First time using Alpha amylase and doing a pastry stout and I have a question about how my beer came out. An issue during mash resulted in likely denaturing the enzymes. When I fermented, the FG initially came down to 1.040 points, but it was calculated to finish at 1.026 so I added some alpha amylase and the FG dropped to 27. However, now the beer is finished and it pours like soda. There is good carbonation but no head whatsoever. The body is thin. Keep in mind I had 2 1/2 pounds of flaked oats along with a pound of lactose in this recipe. I understand the alpha amylase can negatively affect head retention, but would that also include why my beer is so thin despite the lactose and oats? I didn’t think it would affect the unfermentable lactose and the proteins of the oats.
Alpha amylase question
- Thread starter jeeppilot
- Start date
Help Support Homebrew Talk:
hungupdown
Well-Known Member
Was the OG on target?
Alpha-amylase breaks down large, insoluble starch molecules, into smaller soluble molecules. So if enzymes were denatured too soon, that might lead to a lower OG.
Though any dissolved (unconverted) starches, which could be converted later, would increase OG.
If enzymes were denatured too soon, Beta-amylase, which produces most of the fermantable sugars, would have packed in first.
A late addition of Alpha-amylase, should produce mostly unfermentable sugars (that give body), from any dissolved starch. But it wouldn't correct for low initial extraction, and low ABV (due to lack of fermentable sugars).
Alpha-amylase breaks down large, insoluble starch molecules, into smaller soluble molecules. So if enzymes were denatured too soon, that might lead to a lower OG.
Though any dissolved (unconverted) starches, which could be converted later, would increase OG.
If enzymes were denatured too soon, Beta-amylase, which produces most of the fermantable sugars, would have packed in first.
A late addition of Alpha-amylase, should produce mostly unfermentable sugars (that give body), from any dissolved starch. But it wouldn't correct for low initial extraction, and low ABV (due to lack of fermentable sugars).
This bit of convetional wisdom is actually not correct. Which means that this...
...is also not correct. Alpha amylase breaks (1→4)-α-D-glucosidic linkages in polysaccharides containing three or more (1→4)-α-linked D-glucose units. So it doesn't act only on insoluble starches, but also converts dextrins and higher molecular weight polysaccharides into glucose, maltose and maltotriose. All of those are fermentable. It can even convert maltotriose into glucose and maltose. The fact that it cleaves polysaccharide chains at random internal bonds doesn't mean that it only cleaves long chains into slightly smaller chains. Given enough time to act on fully gelatinized starch, alpha amylase can convert all of it to fermentable sugars and limit dextrins. Many posters here have reported good results adding alpha amylase to the fermenter, and it appears that the OP saw an increase in the fermentability of his wort after adding alpha amylase to the fermenter.
Again, commonly believed but not correct. The primary determinant of wort fermentability is neither alpha nor beta amylase, but limit dextrinase, which is optimally active at 140-145F and denatures at slightly lower temperatures than beta amylase. Limit dextrinase is the only enzyme present in the mash that can hydrolyze the (1→6)-α-D-glucosidic linkages at the branch points of amylopectin. If those branch points are not broken, all the alpha and beta amylase in the world (and all the time in the world for them to act) will leave unfermentable limit dextrins in your wort.
https://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.1999.tb00020.x
Last edited:
JJFlash
Well-Known Member
- Joined
- Aug 2, 2008
- Messages
- 62
- Reaction score
- 37
From your link:
"A New Approach to Limit Dextrinase and its Role in Mashing*"
" It was demonstrated that typical conversion temperatures of 63–65 °C and a mash pH of 5.4–5.5 favour the action of malt limit dextrinase. The temperature optimum for the limit dextrinase of a malt extract was 60–62.5 °C, as opposed to 50 °C for purified limit dextrinase. Lowering the mash pH from 5.8 to 5.4 increased wort fermentability due to increased limit dextrinase activity."
Correct me if I am wrong. My interpretation of this statement:
Limited dextrinase is most active in grain mash 145.4-149 degrees F. All grain brewers.
