Electric Brewing Supply 30A BCS Giveaway!


Home Brew Forums > Home Brewing Beer > Fermentation & Yeast > Do you know how to make a yeast starter? Then why not farm yeast and freeze it?
Reply
 
LinkBack Thread Tools
Old 12-30-2011, 05:01 AM   #91
orangehero
Feedback Score: 2 reviews
Recipes 
 
Join Date: Apr 2010
Location: Northeast
Posts: 986
Liked 121 Times on 68 Posts
Likes Given: 196

Default

Yes the data is a little confusing and the samples are very small. The study only gives a rough idea, but note the benefits of the addition of ascorbic acid along with the hypothesis that having enough glycerine to prevent freezing at -20C should reduce loss of viablity and mutagenic effect due to the freeze/thaw cycle. Although they were using wine yeast strains, they were all Saccharomyces cerevisiae.

Mutagenic effect of freezing on mitochondrial DNA of Saccharomyces cerevisiae.

Here they emphasize incubation at 4C for 2-3 days to build intracellular trehalose as a cryoprotectant.

__________________
orangehero is offline
 
Reply With Quote Quick reply to this message
Old 12-30-2011, 05:47 AM   #92
Brewitt
Senior Member
HBT_SUPPORTER.png
Feedback Score: 0 reviews
 
Brewitt's Avatar
Recipes 
 
Join Date: Jun 2011
Location: Encinitas, CA
Posts: 817
Liked 67 Times on 61 Posts
Likes Given: 9

Default

Skip to end of post for my recommendation based upon observations and reading to date.

I don't have access to that full article so I don't know what the composition of the medium was and how they did their freezing and thawing. However, if their "orders" refer to "orders of magnitude" rather than "fold", I am amazed at how poorly their yeast did.

I don't have viability counts but from a quick look at the plates it is clear to me that cells settled out and then resuspended in 15% glycerol (at either 70F or 40F) and then allowed to sit at 40F for several hours followed by chilling to -20F (-30C non-frost free freezer, about 15F lower than a standard freezer) for about 2.5 months survived better than cells shifted straight from 70F to -20F after adjusting to 15% glycerol. It also seems that 15% glycerol is better than 30% glycerol (but the effect is not as great). As I stated yesterday, all of the cultures were beginning to grow and divide after 3 hours. All were at full density (although I don't have the numbers yet after 18 hours (I didn't monitor in between). I guessing viability, 50% or better, in the 15% glycerol cultures. I will try to get a more solid number for you.

I did take advantage of all the excess cells from my frozen tubes (about 20 mls of concentrated cells in an equal volume of 15% or 30% glycerol). I inoculated them into 3 liters of fresh wort and let them go overnight with constant shaking. I harvested about 60 mls of packed cells (1/4 cup containing approximately 4x10e11 cells based upon past experience) and resuspended them in an 100 mls (a bit less than half a cup of 15% glycerol, split them into four 40 ml tubes and cooled them stepwise to -112F for later use. These will be each be grown overnight in 2-3 liters of wort for a 5 gal fermentation. Keep in mind that if they retain 50% viability they will only have to go through three doublings (no problem even with 3-4 hours of recovery time) to get to full density.

HERE is my recommendation:

With the caveat that my freezer is not self-defrosting and somewhat colder than yours, I would feel comfortable recommending the following procedure. Keep in mind that I am starting with small volumes of cells from a freezer but could be starting with the yeast from a beer bottle or a small amount of a smack pak, a White Labs vial, or a starter prepared left over after pitching a fermentation. If you start with yeast from a fermenter then you could go straight to step 3 splitting as appropriate for the amount of yeast.

1. Grow yeast with continuous shaking or stirring until full density (after carefully stepping up to the desired volume to avoid contamination) and then move the cells to the refrigerator and allow to settle for 4 hours to overnight. Your cells should be about 1-2x10e11/liter. Grow about a gallon and you will have enough for about six starters.

2. Pour off spent wort retaining as many cells as possible. The bed of cells should be fairly solid, especially after overnight.

