Fermentation Performance of Refrigerated Yeast Slurry

[WoodlandBrew]

There seems to be room for research on the effects of refrigeration on yeast slurry. There are online calculators that will produce a viability number based on storage time , but the research behind these calculations seems to be lacking.[1] Work has been done examining the effects over the course of several days[2][3] but storage over several months has not been well documented that I know of.

The goal of this experiment would be to better assess both the viability and vitality of stored yeast so that pitch rate can be adjusted accordingly.

Is there a study that has been conducted on the viability and vitality of yeast stored for several months?

What conditions should be emulated?

What metrics might be important to evaluate?

My current plan is to rehydrate[4] and store four different strains of ale yeast. These will be used to inoculate wort at intervals ranging from one week to one year. Viability [5]will be measured prior to inoculation of each test. Cell density [6] will be monitored daily. Residual extract will be measured after fermentation completion.

[1] Mr. Malty, Yeast Calc, Brewers Friend
[2] Murray, "The Effect of Yeast Storage Conditions on Subsequent Fermentations" 1984
[3] www.woodlandbrew.com/2012/12/refrigeration-effects-on-yeast-viability.html
[4] rehydrated yeast provides a more repeatable source than cultures that have previously been stored for an indeterminate amount of time before reaching the consumer.
[5] Viability will be assessed using Methylene Blue staining www.woodlandbrew.com/2012/11/counting-yeast-cells-to-asses-viability.html
[6] Cell density will be measured with an optical cell density meter www.woodlandbrew.com/2014/07/cell-density-meter.html

 

[skunkfunk]

There has been a study done but not really along your exact lines as I believe it was done on this very forum with a vial of liquid yeast. The findings were exactly in line with viability assumptions made on the mr malty calculator. Things like ABV when storing under beer greatly affect the longevity of the yeast, that much is known but I don't think quantified well.

 

[slarkin712]

I would be interested in seeing what some techniques do to improve storage life compared to storage under beer. Maybe store under water with pH adjusted to 4 or so.

 

[WoodlandBrew]

There has been a study done but not really along your exact lines as I believe it was done on this very forum with a vial of liquid yeast. The findings were exactly in line with viability assumptions made on the mr malty calculator. Things like ABV when storing under beer greatly affect the longevity of the yeast, that much is known but I don't think quantified well.

That would be great to see. Do you have a link?

I agee that storing yeast in alcohol will lower viability as a function of time. Here are my results: http://www.woodlandbrew.com/2013/01/abv-effects-on-yeast.html

 

[skunkfunk]

That would be great to see. Do you have a link?

I agee that storing yeast in alcohol will lower viability as a function of time. Here are my results: http://www.woodlandbrew.com/2013/01/abv-effects-on-yeast.html

I do not have a link and my two-page google search didn't turn it up. Sorry. IIRC he went and figured out the viability % with a microscope and some dye periodically, can't remember the exact results or obviously the post.

 

[ColoHox]

What a great idea! I haven't seen any study evaluating long-term storage, and indeed most of the studies involve commercial yeast handling, so this should be really useful.

Based on some of the arguments/debates around here, I can think of a few things:

Comparison of storage conditions, specifically looking at the effect of long-term storage in a frost-free freezer, or something with similar temperature cycling conditions that homebrewers would be likely to cold-store stocks in would be really useful. Or in general, comparison between fridge, freezer, and as cold as you can go (-80C ideally, but that's not really practical).

Of course, there is loads of chatter about the effect of the storage medium, ie. under low-density wort or after water washing. However, that would increase your experimental load pretty significantly.

Also, in addition to the measurements you have described, at the end of the process the inclusion of a simple, standardized flavor profile assessment would be useful. I, of course, appreciate the performance metrics, but most of us are brewing for flavor.

 

[trentm]

Great idea. A lot of homebrewers are now saving their yeast slurries and repitching at a later date, often weeks or months after the initial fermentation and reporting good results. I have seen some of your work on yeast viability in storage and from this standpoint, yeast seem to remain viable for quite some time. Even though, commercial breweries who often have better yeast harvesting capabilities compared to homebrewers, often limit storage to 1 week. Considering the expense of a new pitch at the commercial level, there must be solid reasoning behind this approach. Could it be yeast health? Here is a link to a presentation given by William Maca to the Michigan Brewers Guild:http://www.mbaa.com/districts/michigan/events/documents/2011_01_14yeaststoragefermentation.pdf. Of special interest are slides 7, 26, and 27. I wish I could get my hands on the source of these data. Meca is a Senior Yeast and Fermentation Scientist with MillerCoors LLC. and a faculty member at the Siebel Institute of Technology.

