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	FORUM ON BEER, HOMEBREWING, AND RELATED ISSUES
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Contents:
  Response-FOY, 05-Yeast Starter ("Rob Moline")
  FOY Panel Expands ("Rob Moline")
  Response-FOY, 05-Viability, fructose metabolism and acid production-Part 1 ("Rob Moline")
  Response-FOY, 05-Viability, fructose metabolism and acid production-Part 2 ("Rob Moline")
  FOY-2005: Mead/Wine - Oskaar ("oskaar@cyberclan.net")
  RE: That other yeast company ("Brian Lundeen")
  Mel Allen or Mel Gibson? (Jeff Renner)
  Ballantine XXX (pulsarxp)
  Santizing Aluminum Block from JockeyBox ("Steven J. Owens")


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----------------------------------------------------------------------


Date: Fri, 22 Jul 2005 22:24:33 -0500
From: "Rob Moline" <jethrogump at mchsi.com>
Subject: Response-FOY, 05-Yeast Starter

		Response-FOY, 05-Yeast Starter
Thanks again to those responsible for this valuable contribution to
"craftbrewing" knowledge.
My question might have obvious answers for some readers but here goes
anyway.
When making a starter for an ale with liquid yeast the instructions are
typically to keep the starter at 20-24degC. I realise that this helps build
up yeast numbers quickly, which is usually part of the goal.
My question is :- does doing an ale yeast starter at say 14 deg C have any
detrimental effects on the health or viability of the yeast produced? Or to
put it another way are there downsides to doing low temperature starters
other than the obvious lengthened time taken to build up a large population.
Thanks
Grant  Stott
[9906, 260] AR (statute miles) or [15942.2, 260] AR [Km] (Australia)

Tobias: No there is no harm to your yeast if you grow it at colder
temperature. In fact some lager beer producers propagate their yeast as low
as 10C. Just make sure that you aerate your yeast well.
Tobias
- --

"The More I Know About Beer, The More I Realize I Need To Know More About
Beer!"
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Date: Fri, 22 Jul 2005 23:27:40 -0500
From: "Rob Moline" <jethrogump at mchsi.com>
Subject: FOY Panel Expands

			FOY Panel Expands

     We are truly fortunate to have another heavy hitter join the FOY this
year. While the presence and legacies of Dr's Cone, Fischborn, Waldrop, and
Lemcke are widely known and respected...we are aided in FOY, 05 by the
addition of Dr. Chris Powell.
     Chris Powell obtained a BSc in Biology and Environmental Biology in
1996. Subsequently, he occupied a variety of research positions
investigating aspects of heavy metal toxicity in fission yeast and
oxidative stress in brewing yeast strains. In 1997 Chris moved to Bass
Brewers (now Coors Brewers) to work as part of the Research and
Development team. Chris began his Ph.D. later in the same year at Oxford
Brookes University, in conjunction with Bass Brewers, investigating the
impact of yeast cellular ageing on fermentation performance. Having
obtained his Doctorate in 2001, Chris became involved in a project
funded by the European Commission, exploring mechanisms for the rapid
detection of microbial contaminants within breweries.
     Simultaneously Chris maintained interest in the study of yeast,
supervising projects on wild yeast strains and also flocculation in
brewing yeast. In 2004 Chris joined Lallemand as a consultant in the
Genetic Identification Laboratory. At Lallemand his current role is to
develop molecular techniques for the characterization of a variety of
micro-organisms utilized within the food and beverage industry. Chris is
a member of the ASBC and Chairman of the subcommittee for the
'Determination of Yeast Viability by Fluorescent Staining'. Chris is
also a regular reviewer for several academic journals.
Gump

"The More I Know About Beer, The More I Realize I Need To Know More About
Beer!"
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Date: Sat, 23 Jul 2005 00:05:52 -0500
From: "Rob Moline" <jethrogump at mchsi.com>
Subject: Response-FOY, 05-Viability, fructose metabolism and acid production-Part 1

	Response-FOY, 05-Viability, fructose metabolism and acid production-Part 1


*** Rob here...this response included html and charts....conversion to plain
text  has excluded those features.***

Fredrik,
1) Yeast viability drop during storage and measurement methods
  To make a long story short, last year I did some experiments
and viability drop tests with the methylene staining procedure and I
posted the results to this list for comments some time ago, and come
up with to my limited understanding some conclusions but I really
would like your more authorative opinion on this. The main reason for
my confusion was that the amazingly high apparent survival rates I found
in wyeast smack packs! I did a viability drop experiment with collected
primary yeast and found a quick viability drop. I definitely expected
the smack pack to last longer, but the difference surprised me.

