The Fresh Loaf

News & Information for Amateur Bakers and Artisan Bread Enthusiasts

Does barley have exceptionally high enzymatic activities?

Elsie_iu's picture

Does barley have exceptionally high enzymatic activities?

So far, I've worked with barley flour (pearl barley ground from scratch) for 4 times. Every single time, the resulting dough ended up with broken gluten. The gluten was so short that it's impossible to support itself. Therefore I got flatbread every time.


The % of barley flour I used was 30% while the rest was some kind of whole wheat or spelt flour.


Have anyone experienced the same situation? I suspect this is attributed to the action of protease, which speeded up the break down of gluten into amino acid. Usually I allow the dough to bulk ferment for 6-12 hours at room temperature. The situation seemed to got worse as time passed, which is somewhat similar to how sprouted spelt flour affects the dough.


Some would probably suggest that this is a result of the low gluten property of barley. However, I'm pretty positive that it's unlikely the major cause. I've incorporated a variety of gluten-free flour (mass harina, buckwheat, both glutinous and regular rice flour) into my formula for several times. They did not impose the same effect as barley did at similar percentages.


Thanks ahead for your ideas!


clazar123's picture

Hard to tell what happened at the party if we don't know who was at the party. A recipe is helpful. The answers could be totally different. We don't even know if this is sourdough or commercial yeast. If it is sourdough, knowing the culture maintenance is also necessary for this type of problem. Help us to help you.

Elsie_iu's picture

I had to specify that they're sourdough formulas.

Here are two recipes I tried:’s-cream-cheese-rolls

Today I baked another loaf using the following formula@ 90% hydration excluding the porridge :

30% pearl barley flour                                             

30% sprouted white wheat flour                           

40% whole white wheat flour                                             



10% basmati rice  

60% water                                                                       

20% toasted soya powder


The procedures are pretty much the same as the previous two recipes.

My starter is half whole wheat half whole rye. I feed it weekly and let it rise to 1.5 times of the original size at room temperature before refrigerating.

Thanks a lot!                                                



alfanso's picture

hi Elsie,

Joze posted his barley bread here a few months ago. And had no issues with rise or oven spring or or or...

I also did this as baguettes (surprised?), without issues  

According to lechem/Abe, who seems to be well versed in barley, as well as my singular experience, the grain takes a high hydration as it is a thirsty one.


Elsie_iu's picture

I actually read both Joze's and your posts some times ago. In fact, Joze's post was what inspired me to try barley flour. Yet, I somehow forgot about his recipe when I finally tried it myself... Silly me! 

The key here seems to be carrying out a very brief bulk fermentation, only let the dough rise for a bit then retard directly. Autolyse should probably be skipped as well. 

The hydration isn't really an issue for me as I tend to over-hydrate...

And I wasn't the least bit surprised that you used Joze's formula for baguettes. After all, baguettes are your expertise :) Thanks so much for the reminder!


dabrownman's picture

catalyst ( an amino acid protein" that helps to speed up the breaking of the protein bonds in gluten.  It seems that protease activity of barley and wheat and several other grains are very similar per the attaches.  We also know that the amylase activity is also similar to wheat and they have the same diastatic power.  I'm guessing that the problem might be more to do with your processes and timing..  Happy baking

Elsie_iu's picture

Perhaps that would be my year 4 research topic :) That way I can learn more about the biological and chemical properties of different grains...

Thanks. I'll try Joze's recipe to see if the situation improves for the time being.'s picture

A quick note on "protease".  These poor enzymes find themselves implicated every time someone's dough falls flat.  And rightly so, much of the time.