Also of concern on yeast health for homebrewers could be the extended time we use in the primary before yeast harvest.

 

[WoodlandBrew]

Spot on ColoHox!

Perhaps I'll add yeast from an equivalent 2 liter starter as those seem to be popular and that would also suffice as storage in beer. I could also add one with glycerin freezing. That would be a total of six fermentations at each of the storage periods.

Some sensory analysis would be feasible.

 

[WoodlandBrew]

Also of concern on yeast health for homebrewers could be the extended time we use in the primary before yeast harvest.

I've look at that in terms of viability, buy not on vitality or performance of subsequent pitches.

Thanks for the link to the presentation. Those charts are actually from the paper I cited in my initial post.

 

[skunkfunk]

Spot on ColoHox!

Perhaps I'll add yeast from an equivalent 2 liter starter as those seem to be popular and that would also suffice as storage in beer. I could also add one with glycerin freezing. That would be a total of six fermentations at each of the storage periods.

Some sensory analysis would be feasible.

Well, starter beer isn't really beer, is it? Hopped wort will slow yeast growth, maybe it either enhances or diminishes the storage viability?

 

[WoodlandBrew]

Well, starter beer isn't really beer, is it? Hopped wort will slow yeast growth, maybe it either enhances or diminishes the storage viability?

Your right, using the strict definition if it doesn't have hops it's not beer. The intent of using yeast from a starter stored in the "beer" would be to mimic saving part of a starter for a subsequent batch. I would also hope that it may give some incite into storing yeast slurry in a more normal batch of beer.

Starter beer would have a similar alcohol content and pH of normal beer, but would not have the antimicrobial properties from the hops.

 

[WoodlandBrew]

I would be interested in seeing what some techniques do to improve storage life compared to storage under beer. Maybe store under water with pH adjusted to 4 or so.

That's a good idea, although I'd like to limit the number of storage methods to keep the experiment manageable.

Do you have a commonly used method in mind?

 

[Yooper]

I think most homebrewers either store the saved yeast under water (ala yeast rinsing) or in the beer that was made, so it'd vary from nearly 0% ABV to 6-8% ABV.

I think the best practical experiments would use the yeast rinsing method, and use one beer of say, 5-6%, a common ABV for many of us.

I think the storage vessel might matter- I'd suggest mason jars, since that seems most common.

I'm not sure if a deep yeast bed means more breaking down or not- my gut says it would, due to pressure on the underlying yeast, but I don't think it would be a factor. I have jars that are quart size, and full almost to the top sometimes.

I'd also be interested in the growth of microbes during storage, as we don't have sterile conditions at home. I'd love to see under a microscope the same sample of yeast at 4 weeks, 4 months, and 8 months. I'd like to see how many microbes survive and thrive, as well as determine the viability of the stored yeast.

 

[boot_hill_brewery]

I make a start bigger than needed. Pour of the extra into a large mason jar with boiled cooled water to top off. Once that has settle I do the same thing "decant" the larger jar and put the yeast into smaller jars topped off with filtered,boiled and cooled water. All stay in my kegorater around 40.

 

[jahdub]

I make a starter that is about a pint larger than needed, and store that in a pint mason jar. I always make a later starter from that pint, though. I consider the pint to essentially be the same as a store bought vial of yeast. The longest I've gone with a stored pint is about 2 months in the fridge, though. Probably will do a brew with a harvest that is pushing 3-4 months soon. Not really concerned about it, but that isn't really long-term storage, either.

 

[WoodlandBrew]

I think most homebrewers either store the saved yeast under water (ala yeast rinsing) or in the beer that was made, so it'd vary from nearly 0% ABV to 6-8% ABV.

I think the best practical experiments would use the yeast rinsing method, and use one beer of say, 5-6%, a common ABV for many of us.

I think the storage vessel might matter- I'd suggest mason jars, since that seems most common.

I'm not sure if a deep yeast bed means more breaking down or not- my gut says it would, due to pressure on the underlying yeast, but I don't think it would be a factor. I have jars that are quart size, and full almost to the top sometimes.

