Get into O2 guys, if you're serious about nicer beer

[Jack of all biers]

The entry cost for H2O2 method is under $10 for at least 10 batches, if you are happy with pharmaceutical grade H2O2.

The Gold Cross brand uses phosphoric acid as the main stabiliser. It also contains phenacetin (precursor to paracetamol), which used to be sold as a pain-reliever up until to 80s. Phenacetin is no longer used as a pain reliever and has been replaced by paracetamol, due to concerns that in large doses it can cause kidney damage. I am not too concerned about the phenacetin content while I experiment with the technique. The exposure will be fairly minimal given the dilution rate and rate of consumption. If I decide to continue with the H2O2 technique, when I finish the bottle of H2O2 that I have, I'll try to source something without stabilisers. This will probably increase the cost and require more careful handling, thus defeating some of the advantages of the H2O2 method. It's still likely to have lower entry cost than compressed O2 and require less equipment.
.....
More experimentation is required. I have batch of MO/Columbus SMaSH ready for pitching tonight. I'll do the main batch with H2O2 and get a small sample without H2O2 next to it in the fermentation chamber. There's not much spare room, but I'll see what I can do.

Thanks for posting that Peteru. You're a champion and I am happy to conduct my experiments with the Gold Cross brand now as it's sold at my chemist. I have the capacity to brew 2 x 25L batches temp controlled, but just need some time to organise the time. You know what I mean.... :blink:

I'd also point to the enormous shit storm that occurred around the belief of cubing/no-chill being impractical/dangerous until enough people tried it and survived, and went on to produce award winning beers. Botulism, anyone?

I was thinking the same thing.

I don't think any of us as yet have reported a good comparison of results between an H2O2 batch and one without (& preferably one done with O2).

I plan to and want some advice (see below)

EDIT: @Adr, just went through that paper. V interesting, and thanks v much for linking it. What i found most relevant was the speed of the yeast response - all reacting within minutes, and production of the enzymes done and finished within 30-60mins.
Seems like the TCA cycle, GMP, polyamine & ethanol are repressed; whereas Glycerol metabolism & NADPH is increased.
TBH, i'm not sure what the consequences would be of all that, with regards to what we're interested in. But the fact ethanol is repressed and that also what occurs during the growth/adaption phase (which is what we want to enhance) is maybe slightly reassuring it's not too bad. :mellow:
Probably the main reassurance is the fact the response is so rapid and, most importantly, the production of the enzymes relating to the stress response drop back within an hour or so. To me this suggests the yeast is likely to be only temporarily affected or "stressed" by the presence of H2O2, after which it returns to a "normal" state and proceeds to adapt & multiply B)

Thanks for roughly translating that paper. It was really hard going there and my understanding was at about 5% level. Not sure it is all that definitive an answer one way or the other though. It of course also doesn't answer the question of potential oxidation of the wort that I have, but experimentation with different periods of storage, taken into account as one variable, may answer that to some extent (to the extent of home brew standards anyway). Hope this makes sense.

I still go back to my dosage argument - it's a little similar to zinc: too much zinc is toxic to yeast isn't it?

I would like to get my hands on this study as it sounds interesting:
http://www.jbc.org/content/273/35/22480.full

The gist of that one is that quite a lot of eukaryotes have defense mechanisms for H2O2 as it's something they encounter quite a lot in nature, believe it or not. They need to still be able to function under oxidative stress. That article appears to suggest Saccharomyces cerivisae is one of those beasts.

The catalase enzyme in yeast is the sucker wot grabs the 2H2O2 and breaks it down into 2H2O + O2. The rest of the cell then absorbs the O2. Being a catalyst, it's not strictly a reactant and should not change - except perhaps for extremely high doses of H2O2... but I am curious about this.

I'll try and do a split batch shortly, but also trying to do one thing at a time...