But just to keep the conversation on top of the biology, all should understand that "protease" is not a single unique enzyme, as appears to be widely and understandably assumed by the manner in which the word is deployed in many TFL posts.  As a biologist, I experience a nano-cringe every time I read "protease is responsible" or "barley has protease".  So just for the record:

Plant (and animal) genomes encode hundreds, even thousands, of different protease enzymes -- all of which de-polymerize the strings of amino acids that constitute protein molecules, including gluten's component proteins.  And different proteases accomplish this protein degradation task in different ways, for ultimately different purposes at different times during the organism's life cycle.  For example, and most relevant to TFL interests, plants store protease enzymes in their seeds in order to break down the proteins mom (the parent plant) stored there for junior (the attached embryo) to grow when it's germination time.  Plants can no better directly assimilate intact proteins than we can.  We all have to break down nutritionally supplied proteins in order to use the resulting amino acids to synthesize the proteins we need to grow and run our bodies, proteins that not surprisingly bear no resemblance, structurally or functionally, to the proteins stored in seeds.  The physiology of seed germination also induces the synthesis of scads of freshly made proteases (in addition to those mom stored there), and other hydrolytic enzymes, all for the same purpose of breaking down what was stored (proteins, fats, carbohydrates, nucleic acids) to make their component subunits available for junior to re-polymerize them for her/his growth and development needs.

Different proteases work differently.  No enzyme is a multi-functional swiss army knife.  Each enzyme is very limited in what it can do.  Some proteases chew up proteins from the end(s), Pacman style.  Others attack (scissor) protein molecules internally along their length, targeting specific sequences of amino acids.  For example, serine proteases are a large, important and diverse group that cut proteins at specific amino acid sequences containing the amino acid serine.  The net result of all these proteases' activity is to demolish the stored proteins, thereby freeing up millions of single amino acids for making new proteins.

Re: Barley -- This thesis from 2012 cites prior work suggesting that there are as many as 42 proteases laboring away during the barley malting process.

Finally, if you stop to think about it, a curious aspect of protease chemistry is that proteases, being enzymes themselves, are generally not particularly susceptible to being cut up ("cleaved", as it's called) by their own activity.  These snakes cannot bite their own tails very efficiently.  But they are, after all, enzymes more or less like any other and they do "turn over" in cells.  So molecular evolutionary selection has rendered proteases sufficiently resistant to their own collective activity as to give them serviceable half-lives to get their jobs done in cells before they fall victim to the same fate as their own targets - getting degraded and recycled.  I've always thought that bit of evolutionary tweakage was really cool.

Class dismissed.


Elsie_iu's picture

I've some basis knowledge of protease as a food science student, so I understand most of the concepts you introduced. That being said, of course it doesn't even come close to how much profound biologists like you know.

Mostly I tried to use simple terms for non-biologist here to avoid confusion on TFL. Yet I forgot that using vague terms could lead to misunderstanding as well.

It's indeed easy to get amazed when one learn of how species are evolved. Through different mechanisms like natural selection, the chance of survival is greatly enhanced by adapting to the environment. 

Baking is all about science. Maybe that's why there's a disproportionate share of biologists on this site. I don't think it's a coincidence that you and Dabrownman, with some background of biology, are among the top bakers here.

Thank you for your teaching. It's an educational post that I believe is very useful for bakers on TFL.

dabrownman's picture

science is math.  The  entire universe is math and science.  If you know the science and math of baling then it is much easier to get right.  You are too kind.  Good luck with your studies in food.  My thesis in Architectural school was about how to design and build a sustainable city.  Here it is 40 odd years later and we are finally beginning to actually do some of these things even if they are in China and the UAE.  You are young and will live long enough to see some of your dreams happen too.  Keep at it and it will happen.

Lazy Loafer's picture
Lazy Loafer

Thanks for the lesson, Tom, that's all very useful! It's like trying to explain to people who say "I'm allergic to gluten" what gluten actually is and why their tummy might get upset if they eat bread made quickly.

I do wonder, though, if enzyme activity is the culprit here as the OP didn't mention malting the barley. I'm not even sure you can malt pearl barley, as it has been processed to remove the bran.

I mill whole, hull-less barley and use this flour in some of my breads. One of those breads has a fairly high percentage of whole barley and whole spelt flour, both of which are quite weak. I took Joze's advice and now use a stiff starter and no autolyse for that bread (sourdough), and the dough is much stronger.

Elsie_iu's picture

to my knowledge since without the bran, it cannot even be sprouted.