In malt extract (LME or DME?) limited dextrinass is most active 140-144.5 degrees F.
"A New Approach to Limit Dextrinase and its Role in Mashing*"
" It was demonstrated that typical conversion temperatures of 63–65 °C and a mash pH of 5.4–5.5 favour the action of malt limit dextrinase. The temperature optimum for the limit dextrinase of a malt extract was 60–62.5 °C, as opposed to 50 °C for purified limit dextrinase. Lowering the mash pH from 5.8 to 5.4 increased wort fermentability due to increased limit dextrinase activity."
Correct me if I am wrong. My interpretation of this statement:
Limited dextrinase is most active in grain mash 145.4-149 degrees F. All grain brewers.
In malt extract (LME or DME?) limited dextrinass is most active 140-144.5 degrees F.
Ok, I'll correct you.
When the paper talks about "malt extract" they are talking about wort produced from malted grain. In professional and academic circles "extract" is not the dry powdered or concentrated liquid wort that you buy from the homebrew store, but rather "extract" is all dissolved solids created in the mash - all of which contribute to increasing the SG. In the homebrew world, we tend to get a little sloppy with our terminology and just call the dissolved solids in the wort "sugar", but extract is only about 90% +/- carbohydrates (soluble starch, dextrin, and sugar), with the balance being proteins, lipids, and lots of other minor components.
The purified limit dextrinase experiments were conducted in buffer solutions, and did not contain dissolved carbohydrates (or the other stuff in wort.) Under these experimental conditions, 50°C was the optimal temp for limit dextrinase activity. But these experimental conditions were nothing like the conditions in a real mash. The authors of this paper found that in actual wort, the denaturing of limit dextrinase was pushed to higher temperatures, so the optimal temperature for limit dextrinase activity was higher than for the non-realistic conditions. It's not about DME/LME vs. all-grain.
Brew on
Sounds just like many posts I have made on the same topic. Well done. I think it's good that the myth busting is taking hold.
Brew on
![Craft A Brew - Safale BE-256 Yeast - Fermentis - Belgian Ale Dry Yeast - For Belgian & Strong Ales - Ingredients for Home Brewing - Beer Making Supplies - [3 Pack]](https://m.media-amazon.com/images/I/51bcKEwQmWL._SL500_.jpg)
Please accept my most sincere apology.
Some thoughts on the above:
- The linked paper does show a stronger statistical correlation between free limit dextrinase activity and fermentability of the wort, when compared to the other amylolytic enzymes.
- The data they present in support of this, in Figure 6, is ... not so good.
- The paper is also 12 years old, and I do not know that this conclusion -- that limit dextrinase is the most important enzyme for fermentability -- has held up since this time. My first impression is that it has not.
- I do think that a more cautious statement, that limit dextrinase is an important amylolytic enzyme in the mash, is now fairly widely accepted. See the more recent article at this link. On the other hand, maybe not -- limit dextrinase doesn't make it into the 6th edition on Kunze at all. (Not that Kunze is God, or that the statement is wrong, but just that "widely accepted" does imply "appears in important textbooks," and the contrapositive.)
- The article I linked also points out that the situation is significantly more complicated than just "higher temperature gives faster reactions, which then eventually stop as the enzyme denatures." One big factor is that other proteins in the malt can and do bind to these enzymes and render them catalytically inactive. Most of the limit dextrinase ends up in an inactive, bound form.
- Limit dextrinase clearly never gets a completely free hand, as some branched dextrins remain in pretty much any wort, regardless of mashing conditions, unless enzymes are added.
AdjunctBrewer
Well-Known Member
My thoughts exactly, as how would one conduct a mash to optimize limit dextrinase activity?
It is more complex than simply lowering the mash pH to 5.4-5.5 and the mash step temperature to 140F-144.5F for a certain period of time.
