Frozen yeast bank procedure

[Broothru]

Just going by the "Yeast" book by Chris White and others' experiences.

It hard to find fault or argue with a PhD Microbiologist!

The goal is to have sufficient glycerin to prevent bursting of yeast cells during the freeze/thaw process. Both sterile water and the liquid in wort dilute the glycerin to a concentration optimal to give this protection: 15% for sterile water, 25% for wort, though the other components in wort would lower the exact composition to something closer to 15%. Fifteen percent concentration of glycerin appears to give better cell protection than higher (or lower) concentrations.

In the final analysis it would seem that both processes give similar levels of protection. One difference I can see that favors using a sterile glycerin/water media verses wort is the
sterility. Wort by its very nature is a growth media for not only yeast but other spoilage colonies as well that would compete with thawing yeast cells. Infection risks would be less likely in a (mostly) sterile environment when attempting to propagate a starter from a thawed sample, even though the risk is probably quite low for either process.

 

[Bassman2003]

It is tough track the impact upon re-entry with regards to impact at our level. I think wort is favored for the journey as it gives some sustenance over the long nap. The whole trick to freezing yeast is getting them to the other side in good shape. Cells still die along the way, just at a slower pace. Reviving tired, undernurished cells just leads to poor outcomes in the end. They say in the book that freezing at consumer temps is an in-between process. Not cold enough to truly preserve the cells. So they actually do not recommend it but it can work to some extent.

I am trying what I call 'extended brinking' or 'long term repitching' now. Which is harvesting yeast from your fermenter aseptically, storing in CO2 at cold fridge temps and then using like a repitch. This is less stressful on the cells than freezing and due to the lack of oxygen, the cells are kind of in a state of stasis. I am hoping to get 2-4 months between pitches and still have plenty of healthy cells in the 2 liter bottle with the Kegland "Tee" on the top. I will make a video about it if I see success. My goal is to be able to keep a slurry for 4 months and direct pitch it in a batch with a great fermentation outcome.

 

[dhoyt]

Ive found that it definitely takes 48hrs to fully propogate enough yeast from the small amount in a vial. I make a 1L ( 101g DME) starter for Kveik yeast, a 1.5L starter for standard ale yeasts and haven't yet tried a lager yeast, but would plan on either a 2L ( 202g DME) starter or 2 step starter for that.

I tried double checking brewfathers yeast starter calculator and the only options are for packages of yeast. I guess my question is, would there be any reason why these numbers would change, like the age of the vile? If so how did you get your numbers? Did you use a calculator and which one?

 

[IslandLizard]

I tried double checking brewfathers yeast starter calculator and the only options are for packages of yeast. I guess my question is, would there be any reason why these numbers would change, like the age of the vile? If so how did you get your numbers? Did you use a calculator and which one?

Many of us use this yeast starter calculator (it's free):
https://www.brewunited.com/yeast_calculator.php

 

[Tony B]

Yeast starters. Make sure you have a plan B , but otherwise, … relax, have a home brew.
This is about 24 hours after pitching a previously frozen vial of 6ml yeast slurry combined with 6ml cryo-preservative. WLP-521 Hornindal. This yeast is HUNGRY !

 

[Tony B]

Last week I made about a 1,700ml starter from one if my frozen vials of WLP-521 Hornidal. I’ve been liking this yeast a lot for my no ferm temp control ales.
Anyhow, I was planning to split the starter for two batches, but life got in the way and I was only able to brew one. So, lemons for lemonade, I poured about 1,000ml into my batch and stuck the remainder in the fridge for a few days with the plan to make some more vials for freezing.
I did so tonight. After decanting, I was able to make 12 vials with the remaining slurry. Will post here if I see any difference in gen 3.

 

[Tony B]

I have a few vials of WLP-521 from my original session but decided to see how the recent generation works out. I originally started from a lab packaged pouch and made about 10 vials. As stated above, I recently made 12 more vials. Since the first set of vials were made from a starter that originated from a retail package, lm saying those were gen 2. The new vials should then be gen 3 and a starter from those should be gen 4. Is that correct?
Anyhow, I made a starter Thursday night and it was blowing up by the time I got home from work on Friday and looks more than ready to pitch when I brew a batch tonight.

 

[Antti]

I'm really interested with storing and growing up self-sufficient yeast bank!

Is there anything that I could use instead of the DME?
Or could I make too big batches of wort for a brew and freeze the excess wort in small containers, melt it and grow the yeast in there? So instead of using DME?

