Why doesn't mash heating denature amylase enzymes ?

[brewman !]

All the brewing sources mention that enzymes are easily deactivated or denatured by temperatures much outside their range. Ie 131 to 150F. (http://www.howtobrew.com/section3/chapter14-5.html)

So what happens when one adds heat to a mash via a flame under the kettle, an electric heating element or a HERMs coil ? If the mash is at, say, 140F and you want to heat it up to 150F, the heat source needs to be quite a bit higher than 150F. 160F ? 180 F ? 200F ?

When the amylase touches the heat source, won't it be ruined ?

Is the amylase dissolved in the liquid part of the mash or is it stuck to the solids or both ?

Are some heat sources better than others in this respect, in that they have more area and run on a lower temperature differential ?

 

[Horseshoot]

The way I think of it is that the enzymes are active in their range, and deactivated out of their range. But once back in their range, they are activated again. I could be wrong.

 

[passedpawn]

The way I think of it is that the enzymes are active in their range, and deactivated out of their range. But once back in their range, they are activated again. I could be wrong.

Nope. Once enzymes are denatured, it's permanent.

 

[passedpawn]

All the brewing sources mention that enzymes are easily deactivated or denatured by temperatures much outside their range. Ie 131 to 150F. (http://www.howtobrew.com/section3/chapter14-5.html)

So what happens when one adds heat to a mash via a flame under the kettle, an electric heating element or a HERMs coil ? If the mash is at, say, 140F and you want to heat it up to 150F, the heat source needs to be quite a bit higher than 150F. 160F ? 180 F ? 200F ?

When the amylase touches the heat source, won't it be ruined ?

Is the amylase dissolved in the liquid part of the mash or is it stuck to the solids or both ?

Are some heat sources better than others in this respect, in that they have more area and run on a lower temperature differential ?

Even though you see the flame licking the bottom of the pot, the wort on the surface INSIDE the pot is not that hot. If it were, it would boil and gas would shoot upwards.

There exists a gradient within the thickness of the pot that goes from the temp of the flame to the temp of the wort. I know that might not seem likely, but it is so. Temperature is the accumulation of heat. So a lot of heat has to be transferred into the wort to increase its temperature.

This goes for electrical elements too. If you reach into an electric boil kettle full of cool wort and briefly (!) touch the 240V 5500W element, it won't feel hot. Don't grab it though, you'll find out some other principles of heat right away.

 

[fastengine]

Most base malt has more than enough diastatic power to convert the starches in the mash to sugars. You would have to overheat a pretty big majority of the mash to denature enough of the enzymes to make a difference in the mash. Not just the mash that comes in contact with the heat source. That is why you stir while heating, to NOT overheat or burn the mash.

 

[Gavin C]

Enzymes are catalysts. They work like a key and lock to get components of a chemical reaction close together. The enzyme is the lock, startches the key.

When you heat the beyond a certain point there is enough energy for the reaction to proceed. Heat them too much and their delicate structure breaks down as the amino acid chain unwinds. The lock is screwed in other words and no longer effective.

As the laws of thermodynamics dictate entropy will always increase so an ordered structure like an enzyme won't spontaneously reform out of disorder. The destruction of the lock; the denaturing of the enzyme is irreversible.

I'm sure there is a lot more to it but this is my rudimentary crude understanding. I probably should have paid more attention in biochemistry lab.

 

[mabrungard]

When the amylase touches the heat source, won't it be ruined ?

Yes, the enzymes are contained in the wort after the dough in. This is one of the reasons that the thick part of the mash is the part that is decocted. Most of the enzymes have been removed from that mass and are safe in the wort.

Yes, enzymes are very quickly denatured if they are heated. If there is some part of your wort that is overheated, it will lose a portion of its enzymes. Back in my early brewing years, I used a RIMS without a PID controller. I'd just turn on the switch when the mash needed heat. It took me a few batches to figure out why my beers weren't attenuating very well and seemed to taste worty. I had been denaturing the enzymes in the wort before they could do their conversion job. I switched to a PID controller and very carefully controlled the wort temperature in the RIMS chamber. Everything is good now.

 

[brewman !]

Yes, the enzymes are contained in the wort after the dough in. This is one of the reasons that the thick part of the mash is the part that is decocted. Most of the enzymes have been removed from that mass and are safe in the wort.