So, I plan to do a bit of experiment with H2O2 on my next batch (which may take a couple of weeks to find the time to do, so my results may take a while). I'm looking for any tips or pointers or variables to take into account.

I plan on doing a simple SMaSH with either Vienna or Pils malt to OG1050, Hallertau Pacific to ~25IBU and 2 x identical liquid Ale yeast packs (undecided at this stage which, so am open to suggestion)*

I will make up 50L wort using my usual equipment and process. A single infusion mash at 65C. After the wort is boiled and cooled to pitching temp it will be split into 2 x 25L batches in separate fermenters. 1 x fermenter will be shaken for aeration (4 mins ish) and 1 x yeast packet will be pitched (further thorough shaking). The other batch won't be shaken at all and all care will be taken to get the wort from kettle to fermenter without splashing or aeration (hose from kettle to bottom of fermenter). It will have yeast pack added, stirred and then add 10gm H2O2 (apprx. 11ppm O2 as per Adro's calcs)**.

Ferment both brews at 20C in temp controlled fridge, making notes at 12 hourly marks. Once brews are finished, bottle*** as per usual method (bulk prime). Test brews when bright (apprx 2 week mark) and compare. Compare a bottle of each every two weeks until they run out (I will need a lot of self restraint for this if the brews turn out good). This last one is to see if any oxidation shows itself over a period of time in either of both brews.

* I'm wanting to use Ale yeast for two reasons. To keep it simpler and relevant for most home brewers, but also to counter any chance of faults presenting because of under oxygenated wort in the shaken fermenter being a massive variable to the experiment.

**Can this be checked please.

***Bottling because my keg set up is still only a dream, but also because it is more relevant to most HB's (although that is changing quickly I've noticed). I know this adds a variable of increased risk of oxygenation when bottling, but both will go through the same process and I've never detected an oxidised beer yet. 5 years for a RIS was the longest storage so far. Although, maybe 5L of each will go into my 2 x mini kegs to christen them, but we'll see how organised I am.

 

[Zorco]

I'd also point to the enormous shit storm that occurred around the belief of cubing/no-chill being impractical/dangerous until enough people tried it and survived, and went on to produce award winning beers. Botulism, anyone?

Hops fucks bots

 

[MHB]

**
I did a rough check on the calculation, I get 9mL to add 10ppm of O2 to 25L using 6%W/V, 10mL would add about 11ppm.
I think the W/V (weight by Volume) which appears to be the standard way of expressing the strength of an H2O2 solution, rather than the more common W/W or V/V might be causing a small difference in the calculations, but not enough to worry about.

Considering how rapidly H2O2 degrades when in contact with organic chemicals, I would consider adding the peroxide 10 minutes or so before pitching the yeast - rather than after pitching. This should cause the peroxide to break down and supply dissolved O2 by the time the yeast is added, I would expect this to reduce the chance of the peroxide attacking the yeast.

NB - I still have questions about how good an idea it is to add a really powerful oxidiser to a wort, chemically a lot different to adding gaseous Oxygen. Someone with a DO meter needs to do some experimenting. If the peroxide is reacting with wort constituents differently to O2 gas it should show up as lower than expected dissolved oxygen readings.
I still have questions about the reaction with lipids and the effects on staling and head formation/retention. Something that we will only find out by experimenting and testing.
Mark

 

[Jack of all biers]

Thanks Mark, I thought I was around the mark and yes 6% solution if I can get it. I'm pretty sure the Gold cross brand is 6% W/W not W/V.

I was thinking that by adding the H2O2 after pitching the yeast it would have less time to potentially oxidize the wort. I was planning on adding it whilst the stirring in of the yeast was still going on (TBH the main reason for stirring).

I too have some questions about the method, but without a DO meter, I won't be the guy who can answer that one for everyone. I hope to see what happens with as little bias as possible (obviously my taste buds will be one of the most subjective parts of the test). I'll make sure I take note of head formation/retention in the finished product for each bottle tested.