Small background info, for perspective:

I'm quite new into this, but doing everything from scratch (malting, drying, kilning, toasting).

Next year we will be growing our own barley in this small old farm.

If I manage to grow yeast, then we'd have grown everything scratch. But DME is store-bought that's why I'm asking.

 

[khannon]

I'm really interested with storing and growing up self-sufficient yeast bank!

Is there anything that I could use instead of the DME?
Or could I make too big batches of wort for a brew and freeze the excess wort in small containers, melt it and grow the yeast in there? So instead of using DME?

Small background info, for perspective:

I'm quite new into this, but doing everything from scratch (malting, drying, kilning, toasting).

Next year we will be growing our own barley in this small old farm.

If I manage to grow yeast, then we'd have grown everything scratch. But DME is store-bought that's why I'm asking.

About once a year I brew a batch of "wort" with some added nutrients and no hops. I then pressure can in ball jars in a couple of sizes to make starting and stepping up yeast easy. That way I don't need freezer space.

If doing it this way, I suggest boiling the wort to get protein break before canning(which will boil it again). If you don't it all ends up in the jars.

You can also make it whatever OG you want, .020 for starting and .040 for once it is going is what I do, I make the whole batch at .040 and dilute in the smaller "jam" jars.

 

[Bassman2003]

Is there anything that I could use instead of the DME?
Or could I make too big batches of wort for a brew and freeze the excess wort in small containers, melt it and grow the yeast in there? So instead of using DME?

It is not food safe to boil, freeze and thaw wort. Its pH is above pH 4.6 which is the divider line for allowing dangerous substances like Botulism to grow. Pressure canning is the only safe way to store wort and works well. Best to add some yeast nutrients to it as well when you grow the yeast.

 

[Miraculix]

I have no idea how anything should grow in frozen wort. Just do not let it stand around for ages after thawing and you are good.

 

[Bassman2003]

Science is science and it happens if we believe it or not. It is important to state the facts when dealing with food safety on a homebrew forum where convenience usually wins out over solid practice. Since Antti stated they were new to the hobby, might as well learn things the right way from the start rather than start bad habits.

 

[Miraculix]

Science is science and it happens if we believe it or not. It is important to state the facts when dealing with food safety on a homebrew forum where convenience usually wins out over solid practice. Since Antti stated they were new to the hobby, might as well learn things the right way from the start rather than start bad habits.

Science is not a religion.

There is nothing growing in frozen wort.

 

[Tony B]

On another note, I pitched the starter above into a batch around 11:30pm on Saturday night. It was going nuts by next morning. I just got home from work and took a look. No more yeast activity. It still blows my mind how fast that stuff gets the work done.
I’ll let it ride a few more days before kegging.

 

[PCABrewing]

Olive oil. Optional.

Just curious, what is the Optional olive oil for?

 

[day_trippr]

From a BYO article:

"The idea behind olive oil aeration was to directly give the yeast the UFAs that they would normally synthesize from oxygen. This would eliminate the need to aerate the cold wort (which primes the beer to stale more quickly) or the yeast in storage (which causes them to burn up their glycogen and trehalose reserves)."

Cheers!

 

[Tony B]

That sounds a lot better than what I was going to say. Haha
When doing my own research, prior to starting my frozen yeast bank, I read or heard something about olive oil helping to reduce the yeast’s requirement for oxygen and that was good enough for me. I always have olive oil on hand and have seen no ill affects from using it. I want to make the yeast as happy as possible.

 

[day_trippr]

It takes a vanishingly small amount of EVO, too.
Like the coating on the tip of a toothpick. Remarkable...

Cheers!

 

[Miraculix]

It takes a vanishingly small amount of EVO, too.
Like the coating on the tip of a toothpick. Remarkable...

Cheers!

I don't remember where exactly I saw it, but I've seen people getting mixed results with this practice. I wouldn't count on this technique alone tbh.

One question which needs to be addressed and Which usually is omitted within the olive oil crowd, is ufa synthesis really the only thing that yeast does with the oxygen? My guess is that it's not the only thing.

 

[IslandLizard]

Or could I make too big batches of wort for a brew and freeze the excess wort in small containers, melt it and grow the yeast in there? So instead of using DME?

Sure, I do that all the time.*
I too doubt anything can grow in frozen wort. But I would definitely reboil the wort after it has been stored frozen, just for all security. Something could have gotten in during packaging, storage, defrosting, and handling.

That said, depending on how much extra wort you make and store, it could be more efficient, storage-wise, to freeze concentrated (high gravity) wort. Then dilute, reboil, and chill before use as starter wort.