Yes, enzymes are very quickly denatured if they are heated. If there is some part of your wort that is overheated, it will lose a portion of its enzymes. Back in my early brewing years, I used a RIMS without a PID controller. I'd just turn on the switch when the mash needed heat. It took me a few batches to figure out why my beers weren't attenuating very well and seemed to taste worty. I had been denaturing the enzymes in the wort before they could do their conversion job. I switched to a PID controller and very carefully controlled the wort temperature in the RIMS chamber. Everything is good now.

This makes sense. If you used a heat source with not a lot of surface area and relied on a big temperature differential, all the enzymes that touched the heat source would be denatured and ruined.

With a RIMS or HERMS the wort flow rate past the heating source is going to have to be high enough that it keeps the surface of it low enough that the enzymes aren't denatured. This must be less than 160F or so from what I can tell.

If the heating element temperature is limited to 160F, the amount of heat added to the wort per unit time (heating rate) is going to be solely dependent upon the heating area. (Q ~ Area X dt) This has implications for how the heat sources in RIMS and HERMS are designed.

I brought this question up because I have never heard anyone discuss it before.

 

[Horseshoot]

Nope. Once enzymes are denatured, it's permanent.

I stand corrected. Actually, I sit corrected. Because I am typing.

 

[orangehero]

To expand on the previous comments, above a certain temperature the diastatic enzymes are being denatured. Some are more heat labile than others. I don't know exactly what the minimum temperatures are for the different enzymes but it is less than mash temperatures. At 150°F ß amylase will still quickly become denatured. The optimum ranges are just that, the extent and rate of the reactions are optimal for brewing.

 

[brewman !]

To expand on the previous comments, above a certain temperature the diastatic enzymes are being denatured. Some are more heat labile than others. I don't know exactly what the minimum temperatures are for the different enzymes but it is less than mash temperatures. At 150°F ß amylase will still quickly become denatured. The optimum ranges are just that, the extent and rate of the reactions are optimal for brewing.

If this is true, what does it say about the need for mash heating systems to be ultra gentle in their heat application ? I think lots of surface area and keeping the wort moving over the heating area is key.

 

[orangehero]

No, I don't think think being particularly gentle is necessary. Considering the diastatic power of a typical pale beer mash using well-modified malt is quite high it is my impression the mash will still turn out perfectly fine despite some rough handling regarding temperature. I haven't seen any reports indicating problems on systems that theoretically are rough on the enzymes through heat and shear stress. Certainly there's a limit, but you'd have to be using something unusual.

 

[RM-MN]

Something most of you aren't considering is how fast the enzymes work. I've used iodine to test for starch and when it no longer changes color I consider that conversion is complete. That may not be completely true because the beta amylase may be still working as it is a little slower than the alpha amylase but it gives an indication. I milled the grains quite fine (since I am BIAB brewing I can get away without a stuck sparge) and stirred them into the water and immediately took a sample and tested it with iodine which turned dark blue, a sure indication of starch. I continued to take samples and test them at one minute intervals and at 3 minutes the iodine no longer would change color because there was no starch left. With coarser milling, it will take longer for the grains to wet through and there is no conversion until the grains do get wet so you need to mash longer to account for that but you may not be denaturing the enzymes when you add heat because they are no longer working.

 

[govner1]

Nope. Once enzymes are denatured, it's permanent.

+1 on this. Once you've denatured the enzymes there is no going back.

 

[madhatt3r]

For the practical purposes of brewing, this doesn't apply but the over generalizations were bugging me.

So I've seen at least three posts declaring enzyme denaturation an "irreversible" process which is an oversimplification of the issue.

Denaturation is not only an effect of heat but also of time. There's 4 layers of three dimensional structure a protein needs to devolve through in order to be completely unfolded. This is why most enzymatic reactions in molecular labs need to be held at a denaturation temperature for a fixed time before you can say that all of the enzyme in a solution has lost all catalytic activity. Just because an amylase is heated to 160 for a few seconds doesn't mean it has overcome the energy needed to completely unfold. It may have begun to lose a bit of tertiary structure, but whether or not that affects the binding domain, your guess is as good as mine. A few minutes of that heat, yeah it's toast.

