In my last blog entry, I wrote how I am baffled and intrigued by the math of bread dough science. There is much and more than just math that has roused my curiosity with bread. Other sciences have challenged me too.
I spoke of how science has taken apart the whole in an effort to understand the whole. Here is a recent example of something I struggled to learn in this way. While researching bread bubbles, I stumbled upon the term "puroindolines" which refers to a molecule found in the starchy endosperm of most wheat. "Puro" is Greek for "wheat" and "indolines" refers to the Tryptophan ring of the molecule, which sits on a cysteine backbone. Over the last decade or two, there has been a lot of interest in the puroindolines, both from a genetic standpoint as well as from the point of view of biology and chemistry and agriculture -- and end-users such as millers and bakers are interested too. It seems that there is a direct relationship between the puroindolines in wheat, and the quality of hardness of the wheat (and thus the texture of the resultant flour and bread). But there are still mysteries in puroindolines, and despite the number of articles I've read, I can't seem to get a handle on the many contradictions.
Wheat is currently graded and sold based on its hardness. In general, the harder the wheat, the less starch and the more protein it will have: so a hard wheat may have up to 15% protein, good for bread, while soft wheats may have only 10% protein, better for cakes and pastries. But durum is different: it is considered very hard, and it is used mostly for pasta. Puroindolines are "lipid-binding proteins" and the number and type of them determine the texture of the flour, and it is postulated that their existence allows for the creation of good foamy bubbles in bread dough.
Enter the puroindoline mystery. Durum is a tetraploid wheat (4 sets of chromosomes), while many of our other commonly used wheats are hexaploids (six sets of chromosomes). It turns out that durum doesn't have puroindolines. My understanding is that the softness of the non-durum grain is regulated by something called friabilin, a starch surface protein complex made up of (usually only) two kinds of puroindolines (Pina and Pinb) and a protein called "grain softness protein 1" (Gsp-1). Mutations of the genes that make Pina and Pinb determine the varieties of hardness by which wheat is graded. The more puroindolines you have, the softer the wheat. But here is another mystery: generally you want higher protein content for your bread, because the protein is where you find the gluten content, which gives wheat bread its unique properties. But in fact, some people feel you need some puroindolines to provide a nice texture of foam in the dough and the bread.
You can in fact make decent bread with durum alone (for example, see the Pane di Altamura ). Durum has good moisture retention and some interesting slow-staling properties. It won't rise as high as loaves made with bread flour, but it still works. Many people seem to prefer a bread made with a combination of durum and bread flour, rather than durum alone.
Geneticists have pinpointed the very spot on the genes where puroindolines originate in our hexaploid wheats (the short arm of chromosome 5D, in a spot now named the "Hardness (Ha) locus", comprising some 64kb of genetic code). Some molecular biologists and genome-mappers who are interested in taxonomy and the evolution of life itself have used puroindolines as a marker to trace sixty million years of grass evolution:
Grasses (Poaceae) have 10,000 species worldwide. Fossil records suggest grass subfamilies diverged from a common ancestry 50-80 million years (My) ago. Comparison of various genes in grasses has further pinpointed the divergence time of many important grass lineages: 60 My for Paniocoideae (Sorghum, Zea) and Ehrhartoideae (Rice), and that Pooideae (wheat, barley, Brachypodium) and Ehrhartoideae (rice) further diverged at 50 My. Among the Pooideae, Brachypoidieae (Brachypodium) and Triticeae (wheat, barley) tribes diverged about 35 My. Brachypodium is a diploid, far simpler than wheat (and its genes have been catalogued -- see the database here), and so it has become the touchstone to compare Pooideae genes. Pina and Pinb genes are found in all Triticeae species in which soft endosperm is a dominant trait (wheat, barley rye, oats). It is not found in maize or sorghum at all, and only a vestigial memory is to be found in the rice's genome (at the spot now named Ha-rice-relic).
What does the plant use the Ha-locus for?