You can also reclaim wort that's left behind in the kettle after filling the fermenter. You'll have to strain/filter it to separate it from the trub. I would reboil it to pasteurize, before freezing.

* On a somewhat larger scale, I brew 3-5 gallons (11-19 liter) of concentrated starter wort (1.090-1.115), hopped to about 50-60 IBU, stored in 48 oz (1.4 liter) stubby, plastic cottage cheese containers. They stack well.

 

[Miraculix]

Sure, I do that all the time.*
I too doubt anything can grow in frozen wort. But I would definitely reboil the wort after it has been stored frozen, just for all security. Something could have gotten in during packaging, storage, defrosting, and handling.

That said, depending on how much extra wort you make and store, it could be more efficient, storage-wise, to freeze concentrated (high gravity) wort. Then dilute, reboil, and chill before use as starter wort.

You can also reclaim wort that's left behind in the kettle after filling the fermenter. You'll have to strain/filter it to separate it from the trub. I would reboil it to pasteurize, before freezing.

* On a somewhat larger scale, I brew 3-5 gallons (11-19 liter) of concentrated starter wort (1.090-1.115), hopped to about 50-60 IBU, stored in 48 oz (1.4 liter) stubby, plastic cottage cheese containers. They stack well.

Hops stress yeast so it's probably not the best idea to hop the starters. Otherwise, that's the procedure that I would also follow if I would freeze wort. Concentrating it is a good idea to max out available space in the freezer to omit colliding with the boss of the house and her weird idea that the freezer is supposed to be holding food.

I do my starters on the stove top. I grind pale malt with 30% oats for yeast health, weigh portions for 2l of 1.04 og, Keep them and make the starter wort on the stove top when needed. No hops in there, doesn't go sour, I've tried it. Also, pale wort beer tastes funny without hops. Not exactly bad, but a bit weird. I've even carbonated it once...

 

[IslandLizard]

Hops stress yeast so it's probably not the best idea to hop the starters. [...] No hops in there, doesn't go sour [...]

I read somewhere that (low) hopping (starter) wort prevents, or at least slows, lactobacillus from growing. After diluting the concentrated wort to starter gravity (1.037) it has only around 20 IBU. I've not noticed that level impacting yeast growth, but maybe doing a side by side can shine some light on it.

I grind pale malt with 30% oats for yeast health

Interesting, I wasn't aware of oats helping with that. Unsaturated fatty acids perhaps?

Ah, I found a link to Scott Janish's research:
http://scottjanish.com/case-brewing-oats/

 

[Miraculix]

I read somewhere that (low) hopping (starter) wort prevents, or at least slows, lactobacillus from growing. After diluting the concentrated wort to starter gravity (1.037) it has only around 20 IBU. I've not noticed that level impacting yeast growth, but maybe doing a side by side can shine some light on it.


Interesting, I wasn't aware of oats helping with that. Unsaturated fatty acids perhaps?

Ah, I found a link to Scott Janish's research:
http://scottjanish.com/case-brewing-oats/

Yes, Scott was the reason I started doing that.

Regarding hops, I think they actually boost mutation rates which is not so nice. And what low rates also do is that they train the few lactos that manage to still multiply in hops influence, to tolerate hops. So what you might end up with is lacto stains that are pretty hop tolerant. That's also not so nice. It's probably not a game changer but I keep hops out of my starters.

 

[bwible]

About once a year I brew a batch of "wort" with some added nutrients and no hops. I then pressure can in ball jars in a couple of sizes to make starting and stepping up yeast easy. That way I don't need freezer space.

If doing it this way, I suggest boiling the wort to get protein break before canning(which will boil it again). If you don't it all ends up in the jars.

You can also make it whatever OG you want, .020 for starting and .040 for once it is going is what I do, I make the whole batch at .040 and dilute in the smaller "jam" jars.

How long do you process mason jars of wort? I know different things say different times. We’ve done pickles and tomato sauce. Each of those says boil for different times.

 

[bwible]

If you’re lazy they make this canned wort you can buy and use to make starters. I have a couple of these on hand.

https://www.morebeer.com/products/o...MIwqSe_NCziQMVFUT_AR3r9g6cEAQYASABEgKp9vD_BwE

 

[rlrohan]

It is not food safe to boil, freeze and thaw wort. Its pH is above pH 4.6 which is the divider line for allowing dangerous substances like Botulism to grow. Pressure canning is the only safe way to store wort and works well. Best to add some yeast nutrients to it as well when you grow the yeast.