Some proteins are able to refold after high salt denaturation. This is how biochemists get insoluble protein into solution, then refold by lowering the salt gradient gently to regain catalytic activity.

Like I said, not practical for brewing purposes, but possible.

 

[passedpawn]

For the practical purposes of brewing, this doesn't apply but the over generalizations were bugging me.

So I've seen at least three posts declaring enzyme denaturation an "irreversible" process which is an oversimplification of the issue.

So... you agree that once denatured, there's no return, right?

But you're pointing out that that it takes some time, at a given temperature, to completely denature or "unfold" the protien, right?

Maybe we can come up with a graph for this. I'd like to see the time vs. temperature for denaturing alpha and beta amylase.

 

[brewman !]

So how does this all apply to mash heating systems ?

Lets say we want to design a mash heating system to do step mashes and we want to step between mash temp setpoints (including sparge out) as fast as possible, without damaging the mash. What is the best design ?

We've got several choices.

- direct heat applied to the base of the mash vessel, by fire, induction, conduction, etc. With or without recirculation of the mash via a pump.

- direct heat applied by inserting a hot surface into the mash (under the false bottom ?) The hot surface could be electric element, a hot water coil or a steam heated coil. With or without recirculation of the mash liquid with a pump.

- apply heat indirectly by recirculating the mash liquid via a pump through a loop past a hot surface. (RIMS or HERMS) The hot surface could be electric element, a hot water coil or a steam heated coil. The recirculation coulld be arranged in a counter flow heat exchanger fashion for the water and steam or just past the hot surface for the heating element.

Not considering mash flow, there are two variables at work here. heat flow is determined by contact area and surface temperature. When you add mash flow to the equation, heat flow is determined by mass flow rate, area and temperature. The mass flow rate determines the contact time of the mash to the high(er) temperature.

So what are the safe operating bounds of these parameters ?

Obviously if one puts an electric element into a static mash and runs it at a high enough power (surface temperature) to cause local boiling, the local enzymes are going to be damaged by temperatures exceeding the boiling point.

On the other extreme is heating the mash via a HERMS setup whereby the HLT water is 160F with a setpoint of 152F. It will take a long time to reach the setpoint, but there is almost no chance of damaging the mash.

Where is the optimum point ? Is it better to heat the mash very slightly for a long period of time (direct fire mash vessel) or heat it significantly but for a very short period of time (RIMS where the mash may exit the RIMS cavity at 180F and stay there for a few seconds until its pumped back into the mash which is at 152F ?)

Thoughts, comments ?

 

[passedpawn]

Does anyone know the effect of pH on enzyme activity and denaturing? I'm assuming lower pH (i.e., 5.2) improves rate of reaction and also improves the duration of the enzymes at high temps, but it sure would be nice to see numbers / graphs.

 

[RM-MN]

So... you agree that once denatured, there's no return, right?

But you're pointing out that that it takes some time, at a given temperature, to completely denature or "unfold" the protien, right?

Maybe we can come up with a graph for this. I'd like to see the time vs. temperature for denaturing alpha and beta amylase.

I would too, with some good marks as to the time. I've heard that alpha amylase works quickly but beta is a bit slower. How fast? How much slower?

I've also heard that at the mid 150's range, beta amylase is quickly denatured. How quickly?

With a bit of iodine and some finely milled grains I determined that the starches were converted to something other than starch and it happened quickly. How quickly? My iodine quit changing color in under 3 minutes with malted barley. Today's experiment had some white rice and corn meal in the mix and it took over 5 minutes. Was all the starch turned to sugar? When did the beta amylase get denatured? What happened to the alpha amylase when the iodine quit changing color? Did it quit? Was it too denatured?

How long do we really need to mash our grains? 90 minutes? 60? 30? Maybe more? How about less?

 

[madhatt3r]

So... you agree that once denatured, there's no return, right?

I agree that once a single protein molecule is denatured, it is very difficult through common means to get back a protein with any catalytic activity. Not impossible, just NEXT to impossible as it relates to brewing. Sorry to nitpick.

I was able to track down this graph from a study involving sorghum alpha amylase thermostability. Barley is on there as well. Unfortunately for brewing purposes, it has the sharpest denaturation kinetics. You can see from the graph though, they held the temperatures for 15 mins before achieving the respective level of residual activity. Also keep in mind that the curve for beta amylase will be shifted a bit from this one, so your still seeing hydrolysis of starches beyond these temp ranges.