Using a stiff starter instead of a liquid one is a good advice that's worth a try!  Thanks for the suggestion.'s picture

Thank you for your kind words.  Too kind, actually.  Believe me, I am hardly one of the "top" bakers on TFL.  I aspire to expertise at the craft, but still have many bakes yet before I earn such a rank.

Good point, Elsie:  the pearling process (= hulling and polishing) strips enough essential tissue from barley as to render it "dead" as a functional seed.  Soaking pearled barley likely initiates what's left of its germination chemistry, but having lost its outermost layers (especially the aleurone), pearled seeds are irreparably handicapped for germination.

One final (oh, probably not) rumination on proteases:  The fact that plant and animal genomes have hundreds or thousands of genes encoding a wildly diverse arsenal of proteases is a reflection of how important protein degradation is to life.  That's a lot of genetic resources devoted to taking apart proteins!  We think of life and growth as 'building' things, putting them together.  But disassembling molecular structures and (often but not always) re-using the parts is a huge and indispensable part of life and growth.


Bigblue's picture

Is protease and amylase enzymatic activity reduced and inhibited by refrigeration at 3c the same way yeast and bacteria are?

Thanks.'s picture

Yes absolutely. Every enzyme in nature has a "temperature optimum" that happens to be, not surprisingly, at or near the temperature at which the organism carrying it 'prefers' to grow and reproduce.  There are microbes that live in arctic ice and others in Yellowstone's hot springs.  They have evolved cold-adapted and heat-adapted enzymes, respectively.

So the reason baking yeast and bacteria slow down at 'fridge temps is that their enzymes slow to a crawl at those temps. The collective effect of having most or all of their enzymes barely functioning is that microbes cannot complete their life cycle as fast as they can at their 'preferred' temperature (70-90˚F for our baking bugs, varying among species).

Have you noticed that your refrigerator stock of starter becomes increasingly liquid after days or weeks at 4˚C?  That's evidence that proteases are still functioning, albeit slowly, nibbling away at the gluten, even at a decidedly sub-optimal temperature for them.  If you want to completely shut them down, you have to put them in the freezer.  But then you run into the problem of impaling them with ice crystals...

Make sense?


Elsie_iu's picture

If what I am thinking is correct, enzymes present in the dough can be roughly grouped into two: those from the microbes (yeast and bacteria) and those from the grains themselves. The optimal temperature of each species' enzymes can be very different depending on their living environment. For instance, enzymes in human body functions at peak rate at around 37°C. Homeostasis is necessary for maintaining stable body temperature for this reason.

Enzymes of microbes are used to ease their growth, repair and reproduction. At their optimal temperature, they are more active and thus the rate of anaerobic respiration of microbes rockets. More CO2 is produced to lift the dough.

In the grains (flour), the enzymes are activated once they hit the water. Amylase catalyzes the hydrolysis of stored starch into glucose. Different protease catalyzes the breakdown of specific protein/polypeptide/peptide into one of the twenty amino acids. These processes are responsible for the change in structure of dough.

To my knowledge, microbes also possess protease and amylase, but their main function (I am only referring to the significance they have on the bake) is to digest the food they obtained from the dough(flour). Their have little effect on the dough performance.

When I retard the dough, I noticed quite a bit of rise after the retard. The speed of gluten degradation slowed down remarkably in contrast. I understand that the dough temperature takes hours to drop to the fridge temperature so it makes sense to me the dough got some measurable rise. However, the gluten degrading enzymes seemed to be dormant soon after the start of retard, even though it went through the same slowly-cooled process.

Here is my theory: some of the yeast species (e.g those living in arctic ice as you mentioned) can tolerate fridge temperature and remain a certain degree of activity. On the other hand, grains, being plants, are less adapted to cold environment. Despite their ability to remain alive at cooler temperature (e.g -20°C for winter wheat), their growth ceases as their enzymes become dormant. That is how cold retard offers its advantage of preserving dough structure while allowing the dough to proof at the same time.

Correct me if I am wrong. Thanks!

Bigblue's picture

"When I retard the dough, I noticed quite a bit of rise after the retard. The speed of gluten degradation slowed down remarkably in contrast. I understand that the dough temperature takes hours to drop to the fridge temperature so it makes sense to me the dough got some measurable rise. However, the gluten degrading enzymes seemed to be dormant soon after the start of retard, even though it went through the same slowly-cooled process.