My preliminary conclusions was that

  - Viability as per methylene blue staining seems to generally give
slightly higher numbers as compare to platings, even accounting for
the fact that one colony/count may be generated by a small flocc of
cells.
  - The viability was higher than I though in the 19 month old pack.
Though possibly 86% was and overestimate. The question is exactly how
much?
As it seems, when the health drops, some cells that are technically
alive, or at least enough "alive" to reduce the dye, are not healthy
enogh to complete a budding cycle - thus the deviation between method
are expected to increase with poor health and low viability. In
principle this made sense, but the question is

Q1. How big deviation in numbers can you expect? typically?

Forbes: Methylene blue relies on the yeast being able to exclude the dye
then if the dye does enter the cell the ability to decolourise the Methylene
blue by using it as an electron acceptor (like O2 in respiration).  However,
there are many peculiarities with Methylene blue staining that still escape
me.  It is considered unreliable when the viability is less than about 85%.
Counting under the microscope often seems to give higher numbers than you
see with plating and this goes beyond flocculation/clumping of the cells.
For flocculation to be the reason for the discrepancy between sight and
plate then every cell has to clump with other cells.  This is unlikely even
in the worst cases.

Chris: Methylene blue is widely believed to overestimate cell viability.
However, this is a simplistic view of the matter and the subject is far from
being this straightforward.  In reality, the accuracy of viability
measurements by staining with methylene blue decreases as the number of
viable cells drops below 85-90% (sometimes higher according to the strain).
Unfortunately there is no accurate way of knowing the standard error of this
deviation.  The error is unlikely to be large (e.g. more than 20%), but
enough to reduce confidence in results.  For an example, see the Figure
below which indicates the performance of a  variety of methods.  The main
thing to note is the size of the error bars for methylene blue.  It should
be noted that these experiments were performed under laboratory conditions
and factors such as the vitality of cells, ethanol, wort composition, pH and
beer were not taken into consideration, which may act to skew the data (and
increase error) further.

It is also worth commenting that you are correct in your supposition that
the vitality of cells also influences the ability of yeast to reduce the
stain to its colorless form.  However, it should be noted that the principle
of methylene blue staining is still a subject for debate.  It has been
proposed that the cell membrane may also play a lesser role by acting to
prevent the stain from entering live cells, particularly under alkaline
conditions (in alkaline conditions the proton differential becomes
impaired).

Out of interest, many people observe a variety of shades of colors when
using methylene blue.  This is because the commercially available methylene
blue also contains impurities in the form of azure B (light blue color),
Bernthsen methylene violet and several other lower azures (which arise from
the oxidative demethylation of methylene blue).  The presence of these
compounds becomes more apparent when studying a population of lower vitality
and this can result in differences in staining intensity, leading in turn to
increased subjectivity.
***CHART HERE***
The first test I did was to stain a 19 month old koelsh pack. I
opened the bag, without smacking the nutritient bag, and the
viability was ~86%.

Q2. Is this a normal survival rate if cells are good and O2 kept
away, or is this to be considered exceptional? OR is something wrong
with the staining method? The pack was stored refridgerated the
whole time, around 5C or so.

Forbes: I would think that 86% viability in anything at 19 months old would
be a pretty good figure.  You get what you get with methylene blue. The
drawback may be that you have 86% viability but most of those cells are
hanging on,  viability and vitality (activity) are two different things,
they are related in some ways, hurt the viability and you damage the
vitality, however poor vitality need not mean poor viability.

