micrococcus lysodeikticus, not LAB. ) The researchers reported a 75% increase in lytic activity at 22 °C compared to 5 °C. So it sounds like it worked somewhat, but not very well, at the lower temperature.

I've been keeping my cellar temp at 56 °F (13.3 °C). So I'm wondering if I should raise it to 60 °F / 15.5 °C while adding the Lysozyme and waiting for it to work, just to be safe. Any opinion on this? I'd rather not raise it any higher than 60 °F.

 

[Ohio Bob]

It sounds like you’ve researched this more than most.

I would not go against Daniel’s advice. OTOH, if Lysozyme is in the bottles since you decided to bottle immediately, I can’t see that the lysozyme will stop working. I would think its ok to bottle early.

If you’re concerned about the lysozyme not working at cooler temps, why not move the wine to a warmer area for two weeks or so, then move back to your cooler cellar?

 

[Rice_Guy]

Lysozyme / killing bacteria / making as stable food, is a numbers game. There is a calculated risk with every can or plastic retort pouch you buy. ,,, AND every wine.

You mention lactic acid bacteria. (not Oneococcus/ MLF) Have you had a historical problem with them? In the old days, ie pre 1950 lysozyme wasn’t a choice and folks could make wine.

Infection is a numbers game. I would be concerned if you did sloppy technique as making kombucha with the same equipment. Enzymes are expected to react twice as fast with a 10C increase in temperature. Yes it will still react at lower temps. Takes longer. ,,, Note the university research folks may be saying 99.9% clean is good enough when they say two weeks.

 

[winemaker81]

Why are you worried about MLB? Did you inoculate?

I've had folks tell me that every red wine undergoes MLF. This does not appear to be true, given the difficulty folks have getting MLF going when they inoculate for it. Add in the common usage of K-meta in amounts that is contra-indicated for MLB -- spontaneous MLF does not appear likely.

 

[HoCo Al]

Why are you worried about MLB? Did you inoculate?

OK, a little background. I have three varieties of whites that are ready for bottling, all made from grapes grown in my own vineyard. I age my whites longer than most folks, so these have been bulk aging for two to three years. None were inoculated for MLF. I also have been a little lax in keeping up with sulfite additions, so the molecular SO2 levels were lower than they should have been. Last week I did add more sulfite to bring them up to .8 molecular.

To be safe I did chromatography tests for all of them a couple of weeks ago. One of the varieties - Traminette - is in 4 containers: 2 5-gallon carboys, 1 full 1 gallon jug, and one partially filled gallon jug with 3 liters. I took samples from all 4 containers for the chromatography tests, along with samples for the other two varieties (both of which were also in multiple containers). All samples except for one showed a distinct malic acid spot and no lactic acid. One -- the full 1 gallon jug of Traminette -- had no malic spot but a prominent lactic spot, indicating it had been through MLF. How that happened I have no idea. I try to be careful sanitizing all my equipment and avoiding any cross-contamination. However since this came to light I have started to segregate and color code all my utensils and tubing for red or white, and purchased a second wine thief and stirrer specifically to lessen the chance that I would cross-contaminate in the future.

After bottling I plan to save set aside some of these wines to use for a May wedding of one of my grandchildren. It would be a minor disaster if we opened the bottles at the wedding and found they had been spoiled with the dreaded geranium odor that supposedly is a danger if you use sorbate and there is any LAB in the wine. Since I plan to back-sweeten two of these varieties, including the Traminette, and the third - Petit Manseng - has some residual sugar, and since I am not equipped to sterile filter the wines, the sorbate is necessary to prevent a re-fermentation in the bottle. Hence the need for the lysozyme to kill any LAB and prevent an unwanted MLF from occurring again.

 

[Rice_Guy]

Oneococcus (malic fermenting bacteria) are very sensitive to SO2 they are also sensitive to temperature below 50F / 10 C. That gives you two options.