Currently, the plan is to use eight different storage methods:
Fermentis S-33, stored in water, 5°C (40°F)
Fermentis S-33, stored in 50% glycerin solution, -15°C (5°F)
Fermentis W-34/70, stored in water, 5°C (40°F)
Fermentis W-34/70, stored in 50% glycerin solution, -15°C (5°F)
Danstar Nottingham, stored in water, 5°C (40°F)
Danstar Nottingham, stored in 50% glycerin solution, -15°C (5°F)
Danstar Nottingham, stored in beer, 5°C (40°F)
Danstar Nottingham, stored in 50% glycerin/beer solution, -15°C (5°F)

These will be tested after 10 time intervals of storage. That's 80 containers to store between the freezer and fridge. So I'll be using sealed glass 1 dram vials to emulate storage in mason jars.

I'd also be interested in the growth of microbes during storage, as we don't have sterile conditions at home. I'd love to see under a microscope the same sample of yeast at 4 weeks, 4 months, and 8 months. I'd like to see how many microbes survive and thrive, as well as determine the viability of the stored yeast.

During the viability count I'll make sure to note any microbial presence.

A large amount of data needs to be collected so that outliers can be identified. Each test set will contain 10 growth measurements, two sugar content measurements and one viability measurement. It will be over 1000 measurements in total. You can see why I need to limit the number of storage methods that will be tested to just a few that emulate popular methods. Thank you for help in identifying these methods.

 

[Calder]

I think this is a great idea. Not sure the testing of the frozen samples will provide much new data, since I would expect everyone who freezes yeast will treat it as thou only few survive and go thru several starter steps to revive and build the yeast. Your experiment, but I would think more beer samples, or different waters (like distilled vs RO, etc) would give more information.

There is always a lot of debate on slurry, with people either taking Mr.malty as gospel, or strongly against it (I'm one of the ones that think it is overly conservative on it's viability numbers). It would be nice to know what approximates to reality.

I make a starter that is about a pint larger than needed, and store that in a pint mason jar. The longest I've gone with a stored pint is about 2 months in the fridge, though. Probably will do a brew with a harvest that is pushing 3-4 months soon. Not really concerned about it, but that isn't really long-term storage, either.

I do the same, but for longer-term storage. I use slurry for a series of beers. I do not recommend regularly leaving the samples this long, but per the method you noted above, I have revived yeast stored in the fridge after 26 months. I have one that was left way too long, and I put in a starter last night - it was stored for 33 months - looked OK, but no activity so far after 24 hours. I had another that failed; Duvel yeast after 17 months. The yeast and liquid looked OK, but there was obvious mold growth on the rubber of the Mason Jar seal, so I just tossed it.

 

[WoodlandBrew]

I think this is a great idea. Not sure the testing of the frozen samples will provide much new data, since I would expect everyone who freezes yeast will treat it as thou only few survive and go thru several starter steps to revive and build the yeast. Your experiment, but I would think more beer samples, or different waters (like distilled vs RO, etc) would give more information.

Good point. My motivation for comparing different temperatures may be driven a little too much my just personal curiosity. It would be nice to be able to create some relationship between temperature and vitality. Perhaps one frozen, one refrigerated and one ambient. Further consideration is needed.

There is always a lot of debate on slurry, with people either taking Mr.malty as gospel, or strongly against it (I'm one of the ones that think it is overly conservative on it's viability numbers). It would be nice to know what approximates to reality.

Absolutely. That's another reason for the experiments.

 

[tagz]

The difference between Jamil's and Kai's calc is crazy sometimes. On my last batch, Jamil was telling me I made 125B cells and Kai was telling me 240B. It really end up feeling like a shot in the dark.

Edit: And I'm glad to see Woodland back in action. I look forward to updates.

 

[WoodlandBrew]

The difference between Jamil's and Kai's calc is crazy sometimes. On my last batch, Jamil was telling me I made 125B cells and Kai was telling me 240B. It really end up feeling like a shot in the dark.

Characterizing biomass yield between different strains is another thing I hope to get out of this experiment, although the fermentation will only be aerated at time of inoculation as is typically done with beer fermentation instead of continually aerated as you would get with a stir plate.
Chris White's experiments were done without any aeration and then scaled by some kind of magic number. Kai's were actually done a using a stir plate. He also had other people duplicate his experiments. If you're using a stir plate I would recommend using Kai's equations.

And I'm glad to see Woodland back in action. I look forward to updates.