Once the pack was opened I stored the remaining slurry in the fridge,
however it was unavoidable to expose it to oxygen. Once the pack was
opened, and I assume, exposure to oxygen. The viability drop rate
quickly adopted that of the previous fridge slurry test I did.
(http://hem.bredband.net/frerad/beer/modelling/gallery/graph_82days.jpg)
After another 45 days, the 19 month pack originally 86%,
dropped to 50% *as per the methylene vlue staining method*. But after
this 45 days I correlated with a plating and found the colony
count / #plated cells to be 15%. Then I found by microscope inspection
that the average floc size was around 1.8 cells.

So correcting for this, the 15% would actually be 27% as compare to
the 50% from the staining procedure.

All of the numbers are averaged, so I am a little doutful that all
of the missing 23% are due to measurement errors. And the flocc
size is already corrected for.

Forbes: I would not be so sure the 23% is not measurement error.  In
experience it is quite easy to have this error consistently on a yeast when
comparing plate counts with microscopic observation.

END OF PART 1
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Date: Sat, 23 Jul 2005 00:19:35 -0500
From: "Rob Moline" <jethrogump at mchsi.com>
Subject: Response-FOY, 05-Viability, fructose metabolism and acid production-Part 2

	Response-FOY, 05-Viability, fructose metabolism and acid production-Part 2

Q3. The question is now, if this deviation could be expected due
to differences in the methods? Methylene blue staining vs plating?
Can you please comment?

Forbes: Counting under the microscope often leads to overestimation of cell
numbers as compared with plating.  Why is less easy to understand.

Chris: From personal experience I never saw an overestimation of (total)
cell numbers using microscope counts, so I presume this is just a viable
cell issue.  It is possible that plate count errors can arise as a
consequence of poor/inaccurate dilutions.  However, I believe that the
difference you are seeing is probably due to methylene blue (see my answer
to Q1).

(4) I'm trying to simply the some regulatory control of
the main parts of metabolism and in standard bio text you read about
some of the main regulatory step, but I think I also read a claim that
the main regulating step for glycolysis in yeast is the uptake of sugars,
or transport of sugars through the cell membrane? Is this so, in the
sense that the regulatory role of the hexokinases are negletcable? For
example, I've read general, non-yeast specific biotexts says that the
typical hexokinase enzymes for phosphorylation of hexoses have about
20 times higher affinity for gluseo than for fructose.

Q4. Now my questions in this example is if yeast would ferment fructose
significantly slower than glucose? If not, is this because the
phosphorylation of fructose or glucose is not main limitors, and
that the sugar transport through the cell wall is limiting?

Forbes: This will depend on the yeast strain.  In general the yeast
transporters for glucose and fructose have different sensitivity to the two
sugars and as such glucose my be taken up faster than fructose.  I am not
certain of the hexokinase kinetics but glucose has the advantage of a
glucokinase which is more specific for glucose, whereas hexokinase is less
loyal to glucose.

I had planned to make a comparasion with a 100% glucose batch,
and 100% fructose batch, do you think there would be any
differences in the fermentation profile or acid production?

Forbes: Would be interesting to see your results....on average I would say
that on 100% glucose or 100% fructose you would see little difference.  The
differences may become more apparent when you mix these two sugars together.
Then you would see the preference for either sugar at the level of the
transporter.  To enter glycolysis everything has to pass through some form
of transport mechanism.  Glucose has an advantage in that it can encourage
the yeast to produce everything that it requires to cope with it while
excluding the use of other sugars.  It can vary from yeast to yeast but
fructose can use the same systems as glucose so will compete with glucose at
the level of the transporter, if I recall correctly glucose normally wins
this battle, and in wine fermentations where both these sugars are found,
along with their parental disaccharide sucrose, fructose normally is always
left behind in the fermentation.  For some yeast strains this slower
utilisation can lead to problems in the fermentation and only addition of
glucose allows the residual fructose to be consumed.

Q5) Can you elaborate on factors that affect the production of
organic acids during fermentation? I have made a few sugar brew
experiments some time ago and found a clear correlation between
final pH and amount of sugar used. I am assuming that this may have
more than one reasons. Lacking amino acids might be one? but would
ammonium nitrogen prevent excess acid production? Or does the simple
and "quick" sugars cause a higher flow into the glyolysis pipelines
than the yeast can get rid of? and that this overflow in glycolysis
and perhaps acyl-CoA pools cause elevated acids beeing releasedi into
the beer?