Risk, I am a wine judge and will judge roughly 120 wines a year. In ten years I have tasted geranium twice, the first time was from a different table, they were calling everyone over to learn what the defect was. On a numbers basis I am in two vinters clubs so the actual number of home made wines tasted over a year is three times the contest total.
K sorbate will make an esther, ethyl sorbate. This is bubble gum flavor. Not too bad, kinda like candy at low levels. I taste bubble gum every year.
Risk, I taste oxidation every contest. Low level at 1 / 25, high nutty about once a year. Having 3 liters in a one gallon means that bottle should have that defect. VA also will be a risk with the partial gallon.

All in all the risk of geranium is lower than the risk of bubble gum. If the wine is for a wedding use sorbate. Natural reaction of sorbic acid with ethanol is low, like year or five to get high enough to name it.

 

[mainshipfred]

Here's my unprofessional response without any tested results. I too prefer not using sorbates and except for a rare occasion don't back sweeten my wines. Unless I'm missing something you have not added Lysozyme yet and the wine has been bulk aging for 2-3 years. Depending on how heavily you initially sulfated the wine the native LAB was probably already killed. I do find it odd the one vessel showed high lactic. So my question is why add the Lysozyme if you are going to add additional sulfites to the wine. I wouldn't think there would be any chance any MLF would take place.

It's a matter for personal preference but I like the harsher malic acid in my white wines so I add Lysozyme shortly after the wine begins to clear. Again, I don't regularly back sweeten or use sorbates so based on my understanding of your process I don't see the geranium issue being a problem.

BTW, where are you located? Petit Manseng is one of my favorite white wines and except for here in Virginia I've never found another source.

 

[HoCo Al]

Here's my unprofessional response without any tested results. I too prefer not using sorbates and except for a rare occasion don't back sweeten my wines. Unless I'm missing something you have not added Lysozyme yet and the wine has been bulk aging for 2-3 years. Depending on how heavily you initially sulfated the wine the native LAB was probably already killed. I do find it odd the one vessel showed high lactic. So my question is why add the Lysozyme if you are going to add additional sulfites to the wine. I wouldn't think there would be any chance any MLF would take place.

It's a matter for personal preference but I like the harsher malic acid in my white wines so I add Lysozyme shortly after the wine begins to clear. Again, I don't regularly back sweeten or use sorbates so based on my understanding of your process I don't see the geranium issue being a problem.

BTW, where are you located? Petit Manseng is one of my favorite white wines and except for here in Virginia I've never found another source.

A little more background: These wines have a relatively high alcohol content for white wines - 13% - 14% abv (based on calculators, not lab tests). I let the grapes hang and got 23 - 25 brix grapes at harvest. I also cold soak them for 12 - 24 hours at about 35 °F, which I find gives them more varietal flavor, complexity and a deeper golden color. That also does bring in some tannins from the skins & seeds, so the longer bulk aging is done with the idea that it will soften the tannins before bottling. So far this process seems to have worked for me.

I generally prefer dry wines, but with the high alcohol conent they will taste a bit "hot" if left completely dry. I find that a little residual sugar -- typically about 1% w/v, helps to balance them better for the average palate. My wife agrees, and she does get a vote. So off-dry it is, at least for the high-alcohol whites.

The lysozyme was an insurance policy against the geranium effect, though I've never experienced that yet even when I didn't use lysozyme but did use sorbate. Sounds like it's probably overkill, but from all I've read it won't hurt anything to use it.

I'm in Central Maryland. Got the Petit Manseng vines about 5 years ago from a nursery in Upstate New York -- Amberg Grapevines. The winegrape specialist Extension Agent here in Maryland was encouraging Maryland growers to try it. So far it has worked out well in my vineyard.

 

[winemaker81]

To be safe I did chromatography tests for all of them a couple of weeks ago.

Your explanation makes perfect sense.