Based on? The paper you linked? Seems to me that the information that can be obtained from isothermal mashing is necessarily limited since all of the enzymes are active at overlapping temperature ranges. The only way to truly isolate the impact of each enzyme would be to dough in with boiling water to denature everything and then add pure enzymes individually.
OP
OP
jeeppilot
Well-Known Member
All of this info is terrific and I need some time to break it down into smaller bits I can understand. (So horribly sorry for the pun).
For my grade level understanding of this, I thought I understood that alpha amylase would not effect lactose because lactase is the enzyme needed to break down milk sugar. I also thought the alpha would not affect the oats mainly in the sense the proteins would not be touched, which is how oats serve to increase head retention.
The above posts make clear the starches from the oats become more fermentable sugars with alpha amylase, but I’m still missing the protein interaction and the lactose connection.
Thanks for holding my toddler hand through this.
For my grade level understanding of this, I thought I understood that alpha amylase would not effect lactose because lactase is the enzyme needed to break down milk sugar. I also thought the alpha would not affect the oats mainly in the sense the proteins would not be touched, which is how oats serve to increase head retention.
The above posts make clear the starches from the oats become more fermentable sugars with alpha amylase, but I’m still missing the protein interaction and the lactose connection.
Thanks for holding my toddler hand through this.
That probably wouldn’t work either — a major factor in limit dextrinase activity, for example, is the presence of other proteins in the malt that bind to it. So denature those and…
That's why you do the regression analysis. It gives a quantitative result from data that may look confusing.
Age by itself means nothing. I haven't seen anything yet (including the paper in the next quote) that refutes what Stenholm and Home wrote in their 1999 paper. If interested, here is a link to Stenholm's PhD dissertation, which appears to have even more info on limit dextrinase.
I don't see anything in this link (after a quick first reading) that contradicts what is in the Stenholm and Home paper. If you can point out a specific instance that you think does contradict, please post it and we can discuss. I agree that Kunze is not god, and even experts have blind spots - that's why science depends on lots of experts.
My impression of the paper is that it makes a bunch of unwarranted logical leaps, where the a specific result is consistent with a preconceived conclusion, and alternative explanations for observed behaviors are not even entertained.
Yes, things are usually much more complicated than can be expressed in a single sentence. A huge complicating factor that doesn't get a mention in either of the papers we have been discussing is the interaction of gelatinization rate (as a function of temperature) with the rate of action of the various enzymes (as a function of temperature.) Some level of gelatinization must occur before hydrolytic enzymes can do anything, and solubilization of starch cannot reach 100% until gelatinization has reached 100%. Maximum hydrolysis then occurs after maximum solubilization, if the mash is continued long enough, and the enzymes have not been completely denatured. Gelatinization takes longer at lower temperatures vs. higher temperatures, so isothermal mashes at various temperatures for fixed times are deceptive because levels of gelatinization are ignored, as are the different rates of enzymatic action as temp changes.
Doesn't matter if some, or even most, of the limit dextrinase is inhibited in the mash. The fact that there is some amount of free and active enzyme is what is important. And it is the effect that the active limit dextrinase has that matters.
Inhibition is different from denaturing. Once an enzyme molecule is denatured it is dead forever. Inhibition is just another molecule latching onto the enzyme molecule so that it is not free to interact with its target substrate. There will be an equilibrium between free and inhibited enzyme molecules. A reduction in the amount of free enzyme will unbalance the equilibrium, causing more inhibited enzyme to become uninhibited in order to bring things back into equilibrium. Inhibition is a reversible process.
Brew on
The proposed experiment uses only endogenous (the naturally occurring) limit dextrinase in one cell. This way, any limit dextrinase inhibition will have the same effect as in a typical mash. The question is whether beta amylase or limit dextrinase controls maximum fermentability. For all cells the mash pH target is 5.4 (at room temp), and the mash water should have ~100ppm Ca (Ca is known to aid the stability of some enzymes) The experiment has three essential cells:
- Max fermentability mash schedule - mash in at 149°F (65°C) and hold until SG does not change after 15 minutes. This lets both limit dextrinase and beta amylase have the chance to do as much as they can. Then heat to 162°F (72°C) to promote more complete gelatinization of any harder to gelatinize, and hold until SG does not change after 15 minutes. Then heat to ~190°F (~88°C) to insure complete gelatinization of all starch. Measure and record SG and attenuation.