I agree with safe canning practices but Botulism needs the proper environment to grow. It's not going to grow in the freezer. If the wort was water bath canned and stored at room temperatures then the concern for Botulism is real especially if the ph is too high. We blanch and freeze fruits and vegetables all the time, so if Botulism could grow in the freezer we would all be dead.

Just my observations from the real world, no disrespect intended.

 

[bwible]

This is another article:

https://www.homebrewtalk.com/threads/maintaining-a-healthy-yeast-bank-long-term.678997/

 

[khannon]

How long do you process mason jars of wort? I know different things say different times. We’ve done pickles and tomato sauce. Each of those says boil for different times.

Honestly, I don't know. My wife is the one with all the canning skills, I just fill the jars and fit the lids.. I'll ask her though.

 

[Bassman2003]

I agree with safe canning practices but Botulism needs the proper environment to grow. It's not going to grow in the freezer. If the wort was water bath canned and stored at room temperatures then the concern for Botulism is real especially if the ph is too high. We blanch and freeze fruits and vegetables all the time, so if Botulism could grow in the freezer we would all be dead.

Just my observations from the real world, no disrespect intended.

No disrespect taken as I posted the best practice. Other avenues will be taken from that for sure. One can not argue with the best practice, only choose to deviate from it. What's important to me is boiling does not kill the spores & microbiology often seems to find a way. Throw in some laziness and unexpected behavior then suddenly there is risk.

People gonna do, but in a forum environment, I believe it is important not to overly discuss less than safe procedures. New brewers might read this and take more liberties and be in a dangerous situation. For example: What if a no chill brewer decided to start freezing their wort? Then when they thawed the wort and put some yeast on it, maybe the yeast could be old and not drop the pH. After a few days they add more yeast and pitch the starter. Safe or not?

If they pressure canned there would be zero risk.

 

[rlrohan]

No disrespect taken as I posted the best practice. Other avenues will be taken from that for sure. One can not argue with the best practice, only choose to deviate from it. What's important to me is boiling does not kill the spores & microbiology often seems to find a way. Throw in some laziness and unexpected behavior then suddenly there is risk.

People gonna do, but in a forum environment, I believe it is important not to overly discuss less than safe procedures. New brewers might read this and take more liberties and be in a dangerous situation. For example: What if a no chill brewer decided to start freezing their wort? Then when they thawed the wort and put some yeast on it, maybe the yeast could be old and not drop the pH. After a few days they add more yeast and pitch the starter. Safe or not?

If they pressure canned there would be zero risk.

I agree with the pressure canned way. The temp is high enough in that process to kill Botulism and it is not in water bath canning. But if the water bath canned wort is stored in the freezer and thawed properly it would be safe. As for the situation "What if a no chill brewer decided to start freezing their wort? Then when they thawed the wort and put some yeast on it, maybe the yeast could be old and not drop the pH. After a few days they add more yeast and pitch the starter. Safe or not?", No I don't think it would be safe. In fact I believe there is a great risk in "no chill" and also I believe there is a great risk for all brews if the yeast doesn't start in a timely manner. Not just if canned frozen wort is used.

 

[dhoyt]

I did my first batch for the yeast bank yesterday. Things I noted and also questions... I apologize ahead of time for my ignorance.

It seems that you need about a full 1 - 1.5 L starter to get the amount of slurry you need to do 10 - 15 mL tubes. I realized this after I did a 1.5 L starter for my upcoming batch and only had about 100 mL of slurry. So next time I will overbuild... So I would like to have a general idea of the amount of yeast is in each tube for using them for starters later. That being said if I use a yeast calc and it say a 1.5 l starter will yield 212 billion cells which is calls out as 9 billion cells per mL that is the full volume of the starter correct? So if I decant all of the wort from the top of my flask and what is left of slurry is 100 mLs then the count would be 2.12 billion cells per mL right? Only thing is that math doesn't seem to math. 9 million cells per mL in 1.5 L isn't 212 billion cells. So obviously I am not looking at this or thinking about this properly. What exactly am I missing here? I am using brewfather by the way.

Other question - Is there not much concern about the yeast setting in the cryo liquid before freezing? I noticed when I checked them today that there was pretty good amount of separation.

 

[Tony B]

I did my first batch for the yeast bank yesterday. Things I noted and also questions... I apologize ahead of time for my ignorance.