EDIT - No, just checked it's W/V, but as you said, not much diff

 

[technobabble66]

... I would consider adding the peroxide 10 minutes or so before pitching the yeast - rather than after pitching...

Wrong. Needs to be added AFTER PITCHING the yeast. The idea is the yeast actually protects the wort. It responds to the presence of the oxidising H2O2 by producing catalase, which rapidly breaks down the H2O2 to form O2 in solution.

I was thinking that by adding the H2O2 after pitching the yeast it would have less time to potentially oxidize the wort. I was planning on adding it whilst the stirring in of the yeast was still going on (TBH the main reason for stirring).

Correct. I'd be more tempted to add the H2O2 more like 10mins after adding the yeast, so it has a chance to wake up a little.


Now in terms of your out-lined experiment, a few points spring to mind.
1) Good choice of recipe. (Unlike Brulosophy,) it's seems smart to use a simple recipe that isn't hopped to the hilt, to allow any flaws to be easily apparent.
2) Use a liquid yeast or yeast cake. I'd agree with Galbrew's comment above, it's suggested/recommended (by the manufacturers) that dry yeast is prepared in a way that it doesn't need oxygenation, and in fact oxygenation is likely to impede the yeast.
3) i'd be tempted to use a lager yeast, as a simple lager is likely to show flaws more readily. However, it also means more other stuff could go wrong, so maybe the first comparison should be kept simple as an ale. Either would be fine, really.
4) Why limit the estimated addition of O2 to 10 or 11ppm? I believe (from memory) the saturation limit is 12ppm, so why not take it to that? (so basically i'd take it to 11-12mL of your 6% solution.

Everything else seemed fine, i think.

Not sure what the best choice of yeast would be. I'm thinking the options would seem to be either a simple neutral finish, like WLP-001 or 1056; or go for one that has more character (to show the yeast performance more?) and possibly is a little harder to get to attenuation, such as the English yeasts like 1469/1187/1028/1275/wlp023/wlp005. One other that springs to mind that might suit the recipe well is a kolsch like WLP-029. Sorry, my breadth & depth of knowledge on yeasts is limited

 

[MHB]

Wrong. Needs to be added AFTER PITCHING the yeast. The idea is the yeast actually protects the wort. It responds to the presence of the oxidising H2O2 by producing catalase, which rapidly breaks down the H2O2 to form O2 in solution.

I think you might find that the situation is a lot more complex than it looks. At least too complex to be so emphatic - try both options before laying down the law.
Mark

 

[technobabble66]

Fair enough [emoji4]
The caps lock is not meant to be a put down on you MHB (apologies for it sounding like that!).
Rather, it's meant to highlight what *seems* to *most likely* be the better practice, as it seems to be forgotten every second page of this thread.


Though it's definitely only theoretical, the issue *seems* fairly cut & dry.
Adding before pitching will likely infer no benefit and all of the risks: H2O2 will potentially react with the wort and still likely be present in high amounts to stress the yeast if that's what will happen, only now the yeast is possibly in a more dormant state and might take longer to produce/release catalase.
Adding after pitching (especially a little time after pitching) should at least minimize the risk of oxidation to the wort, if not also minimize the risk to the yeast.
I appreciate it sounds wrong given the antimicrobial uses of H2O2, but we've got to keep in mind the dilution were using combined with the function of the catalase.

Having said all of that, again I'd agree: currently it's still all just theory. A variety of good comparisons need to be done to verify any of this. [emoji6]

 

[Adr_0]

I think you might find that the situation is a lot more complex than it looks. At least too complex to be so emphatic - try both options before laying down the law.
Mark

Indeed.

I don't have the facility for O2/air injection, so I'm going to try for (massive) 3L batches with the following tests:
- adding H2O2 before pitching yeast
- added H2O2 after pitching yeast
- just pitching yeast... perhaps some sort of shaking should happen with this one.

