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Saturday, August 18, 2012

Experimental Mushroom Flour and Whole Wheat Bread

Adding mushroom flour to sourdough whole wheat bread to enhance phytase

I've discussed phytate before, and even a little about phytase (although I think now that what Laurel Robertson said about sourdough increasing phytase is wrong; a longer fermentation may simply allow existing phytase to work longer, it will not increase the amount of phytase).

But it was only the other day that I learned about fungal phytase.  Since then, I've been reading Patrick Collopy's 2004 thesis, "Characterization of phytase activity from cultivated edible mushrooms and mushroom substrates" with great interest.  Thanks, Patrick, for providing this thesis for free to interested people.  I have gleaned some interesting info, which I give here in point form.  I would refer questions about this to the original work:

  • Plants store their phosphorus as Phytic acid (myo-inositol-hexakisphosphate)
  • Ruminants can digest phytic acid, but humans and other single-gut animals can't
  • Phytic acid binds calcium, iron and zinc, reducing their bioavailability
  • Phytase (myo-inositol hexakisphosphate 3-phophohydrolase) breaks down phytic acid into my-inositol phosphates, myo-inositol and inorganic phosphates
  • most seeds contain phytase, but in addition to this, many bacteria, yeast, and other fungi produce extracellular phytase.
  • Microorganisms produce 3-phytase, and plants contain 6-phytase, which perform differently depending on the pH.
  • Optimal pH of wheat phytase is 5.15.   The human gut has a pH of 1.2-3.0, so whole wheat's own enzyme will not help us break down the phytate
  • Microbial phytase is active over a wider range, and would remain active in the human gut; but it is not likely to survive baking temperatures.
  • Some fungi may be sources of phytases that are stable in high heat
  • Many mushroom phytases are most active at pH 5.5, so they would be most active in the mixing stage of breadmaking
  • The addition of 285 U/kg phytase, and 3.125g/kg to bread dough can enhance the absorption of iron 15x.  ("One 'phytase unit' (U) is defined as the amount of enzyme which liberates 1 micromole inorganic phosphorus per minute from 0.0051 mol/L sodium phytate at 37.0 degrees C and at pH 5.50")
  • Collopy's purification of phytase was accomplished by extracting protein by homogenization in a solution of sodium acetate with phenylmethylsulfonyl fluoride, centrifuging and filtered through 0.2 μm polyethersulfone membranes.  This filtered protein extract is then placed in an ion-exchange column with piperzine buffer. Unbound phytase-inactive protein is eluted, and then NaCl is added to supply a linear gradient of salt, to collect further fractions.  Those showing phytase activity were then pooled and concentrated by ultrafiltration.  Results were calibrated and tested with Gel filtration chromatography.  Collopy indicated that his method was unwieldy and was not going to be used by industry, as it was not cost-effective or fast.
  • Following the multi-stage process of purifying phytase, 100g of mushroom material resulted in the purest yield of only 1g phytase; earlier steps resulted in inpure forms but as much as 5g of phytase.
  • Collopy has identified the mushroom genes responsible for production of phytase, and there is a suggestion that one day it may be possible to insert this gene into yeast, or enhance it within yeast that already have it, for bread production.  (But why stop there?  One could insert it into other DNA -- perhaps even into humans.  Then we would have no trouble digesting phytate).

So it certainly behooves us to try to get some extra phytase into our sourdough (or allow it to work longer), so that the phytate is broken down and we can better metabolyze crucial vitamins and minerals.  Could adding phytase to dough be done during the mixing period of breadmaking?  Would that be enough to increase the bioavailability of the whole grain's nutrients?

Questions proliferate
I wanted to know: 
  • if I ground up some dried mushrooms and put it in my flour, would I get the benefit of the phytase?
  • Would phytate survive the mushroom drying process?
  • How much mushroom would I actually require to have an effect?
  • How long would the enzymes need to do their work, converting phytate?
  • Would mushroom phytase work at the pH of my sourdough?
  • What would happen to the viscosity of the dough?  
  • Would I still be able to have a bread that held its shape while baking?

My Mushroom Bread Experiment
I had some dried mushrooms on hand.  I think that these were ordinary Agaricus bisporus, button mushrooms, described by Collopy -- but they may have been portobellos, I can't be sure now that they are dried.  I read his thesis fairly carefully, but I couldn't find anything that would lead me to believe that the mushrooms in dried form would, or would not, have any phytase in them.  He indicates that the phytase is stable at 4 degrees C (but only for the 2 weeks he kept them), but that at 60 degrees C, they are denatured.  I am not sure how hot the mushrooms got during the drying process in my excalibur dehydrator.
The dried mushrooms I used are on the right

Mushroom 'flour'

Two doughs: 75% hydrated mushroom-fortified dough on the right: should be more viscous but it isn't.
80% hydrated whole wheat and 20% whole spelt Pizza dough on the left

Dough is too relaxed, holes appearing in the gluten structure
I ground them in the food processor until I had some mushroom dust, or "mushroom flour".

I mixed it with an ordinary Tartine 100% Whole Wheat Dough, supplemented with 5% wheat germ, at 75% hydration.  By itself, this is usually a fairly nice dough to work with.  I can bake a pretty decent loaf with this mixture, and have it stand up on its own.  I can even add more water to it, and I have had pretty good results with dough at 80% and 85% hydration.

Compare this dough to one without mushroom flour that I was making pizza with.