- Alpha amylase only - mash into boiling water, and hold above 200°F (93°C) for 15 minutes to insure 100% gelatinization, and complete denaturing of all endogenous enzymes. Reduce temp to 149°F (65°C), add alpha amylase enzyme, and hold until SG does not change after 15 minutes. Measure and record SG and attenuation.
- Alpha and beta amylase - mash into boiling water, and hold above 200°F (93°C) for 15 minutes to insure 100% gelatinization, and complete denaturing of all endogenous enzymes. Reduce temp to 149°F (65°C), add a mixture of beta and alpha amylase enzyme, and hold until SG does not change after 15 minutes. Measure and record SG and attenuation.
If the hypothesis that limit dextrinase controls fermentability is correct, then cell #1 will have the highest fermentability, and cells #2 and #3 will be less fermentable, and pretty much equal to each other. If cell #3 has significantly higher fermentability than cell #2, then beta amylase does have a significant effect on fermentability.
Brew on
The two sayings that come to mind are "garbage in, garbage out" and "there are lies, damned lies, and statistics." The regression analysis does give you a number, but it is always necessary to look at the raw data and decide whether that number means anything, or whether it means what you think it does.
There's a ton of scatter in the data in Figure 6, comparable in size to the correlation effect. What bothers me more is that there actually seems to be a decent correlation between fermentability and beta-amylase activity, but that whatever correlation that's there is ruined by the first two data points -- if they hadn't chosen to do those two experiments, a major conclusion of their paper might have been different. And then there's the last sentence of the paper: "The higher the free limit dextrinase activity was, the higher was the fermentability." I understand that a conclusion section is prone to fluff and grandiloquence, but this is flat wrong, if you look at Figure 6C, which is clearly not monotonic: if you compare the second and ninth data points, a nearly twofold increase in free limit dextrinase activity is accompanied by a slight decrease in fermentability.
Age in itself is not disqualifying ... but it certainly means more than nothing. The older the paper is, the more likely it is to be outdated -- that's tautological. And I see I missed the actual age of the paper, which is 25 years old. I also see it's been cited 63 times since then, so roughly 2.5 citations a year. I don't publish in brewing science, so it's possible that this is actually a high number, but it doesn't seem like a lot to me. This doesn't mean the paper is wrong, but it strongly implies that it has not been influential among academics.
The article I linked doesn't (again, after a quick first reading) provide evidence that refutes Stenholm, but it seems clear that the authors do not buy Stenholm's conclusion, as they make a few statements along the lines of "Long-chain starch molecules in the form of amylopectin and amylose are cleaved by the amylolytic enzymes α- and β-amylase, and to a lesser extent by limit dextrinase, to form simple glucose molecules, maltose, maltotriose, and dextrins." This sort of statement (along with the absence of limit dextrinase in Kunze) is what makes me suspect (as I said was my first impression) that "this conclusion -- that limit dextrinase is the most important enzyme for fermentability -- has held up since this time. My first impression is that it has not."
As for Kunze, when someone writes a textbook, and the textbook is generally highly regarded, the assumption is that on most points they've managed to accurately represent the field's general consensus on major issues. Of course he'll get some things wrong, but I think it's a safe assumption that (1) he's aware of the Stenholm paper, (2) he didn't find it convincing enough to include its conclusions in his sections on enzymes in the mash, and (3) his judgment on that is probably at least as good as anyone's on this forum. This is sketchy and circumstantial evidence that Stenholm's conclusions are overstated, but it's much better evidence that academic brewing has not gone all in on those conclusions.
