Author Topic: Yeast Starter calculation in BS3  (Read 357 times)

Offline thebeershack

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Yeast Starter calculation in BS3
« on: May 16, 2022, 02:34:21 PM »
Where does the %Viability data come from? And how can I set my desired pitch rate? I'm seeing big differences in viability across a variety of calculators. For a recently manufactured PP of WLP001 I'm seeing values ranging from 71% to 96% (BS3). Not knowing the pitch rate used in BS3 really leaves me wondering how accurate it is.

Offline GigaFemto

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Re: Yeast Starter calculation in BS3
« Reply #1 on: May 16, 2022, 05:54:56 PM »
Options->Yeast Starter has pitch rates, viability and aging settings. You need to enter the yeast packaging date and the brew date for the viability calculation. BeerSmith3 assumes the yeast starter is made on the brew date.

Offline Oginme

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Re: Yeast Starter calculation in BS3
« Reply #2 on: May 16, 2022, 06:29:38 PM »
Where does the %Viability data come from? And how can I set my desired pitch rate? I'm seeing big differences in viability across a variety of calculators. For a recently manufactured PP of WLP001 I'm seeing values ranging from 71% to 96% (BS3). Not knowing the pitch rate used in BS3 really leaves me wondering how accurate it is.

The accuracy of the model is highly dependent upon the age of the yeast package, the conditions under which it was stored, actual viable yeast at packaging, yeast strain, etc.  It is never perfect, but designed to get you in the ballpark.  There is a pretty wide range of cell counts which the yeast can and will perform consistently and that is highly strain dependent as well. 

Then you get into the models which have mostly been developed decades ago and do not take into account the improvements which have been made in yeast health, packaging integrity, and overall vitality of the yeast over the past number of years. The rate of decrease in viable cells over the age of the yeast package tends to be extremely conservative in the estimate of % viability, so the actual percentage of viable cells will tend to be greater than the models predict.

This is one reason that some of the yeast companies have switched from printing the manufacture date to printing the best-by date on the packages.  I use yeast packages as a means of calibration on my cell counting technique and personally, I have never had a Wyeast package check in at less than 100 billion active cells even at 4 to 6 months old.  Most clock in at a consistent count of 108 billion to 112 billion cells total (counting both dead and live cells).  This correlates fairly well with the information I was able to squeeze out of the Wyeast people at HBC back in 2019.

I cannot comment too much on White Labs other than to say the two tubes I tested back a number of years ago were just a bit over the 100 billion total cells. Their new packaging probably negates anything tested in the prior packaging in terms of viability and total cell counts.
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Offline thebeershack

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Re: Yeast Starter calculation in BS3
« Reply #3 on: May 17, 2022, 08:34:09 AM »
Options->Yeast Starter has pitch rates, viability and aging settings. You need to enter the yeast packaging date and the brew date for the viability calculation. BeerSmith3 assumes the yeast starter is made on the brew date.

AHA, thanks for this! I knew the rest.  I usually make the starter 2-3 days ahead of brew day. I can correct for those days by just adding those days to the yeast packaging date.
As an interesting aside, White Labs current PurePitch packaging does NOT contain 100B cells. Rather, closer to 0.86B cells. This was confirmed by them during a chat I had with them yesterday. So be sure to correct the # cells/pack before the calculation.

Offline thebeershack

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Re: Yeast Starter calculation in BS3
« Reply #4 on: May 17, 2022, 09:16:07 AM »
Where does the %Viability data come from? And how can I set my desired pitch rate? I'm seeing big differences in viability across a variety of calculators. For a recently manufactured PP of WLP001 I'm seeing values ranging from 71% to 96% (BS3). Not knowing the pitch rate used in BS3 really leaves me wondering how accurate it is.

The accuracy of the model is highly dependent upon the age of the yeast package, the conditions under which it was stored, actual viable yeast at packaging, yeast strain, etc.  It is never perfect, but designed to get you in the ballpark.  There is a pretty wide range of cell counts which the yeast can and will perform consistently and that is highly strain dependent as well. 

Then you get into the models which have mostly been developed decades ago and do not take into account the improvements which have been made in yeast health, packaging integrity, and overall vitality of the yeast over the past number of years. The rate of decrease in viable cells over the age of the yeast package tends to be extremely conservative in the estimate of % viability, so the actual percentage of viable cells will tend to be greater than the models predict.

This is one reason that some of the yeast companies have switched from printing the manufacture date to printing the best-by date on the packages.  I use yeast packages as a means of calibration on my cell counting technique and personally, I have never had a Wyeast package check in at less than 100 billion active cells even at 4 to 6 months old.  Most clock in at a consistent count of 108 billion to 112 billion cells total (counting both dead and live cells).  This correlates fairly well with the information I was able to squeeze out of the Wyeast people at HBC back in 2019.