Thanks, I'm glad I have a little time these days for experiments, but things will probably change again in a few months. The bulk of the work for this experiment is up front, so I'll be able to run this to completion.

 

[Cyclman]

Awesome experiment. I am highly interested in the results related to microbes- at the homebrew level this may be why homebrewers can use "old" yeast a big brewer wouldn't use- but big brewers recycle yeast more times than homebrewers typically do. I think it well might be a cumulative thing.

 

[WoodlandBrew]

Here are some preliminary results:

One strain, US-05, was tested using three storage methods:
1) Frozen, Glycol – 10% glycol added to yeast suspended in water. Temperature of storage is 0°F in an insulated box in a freezer without auto-defrost.
2) Refrigerated, Water – yeast suspension stored in 1 dram vial in the back of a refrigerator at 40°F
3) Refrigerated, Beer – 70% 5% ABV beer and 30% yeast suspension stored in 1 dram vial in the back of a refrigerator at 40°F.

Storage Method     | Storage Time | Lag | Yield | Growth Rate
=============================================================
Not stored           0              0.0   12.5    0.5
Frozen, Glycol       3.06           0.8   12.14   0.7
Refrigerated, Beer   3.06           0.63  11.35   0.5
Refrigerated, Water  3.06           0.65  10.33   0.5
Frozen, Glycol       8.82           0.4   10.86   0.5
Refrigerated, Beer   8.82           0.5   9.85    0.5
Refrigerated, Water  8.82           0.35  12.44   0.5
Frozen, Glycol       16.72          0.5   10.5    0.5
Refrigerated, Beer   16.72          0.5   10.35   0.5
Refrigerated, Water  16.72          0.4   12.82   0.5

All time units are days.
Yield is Billions of Cells per Liter Degree Plato

Of the fermentation performance metrics measured, only lag time was effected by storage. Lag time was zero without storage and about 12 hours after storage. The length of the lag time was independent of the storage method and the storage time. Viability, microscopic morphology, bacteria contamination, yield, and growth rate were unaffected when comparing metrics from zero to 17 days of storage.

I plan to continue collecting data for a year to evaluate the long term effects.

 

[Weezy]

Lovely test! Thanks for tackling it and sharing.

Are you pitching the frozen and refrigerated samples straight from the fridge or freezer or are you warming them first? Are you proofing them?

could you please define your 'yield' for us? Is that viable cell density after lag phase in the wort or something else?

How are you tracking viability and morphology?

I'm looking forward to following this.

 

[flars]

Excellent data. Are you planning the same data collection for yeast stored 3 to 6 months?
I have a fermentation going right now with WY 1056, that had been stored under beer for 6 months. Pitched the yeast without a starter as an experiment. Getting a very good fermentation. The difference I have seen is that lack of an aggressive start tapering off within a few days. This ferment is holding very steady with stable wort temperature.

 

[ColoHox]

These first three timpoints are a great teaser. What are your upcoming timepoints?

Did you expect to see a difference among samples after only 2ish weeks of storage? Doesn't seem that surprising to me. I am excited for those long term timepoints. Are you testing a specific hypothesis or is this a descriptive study?

 

[WoodlandBrew]

Thanks everyone for your interest.

The results from the first couple of weeks are about what I expected based on my experience, but a paper I read on the subject seemed to suggest that fermentation performance would degrade significantly over this time period. ( Murray, C.R., 1984. The Effects of Yeast Storage Conditions on Subsequent Fermentations: MBAA Technical Quarterly, Vol. 21, No. 4)


This is what I have for my Materials and Method section. Hopefully it will answer the other questions:

Materials and Methods
Samples will be removed from storage on nine different days. The storage time will range from 1 week to 1 year.
Yeast: US-05 will be rehydrated per the manufactures directions.
Wort: 9°P solution of Briess Extra Light DME and sterile water. Wort will be autoclaved and refrigerated for the extent of the experiments.
Storage Solutions: Refrigerated storage will use autoclaved tap water or beer containing 5% alcohol (v/v) and 10-15ppm isomerizes alpha acid. Frozen storage will use the same water and beer but be combined with 10% (w/w) glycol.
Storage: Domestic grade refrigerator and freezers will be used for storage. Each of the samples will be in 1 dram vials. Temperature of storage will be 5°C (40°F) and -15°C (5°F) which will be measured with an IR thermometer and recorded. The vials will be contained in an insulated ABS plastic box.
Inoculation: Temperature of stored samples and wort will be equalized prior to inoculation by placing them in a water bath for 30 minutes. 2ml of 12.6°P wort will be added to the 1ml yeast suspension to create an inoculated 8.4°P wort. Initial cell density is 10 Billion cells per Liter. Sample will be shaken for 30 seconds to aerate.
Cell Density: Transmission measured at 624nm will be compared with known samples.
Residual Extract: Refraction index will be measured at the time of inoculation and after fermentation completion with a refractometer. Tables for maltose solution refraction index, ethanol solution refraction index and stoichiometry of anaerobic respiration will be used to calculate sugar content.
Viability: A 0.5% Methylene Blue stain will be used at the time of inoculation to assess viability. A minimum of 100 cells will be counted for each viability assessment.
Morphology and Bacteria Content: Microscopy will be used to assess morphology and bacteria content. Coci cells that are smaller than 1µm, and rod shapes smaller than 5µm will be noted as visible bacteria. Rod shapes and chains longer than 5µm will be noted as stressed yeast. Round cells from 5µm and larger will be considered yeast. Cells that do not match any criteria will be noted as uncategorized. Material that is not round or rod shaped, such as protein, will be ignored.
Lag: Immediately following inoculation, before the yeast becomes sufficiently active, there will be a lag period. During this time the yeast fall out of suspension. Once the yeast become active they produce carbon dioxide and become suspended. Lag time will be defined as the point at which the yeast are entering suspension faster than they are falling out of suspension. This will be evident as a low point in cell density normally occurring within one day of inoculation.
Yield: Biomass yield will be calculated as the change in cell density (Billions per Liter) divided by the wort gravity (°P.) Most scientific publications use grams CDW per gram extract (g/g) although in brewing cell density and °P are more widely used.
Yield=(CDF-CDI)/GI
Yield is the biomass yield. CD is the cell density in billions per Liter. G is the gravity in degrees Plato. Subscripts: F is the final value after 10 days of fermentation. I is the initial value.

 

[tagz]

Love it! Keep up the good work.

 

[Weezy]

So these are test tube sized batches? Should we be concerned with how this translates to home brew and commercial scale batch sizes? We've all heard stories about yeast death being faster and autolysis happening faster when the yeast are under higher fluid pressures of commercial fermentation vessels?

 

[WoodlandBrew]

So these are test tube sized batches? Should we be concerned with how this translates to home brew and commercial scale batch sizes? We've all heard stories about yeast death being faster and autolysis happening faster when the yeast are under higher fluid pressures of commercial fermentation vessels?

Yes, the test size is quite small. It's a 1 dram vial with 3ml of wort. I've also heard the stories about high pressure in the cone of commercial conical fermenters leading to faster degradation of the yeast. I have not found any papers on this subject. I really can't say for certain how size would translate in this application, but my hunch is that for homebrewers using mason jars and even commercial brewers that use kegs for a yeast brink that size wouldn't play a role in fermentation performance after storage.

 

[Weezy]

I agree for the home brewer. I can imagine their being a concern as confining pressure increases and further inhibiting the yeast access to nutrients...but on the mason jar scale? Meh. Agreed.

 

[Tinpanharry]

Woodlandbrew Thank You! I am buying your book tonight to support everything you do and all of the time you put in to help us improve our homebrew.

Cheers to you

TPH

 

[WoodlandBrew]

Thanks! I appreciate it!

 

[Tinpanharry]

Awesome work Woodlandbrew! Legend-(wait for it....) - Dary!

 

[fakeuser]

This thread might be relevant for you:
https://www.homebrewtalk.com/f163/d...tarter-then-why-not-farm-yeast-freeze-269488/

https://www.homebrewtalk.com/freezing-yeast.html

If I can remember correctly, the TLDR is:

7.5% glycerin/yeast slurry performed best.
Cool slow if possible, left for 1-3 days in a fridge then in the freezer in an iso-propol bath to buffer cooling.
Use froze free if possible, if not use gel packs / insulation.
Thaw rapidly in 37C hot water, tip off solution.
Pour in an overnight starter.

I think I read the slow cooling has something to do with a build up of trehalose to protect the yeast, I've only seen in one other place the question asked whether or not it is possible to add it to the solution itself but it hasn't been answered.

(tested for up to 15 months storage).