I'll have to try this DO standard too, though I'd probably go 2, 4, 6, 8, 10 if possible:
http://aussiehomebrewer.com/topic/91478-low-dissolved-oxygen-brewing-techniques/?p=1388803

I need to work on the structure of it and exactly how I demonstrate one thing or another. I'm particularly curious about the decomposition without yeast/catalase, but it's also conflicting with DO measurement if yeast is added first - perhaps it will be captured with high resolution (20 minute?) DO samples.

SG should be easy enough to do.

On top of the DO, there have to be sensory tests: perhaps visual indication will be good in the second test, but tasting/smell should be part of it too.

I also need to figure out a mutation test, and how to ensure the right amount of yeast to add to the 2nd/3rd generation, and do a sensory comparison of a 1st gen for example.

 

[manticle]

Also need comparison between o2 and h2o2.

 

[Jack of all biers]

Also need comparison between o2 and h2o2.

Absolutely. Any volunteers? Gold cross H2O2 only costs about $4. Or alternatively, anyone know someone willing to lend an O2 setup for experiments?

 

[Moad]

I was thinking today the thread title needed to be changed, thanks to whoever fixed it up!

I will volunteer to run side by side but won't be able to do it for a month or so

 

[Jack of all biers]

We have one volunteer. Everyone loves a volunteer. Good on you Moad. It's all good re a month or so. We're not getting paid for this and we all have commitments. My results may not be fully known for some months (re any oxidation) unless of course it happens quickly.

 

[Jack of all biers]

Correct. I'd be more tempted to add the H2O2 more like 10mins after adding the yeast, so it has a chance to wake up a little.

Will do.

Now in terms of your out-lined experiment, a few points spring to mind.
1) Good choice of recipe. (Unlike Brulosophy,) it's seems smart to use a simple recipe that isn't hopped to the hilt, to allow any flaws to be easily apparent.

My thoughts also. Keep it simple and less to influences on the end result.

2) Use a liquid yeast or yeast cake. I'd agree with Galbrew's comment above, it's suggested/recommended (by the manufacturers) that dry yeast is prepared in a way that it doesn't need oxygenation, and in fact oxygenation is likely to impede the yeast.

Yep. Already posted I'd use 2 liquid yeast packs.

3) i'd be tempted to use a lager yeast, as a simple lager is likely to show flaws more readily. However, it also means more other stuff could go wrong, so maybe the first comparison should be kept simple as an ale. Either would be fine, really.

Yeah, I really wanted to keep it as simple as possible with less variables. I see doing a lager comparison maybe more appropriate with an O2 comparison. That way it is comparing apples with apples, as it can't be said that the comparative sample didn't have enough dissolved oxygen from shaking.

4) Why limit the estimated addition of O2 to 10 or 11ppm? I believe (from memory) the saturation limit is 12ppm, so why not take it to that? (so basically i'd take it to 11-12mL of your 6% solution.

Purely because a 100ml bottle will make 10 brews. Given it's an Ale, maybe I should go for less to make it a fair comparison to the purely aerated batch, that might if I'm lucky get to 6ppm. But then if I push the H2O2 higher, there is more chance of things going wrong with the yeast or oxidation of wort. So with the 11ppm O2 conversion level, I will more likely detect any faults easier. So my logic is that if I can get away with a value higher than the ideal 8ppm for Ale, and don't taste any faults, then we will know we can scale it back for the different needs of top or bottom fermenting yeasts. Make sense?

Everything else seemed fine, i think.

Not sure what the best choice of yeast would be. I'm thinking the options would seem to be either a simple neutral finish, like WLP-001 or 1056; or go for one that has more character (to show the yeast performance more?) and possibly is a little harder to get to attenuation, such as the English yeasts like 1469/1187/1028/1275/wlp023/wlp005. One other that springs to mind that might suit the recipe well is a kolsch like WLP-029. Sorry, my breadth & depth of knowledge on yeasts is limited

After a bit of thought, I'm thinking Wyeast 1469-West Yorkshire. Not used it, but purely because of the description of creating malty balanced beers and is highly flocculent. I may also go with Maris Otter as I can get it cheaper than the Vienna.

