Adr_0
Gear Bod
- Joined
- 4/4/13
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Two guys walk into a bar. The first says "I'll have a great big glass of H2O" - he drinks it and walks away. The second guy says "I'll have a great big glass of H2O, too"... but we don't know what happened to him.
Like a few other people, I have been curious to see if H2O2 is a possible alternative to using bottled oxygen or shaking. The cost and simplicity is appealing, but there is possibly a negative impact on yeast health (counter to the point of oxygenation) and flavour/aroma effects.
There have been some great experiments in the O2 thread, and like these guys I wanted to see for myself rather than endlessly hypothesise. So I started a quick experiment yesterday.
A few points on the beer and common points:
- Wort was base, some German caramels, all Challenger to 1040 and 35-40IBU's. SG was a bit down due to a doughball issue.
- Yeast was frozen 1275, which had been slowly unfrozen and 'revived' with 500mL of weak starter. If ever there was a bunch of cells that needed O2 I would say this would be appropriate.
- The slurry was then added to a 1L starter, mixed for about 15min (by hand) and split in the following manner:
> 500mL onto stirplate into 12L of wort for control batch
> 150mL into 3 x glasses for 3 x tests into 2L test batches
> Yeast in the 3 x test batches was static, expecting oxygenation from H2O2 vs the control which was on a stirplate
I framed the experiment around the following:
- Control, being batch with yeast on stirplate for ~6hrs, hopefully showing oxygenation but not significant growth
- 1, being 150mL yeast starter with 4mL of 1.2% H2O2(12% diluted as 20g made up to 200g with distilled water) straight into the yeast
- 2, being 4mL of 1.2% H2O2 added to the wort, adding 150mL yeast starter 40min later
- 3, being 150mL yeast starter added to wort, 20min later adding 4mL 1.2% H2O2 solution
I used Lyrebird_Cycles' method detailed here
The 4mL of 1.2% was based on approximately 12ppm O2 target in 2L of wort, but also diluted to aid mixing. Test batch 1, given it was added to 150mL of starter, copped 160ppm H2O2 which would either make or break it....
I wanted to evaluate H2O2 impact in four ways:
- Dissolved oxygen measurements
- Visual indicators, largely krausen or flocculation
- Gravity measurements
- Sensory (aroma/flavour) indicators
A 3200ppm methylene blue solution was made up (3.2g diluted up to 1kg with distilled water), which was then diluted down to 1600ppm with 100g made up to 200g, then 160ppm with 20g made up to 200g – which should represent 16ppm O2. This was then serially diluted into 12ppm O2, 8ppm O2, 4, 2, 1ppm O2 standards:
A few introductory pics:
Test containers for each test wort/yeast/H2O2
Test jars for DO samples:
Yeast splits:
Unfortunately my beer was yellowy red (********!) so I should had added some yellow food colouring to make the standards a little more representative. Anyway.
Measurements of the base wort for each one were taken:
- Control was 0-1pppm
- Batches 1, 2, 3 were all 1-2ppm which is likely from transfer to each of the test containers. The below pic shows a sample with no methylene blue indicator (left) vs indicator in, and the 1ppm standard in the background. All three tests were similarly, likely 1-2ppm.
DO measurements were taken every 20min, with events – yeast or H2O2 additions – shown on the graph. In each, a strong methylene blue solution was added – approx. 0.5mL – and lightly swirled, with resulting colour assessed. Colour indication was a little hard, so I also made up a ‘saturated’ sample with 12% H2O2 dumped in, and a ‘reduced’ sample with ascorbic acid dumped in. I also had a ‘no indicator’ vial to illustrate the difference.
Measurements are below:…
A few example pics. Test 1, comparing 20min after pitching H2O2'd yeast starter into the wort vs the reduced sample (left) and the saturated sample (right). I'd say 1ppm or less - basically no blue/green there at all:
Test 2, 40min after adding the H2O2, just before yeast pitch, as compared to the reduced sample (left) and saturated sample (right). I'd say 12-16ppm:
Test 3, 20min after adding H2O2 where the yeast was already in suspension. Left one is 35min after adding H2O2 with yeast, while right is only 20min after. Possibly 4ppm on the right vs <2ppm on the left?
Gravity readings I will update over the coming days, but appear to be similar: essentially all three have gone from 1040 to 1032 in 12hrs, with batch three potentially only at 1033.
Sources of error:
- Sample collection was actually pretty good. My LODO method, post boil, seems to not pick up any O2 and this held on for 60min to not indicate any blue/green tint when compared with the reduced sample:
- Colour indication was a big one. For some stupid reason I decided to make a tasty, malty ale rather than a wit bier. A control for this would have been adding some yellow food colouring to the standards.
- DO measurement would have been very accurate with a DO meter. I could have potentially hired one, but I wasn’t able to arrange one unfortunately.
- Gravity readings are being taken with a refractometer. Light source is consistent (only during the day) but there may be a 0.1% Brix error.
- Mixing in the samples. I did do a decent rock back and forth and try to wait 20min or so for mixing to occur, but it is quite possible that there was not adequate mixing.
Conclusions – so far:
- It seems like adding H2O2 does increase the O2 content. There is an evident spectrum of blue from a completely reduced solution to a saturated solution.
- Yeast does indeed reduce the O2 as every mark past 40min was lower in O2.
- There did not seem to be a visual effect of adding H2O2 directly to the yeast.
- A 160ppm dose to the 150mL starter didn’t seem to kill the yeast – given gravity has dropped in line with the other three batches.
Next points:
- Gravity over the next few days may reveal something
- Sensory tests will be a really big test. Batch 2 had H2O2 added and floating around for 40min before yeast was added and managed to get up to 12-16ppm, so this may taste different to other batches. Inversely batch 1 didn’t really get above 1-2ppm IN THE WORT at any stage, though it is presumed the starter was quite high.