Ragupathy and Cloutier at Winnipeg's Cereal Research Center found that even in the hexaploid wheats, the Ha locus is only found in the D-genome, and it has been replaced by curious retro-elements in the A- and B- genomes. Turnbull and the team of plant geneticists from Australia showed that Aegilops tauschii (the wild goat-grass) is the origin of wheat's D-genome. How these species shared genetic material in the wild is somewhat of a mystery, but there it is.
There are some bits and pieces of genetic code at the Ha Locus that are all mashed together, and reading the work of the scientists who painstakingly sift out the important bits is like reading a really bad detective novel. Why do all the genomes of all tetraploid and hexaploid Triticum wheat species not carry all the Ha genes? And why, where it does appear, scattered among the genes responsible for soft textures of endosperm, are there are junk encodings, other relics of evolution, some wilder forms, and several unknown genes? The mystery unfolds and continues. But the molecular biologists have surmised that the Ha-like genes emerged after the whole-genome duplication and after the divergence of Pooideae and Ehrhartoideae from Panicoideae.
Massa and Morris from Washington State University have crunched the numbers and have determined that the proteins encoded for at the Ha locus were not just a useful random mutation, but that they were positive adaptations that resulted from "Positive diversifying selection" . This is beyond Darwin's concept of negative selection or natural selection. The locus itself appears to be in evolutionary flux, but the Pin mutation is being selected for. Even if mankind is now also involved in the selection process. Thus, it is supposed that the plant finds it very useful indeed -- again suggesting an anti-microbial reason for its existence.
Using transgenic methods, geneticists have further proved what manipulating these genes will do. They can express them or silence them with ease, as Gasparis' team showed. They can insert this gene into other plants, like rice and perhaps corn. Why would they do that? Because they believe that puroindolines have interesting anti-fungal and anti-bacterial properties, and protect the growing plant against disease.
This appears to be mostly conjecture at this point. Precisely how puroindolines protect the growing grain remains to be elaborated. Lesage's team investigated the function of the puroindolines and found that harder wheat, with fewer puroindolines, have more stress-related foldings of proteins. The puroindolines may be somehow protecting the softer wheats from an early endosperm-cell death. Whether this is because of the protein-folding that the harder wheat does, or because the puroindolines do in fact have anti-bacterial properties is unclear.
Plant breeders have found that they can manipulate the puroindolines, even creating new mutations of Pin b. Pin a, they found, is well conserved in the genetic code, but Pin b is highly mutable. Feiz and his team have found or caused several new Pin b mutations to form, and in the process have learned that it is largely the variation in Pin b that determines the overall softness of the endosperm. But some mutations that affected the Tryptophan rings destroyed the structure of the plant entirely. Day's team at the University of Reading found that there are several puroindoline b variants, and Clifton's team at the same facility found that by substituting different amino acids in Pin b's sequence, they could alter its lipid binding properties. They therefore conjectured that the antimicrobial feature of their man-made Pin-b mutants would not be as efficient as the wild Pin-b type -- the one that evolved on its own.
Rumors of Puroindoline Toxicity
This too is a puroindoline mystery. The team of Llanos and his researchers in Chile found that puroindoline-a and the endosperm protein alpha1-purothionin are toxic because they dissipate ion concentration gradients of cells. How toxic is it? Is it toxic only to bacteria, or will it damage our own body's cells? Mattei and his French Neurobiologists studied the effect of puroindoline-a and alpha1-purothionin on frog myelinated axons using confocal laser scanning microscopy. Exposed to these wheat proteins, the nodes of Ranvier became swollen when salt was present in the external solution. Mattei conjectured that these proteins bind to the lipids of the axonal membrane and create ionic gradients to induce water influx. So it does have the potential to cause significant damage.
Le Guerneve, Seigneruret and Marion wanted to know how puroindolines actually interact with phospholipid membranes. Using proteolysis experiments, this French team of Biochemists decided that these molecules penetrate into cell membranes to various depths. It was chilling to read that puroindolines resemble cardiotoxins in the way they work.