There is not a lot of LD, and most of it is inhibited. My first expectation, based on this, would have been that it was unimportant. Props to Stenholm for providing some evidence that it does indeed play a role in starch digestion in the mash. I don't think it's a reasonable read of Stenholm to say that LD is the major player, based on the quality of their data and the limited scope of what they were actually able to show statistically.
The other reason to think about inhibition is that the reality is that the mash is a big ol' mess. For instance, some of LD's main inhibitors are proteins, so proteases that are also in the mash can cut them and indirectly increase LD activity. But the proteases also have their own temperature dependence for reaction, and denaturation temperature ... and their denaturation presumably also depends on other components in the mash.
It's an interesting proposal.The proposed experiment uses only endogenous (the naturally occurring) limit dextrinase in one cell. This way, any limit dextrinase inhibition will have the same effect as in a typical mash. The question is whether beta amylase or limit dextrinase controls maximum fermentability. For all cells the mash pH target is 5.4 (at room temp), and the mash water should have ~100ppm Ca (Ca is known to aid the stability of some enzymes) The experiment has three essential cells:
The goal of all mashes is to get 100% starch gelatiniztion, and similar levels of malt component solubilization among the mashes. Only cell #1 will have any limit dextrinase action. If the SGs of the three cells are the same, then the same amount of extract was created by each mash, although the carbohydrate molecular weight distributions will likely be different. If there are significant differences in SGs across the mashes, then the cause of this must be understood before solid conclusions can be drawn from the experiment.
- Max fermentability mash schedule - mash in at 149°F (65°C) and hold until SG does not change after 15 minutes. This lets both limit dextrinase and beta amylase have the chance to do as much as they can. Then heat to 162°F (72°C) to promote more complete gelatinization of any harder to gelatinize, and hold until SG does not change after 15 minutes. Then heat to ~190°F (~88°C) to insure complete gelatinization of all starch. Measure and record SG and attenuation.
- Alpha amylase only - mash into boiling water, and hold above 200°F (93°C) for 15 minutes to insure 100% gelatinization, and complete denaturing of all endogenous enzymes. Reduce temp to 149°F (65°C), add alpha amylase enzyme, and hold until SG does not change after 15 minutes. Measure and record SG and attenuation.
- Alpha and beta amylase - mash into boiling water, and hold above 200°F (93°C) for 15 minutes to insure 100% gelatinization, and complete denaturing of all endogenous enzymes. Reduce temp to 149°F (65°C), add a mixture of beta and alpha amylase enzyme, and hold until SG does not change after 15 minutes. Measure and record SG and attenuation.
If the hypothesis that limit dextrinase controls fermentability is correct, then cell #1 will have the highest fermentability, and cells #2 and #3 will be less fermentable, and pretty much equal to each other. If cell #3 has significantly higher fermentability than cell #2, then beta amylase does have a significant effect on fermentability.
Brew on
The applicability of #2 and #3 to actual brewing assumes that the enzymes behave similarly in pre-boiled wort. This will not be the case if there are wort proteins (denatured in samples #2 and #3 but still active under normal mash conditions) that act synergistically with either enzyme, and based on this reference (which, admittedly, I have only skimmed, and it looks like a real slog), there are.
I suppose it's also not out of the question that some solubilized starches also modulate enzyme activity. Getting everything into solution and then adding enzymes might not give the same results as letting the enzymes act during starch gelatinization. This would complicate the picture too.
In the case that #1 has substantially higher fermentability than #2 and #3, I think your conclusion is more limited than what you state: you will be able to state that there are important factors influencing fermentability in the mash that go beyond the simple action of alpha- and beta-amylase. Maybe LD is one of -- or even the main -- factor, but the experiment doesn't speak to that directly.
Of the various possible results, #2=#3 would (to me) be the most surprising, and thus provide the most food for thought. I would expect #1 to be higher than #3 (conclusion: the mash process is complicated), but it would surprise me if it were a lot higher.
Similar threads
Latest posts
-
-
-
-
-
-
Need help diagnosing lower than expected efficiency- Latest: Brockness Monster
-