I concur with this observation--well said. And I hadn't read that anywhere else. It does seem like the retard allows the rise from fermentation while slowing the gluten degradation process. Or, perhaps, it could be that the coolness 'strengthens' the dough structure.

And thank-you, Tom, for that detailed response.

Elsie_iu's picture

What gives the dough its stretchy structure is the gluten network. It's a protein matrix composed of protein gliadin and glutenin. When mixed with water, they give rise to the viscoelastic texture of dough. The only things that catalyze their break down are their corresponding proteases. The role temperature plays here is just slowing down or speeding up the activity of protease. At higher temperature, the kinetic energy of protease (e.g one of the gluten degrading enzymes) is increased. It thus has higher chance to collide with the protein they are specified to, and help break them down into the resulting smaller molecules (e.g dipeptide into amino acids). 

Here, I take that what you meant by 'strengthening the dough structure' as rejoining the broken bonds between these amino acids. To achieve that, you need another enzyme that specified at catalyzing the reaction as well as a water molecule. Lowering the temperature, therefore, has little help regarding this.'s picture

Bigblue, You raise some interesting questions about what exactly is going on when we retard our doughs.  You all have motivated me to look back into books I have (does Emily Buehler cover this?) to see what's known.  Levain microbes may release acids during retardation that "strengthen" gluten as well.  But yes it would certainly seem as though proteases that target gluten are especially sensitive to retardation temperatures since we certainly have plenty of gluten structure left after 12-16 hour retardations.

If I dig up something interesting about this, I'll post it.


Tom's picture

Elsie, I do not know of any data documenting the relative contributions of enzymes from the flour vs levain microbes although I assume it exists.  And I would assume that due to their abundance and ongoing reproduction during fermentation, the levain microbes make a substantial contribution to dough enzyme activities.  But again, I don't know the quantitative data on that.

Yes, the enzymology of fermenting dough at suboptimal, retarding temperatures is interesting.  And yes, my doughs definitely move in the fridge during an overnight retard, but also definitely more so in summer when the kitchen is closer to 80˚F than in winter when it is closer to (or below) 60˚F.  Dough is a fantastic insulator so the effect of  refrigeration on dough is very much dependent upon the temperature of the dough prior to refrigeration, in my experience.  I wouldn't assume proteases are completely "dormant" at 4˚C (fridge temp).  But they are slowed down enough to make retardation worthwhile from the cost (crumb structure)/benefit (flavor from fermentation) standpoint.

Yes, I totally agree that plant enzymes are all pretty much inactive in the fridge.  Indeed, plant biochemists shove their test tubes of active enzyme extracts in buckets of crushed ice, in the fridge, when they want to hold them inactive for future procedures.  The bacteria in our levains do continue to metabolize to some extent, giving us the flavor benefits of retardation.


Elsie_iu's picture

Thank you for your detailed explanation. It provoked another question from me... Sorry for being infuriating :)

If I am reading your words right, both protease and amylase of some yeast and bacteria species are somewhat functional in fridge temperature. While the activity of those in flour would be so low that it's almost ignorable. 

Since the rate of gluten degradation seems less significant than that of dough fermentation, I have made the following hypothesis: Under anaerobic condition, yeast and bacteria carry out alcoholic fermentation and lactic acid fermentation respectively (and likely other anaerobic processes that I don't know). During these reactions, only hydrolysis takes place so the ATP produced is limited (2 versus 38 from one glucose). Microbes would tend to obtain their energy from starch rather than protein in flour for efficiency maximization. Therefore, at both room and fridge temperature, amylase catalyzes the hydrolysis of starch into glucose. On the other hand, since protein is not an ideal energy source, the hydrolytic reaction rate of protein into amino acids is much lower.

At room temperature, plant protease is active so the rate of gluten breakdown is high. Nevertheless, as plant protease becomes almost dormant during the retard and microbial protease is inactivated under anaerobic condition, gluten degradation is very low.

Please point out any erroneous concepts I may have. Thanks again for your time!