3. Add half a cup to a cup of 15% glycerol for cells from a gallon of starter and resuspend the cells by swirling for a bit until the solution of cells is homogeneous.

4. Split the cell slurry into 4 to 6 equal portions and then move to the freezer (surround in ice packs if self-defrosting and seal in bag).

5. When ready to thaw, swirl continuously in water at approximately 100F until completely thawed but not allowed to warm for extended period. This is pitched into 2-3 liters and stirred or shaken overnight to prepare your starter. If you don't have a shaker or stirrer, you will have considerably less yeast (see Mr. Malty or similar for differences in yield).

6. Pitch your starter and enjoy a vigorous fermentation after 3-4 hours. I have cold crashed my starters for several hours to overnight and poured off the spent wort to avoid off flavors but that is a matter of preference. Although not recommended, if you pitch without growing a starter, I would use the frozen yeast from a full gallon of starter culture to account for loss of viability and lag time.

I have done three brews using the procedure posted above (two from yeast stored a month and a half at -20F and another with yeast stored a month at -112F). Both worked great. Hopefully folks using this procedure will report back and tell us the longevity of the cells in standard self-defrosting freezers.

Good luck and tell me where this can be improved upon.

__________________
Brewitt is offline
 
Reply With Quote Quick reply to this message
Old 12-30-2011, 05:53 AM   #93
Brewitt
Senior Member
HBT_SUPPORTER.png
Feedback Score: 0 reviews
 
Brewitt's Avatar
Recipes 
 
Join Date: Jun 2011
Location: Encinitas, CA
Posts: 817
Liked 67 Times on 61 Posts
Likes Given: 9

Default

Let me add to the above:

The three brews I have done using this procedure were with two different yeast recovered from California IPAs (maybe the same yeast). I have brewed a high SG Russian Imperial Stout (10% ABV), a 6.5% IPA, and a 6% ginger beer. All had vigorous ferments with trap bubbling within 4 hours and came out very well (well, I'm not so fond of the ginger beer but I think that is simply a matter of taste).

__________________
Brewitt is offline
 
Reply With Quote Quick reply to this message
Old 12-30-2011, 05:59 AM   #94
orangehero
Feedback Score: 2 reviews
Recipes 
 
Join Date: Apr 2010
Location: Northeast
Posts: 986
Liked 121 Times on 68 Posts
Likes Given: 196

Default

They used in %: glucose 2, yeast extract 1, peptone from casein 1, and 50 % (W/V) glycerol; with and without 1 g/L Ascorbic acid, 100 mg/L Catechin, or 1 g/L Ascorbic acid and 100 mg/L Catechin.

Based on your observation that leaving the yeast to sit at 40F for a few hours showed an improvement, I would guess this is due to an increase in intracellular trehalose as found in the last study I linked to. Perhaps allowing it to sit for 2-3 days at 40F would be even better as is also recommended. They also mention that at low temperatures cell permeability is very low for the usual external cryoprotectants.

Would you be willing to compare with an even higher concentration of glycerol that doesn't freeze solid?

__________________
orangehero is offline
 
Reply With Quote Quick reply to this message
Old 12-30-2011, 06:49 AM   #95
Brewitt
Senior Member
HBT_SUPPORTER.png
Feedback Score: 0 reviews
 
Brewitt's Avatar
Recipes 
 
Join Date: Jun 2011
Location: Encinitas, CA
Posts: 817
Liked 67 Times on 61 Posts
Likes Given: 9

Default

orangehero, your link is not working, I get a UConn login page. I found the article on the web but my VPN is not giving me access for some reason. I'll check it out when I get access. The issue with the formation of rho- cells is interesting but probably low frequency and not likely to affect our use. I was surprised that the viability of my 30% glycerol was lower than the 15% (not yet confirmed). I probably should have done all this more carefully but I wasn't able to commit the time. I suspect the effect of the 40F incubation was more on the slow cooling rate than the trehalose accumulation but possible that it is both. There is good evidence for trehalose being protective to stresses of various sorts. To be honest, in the interest of time and having achieved the general goal, I am not able to commit a lot more time to this except as it becomes available, which is sporadic. I am curious about a lot of aspects of this issue from both an academic and brewing perspective. Interesting, but not critical for the current use.