 >From the pH/sugar data I had the impression that there was a treshold
of simple sugars where pH started to drop. How does the cell respond
when pH drops? Do it increase secrection of acids into the cytosol
to maintain a pH gradient? Could this be a factor, accelerating
acid production in the case of pH drop due to poor buffering capacity
of the growth media?

Forbes: pH drops as a response to transporting some of the sugar and many of
the other nutrients.  Reduction of pH outside of the cells becomes one of
the many stresses that the yeast has to endure in poorly buffered
fermentation medium.  The yeast has to maintain a higher pH internally and
thus as transport of nutrients causes a reduction in extracellular pH so the
cell have to expend more energy to keep internal pH high.  Saccharomyces
produces some organic acids, acetate is the biggest one but others can come
from TCA cycle intermediates that accumulate due to the lack of TCA cycle
activity itself.  Nutrition of the yeast can be addressed to alleviate some
of the acetate issues.

Any other factors relevant to acid production?



"The More I Know About Beer, The More I Realize I Need To Know More About
Beer!"
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Date: Sat, 23 Jul 2005 00:00:37 -0700
From: "oskaar at cyberclan.net" <oskaar@cyberclan.net>
Subject: FOY-2005: Mead/Wine - Oskaar

Questions for the panel:

1. Relating to mead/wine:

I see that wine makers are adding their yeast inoculum through the manway
in their fermenters to give the yeast a head start on any already present
microbes/flora. Question: Does the introduction of the yeast into the
fermenter as the juice is filling the tank add value to other roles as
well such as aeration, and does it accelerate the growth dynamics of the
yeast? Would this be beneficial for smaller scale production in the ten to
twenty gallon range for us home mead/wine makers?

2. Relating to mead/wine:

I?ve been reading that yeasts like nitrogen from different sources during
different phases in the early stages of fermentation. I?ve seen
recommendations for using DAP at the end of the lag phase and Fermaid-K at
the 1/3 sugar depletion phase. Does this play a bigger role than
supplementing YANC and FAN already present in the must to ensure optimal
fermentation dynamics? I generally work in ten to twenty gallon batches,
what is the proper dosing of DAP to use when preparing the must for
introduction of the yeast, and at the end of the lag phase?

Thanks,

Oskaar





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Date: Sat, 23 Jul 2005 02:17:28 -0500
From: "Brian Lundeen" <blundeen at mts.net>
Subject: RE: That other yeast company

> Date: Fri, 22 Jul 2005 14:43:35 -0400
> From: Jeff Renner <jsrenner at umich.edu>
> Subject: YeastLab W51 weizen yeast available
> 

> > Bavarian Weizen Ale* Available July/August (WLP351)
> >
> > Former Yeast Lab W51 yeast strain, acquired from Dan 
> McConnell. The  
> > description originally used by Yeast lab still fits: " This strain  
> > produces a classic German-style wheat beer, with moderately high,  
> > spicy phenolic overtones reminiscent of cloves."
> Hope this gets lots of use so it stays in the catalog.
> 

I would love to help out, but I live in the backwaters of Canada, eh? where
Wyeast still dominates the yeast world. You would think in the nearby
homebrewing powerhouses of Alberta and Saskatchewan there would be somebody
selling White Labs products but alas that is not the case. Needless to say,
the locals have never summoned the nerve to nerve to venture beyond the
ubiquitous smack pack. I suppose I could order a minimum of 10 vials from
Scott Labs then brew like mad before they die of old age. But I prefer to
enjoy my brewing as a relaxing hobby, not a race against time. Perhaps
someday White Labs will make a serious push at representation in the Great
Wit North... Perhaps....

Cheers
Brian




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Date: Sat, 23 Jul 2005 09:52:11 -0400
From: Jeff Renner <jsrenner at umich.edu>
Subject: Mel Allen or Mel Gibson?

I wrote:

> the basic Ballantine XXX
> ale (the kind that Mel Gibson used to advertise on Yankee's games and
> which he apparently drank as he broadcast, with evident effect by the
> ninth inning) or Ballantine IPA.

Doh!

I, of course, meant Mel Allen, the longtime Yankee broadcaster.