It seems that you need about a full 1 - 1.5 L starter to get the amount of slurry you need to do 10 - 15 mL tubes. I realized this after I did a 1.5 L starter for my upcoming batch and only had about 100 mL of slurry. So next time I will overbuild... So I would like to have a general idea of the amount of yeast is in each tube for using them for starters later. That being said if I use a yeast calc and it say a 1.5 l starter will yield 212 billion cells which is calls out as 9 billion cells per mL that is the full volume of the starter correct? So if I decant all of the wort from the top of my flask and what is left of slurry is 100 mLs then the count would be 2.12 billion cells per mL right? Only thing is that math doesn't seem to math. 9 million cells per mL in 1.5 L isn't 212 billion cells. So obviously I am not looking at this or thinking about this properly. What exactly am I missing here? I am using brewfather by the way.

Other question - Is there not much concern about the yeast setting in the cryo liquid before freezing? I noticed when I checked them today that there was pretty good amount of separation.

You seem to be on the right track. I would say that if you’re planning to use the majority of your starter for a batch and make vials of the remainder, you should overbuild your starter. I only have a 2L flask, which is why I generally make a 1.5L starter and depending on batch requirements, I sometimes only get 4-6 vials out of the remainder. You could also take the remainder from what your batch requires and make a second starter from which to make vials.
I’m still learning as I go. One thing I have learned is propping up a lager yeast from a vial will require a large starter or a two step.
I hope this helps.

 

[dhoyt]

You seem to be on the right track. I would say that if you’re planning to use the majority of your starter for a batch and make vials of the remainder, you should overbuild your starter. I only have a 2L flask, which is why I generally make a 1.5L starter and depending on batch requirements, I sometimes only get 4-6 vials out of the remainder. You could also take the remainder from what your batch requires and make a second starter from which to make vials.
I’m still learning as I go. One thing I have learned is propping up a lager yeast from a vial will require a large starter or a two step.
I hope this helps.

Yeah I'm thinking 10 vials might be too many really. I think doing 5 should be sufficient and give me more room for a better variety of yeast strains. Do you have any thoughts on the yeast count / calculations? I seem to be missing something with that.

 

[Tony B]

Yeah I'm thinking 10 vials might be too many really. I think doing 5 should be sufficient and give me more room for a better variety of yeast strains. Do you have any thoughts on the yeast count / calculations? I seem to be missing something with that.

I use… http://www.brewunited.com/yeast_calculator.php
For calculations. But they’re really just guesstimates.
There are methods for getting a more accurate estimate. I think they require a microscope and slides. I haven’t gone that deep.

 

[schuchdan]

I have heard it is about 2 billion cells per ml. of well compacted yeast cake

 

[beren]

This thread is pinned now .

It really deserves it.

I’ve been using this for the last 2 years, and I use 15ml boro tubes so I can reuse them, sterilize in a pressure cooker: https://www.homebrewtalk.com/threads/maintaining-a-healthy-yeast-bank-long-term.678997/

As a side note, more yeast to start isn’t better. It can allow more mutated yeast to multiply and form undesirable flavors. Some people use more glycerin to keep it liquid, and only take a drop out of a single vial, then start with a 5ml starter and build it up from that.

 

[dhoyt]

I’ve been using this for the last 2 years, and I use 15ml boro tubes so I can reuse them, sterilize in a pressure cooker: https://www.homebrewtalk.com/threads/maintaining-a-healthy-yeast-bank-long-term.678997/

As a side note, more yeast to start isn’t better. It can allow more mutated yeast to multiply and form undesirable flavors. Some people use more glycerin to keep it liquid, and only take a drop out of a single vial, then start with a 5ml starter and build it up from that.

I also bought boro glass tubes to reuse and limit plastic. I was curious how the caps would do being sterilized in a pressure cooker. Mine have a silicon seal in the cap I think so should be good. You have had no issues with that?

 

[Antti]

I've read about plastic tubes (seen them sold online) that can take 122°C temperature like autoclaves.

Doesn't the Boro glass tubes shatter in the freezer? When the water expands, when turning into ice.

 

[dhoyt]

I've read about plastic tubes (seen them sold online) that can take 122°C temperature like autoclaves.

Doesn't the Boro glass tubes shatter in the freezer? When the water expands, when turning into ice.

Mine are ok, there is only a total of 12 ml in it so there is enough room in there for expansion i think. The caps are plastic though so I don't know how there will sterilize in a canner but time will tell i guess. For these new ones I just soaked them starsan for a day or so, only cause I didn't get to it for that long...

 

[beren]

They are Bakelite caps, which are fine in an autoclave. The first synthetic plastic and heat safe to 285°C depending on the type. Brittle though. You’ll break more caps than vials I bet.