In vitro studies have determined that puroindolines will pierce the membranes of cells, opening an ionic channel. I was fascinated to learn that some of these petrie-jar studies involved the binding of puroindolines to yeast. Apparently the puroindolines bind to the lipids of the yeast cell membrane; and this opens a channel to the cell that allows for the fungus to be destroyed. But if this is true, why do our bread flours rise so well with yeast? The in vitro study I first read suggested that the yeast does not die, despite being pierced by the puroindoline shards. So where did the idea of puroindolines being anti-fungal come from? Are hard wheats, that have fewer puroindolines, more prone to disease? I've never heard that reported. So the mysteries seem to pile up.
Animal scientists want to know more about puroindolines too. We feed grain to cattle, and we have expectations that the animals will grow and thrive on the food we give them. Swan discovered that the more puroindolines there are in the starch, the slower the digestion rate, regardless of how fine the grain is milled. He concluded that puroindolines protect starch molecules from microbial digestion in the rumen of cattle, and therefore the amount of starch available in the small intestine is potentially increased. As a home baker, I find this incredulous. A longer transit-time should mean more time for fermentation in the gut. But If the bacteria and yeast in my sourdough starter are having a harder time ingesting the starch of soft wheat because it contains puroindolines, how will bread made from soft wheat ever rise? Yet it does.
Puroindolines and Texture of flour and bread
Despite the promise of puroindolines as the premier dough texture protein, Salt et. al., working in the Institute of Food Research at Norwich Research Park in the U.K. took a look at dough bubbles using lasers and 2D gel electrophoresis and concluded that it wasn't the puroindolines that were stabilizing the gas bubbles. Mostly the network of bubbles comes from beta-amylase, triton, serpins, and especially alpha-amylase/trypsin inhibitors which do the bulk of the work. "Neither prolamin seed storage proteins nor the surface-active protein puroindoline were found," Salt said.
Undaunted, Dubreil, Biswas, Didier and Marion examined how the puroindolines in the flour and dough affected the texture of bread by looking with a confocal scanning laser microscope. According to the abstract of their article Dubreil et. al, "Localization of Puroindoline-a and Lipds in Bread Dough using Confocal Scanning Laser Microscopy" (2002) Journal of Agriculture and Food Chemistry, 50 (21) pp 6078-6085, this is what they found:
"Wheat lipids were located around gas cells (GC) and embedded within the protein−starch matrix (SPM) of the dough. PIN-a was mainly located in the matrix of dough, where it was associated with lipids. In contrast, in defatted dough, PIN-a was found around GC. Addition of puroindolines in bread dough induced a defatting of the gas bubble surface and a decrease of the lipid vesicles and/or droplet size embedded within the SPM. Therefore, puroindolines control the lipid partitioning within the different phases of dough, a phenomenon that should have important consequence on the gas bubble expansion and GC formation in the further stages (fermentation, baking) of the bread-making process."
Whoa. Did you catch that? GC formation beyond the mixing stage? Two generations ago, Baker and Mize said that never happens (see my last blog entry, where I discuss Baker and Mize's work). But because of the puroindolines, "lipid partitioning" may indeed occur, leading to more bubbles beyond the mixing stage! Whoa!
While it is suggested that puroindolines may be part of the reason why bubbles form in bread flour, they are not the final answer. Further genetic clues can be found in the combined China-U.S. thesis of Feng Chen, Fuyan Zhang, Craig Morris and Dangqun Cui, "A Puroindoline Mutigene Family exhibits sequence Diversity in Wheat and is Associated with Yield-Related Traits". This is a very complex subject indeed.
Food scientists want to know more about puroindolines: they want to know its 3D structure, and how it will function and interact in recipes. Richard Frazier and his team at the Dept of Food and Nutritional Sciences at the University of Reading are methodically pursuing this molecule which has generated such worldwide interest. The Reading Team has done work with neutron-scanning of these proteins, and Frazier has published several papers describing it. He has presented his work at the 2012 Symposium on Neutrons and Food and Frazier teamed up with Luke Clifton and others to publish information on the shape of the structure. Clifton and Frazier and others on the team found that, in solution, 38 of these PinA molecules spontaneously join together by hydrophobic forces to form an ellipsoidal bubble, which is stable over a wide pH range and a large temperature range. Such a bubble can be called a micelle.