Based upon what I know of MLB, the likelihood of the two 2.5 gallon carboys undergoing MLF between now and the wedding appears extremely low. If it were me? I'd keep the two 2.5 carboys segregated and dose them with 75 ppm K-meta, which is good insurance.

The gallon that shows lactic acid? Dose it with Lysozyme and keep it away from the others to avoid problems. You can serve it at the wedding, but I'd bottle it after the others to ensure they're safe.

The partial gallon? I agree that the likelihood of oxidation is high. Keep that one separate as well.

If you're still feeling unsure, AFAIK dosing with Lysozyme won't hurt anything. I'd follow Pambianchi's advice.

As to having separate equipment? IMO that's overkill. If you are regularly using good hygiene and sanitation, it's not necessary. It's very likely that the cross-contamination came from the air. For wines you don't want to undergo MLF, in the future make sure your K-meta regimen is followed.

 

[HoCo Al]

Your explanation makes perfect sense.

Based upon what I know of MLB, the likelihood of the two 2.5 gallon carboys undergoing MLF between now and the wedding appears extremely low. If it were me? I'd keep the two 2.5 carboys segregated and dose them with 75 ppm K-meta, which is good insurance.

The gallon that shows lactic acid? Dose it with Lysozyme and keep it away from the others to avoid problems. You can serve it at the wedding, but I'd bottle it after the others to ensure they're safe.

The partial gallon? I agree that the likelihood of oxidation is high. Keep that one separate as well.

If you're still feeling unsure, AFAIK dosing with Lysozyme won't hurt anything. I'd follow Pambianchi's advice.

As to having separate equipment? IMO that's overkill. If you are regularly using good hygiene and sanitation, it's not necessary. It's very likely that the cross-contamination came from the air. For wines you don't want to undergo MLF, in the future make sure your K-meta regimen is followed.

Thanks for all the good advice, Bryan. Actually it is two 5 gallon carboys, not 2.5 gallon. Should have used the word "two" instead of the number in the original post to avoid confusion.

So here's what I've decided, at least for the Traminette. I already have put 350 ppm lysozyme in the two 5 gallon carboys, and will wait the two weeks Daniel recommends, and then back-sweeten/sorbate as planned. I have also already bottled the full gallon without lysozyme or back-sweetening. We can use that for our holiday season parties over the next couple of weeks. I'll bottle the 10 gallons too, but reserve most of that for the wedding.

Though I haven't done it yet, I'll probably back-sweeten the 3 liters in the partial gallon jug but not worry about the lysozyme or sorbate. It will only be 4 bottles, so we'll use that up quickly here at home, and if it's a little off-flavor from oxidation, not a big deal. It doesn't look oxidized - color is similar to that in the other bottles. Though I didn't keep up with the sulfite as I should have, I did use CO2 to try to remove oxygen from the headspace, and tightly capped it with a teflon-lined cap. Given all that and the fact that it is a glass bottle almost impervious to oxygen infiltration, I think it will be OK.

 

[Shurt1073]

Just a thought ... but you could use two 375ml bottles when bottling ten gallons or (49 bottles of 750ml & 2 bottles 375ml) and prior to the wedding periodically open a small 375ml for testing.

 

[HoCo Al]

Just a thought ... but you could use two 375ml bottles when bottling ten gallons or (49 bottles of 750ml & 2 bottles 375ml) and prior to the wedding periodically open a small 375ml for testing.

That's an interesting idea. I'd have to go out and get a case of 375ml bottles to do that since I don't have any right now. However since I also have pretty good quantities of two other varieties ready to bottle - Petit Manseng and Chardonel - even if I use up a couple of bottles of the Traminette, those will also be available for the wedding as well.

I was going to let my daughter -- the mother of the groom -- taste several of my wines to see which one or ones she would like best for the wedding. Of course in order to do that I'll have to bottle at least some of those two as well. Which was my original plan.

 

[HoCo Al]

Lysozyme / killing bacteria / making as stable food, is a numbers game. There is a calculated risk with every can or plastic retort pouch you buy. ,,, AND every wine.