I cannot comment too much on White Labs other than to say the two tubes I tested back a number of years ago were just a bit over the 100 billion total cells. Their new packaging probably negates anything tested in the prior packaging in terms of viability and total cell counts.

Thanks for all this. I'm aware of the impact of packaging, among other variables, impact on viability. What I didn't know is if someone has independently assessed WL's PP packaging viability and that accounts for the higher viability rates in BS3 compared to say, Brewer's Friend's calculator (which is using data from 2012, predating PP).
All this stems from a conversation I had with WL yesterday around their cell counts and package labelling. Their PP claims "Made to contain over 100 billion cells." Yet checking the QA report for the vial I have on Yeastman.com, instead of an actual cell count, it says it's made to contain 2.15M cells/mL. So a 40 mL PP has only 86B cells, not the 100B claim. Of course one must account for this discrepancy in the starter calculation. Further, this will be their standard packaging density in the future, but they're also going to make the packages slightly larger to account for this. Or so they say. I wonder how/if this will impact viability. I hope to see Chris White at HBC and have this conversation with him. Cheers!

Offline Kevin58

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Re: Yeast Starter calculation in BS3
« Reply #5 on: May 17, 2022, 01:41:55 PM »
After reading posts by Saccharomyces aka Yeast Whisper aka S. Cerevisiae on various homebrew forums I have stopped worrying about cell count. Saccharomyces introduced a starter method called Shaken Not Stirred which relies more on a smaller number of yeast cells at their peak of health rather than a large number many of which are old and tired by the time they are pitched. Here is one that may be interesting...


YEAST CELL COUNT AND WHY STARTERS MAY NOT BE NECESSARY
March 22nd, 2021
Copied and pasted from the AHA forum
Posted by user: Saccharomyces

I have been stating that close is more than good enough when it comes to cell counts for years.  I even wrote a blog entry about the amateur brewing community?s preoccupation with cell counts entitled ?Yeast Cultures are Like Nuclear Weapons? back in 2015 (somehow the publication date got changed when I performed a few edits on the text recently).  People have been so focused on cell counts since Kai Troester published his experiments with different stir plate protocols that the forest has been lost for trees.

Let?s examine the first misunderstanding when it comes to yeast cell counts.  The difference between 200 billion cells and 300 billion cells is insignificant.  The difference between 200 billion cells and 400 billion cells is also insignificant in the grand scheme of things. Lag times and dissolved O2 demands are not significantly reduced until the one pitches in excess of 800 billion cells per 5 gallons of wort.  While I may be wrong, I believe that the confusion stems from brewers believing that the yeast biomass grows linearly, that is, the number of cells at replication period N is N times the original cell count.  The reality is that the cell count is closer to 2^N times the original cell count, where N is the number of replication periods that have elapsed and the symbol ?^? denotes raised to the power of.  That difference means that after replication period 1, the yeast biomass has doubled in size.  After replication period 2, the yeast biomass has quadrupled in size.  After replication period number 4, the yeast cell count is now 16 times the original cell count.  In essence, yeast biomass growth is binary exponential because every cell that is alive during a replication period buds a daughter cell, which results in the cell count doubling during every replication period.

With that said, there are four basic limiting factors when it comes to biomass growth; namely, a yeast culture?s genetically-defined O2 demand, dissolved O2 level, amount of carbon in the medium, and the volume of the medium.  The main area where brewers encounter problems with yeast growth is not the amount carbon in the medium or the volume of the medium.  It is the amount of dissolved O2 needed for the yeast cells to consume the carbon source and turn it into enough cell growth to reach maximum cell density.  At a very simple level, the amount of yeast that needs to be pitched is inversely proportional to the amount of foam on the top of the wort when the yeast culture is pitched because aerating wort results in foam building up on its surface.  For example, the shake/rock wort in an almost full carboy method results in inadequate aeration for all but low O2 demand yeast strains.   The paint stirrer method is marginally better.  However, like stirring a culture in an Erlenmeyer flask, O2 pickup is limited by the amount of surface area a brewer can create.  The two most effective ways I have personally found to aerate wort is via a venturi in the drain tubing from one?s kettle to one?s fermentation vessel or direct O2 injection via an O2 bottle and diffusion stone.  Of the two, direct O2 injection is the gold standard, which why professional breweries tend to use it.  Using an air pump, inline filter, and a diffusion stone works too, but it is not much more effective than a well-designed venturi while adding significantly more complexity to the equation.