 

[peteru]

The last couple of trials I have done were all with reasonably simple SMaSH beers. Gladfield American Ale + US Centennial and Simpsons Maris Otter + US Columbus.

The one thing that I have been keeping constant is the yeast. It's always US-05 that has been harvested from a yeast cake back in June 2016. I have two 1L bottles of rinsed yeast. Each time I do a batch I pick one of the bottles (alternating between them), pour off the clear(ish) liquid from the top then add the white healthy yeast layer to some starter wort (saved from whatever I'm about to ferment) on the stirplate. I then add water to the remainder of the yeast and trub in the storage bottle, agitate well and sit it in the fridge for about an hour or two. This lets the trub settle and I pour the remaining yeast in suspension into the stirplate vessel. I then top up with more wort. I usually run the stirplate for around 10-20 hours, then turn it off and let it settle until I can see a distinct layer of trub vs suspended yeast (sometimes I also get a clear layer on top). If it looks like I have a healthy dense starter, I'll pitch most of the yeast, but save some in the bottom of the starter flask to step up and store in the fridge for future use. If the starter looks a bit thin, I'll fridge it so that I can separate the "beer", trub and yeast layers and then step up the yeast layer with another stirplate session. I no-chill/cube all my wort (because I hate using and cleaning the Grainfather counterflow chiller), which gives me plenty of flexibility as to when to pitch the yeast.

Totally unscientific, but it actually works fairly well in practice. One of these days I'll have a hop and yeast freezer for long term storage of yeast that has gone through fewer generations, but so far what I have has exhibited no flavour degradation or drift. I guess that as a result of this yeast pitching practice, I probably overpitch, or at least start with a fairly good and active yeast population. I fully expect that the addition of H2O2 could cause some potential yeast cell death, but I also suspect that the catalase reaction will work in favour of the yeast and wort, causing the H2O2 to split into O2 + H2O fairly rapidly, thus increasing the DO and effectively getting me to the same point that adding O2 directly to the wort would get me.

I find it strange that people are questioning the oxidative reactions in wort when using H2O2, but not O2. Surely if O2 in the wort is going to cause issues, it doesn't really matter if it got there by bubbles from an airstone or hitched the ride on a molecule of water. Sure, the chemical reactions can be complex (the chemistry force was never strong with this one, and biochemistry is just black magic), but the proof is in the beverage served at the end of the process. And from that point of view, adding 10-12mL of 6% hydrogen peroxide to a 22-25L batch of beer has caused no harm. Now I just need to convince myself that it provides a benefit. It could just be that I'm doing everything else right and the hydrogen peroxide is a placebo! :unsure:

Or it could be that I've actually done the ground work on an alternative oxygenation method for home brewing purposes, in which case I call the naming rights.

P.S. - Although I am a scientist (I do have a B.Sc.), I am also a pragmatic. My approach is that rather than dismiss an idea because there is no literature to confirm that it should work, I'd rather figure out why something appears to work. Sadly, my scientific background is in neither, chemistry, biochemistry nor biology. But hey, if my tinkering can narrow the search space so that the experts can look deeper at the relevant factors, then I'm happy to be a part of the discovery process.

And the beer is good. :chug:

 

[manticle]

We have one volunteer. Everyone loves a volunteer. Good on you Moad. It's all good re a month or so. We're not getting paid for this and we all have commitments. My results may not be fully known for some months (re any oxidation) unless of course it happens quickly.

I'd be willing but don't yet have an oxygenation setup and I really feel measuring DO levels of the same worts using nothing, shaking, h202 and 02 injection is required to get a good indication.

Then blind triangle tasting.

