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Warthaug · 12-17-2012, 08:16 PM

Quote:

Originally Posted by Kaiser

well, this is something I’m looking into and where I already have some data.

Yeast growth on a stir plate is in general limited the nutrients available in that wort and not by the available oxygen. I haven’t done any experiments that would show that, but that’s what I conclude from the results that I have so far.

I've not done the experiments, but I am a biologist whose work frequently deals with lipids & sterols; sometimes even in yeast...

Yeast do indeed need oxygen to produce sterols, which is yet one more reason to use a stirred starter. However, sterols are readily synthesized from simple compounds, and as such are not a limiting factor in aerobic growth. Hence why you see growth in a starter being limited by nutrient availability; to make sterols a yeast's minimum requirement is a carbon source (i.e. sugar) and O2.

Where sterols become important is in the ferment itself. In theory, sterol-rich yeast can undergo more in-fermenter cell divisions. More importantly though, these yeast should be more stable as sterols help to maintain proper membrane fluidity and make membranes more resistant to things like temperature changes and alcohol.

In short, sterol production is more important in ensuring the maximum quality of yeast than quantity. Yeasts with lots of sterols (and unsaturated fatty acids, also produced during aerobic metabolism) have more stable membranes, making them more resistant to temperature changes & alcohol, and making them more resistant to autolysis. Ideal features if you want to make a high gravity beer, or re-use a yeast cake for multiple batches.

Bryan

WoodlandBrew · 12-17-2012, 08:21 PM

Quote:

Originally Posted by Kaiser

WoodlandBrew, that's some mighty low yeast viability. If the 10% was determined with Methylene Blue, the number is pretty much useless since Methylene blue tends to ovestimate cell viability, especially in old cultures.

Thanks Kai,
Yes, absolutly. That was low viability. The reason I was recording the data was to see if the dead cells would sink to the bottom or be suspended by the living cells durring fermentation.

I'm glad you mentioned the discrepancies in cell count with methylene blue. That is not something that I have seen before. I would certainly not want to post false findings. I have done counts with both trypan blue and methylene blue and seen similar numbers. I have also read articles that indicate they both work equally well. White Labs also uses methylene blue as does Wyeast.

But I will have to revisit that, especialy with older slurries.

Kaiser · 12-17-2012, 08:30 PM

Quote:

Originally Posted by WoodlandBrew

Thanks Kai,
Yes, absolutly. That was low viability. The real reason for recording the data was to see if the dead cells would sink to the bottom or be suspended by the living cells durring fermentation.

Interesting experiment. I haven't thought of that.
If you want to be sure about healthy vs dead, you could take a healthy culture, heat 90% of it to 70 C to kill them and then mix with the rest and pitch. That way you know 90% are dead.

Quote:

I'm glad you mentioned the discrepancies in cell count with methylene blue. That is not something that I have seen before. I would certainly not want to post false findings I have done counts with both trypan blue and methylene blue and seen similar numbers. I have also read articles that indicate they both work equally well. White Labs also uses methylene blue. And also by Wyeast.

But I will have to revisit that.

Methylene blue's tendency to overestimate viability is actually well known. But because of its simplicity this stain has become the standard viability test in brewing. Here is some of the info I have on that: http://braukaiser.com/wiki/index.php?title=Microscope_use_in_brewing#Methylen e_blue_staining

Typran blue may have the same issues and may be equally bad as MB.

Kai

WoodlandBrew · 12-18-2012, 12:46 PM

Sorry to get distracted from the original topic of sterols, RockJetty. I commented on your thread because sterol production and use is something that I have been interested in. My understanding from Fix "Principals of Brewing Science" is that there are two metabolic pathways that produce sterols. To summaries my simple understanding: the primary pathway uses oxygen, but there is a secondary pathway that does not use oxygen but uses more energy.

Warthaug mentioned several reasons for the importance of sterols, and that matches what I have read:

Quote:

Originally Posted by Warthaug

Where sterols become important is in the ferment itself. In theory, sterol-rich yeast can undergo more in-fermenter cell divisions. More importantly though, these yeast should be more stable as sterols help to maintain proper membrane fluidity and make membranes more resistant to things like temperature changes and alcohol.

In short, sterol production is more important in ensuring the maximum quality of yeast than quantity. Yeasts with lots of sterols (and unsaturated fatty acids, also produced during aerobic metabolism) have more stable membranes, making them more resistant to temperature changes & alcohol, and making them more resistant to autolysis. Ideal features if you want to make a high gravity beer, or re-use a yeast cake for multiple batches.

Bryan

My real curiosity is how this applies to saved slurries, and what the best way is to revive them. Would it be best to use a high pitch rate and a lower gravity with plenty of oxygen to boost sterol content while minimizing the number of new generations of cells to prevent mutation? Or perhaps mutation and cell selection is not a concern?

Kaiser · 12-18-2012, 01:44 PM

Quote:

Originally Posted by WoodlandBrew

To summaries my simple understanding: the primary pathway uses oxygen, but there is a secondary pathway that does not use oxygen but uses more energy.

I haven't read Fix in a while, but it is my understanding that sterol production takes oxygen or unsaturated fatty acids (UFA). There is no way to make sterols by simply spending more energy.

Quote:

My real curiosity is how this applies to saved slurries, and what the best way is to revive them. Would it be best to use a high pitch rate and a lower gravity with plenty of oxygen to boost sterol content while minimizing the number of new generations of cells to prevent mutation?

I think the best way to revive an old slurry is to take a small amount of it and grew a new population of cells from it.

Kai

Warthaug · 12-18-2012, 08:37 PM

Quote:

Originally Posted by WoodlandBrew

My real curiosity is how this applies to saved slurries, and what the best way is to revive them. Would it be best to use a high pitch rate and a lower gravity with plenty of oxygen to boost sterol content while minimizing the number of new generations of cells to prevent mutation? Or perhaps mutation and cell selection is not a concern?

Kaiser already hit the nail on the head - the best thing is to grab some yeast from the slurry, re-grow it aerobically, and use that. The yeast in a yeast cake will be somewhat sterol/unstaurated lipid-depleted, is typically glycogen depleted, is stressed, and thus is less than ideal for brewing. That said, many brewers will re-pitch the same yeast cake 4 or 5 times without an aerobic growth phase (aside from oxygenating the wort pre-pitch), and seem to not suffer overly from it.

In terms of mutation, no matter what you use for a yeast source there will be mutations (in lab strains, you get 1 mutation for every 3 or so cell divisions; presumable brewing strains will be similar). Re-pitching (vs going back to a low-generation stock) will lead to more mutations, and thus be more likely to lead to changes in the yeast characteristics. That said, the effect over a small number of re-pitchings (3-4) should be minimal. And more than that means you now have a house strain!

Bryan

WoodlandBrew · 12-18-2012, 10:56 PM

Thank you both!