__________________
Brewitt is offline
 
Reply With Quote Quick reply to this message
Old 01-03-2012, 09:39 PM   #96
Brewitt
Senior Member
HBT_SUPPORTER.png
Feedback Score: 0 reviews
 
Brewitt's Avatar
Recipes 
 
Join Date: Jun 2011
Location: Encinitas, CA
Posts: 817
Liked 67 Times on 61 Posts
Likes Given: 9

Default

orangehero, I had a chance to look at the article you pointed out. I shouldn't be critical since I just did a very quick and dirty experiment (Then again, I'm not trying to publish my results in scientific journals). However, I don't learn much from this article. They have surprisingly low viability and the viability is quite inconsistent from preparation to preparation. Sometimes they lose 90% of their yeast in 3 days and sometimes the same yeast is stable for a long period. They seem to lose more than 70% of their yeast early in most experiments and then they are relatively stable. It seems they are freezing by just throwing in the freezer as opposed to pre-cooling. They are working at much higher glycerol concentrations than I am. It doesn't seem that they are getting much benefit from using glycerol concentrations that prevent freezing. However, since they don't compare to lower concentrations under the same freezing conditions it is very difficult to say. Finally, they don't say how they thaw the yeast.

That said, for the purposes of those on this list, I think we can say that the procedure I posted is practical and gives the desired outcome. The missing piece is how it translates to a self defrosting freezer and how long the yeast remain sufficiently viable. Three months seems fine.

__________________
Brewitt is offline
 
Reply With Quote Quick reply to this message
Old 01-06-2012, 02:56 AM   #97
katy_bug
Feedback Score: 0 reviews
Recipes 
 
Join Date: Dec 2011
Location: athens, ga
Posts: 17
Liked 3 Times on 1 Posts
Likes Given: 1

Default

I ran across this thread while researching how to make my yeast library. I am a newbie to homebrewing, but I have been growing microbes in the lab for a very long time and I couldn't resist commenting.

Brewitt has put together a nice recommendation for yeast storage and I thought I could comment on the science behind his findings.

1. Glycerol content.
Glyceral is a cryoprotectant but it is also toxic to the cells. The goal is to add the smallest amount of glycerol possible that will still prevent ice crystal formation, but not so much that it will kill the yeast cells. The industry standard is 15% volume/volume.

Someone mentioned trehalose in a prior post. Trehalose is a sugar that also serves as a cryoprotectant. It can be added to the freezing liquid or the yeast will produce plenty of it themselves if they are healthy. The best way to have plenty of trehalose is to well aerate your culture and make sure they are healthy when you freeze.

2. Slow cooling.
Best viability is obtained by cooling the culture in 15% glycerol for 1 degree per minute until you reach the desired storage temp. The ability of a homebrewer to do controlled rate freezing is not easy but can be obtained by using a Mr. Frosty container that someone linked to earlier or by freezing in a propanol bath (which is basically what Mr. Frosty is). A propanol bath can be made by putting your yeast tubes in a ziplock bag, squeeze out the air and then put your bag into a larger jar containing propanol. Put your jar into the freezer for 24 hours. After 24 hours you can move your tubes to a more permanent storage container. Warning - isopropanol will wash-off permanent marker so use an ethanol-proof marker to label your vials or make sure your ziplock bag is sealed tight.

3. Fast thawing.
Fast thawing is the way to go. Like I said before, glycerol is toxic so when they wake up, you want them out of that glycerol solution as fast as possible. A few minutes in warm water and then into the wort. I think the slow thaw in the fridge and higher glycerol concentrations that people use may be the reason why they are having to store such large volumes to get decent yeast numbers. If I fast thaw, I can pitch at a 200x fold rate and get great numbers of yeast in 24 hours.