Not sure how seriously expensive DO meters are but maybe if smurto were encouraged to be involved somehow we could get some quantitative analysis.

 

[technobabble66]

...I find it strange that people are questioning the oxidative reactions in wort when using H2O2, but not O2. Surely if O2 in the wort is going to cause issues, it doesn't really matter if it got there by bubbles from an airstone or hitched the ride on a molecule of water....

OK, i can chime in here for the nay-sayers. There is a difference in the reactivity between O2 gas and H2O2 delivered oxygen.
The issue is not the O2 itself so much as the reaction of H2O2 --> O• + H2O.
That single oxygen atom is more highly reactive than O2 and cause oxidative reactions quite easily, until in binds to another O• to form O2 gas. This should happen very rapidly, but the tiny fraction of time in between involves a more oxidative state. O2 gas is also highly reactive, but not quite to the same extent as the single oxygen radical.
THIS is why i'd argue that the yeast needs to be added to the wort before the H2O2, because i'd be fairly confident in assuming catalase works by decomposing 2 of the the H2O2's together to allow the smooth transition to 2x H2O molecules and an O2 molecule.
The reason i'd assume this is that if it didn't, there'd be little point in having the catalase in the first place, in fact it'd be quite dangerous for the microorganism as it's suddenly going to be creating a truckload of O• radicals around itself if the catalase simply decomposes a single peroxide by itself (does that make sense?). Furthermore, the creation of O•'s near the catalase enzyme would likely cause the rapid destruction of the catalase. Again, a fairly pointless strategy having an enzyme that destroys itself (in a random manner - given the unpredictability of what the radical would attack). This is obviously an assumption currently on my part as i haven't found a paper explaining the exact reaction kinetics of catalase yet. However, i'm fairly happy with the reasoning behind it: evolution generally doesn't tolerate stupid :lol:

I'm also happy to acknowledge this downside of the peroxide method - that the O• radical could damage both the wort and the yeast (and agree with peteru's comment about it above). However, i'm also happy to think there's a very good chance the damage is negligible if the catalase is allowed to quickly decompose the H2O2 and there is only a tiny amount of H2O2 being used in the first place. Both of which are the case here.

Again, i'm happy with the sciencey stuff, it now really needs to have empirical evidence to indicate the veracity of the process. Namely, the production of clean and fully fermented/attenuated beer, preferably with yeast that can hold true in multiple generations without mutations.
-----------------------------

On a slightly separate note, peteru, that process of your's above sounds totally fine to me. Generally, I think if you're getting successful, clean fermentations from many generations of a yeast that holds true to characteristics of the strain, then you're doing it right.
-----------------------------

FWIW due to the ease of it, another bonus of the peroxide method is i generally try to do a 2nd addition of H2O2 ~8-12hrs after pitching (~1/2 to 2/3 the initial volume). Takes ~60secs, including time to go to the fridge to get the bottle: Whip off the glad wrap, quick squirt, 5 second stir, back on with the glad wrap. Job done.

 

[Killer Brew]

Absolutely. Any volunteers? Gold cross H2O2 only costs about $4. Or alternatively, anyone know someone willing to lend an O2 setup for experiments?

I'm in adelaide and have an O2 set up. Happy to get involved.

 

[GalBrew]

On the topic of catalase, there is no need to guess at the stoichiometry of its reactions, it's pretty well documented. Just google free radical detoxification pathways and it will take the guesswork out. I'm still not convinced by any measure that yeast based catalase which is located intracellularly will get much of chance to do much before oxygen radical react with the wort and or the cell membrane of the yeast themselves.

 

[technobabble66]

The papers I've read indicate the catalase is released extracellularly by the yeast to neutralize it's surroundings.

 

[GalBrew]

The papers I've read indicate the catalase is released extracellularly by the yeast to neutralize it's surroundings.

But will it neutralise oxygen radicals before it oxidises wort components?