 

[passedpawn]

I agree that once a single protein molecule is denatured, it is very difficult through common means to get back a protein with any catalytic activity. Not impossible, just NEXT to impossible as it relates to brewing. Sorry to nitpick.

I was able to track down this graph from a study involving sorghum alpha amylase thermostability. Barley is on there as well. Unfortunately for brewing purposes, it has the sharpest denaturation kinetics. You can see from the graph though, they held the temperatures for 15 mins before achieving the respective level of residual activity. Also keep in mind that the curve for beta amylase will be shifted a bit from this one, so your still seeing hydrolysis of starches beyond these temp ranges.

Can you go back to that resource and find out how the activity of the enzymes is measured? I.e., how does one know when they've denatured the enzyme? I've got a boy going through biology and we want to do a home experiment to measure this exact thing. I just need to know how to measure things without waiting for something to ferment.

 

[ColoHox]

Does anyone know the effect of pH on enzyme activity and denaturing? I'm assuming lower pH (i.e., 5.2) improves rate of reaction and also improves the duration of the enzymes at high temps, but it sure would be nice to see numbers / graphs.

No charts on hand, but changes in pH outside of the optimal range for any enzyme will cause either protonation or deprotonation of the side groups (amino acids) of the enzyme and decrease its affinity for the substrate (starch). A drastic change in pH outside of optimum will affect the folding of the enzyme and deactivate it.

A change in the conformation of the enzyme will cause a decrease in substrate affinity and slow the rate of reaction considerably. However, enzyme changes due to pH are usually reversible.

 

[madhatt3r]

I'm gonna link to the paper here because there is far more useful information in here than I could hope to relay effectively by typing it.

In short, I'm lazy.

http://pubs.acs.org/doi/pdf/10.1021/jf0501701

passedpawn - The methods section on page 1, bottom right column is where they describe their process for determining residual enzymatic activity. Looks like they utilized a spectrometer to calculate micromoles of maltose produced after incubation with a 2% geletanized starch solution. Once the maltose stopped coming, they knew the enzyme was spent. Might be able to find a used spec on ebay for cheap.


There's also an excellent section concerning pH effects and solution additives on thermostability. Enjoy!

 

[passedpawn]

I'm gonna link to the paper here because there is far more useful information in here than I could hope to relay effectively by typing it.

In short, I'm lazy.

http://pubs.acs.org/doi/pdf/10.1021/jf0501701

passedpawn - The methods section on page 1, bottom right column is where they describe their process for determining residual enzymatic activity. Looks like they utilized a spectrometer to calculate micromoles of maltose produced after incubation with a 2% geletanized starch solution. Once the maltose stopped coming, they knew the enzyme was spent. Might be able to find a used spec on ebay for cheap.

There's also an excellent section concerning pH effects and solution additives on thermostability. Enjoy!

I've got a nice spectrometer (Ocean Optics). Thanks. I also have loads of absorbance equipment and light sources and cuvettes and fibers and such. My boy and I will get busy with that paper and figure out a path forward. Thanks!

 

[madhatt3r]

I've got a nice spectrometer (Ocean Optics).

I'll see myself out....

 

[brewman !]

Nice info, MadHtt3r, but the time and temp scales (15 minutes, 70C) don't extend to mash heating conditions. We'd need to see 80 - 120C and 5 seconds to 1 minute to see the conditions involved in heating.

So here is my quandary. I'm building a steam powered brew stand. I've got a "ton" of live steam at 275F available for mash heating. How do I best add the heat from the steam to my mash, quickly, without destroying it ?

My options are:

1) infuse the steam via a manifold or screen.

For those that aren't aware, steam is hot water vapor. The heat from a steam bubble transfers to a liquid via the bubble - liquid interface. This is not a fast process for large bubbles, ie they will travel to the surface before they pop. The heat in the bubble is retained in the bubble until it collapses. Fine bubbles collapse much faster than big bubbles.

I think its easy to get a ton of surface area by infusing steam beneath a (fine) pitched screen. The screen will break the steam up into small bubbles over a large area and the small bubbles will release their heat into the mash in a short distance, a couple inches.