I guess he's been gone too long.

Or maybe my mind has been.

Jeff
- ---
Jeff Renner in Ann Arbor, Michigan USA, jsrenner at umich.edu
"One never knows, do one?"  Fats Waller, American Musician, 1904-1943
***Please note new address***



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Date: Sun, 24 Jul 2005 07:00:06 -0500 (GMT-05:00)
From: pulsarxp at earthlink.net
Subject: Ballantine XXX

Jeff:
Thanks for nice nice note and info you put on the reflector regarding 
Ballantine Ale (XXX and IPA).  I think I may be thinking of XXX.  Back in
the late 50s and early 60s when I was in College at the Univ of Wisconsin
I was addicted to Ballantine.

I can't remember for sure if it was IPA or XXX, but I believe it was
probably XXX. Back then in the 50s I was unaware of there being two
types of Ballantine.  In college, you just asked for Ballantine but I think
I vaguely remember the label showing XXX on it.  Bye the way, almost
every bar or tavern back then sold this Ballantine. 

I am just now getting involved with home brewing but have had an
interest in it for years.  About a year ago I joined the KGB,  a great
brewing club down here in the Houston area.  They are teaching me
a lot and so far I have just been observing and helping other club
members brew.  (I still have a very lot more to learn).  

I am just now getting serious by collecting everything needed to start
brewing on my own.  (Should be there in a couple of months but am
discussing all the internet comments with other club member friends
the Ballantine posts.  Non of these guys know anything about Ballantine
but they will be a great asset cloning it when I get started.

Jeff, this is what has me confused.  (Again, I am thinking I am talking
about XXX, I think).  I remember Ballantine in Wiscionsin back in the
50s to smell like Chrismas Trees or pine needles when you smelled it.
Everyone back then talked about the pine needle aroma.
(A good thing).  Yet today, nobody talks about it or mentions it when
they talk about Ballantine.  Also, back then I was told the pine needle 
aroma was due to Juniper Berries being put into the beer as used in Gin.
If you do a research on the web on Juniper Berries, you indeed can find
reference to them being used in beer but no mention of the brands they
were placed within.  Jeff (Jeff Renner), you and Charles Binkley and
others have told me there were no Juniper Berries used in Ballantine.
However, maybe this was true for IPA and not true for XXX. I still think
Juniper Berries were used in Ballantine XXX because it always had 
a pine needle aroma to it and I know of no hops nor do any of my
friends know of any hops that give off a pine needle aroma.  I really
wish we could find out from someone who really knows for sure one way
or another the story on all this.  An old employee who really has first hand
knowledge on all this would would make me a believer one way or another.
I can't understand how the beer could have smelled like pine needles,
which it did, without the use of Juniper Berries.  To me, without this aroma,
the clone is not Ballantine.  Maybe a good Ale, but not Ballantine Ale.
Lee Bahr
Houston



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Date: Sun, 24 Jul 2005 15:35:37 -0400
From: "Steven J. Owens" <puffmail at darksleep.com>
Subject: Santizing Aluminum Block from JockeyBox

Hi all,

     We're dismantling, sanitizing and rebuilding our jockeybox.  The
heat exchanger is the aluminum block variety.  We're replacing all the
dingy plastic hosing, but how do we sanitize the aluminum block? 

     So far the only thing I can think of is to run pressurized
one-step solution through it, or possibly a mild bleach solution,
though I wonder/worry if there's any chance of that reacting badly
with aluminum.

     We're replacing the picnic cooler component, which has become
cracked and generally fragile from several years of abuse.  We're
considering replacing it with a Coleman Xtreme 5-day cooler (or
similar), if we can find one wide enough to fit the aluminum block.
I'd like to find an old-style coleman cooler, with the metal outer
shell, considering the abuse we put this thing through.  Or possibly a
cooler with a wooden exterior, since we use it at SCA events and the
like.

     Any other suggestions?

- -- 
Steven J. Owens
puff at darksleep.com

"I'm going to make broad, sweeping generalizations and strong,
 declarative statements, because otherwise I'll be here all night and
 this document will be four times longer and much less fun to read.
 Take it all with a grain of salt." - http://darksleep.com/notablog



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---