When a yeast culture is pitched into a medium that is above the Crabtree threshold of 0.2% weight-by-volume (w/v), it will chose fermentation over respiration.  A 0.2% w/v solution has a specific gravity of 1.0008. Unlike humans, yeast cells have two metabolic pathways. One pathway processes carbon sources (sugar is carbon bound to water; hence, the term carbohydrate) aerobically (we will get to this pathway later when we discuss the Crabtree threshold in greater detail).  We can refer to this pathway as the respirative metabolic pathway.  It only plays a minor role in fermentation, which occurs via the anaerobic (fermentative) metabolic pathway.  Yeast cells always consume carbon via the fermentative metabolic pathway in brewing, even in the presence of O2.  However, there is a little twist during the lag phase. Yeast cells shunt a small amount of carbon along with O2 to the respirative metabolic pathway for the creation of ergosterol (the plant equivalent of cholesterol) and unsaturated fatty acids (UFA).  These compounds are necessary to keep yeast cell plasma membranes pliable.  The ergosterol and UFA reserves that are built up during the lag phase are shared with every yeast cell that is budded during the exponential growth phase.  That is an important concept to understand.  It is the reason why we want to pitch a starter at high krausen instead of allowing it to ferment out.  We want to do so because all cell production after high krausen is reached is for replacement only and causes these reserves to be further depleted.  There are also morphological changes that occur at the end of fermentation before a culture settles out that have to be undone when we pitch a culture.  The most significant of is thickening of the cell wall.

Getting back to the Crabtree threshold, dry yeast producers take advantage of the Crabtree effect to milk more cell growth out of the same amount of carbon.  They do so by holding the medium in a steady state below the Crabtree threshold in a chemostat, which is a bioreactor.  Holding the medium at a steady state below the Crabtree threshold prevents yeast cells from switching over from respiration to fermentation as well as undergoing flocculation, which is caused by the exhaustion of mannose, glucose, maltose, sucrose, and higher level saccharides that a yeast cell can reduce to one of these sugars.  New medium and O2 are continuously fed into the process in order to achieve a steady state.  Dry yeast propagation is significantly more complex than liquid yeast propagation.  Liquid yeast is propagated much like brewers propagate yeast when making a starter.  It is just on a much larger scale.

Why do dry yeast manufacturers propagate below the Crabtree threshold even though it is a significantly more hi-tech process? Well, it is because they are taking advantage of the fact that respirative metabolic pathway generates nine times more energy than the fermentative metabolic pathway using the same amount of carbon.  Respiration pretty much results in energy, water, and carbon dioxide gas.  Alcohol, esters, and the VDKs found in beer are the result of the fact that the fermentative metabolic pathway is lossy.  They are basically the result of the yeast cell equivalent of incomplete combustion.

Now, this information brings us around to why dry yeast requires little to no aeration on the first pitch.  It is due to the fact the carbon source is entirely consumed via the respirative metabolic pathway and that is where ergosterol and UFAs are produced.  Unlike fermentation, the generation of these compounds is a not a build in an early phase, consume in later phases situation.  Yeast cells are continuously building/replenishing ergosterol and UFA reserves when they are reproducing via their respirative metabolic pathway below the Crabtree threshold.   The dying process has nothing to do with not having to aerate wort with dry yeast on the first pitch.  It has everything to do with how the yeast biomass is propagated.

Finally, why do we make starters with modern liquid yeast cultures? The amount of cell growth that occurs in a 1L or even a 2L starter is insignificant. The number of viable yeast cells in a modern liquid yeast culture is significantly higher than a first generation Wyeast smack pack.  I would go as far as to state that the average modern liquid yeast culture can be pitched directly into 5 gallons of wort without a starter.  Pitching a first generation Wyeast smack pack without making a starter was a ?pitch and pray? event with lag times measured in days. A 1L starter with a modern liquid yeast culture does two things; namely, it brings the cells out of quiescence and gives them time to reverse the morphological changes they underwent in preparation for quiescence.  The second thing making a starter does is afford yeast cells the opportunity to replenish ergosterol and UFA reserves before going work on a batch of wort.  That is why it is critical to pitch a starter at or as close to high krausen as possible.  Allowing a starter to ferment out, so that the supernatant can be decanted basically a) wastes ergosterol and UFA reserves for replacement cell production during the stationary phase and b) puts the cells back in the same quiescent state they were when the culture was received from the yeast propagator.  Sure, the cell count has been increased slightly, but that is insignificant.  It is definitely not the reason why me make a starter today.










Part 2
March 25, 2021

Yes, I know that pitching a starter at high krausen is nothing new.  I have been doing it since I started brewing the better part of three decades ago.  Back then, pitching at high krausen was common knowledge.  Somehow, the practice has gotten lost in the noise of perfecting the stir plate.  The common practice today is to spin a starter for a couple of days before cold crashing it and decanting the supernatant.  That approach never made sense to me. 