I'm worried about this setup giving a high thermal gradient, ie the solid mash for an inch or so above the screen might be at 180F or something like that, even if one is recirculating the wort.


2) insert a heat exchanger coil into the mash vessel and fill it with steam. A 20 foot coil filled with 275F steam will transfer about 15,000 BTU (6 Kw) to water. This coil would have massive surface area compared to a typical water heater element, even of the ultra low heat density variety (50 watt/in^2). However, its pretty high power too. Area = 240 inches x pi/4 = 188 in^2. 6 KW/ 188in^2 = 32 watt/in^2. Its unclear to me what the surface temperature of the coil would be.

The mash liquid would be recirculated. Given the maximum temp this coil can get to is 275F, the risk of scorching is zero. But it still gets hot enough to kill enzymes !

The coil would be set up below the false bottom. Recirculation would be used.

The thing I like about this is that the liquid mash would only be in contact with the coil for the time it resides under the false bottom. Due to the large heating area the heating would be relatively gentle. The solid part of the mash would be more isolated from the heat than with the steam infusion setup. The pump will be pulling the cooler liquids from above the coil across the solids nearest the coil and out through the bottom of the vessel. The liquids would spend only a brief period of time being "hot" before they are returned to the top of the mash where they will lose their heat to the rest of the mash.

However, nothing guarantees that the flow under the false bottom is uniform and areas with lower flow are going to get pretty hot.

3) pump the mash liquids through an external steam heat exchanger and back into the mash.

The flow rate for this process would be the same as in the other processes.

The contact area could be similar to the coil under the false bottom, ie 20 or 30 feet of copper coil.

The mash liquid volume would be greater because of the coil volume of the heat exchanger , making it slightly harder to get high gravity worts with a fixed kettle above false bottom mash/water ratio, if that makes any sense.

The returning temperature of the mash liquid would be the same.

The main advantage of this setup would be that none of the mash solids are in contact with the heat source, just the mash liquids. Another advantage would be that all the mash liquid flows at the same speed, there are no areas of less flow. And the contact time with the heat is limited to the time it takes to flow through the heat exchanger.

The heat exchanger used would not be a counter flow as we DO NOT want the mash liquids to attain the temperature of the steam (275F). The heat exchanger would probably be a vessel with a length of copper tubing in it. The steam would be injected into the vessel whenever heat is needed and the mash liquids would run in the copper tubing.

Thoughts, comments, ideas, advice ?

 

[brewman !]

FWIW, I've pretty much convinced myself that method #3 is the way to go.

I'm hoping that I can add a second heat exchange coil inside the steam column that will serve to preheat sparge water such that I don't have to have an HLT. I'll have to do mash pH adjustments on the fly, or have a cold water holding vessel, but that is easier and cheaper than setting up an HLT.

 

[VladOfTrub]

"Maybe we can come up with a graph for this. I'd like to see the time vs. temperature for denaturing alpha and beta amylase."

The IOB. Effects of Mashing Parameters on Mash. Effects of pH and Temperature on Malt. All the graphs and info you want.

OP..Take a look at Weyermann's site. They have a few recipes with the rest temperature and rest time noted. You'll notice in some of their recipes, that two beta rests are used at different temperatures and an alpha II and alpha I rest are used, as well.

It is best to reach the next higher rest temperature, ASAP. But, over shooting the temperature or hot spots in the mash should be avoided

 

[govner1]

The way I think of it is that the enzymes are active in their range, and deactivated out of their range. But once back in their range, they are activated again. I could be wrong.

Unfortunately, you are incorrect. Once the amylases ( beta & alpha) are denatured they that is it. No reactivation occurs if the temps are lowered back into their range(s) of activity.

 

[brewman !]

Tonight my local home brew club had a guest speaker speaking on mash chemistry. He was familiar with RIMS. He stated that the do not exceed temp for liquid out of the RIMS chamber was 75C or 167F.

 

[bob1852]

Cool thread.

I've always wondered the effect of heating strike water to ~167-170 and then dumping in 12-16lbs of grain and stirring pretty quick to equalize temps at ~152-156.

I always figured that some of those initial grains enzymes may be toast, but also just assumed it was statistically insignificant at the homebrew scale.