My mushroom flour dough felt different.  I turned it q30min for the bulk fermentation period, but even at the very first stretch and turn I realized the dough was quite different.  It smelled like mushrooms -- some of the pieces of mushroom were still flecks big enough to identify.  But it was how this tiny addition of material affected the dough's viscosity that was interesting.  Holes were appearing in my dough; it remained elastic and extendible, but it tore too easily.  During the proofing period (which I cut short by 30 minutes), the loaf spread out drastically and over-relaxed.  I gently placed the dough into the hot dutch ovens, but it simply flattened out, sagging into the pan.  I scored it, not expecting any drastic rise because the dough seemed to have destroyed its own gas-cells.  Indeed, the loaves were pretty flat.

Is this what phytases will do?  Will they break down gluten proteins as well as phytate?  If so, they may be of little use to bread bakers.

Although I could smell the mushroom while mixing, I could not detect it either by smell or taste during the baking, storing or eating phases of the bread.  Once every so often one gets an aftertaste, that is all.  It is not unpleasant -- I like mushrooms.

Very flat


Stales quickly
I could not bring myself to eat a slice of it on the day it was made, I was so full of pizza.  But the very next morning I sliced into one loaf to get a picture.  And I was disappointed to learn that the bread was obviously staling quickly, far more quickly than my other breads.

I'm not too worried about phytate in my diet.  I believe that I get enough minerals from other parts of my diet, and I don't eat bread at every meal.  

I still believe that my whole grain bread is healthier for me than other kinds of bread.  If you saw the effects of bowel cancer as I do, you'd probably elect to eat more fibre to scrub your gut a bit too.

Furthermore, I believe that even phytate is not all bad.  It has anti-oxidant properties, so may have benefits preventing cancer, and one study suggested it may even protect kidneys, teeth and heart/blood vessels, which are prone to form dangerous calculi.  Prieto R. et al. (2010) "Effects of Mediterranean diets with low and high proportions of phytate-rich foods on the urinary phytate excretion" Eur J Nutr. 49(6) pp 321-6.  But I have also heard that some middle eastern diets high in phytates (a diet heavy in whole grain flatbreads) have caused zinc deficiency (e.g. see Gocmen, D. et al. (2009) "Flat Breads" Bulg. J. of Agric. Sci. 15(4) pp.298-06); however, one must also realize that many soils and plants grown in that region are zinc deficient to begin with (e.g. see Roohani, N. (2012) "Human zinc nutrition in arid regions with zinc deficiency in soils and crops - a case study in central Iran", thesis, Shiraz University.)

I believe that my sourdough and long-fermentation method naturally eliminates a lot of the phytate naturally (see Lopez, below).  

But these are merely my beliefs.  Like those who propose the Paleo diets, who put blinders on when it comes to evidence that excessive protein intake carries its own dangers, I have my own set of blinders.  The fact is, excess phytate in the diet is something to be aware of -- and to watch for things like calcium, iron, magnesium, and zinc deficiency.  Symptoms are easy to look up online.  Blood tests may be required.

Notes to Myself
  • Here is an idea: measure your bread dough as usual, but first remove the bran by sieving or fine bolting. In addition to fermenting a sourdough starter to leaven it, set aside the bran in some or all of the hydration for the bread, and add to this your mushroom flour. The phytase should act on the bran before it is added to the dough. The solution of hydrolyzed phytate & phytase mushroom bran solution would be added to the sieved flour with the sourdough starter, on the mixing day. A rapid fermentation might be better; adding commercial yeast to the sourdough wild yeast would quicken the rising time, and prevent the breakdown of gluten, by the mushroom's other proteinases.
  • You could back the hydration of this dough off to 70% or even 65% -- but would that leave enough water for phytate hydrolysis to occur?
  • If phytase must be chemically extracted from mushroom to be a workable ingredient, it may not be the solution to reducing phytate levels for health-conscious consumers. It will be seen as just another additive, which will be considered suspicious, and avoided. And not being a chemist, I am always suspicious of their claims that something treated with formaldehyde or phenylmethylsulfonyl fluoride or any other possibly dangerous chemical, is entirely free of it, never mind how much filtration it undergoes.
  • Spier has studied the enzyme's viability at cold and room temperatures. The cooler, the better; but some of the enzymes (perhaps over half) will lose their ability to work the longer it is saved. So perhaps my mushroom mixture had no phytases at all. If that is so, what happened to my dough to break it down so?
  • Phytase added to animal feed (pigs, chickens etc.) may be one of the solutions to increased phosphorous pollution downriver from their excrement.
  • There may come a time (or a place, like Iran) when phytase is governmentally mandated as an additive to whole wheat flour -- much the same as governments mandate additives to flour with bran and germ removed, and we call them "enriched".
  • Harland and Harland tested the levels of phytate in bread to find out how much they are reduced by mere fermentation. More yeast and longer fermentation times meant far less phytate. They did not test sourdough, which generally means more fermentation and typically longer rises.

    Lopez H. et al. (2001) "Prolonged Fermentation of Whole Wheat Sourdough Reduces Phytate Level and Increases Soluble Magnesium" J. Agric. Food Chem. 49(5) pp. 2657-2662  Lopez' team found that sourdough fermentation reduces a lot of the phytate in bread dough, almost 2/3. And they also tried treating the bran with microbes containing phytase separately -- hey, that was my idea! They just had it 10 years before I did! -- and found that phytate was 9/10 eliminated, using sourdough and sourdough-like techniques.
  • I've tried to use properly the terms phytate (the storage form of phosphorus) and phytase (the enzyme that breaks it down) throughout this blog entry.  But re-reading it a couple of times, I've found I sometimes interchange them by mistake.  If there are further errors, I apologize.

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