You mention lactic acid bacteria. (not Oneococcus/ MLF) Have you had a historical problem with them? In the old days, ie pre 1950 lysozyme wasn’t a choice and folks could make wine.

Infection is a numbers game. I would be concerned if you did sloppy technique as making kombucha with the same equipment. Enzymes are expected to react twice as fast with a 10C increase in temperature. Yes it will still react at lower temps. Takes longer. ,,, Note the university research folks may be saying 99.9% clean is good enough when they say two weeks.

Perhaps I used the wrong terminology, but by "lactic acid bacteria" or LAB as I abbreviated it, I meant the bacteria normally used to convert malic to lactic, whether intentionally inoculated, or inadvertently inoculated, as seems to have happened with my full one gallon jug of Traminette.

In any case -- and please correct me if I'm wrong, as I'm still in the learning phase about this -- it was my understanding that Lysozyme itself will produce no ill effects in wine, such as bad tastes or odors. And, using Lysozyme can add anti-microbial protection that allows you to use less sulfite to protect the wine (though I also understand that Lysozyme does not provide any protection against oxidation). I was under the impression that the only downside of Lysozyme is that, since it is a protein, its use will require protein stabilization with bentonite or other fining materials before bottling to prevent a protein haze from developing.

Which leads to the 2nd still-unanswered question in my OP (re-stated a little differently): Will fining just before bottling with Super-Kleer or equivalent Chitosan/kieselsol product prevent the protein haze that might otherwise be caused by the Lysozyme?

 

[Rice_Guy]

2) typical protein treatment is to put a charged molecule in and let them combine. My go to would be a finishing tannin. I haven’t intentionally used C/K (but this is in a kit so yeah).

1) Yes losozyme will kill (destroy the membrane of) several bacterial families. At normal levels it will not create off flavors. Likewise if you use normal SO2 levels you will not have a bacterial issue. If you use unusually low levels of SO2 you will reduce the expected shelf life. I would never skimp on meta. As a home wine maker and as a judge I experience oxidation as a bigger issue than refermentation.

 

[Ohio Bob]

I was under the impression that the only downside of Lysozyme is that, since it is a protein, its use will require protein stabilization with bentonite or other fining materials before bottling to prevent a protein haze from developing.

That has not been my experience. In fact in a Riesling I made I would say it clearly stripped out some color from the wine. Flavor seemed ok, just a very pale color.

Literature says the Lysozyme remains active for 3-6 weeks. If you desire to postpone MLF for some reason, as with a Pinot noir, you can use bentonite to drop out the lysozyme and thus enable MLF.

https://digitalcommons.calpoly.edu/...er=&httpsredir=1&article=1062&context=fsn_fac

 

[Ohio Bob]

Perhaps I used the wrong terminology, but by "lactic acid bacteria" or LAB as I abbreviated it, I meant the bacteria normally used to convert malic to lactic

The vast majority of literature reference and industry jargon use Malolactic Fermentation (MLF) and malolactic bacteria (MLB) to describe the bacteria that converts malolactic to lactic.

You’re not wrong, but it seems you created a definition where there already was one that is generally accepted. It’s a good discussion to have as this area of winemaking has only been available to the winemaker for the last 2-3 decades(?).

 

[HoCo Al]

The vast majority of literature reference and industry jargon use Malolactic Fermentation (MLF) and malolactic bacteria (MLB) to describe the bacteria that converts malolactic to lactic.

You’re not wrong, but it seems you created a definition where there already was one that is generally accepted. It’s a good discussion to have as this area of winemaking has only been available to the winemaker for the last 2-3 decades(?).

OK, thanks for correcting me on the accepted terminology. As I said I'm still learning.​

 

[Ohio Bob]

As I said I'm still learning.

Me too. In looking over some of my info I stumbled onto one application... added to the red wine must at crushing (?) in order to allow extended maceration without MLF kicking off. Did not know that.