But Le Bihan and his team at Laval University in Quebec said that puroindoline has quite a different structure when examined by spectroscopy. Here, one finds alpha-helices, beta-sheets, and unordered structures, all highly dependent on the pH. Some time in the distant past, they surmise, a plant evolved a helicoidal protein that it found gave it a defence against bacterial pathogens, and this ancestor of wheat passed it on to the wheat plants that we share the planet with today.
What it means to the home baker
While I find all this fascinating -- scientists don't even seem to agree on the shape of the molecule -- what does it teach me about bread baking? How can I use puroindolines to my advantage in my home loaves?
I use mostly hard red wheat when I bake, thinking that the higher protein will provide a nice gluten structure. If indeed puroindolines impart a nicer crumb structure, I should use some soft wheat in my baking, too, in order to take advantage of the bubble structure that the softer wheat's puroindolines provide (not to mention the finer flour and the unbroken starches, which should provide more body-useful nutrition). This may be why all-purpose flours have gained prominence: they even-out the protein levels by adding different grades of wheat together to get a homogenous effect. I don't use all purpose flours anymore in my recipes because I want whole grains, and I have a suspicion of the additive sources in ap flours. But the idea of mixing (whole-grain) hard and (whole-grain) soft wheat together may be a good one for bakers.
But then again, is it good for health-conscious consumers of bread? I have read the concerns of those who fear that agribusiness is manipulating our grain to provide disease-resistance. Some corn, for example, is genetically modified now to resist disease, or to be non-resistant to herbicides. Some consumers are worried about ingesting this food, wondering what it might do to their bodies: if it is so good at resisting bacteria and other pathogens, what is it doing to the cells of the gastrointestinal tract? This raises a point: should we be eating puroindolines, or avoiding them? If puroindolines have this ability to build ionic channels into cells, and they have this ability to form strong bubbles (virtually creating their own cell surface!), what are they doing to our gut? What are they letting through? What might they enclose when they spontaneously form, and where might they deliver this payload? It might be something as innocuous as a vitamin, or something as deadly as a cardiotoxin or neurotoxin. We don't know.
Perhaps high-protein wheat without puroindolines or with fewer puroindolines are better for you, after all, despite the wonderful texture these proteins may impart to a loaf's crumb. I throw all this speculation out there as my own puroindoline mystery.
No one knows yet, and this seems to be cutting edge science I've stumbled upon. One more thing to try to track.
Spiced Loaf and Beast Rye
I can't post a blog entry without at least some bread.
My experiments with my 'stiff starter' continue. I was going to make my recipe again, to see if I could make it slightly less gummy -- a frequent complaint I hear, whenever I make that panned sandwich bread.
So I made up my stiff starter, as usual, and left it overnight. In the morning, I had 300g of stiff starter, slightly moistened now from fermentation. It was made of 200g of starter at 100% hydration, and another 100g of ww flour.
|The odd kidney-shaped loaves are a result of the dough not fitting into the dutch oven properly|
At the same time I made a ww bread with rye. I emptied my container of ww flour, and it came to 666g. "666? Mark of the Beast Loaf" I mused. I made up the rest of the 1000g with rye flour, making it a 1/3 rye. I gave away both boules: one a rye, the other the bread-spice loaf.
R. chose the rye, D. got the bread-spice loaf by default.
"D. says the bread you gave him was one of the best ever*. He liked the spice flavour and really liked how the bread was softer - much easier to cut. Great texture etc.
* because he likes it so much he assumes you will never make it again…. LOL"
The crust *was* different. It was less crunchy, more flexible, like a good German loaf often is. I had wondered about that crust myself. Why did it happen?
Notes to Myself
- A few of the articles I've read (some I only had access to the abstracts), during
the course of my stumbling around in the dark trying to teach myself
- Boyacioglu, M., D'Appolonia, B. "Characterization and Utilization of Durum Wheat for Breadmaking. III. Staling Properties of Bread Baked from Bread Wheat Flours and Durum Wheat Flours" (1994) 71(1) pp. 34-41
- Charnet, P. et. al. "Puroindolines form ion channels in biological membranes" (2003, Apr). Biophys. J. 84(4). pp 2416-26.