Like a few other people, I have been curious to see if H2O2 is a possible alternative to using bottled oxygen or shaking. The cost and simplicity is appealing, but there is possibly a negative impact on yeast health (counter to the point of oxygenation) and flavour/aroma effects.
There have been some great experiments in the O2 thread, and like these guys I wanted to see for myself rather than endlessly hypothesise. So I started a quick experiment yesterday.
A few points on the beer and common points:
- Wort was base, some German caramels, all Challenger to 1040 and 35-40IBU's. SG was a bit down due to a doughball issue.
- Yeast was frozen 1275, which had been slowly unfrozen and 'revived' with 500mL of weak starter. If ever there was a bunch of cells that needed O2 I would say this would be appropriate.
- The slurry was then added to a 1L starter, mixed for about 15min (by hand) and split in the following manner:
> 500mL onto stirplate into 12L of wort for control batch
> 150mL into 3 x glasses for 3 x tests into 2L test batches
> Yeast in the 3 x test batches was static, expecting oxygenation from H2O2 vs the control which was on a stirplate
I framed the experiment around the following:
- Control, being batch with yeast on stirplate for ~6hrs, hopefully showing oxygenation but not significant growth
- 1, being 150mL yeast starter with 4mL of 1.2% H2O2(12% diluted as 20g made up to 200g with distilled water) straight into the yeast
- 2, being 4mL of 1.2% H2O2 added to the wort, adding 150mL yeast starter 40min later
- 3, being 150mL yeast starter added to wort, 20min later adding 4mL 1.2% H2O2 solution
I used Lyrebird_Cycles' method detailed here
The 4mL of 1.2% was based on approximately 12ppm O2 target in 2L of wort, but also diluted to aid mixing. Test batch 1, given it was added to 150mL of starter, copped 160ppm H2O2 which would either make or break it....
I wanted to evaluate H2O2 impact in four ways:
- Dissolved oxygen measurements
- Visual indicators, largely krausen or flocculation
- Gravity measurements
- Sensory (aroma/flavour) indicators
A 3200ppm methylene blue solution was made up (3.2g diluted up to 1kg with distilled water), which was then diluted down to 1600ppm with 100g made up to 200g, then 160ppm with 20g made up to 200g – which should represent 16ppm O2. This was then serially diluted into 12ppm O2, 8ppm O2, 4, 2, 1ppm O2 standards:
A few introductory pics:
Test containers for each test wort/yeast/H2O2
Test jars for DO samples:
Yeast splits:
Unfortunately my beer was yellowy red (********!) so I should had added some yellow food colouring to make the standards a little more representative. Anyway.
Measurements of the base wort for each one were taken:
- Control was 0-1pppm
- Batches 1, 2, 3 were all 1-2ppm which is likely from transfer to each of the test containers. The below pic shows a sample with no methylene blue indicator (left) vs indicator in, and the 1ppm standard in the background. All three tests were similarly, likely 1-2ppm.
DO measurements were taken every 20min, with events – yeast or H2O2 additions – shown on the graph. In each, a strong methylene blue solution was added – approx. 0.5mL – and lightly swirled, with resulting colour assessed. Colour indication was a little hard, so I also made up a ‘saturated’ sample with 12% H2O2 dumped in, and a ‘reduced’ sample with ascorbic acid dumped in. I also had a ‘no indicator’ vial to illustrate the difference.
Measurements are below:…
A few example pics. Test 1, comparing 20min after pitching H2O2'd yeast starter into the wort vs the reduced sample (left) and the saturated sample (right). I'd say 1ppm or less - basically no blue/green there at all:
Test 2, 40min after adding the H2O2, just before yeast pitch, as compared to the reduced sample (left) and saturated sample (right). I'd say 12-16ppm:
Test 3, 20min after adding H2O2 where the yeast was already in suspension. Left one is 35min after adding H2O2 with yeast, while right is only 20min after. Possibly 4ppm on the right vs <2ppm on the left?
Gravity readings I will update over the coming days, but appear to be similar: essentially all three have gone from 1040 to 1032 in 12hrs, with batch three potentially only at 1033.
Sources of error:
- Sample collection was actually pretty good. My LODO method, post boil, seems to not pick up any O2 and this held on for 60min to not indicate any blue/green tint when compared with the reduced sample:
- Colour indication was a big one. For some stupid reason I decided to make a tasty, malty ale rather than a wit bier. A control for this would have been adding some yellow food colouring to the standards.
- DO measurement would have been very accurate with a DO meter. I could have potentially hired one, but I wasn’t able to arrange one unfortunately.
- Gravity readings are being taken with a refractometer. Light source is consistent (only during the day) but there may be a 0.1% Brix error.
- Mixing in the samples. I did do a decent rock back and forth and try to wait 20min or so for mixing to occur, but it is quite possible that there was not adequate mixing.
Conclusions – so far:
- It seems like adding H2O2 does increase the O2 content. There is an evident spectrum of blue from a completely reduced solution to a saturated solution.
- Yeast does indeed reduce the O2 as every mark past 40min was lower in O2.
- There did not seem to be a visual effect of adding H2O2 directly to the yeast.
- A 160ppm dose to the 150mL starter didn’t seem to kill the yeast – given gravity has dropped in line with the other three batches.
Next points:
- Gravity over the next few days may reveal something
- Sensory tests will be a really big test. Batch 2 had H2O2 added and floating around for 40min before yeast was added and managed to get up to 12-16ppm, so this may taste different to other batches. Inversely batch 1 didn’t really get above 1-2ppm IN THE WORT at any stage, though it is presumed the starter was quite high.