4. Storage volume.
I know you guys really want to make a "pitchable" product, but your best bet is going to be freezing in small volumes and pitching a starter. The extra step is the price you pay for being able to store your yeast in the freezer in large quantities for longe periods of time.
The Pros of a small frozen volume is:

  • space - you can store many more tubes of a smaller volume. I can store 100 x 1ml tubes in the same space you can put 2 x 50ml tubes.
  • washing - pitching the starter allows you to dilute the glycerol and wash it away by the time you pitch your homebrew. Glycerol tastes like soap and I am sure it's not a flavor you want to add to your beer. Also - like I said before . . . it is toxic to the cells so the more you can get out of the culture before you add to your fermenter, the healthier your brew will be.
  • viability is less important - because you are pitching a starter, you don't have to worry about yeast counts and post-thaw viability. You only need a little to live to get the starter going and grow until you get the right amount of yeast. I know everyone struggles to quantify how much yeast you have, but you can go by optical density. You can literally tell how much yeast you have by how opaque it is. Once you get used to culturing yeast, you can tell just by glancing at it.

5. Storage temp.
Yeast is viable in a non-defrosting freezer at -20C for about 9 months to a year, at -80C for many years and in liquid nitrogen forever. This is because the yeast are still slightly metabolically active at -20C and the cryoprotectant you use to prevent ice crystals will slowly kill the yeast over time. They are less metabolically active at -80C and completely inactive in liquid nitrogen.

6. Testing viability.
Because most homebrewers are going to be using a -20C (standard freezer temp) then to get a good measure of your freezing technique, you should test your yeast viability within a couple of days of freezing it. If you wait longer, you are actually testing your freezing technique AND your freezer's ability to maintain a stable low temp and it is impossible to separate the 2 factors.

All that being said, I am about to start making my own yeast libraries and have access to all my lab equipment of you guys would like to put some real numbers to the protocol parameters. I know this is what Brewitt proposed, but it sounds like he has been swamped at work. What do you think? Tell me what you want to test and I will generate the data.
__________________
katy_bug is offline
3
People Like This 
Reply With Quote Quick reply to this message
Old 01-06-2012, 09:45 PM   #98
Brewitt
Senior Member
HBT_SUPPORTER.png
Feedback Score: 0 reviews
 
Brewitt's Avatar
Recipes 
 
Join Date: Jun 2011
Location: Encinitas, CA
Posts: 817
Liked 67 Times on 61 Posts
Likes Given: 9

Default

Quote:
Originally Posted by Brewitt View Post
I guessing viability, 50% or better, in the 15% glycerol cultures. I will try to get a more solid number for you.
Well, my guess was somewhat off. After 10 weeks in the freezer the viability was ~25% in the culture which went into the cold overnight and then was adjusted to cold 15% glycerol, allowed to sit and then moved to the freezer. The cells that were allowed to settle at room temperature, adjusted to room temperature 15% glycerol, allowed to sit and then moved to the freezer retained >10% viability (about 1/3 of the other). The one that was treated the same as the first except adjusted to 30% glycerol fared somewhat worse. Keep in mind that these are rough numbers.

So, my recommendation stands. Even with 25% viability, you should have an expansion of 8 fold or greater overnight yielding a sufficient starter for pitching. The dead cells will make a negligible contribution.

I'm trying two such starters this weekend and will report back.
__________________
Brewitt is offline
 
Reply With Quote Quick reply to this message
Old 01-09-2012, 04:18 AM   #99
Brewitt
Senior Member
HBT_SUPPORTER.png
Feedback Score: 0 reviews
 
Brewitt's Avatar
Recipes 
 
Join Date: Jun 2011
Location: Encinitas, CA
Posts: 817
Liked 67 Times on 61 Posts
Likes Given: 9

Default

Quote:
Originally Posted by katy_bug View Post
I ran across this thread while researching how to make my yeast library. I am a newbie to homebrewing, but I have been growing microbes in the lab for a very long time and I couldn't resist commenting.
Katy_bug,