- Chen F. et. al. "Physical mapping and a new variant of Puroindoline b-2 genes in wheat" (2010, Feb). I Theor Appl Genet 120(4) pp 745-51
- Clifton, L. et. al (2010). "Puroindoline-a, a lipid binding protein from common wheat, spontaneously forms prolate protein micelles in solution" (2011) Phys. Chem. Chem. Phys., 19(13), pp. 8881-8888
- Clifton L. et. al. "Single amino acid substitutions in puroindoline-b mutants influence lipid binding properties" (2007, Feb). Biochemistry. 46(8) pp 2260-6
- Day L. et. al. "Characterization of wheat puroindoline proteins" 2006, Dec) FEBS J. 273(23) pp 5358-73
- Dubreil et. al, "Localization of Puroindoline-a and Lipds in Bread Dough using Confocal Scanning Laser Microscopy" (2002) Journal of Agriculture and Food Chemistry, 50 (21) pp 6078-6085
- Dubreil, et. al. "Effect of Puroindolines on the Breadmaking Properties of Wheat Flour" March/April 1998, V. 75, N.2 pp. 222-229.
- Evrard et. al (2008) "Puroindoline-a and puroindoline-b interact with the Saccharomyces cerevisiae plasma membrane through different amino acids present in their tryptophan-rich domain". (48) pp 379-386
- Feiz et. al. "In planta mutagenesis determines the functional regions of the wheat puroindoline proteins". (2009, Nov). Genetics 183(3) pp 853-60
- Feng Chen, Fuyan Zhang, Craig Morris and Dangqun Cui, "A Puroindoline Mutigene Family exhibits sequence Diversity in Wheat and is Associated with Yield-Related Traits"
- Gasparis et. al. "The RNA-mediated silencing of one of the Pin genes in allohexaploid wheat simultaneously decreases the expression of the other, and increases grain hardness." (2011, Jul). IJ Exp Bot 62(11) pp 4025-36
- Le Bihan, T. et. al. "Determination of the secondary structure and conformation of puroindolines by infrared and Raman spectroscopy". (1996 Oct) 35(39) pp 12712-22
- Le Guerneve, C. et. al. "Interaction of the wheat endosperm lipid-binding protein puroindoline-a with phospholipids" (1998, Dec) Arch Biochem Biophys 360(2) pp 179-86
- Lesage, V. et. al. "Proteomes of hard and soft near-isogenic wheat lines reveal that kernel hardness is related to the amplification of a stress response during endosperm development". (2012, Jan) IJ Exp Bot. 63(2) pp 1001-11
- Massa A. Morris C. "Molecular evolution of the puroindoline-a, puroindoline-b, and grain softness protein-1 genes in the tribe Triticeae." (2006, Oct) IJ Mol Evol. 63(4) pp 526-36
- Mathieu et. al. (2009) "Sixty Million Years in evolution of soft Grain Trait in Grasses: Emergence of the Softness Locus in the Common Ancestor of Pooideae and Ehrhartoideae, after their divergence from Paniocoideae". Mo. Biol. Evol. 26(7). pp 1651-1661
- Mattei C. et. al. "The wheat proteins puroindoline-a and alpha1-purothionin induce nodal swelling in myelinated axons." 1998, Dec. 9(17). pp 3803-7
- Ragupathy R. Cloutier S "Genome organization and retrotransposon driven molecular evolution of the endosperm Hardness (Ha) locus in Triticum aestivum cv Glenlea" (2008, Dec) Mol Genet Geneomics 280(6) pp 467-81
- Salt L. et. al. "The identification of foam-forming soluble proteins from wheat (Triticum aestivum) dough". (2005, Apr). 5(6) pp 1612-23.
- Swan, C. et. al "Increased puroindoline levels slow ruminal digestion of wheat (Tricum aestivum L.) starch by cattle". (2006, Mar). Anim Sci. 84(3) pp 641-50
- Weiguo Jing et al in "Conformation of a Bactericidal Domain of Puroindoline a: Structure and Mechanism of Action of a 13-Residue Antimicrobial Peptide" (August 2003), Journal of Bacteriology, 185. no. 16 pp. 4938-4947