In reallity, what is happening today in commercial liquid yeast is that the average packaged cell count is reaching upward toward 200B cells.  Imperial already ships cultures that contain 200B cells.   In essence, pitching an Imperial yeast package into a batch of wort is like pitching 167ml of thick cropped slurry that contains 40% viable cells.  Making a starter today is not like making a starter 20 years ago.  White Labs? selling point when they entered the market in 1995 was that their preform packaged cultures contained 30B cells, which was significantly more than a Wyeast smack pack from that era.  White Labs considered these cultures to be direct pitch.  However, even with a relatively fresh White Labs preform, lag times could be long.  The longer the lag time, the greater the opportunity for bacteria to gain a foothold in the fermentation because bacteria biomass grows by a factor of 8 in the period of time it takes yeast biomass to double.  A starter back then was as much about increasing the culture cell count as it was about waking it up from quiescence and getting it prepared to go to work.  Today, a starter is about waking a culture up from quiescence and getting it prepared to go to work.

Now, the premise that a package of dry yeast pitched into 500ml of starter wort erases the yeast culture?s ergosterol and UFA reserves is not quite right.   An 11g package of dry yeast contains around 55B cells.  The maximum cell density for 500ml is 100B cells, making the difference in O2 demand when the rehydrated dry yeast culture is pitched almost insignificant because there will not be much in the way of replication in 500ml of rehydration wort.  That is a completely different thing than repitching cropped slurry from a batch pitched with dry yeast.  The yeast cells in a crop have gone through a mininum of 4 to 5 replications and that?s if there was no replacement cell growth during the stationary phase.  Aerating wort that is pitched using this dry yeast rehydration method is a good safeguard, but it is not an absolute necessity when we are talking about 500ml of rehydration wort.

Finally, why is the generally accepted rule of thumb for pitching 0.75M cells per milliliter per degree Plato?  After all, maximum cell density is based on volume, not wort density; therefore, why are we pitching more cells into denser wort?  The answer is that the saturation point for dissolved O2 goes down as gravity goes up.  Lower dissolved O2 equates to reduced ergosterol and UFA production.  Higher gravity beer has higher osmotic pressure and ethanol levels, both of which are hard on cell walls and plasma membranes
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Offline Oginme

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Re: Yeast Starter calculation in BS3
« Reply #6 on: May 18, 2022, 03:26:18 AM »

Thanks for all this. I'm aware of the impact of packaging, among other variables, impact on viability. What I didn't know is if someone has independently assessed WL's PP packaging viability and that accounts for the higher viability rates in BS3 compared to say, Brewer's Friend's calculator (which is using data from 2012, predating PP).
All this stems from a conversation I had with WL yesterday around their cell counts and package labelling. Their PP claims "Made to contain over 100 billion cells." Yet checking the QA report for the vial I have on Yeastman.com, instead of an actual cell count, it says it's made to contain 2.15M cells/mL. So a 40 mL PP has only 86B cells, not the 100B claim. Of course one must account for this discrepancy in the starter calculation. Further, this will be their standard packaging density in the future, but they're also going to make the packages slightly larger to account for this. Or so they say. I wonder how/if this will impact viability. I hope to see Chris White at HBC and have this conversation with him. Cheers!

As Kevin noted in the post from Saccharomyces on the AHA forum, there has been a shift in the recent years away from cell counts.  I have seen several reports supporting this, mostly from the yeast companies.  While I do respect Saccharomyces experience and experience, most of the small breweries in my area are still doing cell counts. The main reason for this is to (a) determine the health of the slurry when repitching and (b) establish consistency in pitch rates from batch to batch.  Note that none of the people I know do cell counts or viability testing on new pitches received from their various yeast suppliers and pitch that straight as received. 

The difference in cell volume from one strain to another and, therefore, the reliance on volume and viability of cells makes a lot of sense on the surface, but it would be interesting to do a side-by-side comparison of repeated batches made using volume and correlated to actual viable cell counts.  My guess is that given the ability of most humans to discern small changes (read that with much sarcasm) in beer tastings, the difference between the two methods is pretty much a wash.  This makes the pitch rate of a yeast slurry containing 2.15 cells/ml versus one at 3.25 cells/ml well inside detectable limits.  Fermentation temperature and control, available FAN in the wort, and degree of oxygenation most likely have a greater effect on our taste versus plus or minus a few billion cells in viable cells pitched.

BTW, the willingness of WL to claim the contents of the packaging at 100 Billion cells while delivering (last I checked -- they may have since changed their claim) a known variability which statistically demonstrates this is not true of a significant percentage of their sold packages is one reason I have stayed mostly with Wyeast and other manufacturers.  That said, WL was on the forefront of presenting actual test results and traceability of lot data to the average user in addition to the commercial brewers. 
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