Very nice writeup that covers lots of ground. I like the propanol bath which I was not familiar with. I also wasn't aware that 30% glycerol as a cryoprotectant was toxic. The only thing I will reiterate from an earlier post is that the goal is to have a pitchable culture after overnight rather than stepping up from a small innoculum. Since we work in a formal laboratory environment, we have the advantage of being able to autoclave large volumes of wort (at least that is what I do) and using ultracold freezers, so some of the issues are moot but I am trying to adapt this to home use. Of course I am storing 1 ml samples of my yeast. But my goal was to store samples of about 40 mls containing on the order of 10e10 to 10e11 cells. I can pitch one of those into 1-2 liters (a large dilution of glycerol) at night and have 2-4x10e11 cells by morning for pitching 5 gal of wort. I pour off the spent wort after cold crashing so put less than 2 mls of glycerol into 5 gal (about 2000 fold dilution). [EDIT: I forgot to say that a home brewer has concerns about sterility leading to the recommendation to step up in 10X steps. Since the sterility of their wort and environment are not great, that is a good recommendation. The approach I suggest creates a culture which is at the last step before pitching.]

I did this last night and pitched cells cold crashed from 1.5 liter stirred starter into 6 gal of a black imperial IPA and it was bubbling actively within 1 hour. That is the fastest fermentation start I have gotten to date (only 12 brews under my belt). I have to admit, although I am meticulous about generation of research data, for my brewing I am settling with "the proof is in the pudding". The procedure I recommend is working and I think is adaptable to home conditions.

However, if you have the time and patience to do the exercise that you propose, my scientific side would love to know that answer. I don't know what your constraints are but I really don't think this issue has been adequately addressed in the literature. The small sample freezing data from at least one study that I referred to in an earlier post looks reasonably good. However, since it has relatively little use in the laboratory environment, freezing large samples has not been carefully looked at. I have no idea whether it is important to you but perhaps a well controlled study could be publishable in a brewing industry or technical journal (just a thought in terms of making your effort have some academic value).
__________________
Brewitt is offline
 
Reply With Quote Quick reply to this message
Old 01-11-2012, 05:16 PM   #100
katy_bug
Feedback Score: 0 reviews
Recipes 
 
Join Date: Dec 2011
Location: athens, ga
Posts: 17
Liked 3 Times on 1 Posts
Likes Given: 1

Default

Quote:
Originally Posted by Brewitt View Post
However, if you have the time and patience to do the exercise that you propose, my scientific side would love to know that answer. I don't know what your constraints are but I really don't think this issue has been adequately addressed in the literature.
I also have access to autoclaves, shakers, stirplates and -80 and -20 freezers so I would love to evaluate some of these parameters and I can do it in a controlled setting and then try to mimic a homebrew setting.

Since I am still a newbie to homebrewing, help me understand exactly what you want to learn. From the conversation in this thread, this is what I have extracted:

Ultimate Goal - An aliquot of yeast stored in the freezer that can be pitched into a starter and be ready to add to a brew in 24 hours.

Parameters to evaluate -
Ideal % of glycerol as a cryoprotectant
Ideal frozen aliquot size
Freezing protocol that maximizes viability
-Flash freeze
-Cold crash
-Controlled rate freeze
-Sequential freeze (room temp, fridge for a while, freezer)
Ideal freezing protocol
-fast thaw at 37C
-slow thaw in the fridge

How to measure effectiveness -
I have the capability to do live/dead cell counts so I can sample and measure viability immediately post thaw and then 24 hours later.

Any requests? Thoughts? Snide comments?
__________________
katy_bug is offline
 
Reply With Quote Quick reply to this message
Reply



Quick Reply
Message:
Options
Thread Tools


Similar Threads
Thread Thread Starter Forum Replies Last Post
Mocktoberfest (marzen)- should i make a yeast starter with harvested yeast? mhayden37 Fermentation & Yeast 12 02-22-2012 01:24 PM
Use Dry Yeast or make Starter? beitzjr Fermentation & Yeast 23 04-06-2011 12:58 AM
When to make a yeast starter? mudhen5 Fermentation & Yeast 5 02-05-2011 11:18 PM
How Little Yeast to Make a Starter? Bmorebrew Fermentation & Yeast 4 06-16-2010 03:05 AM
How to make a yeast starter blackheart Fermentation & Yeast 4 05-26-2010 05:29 AM