Pea Protein, do it yourself

Introduction

Pisum sativum is the Latin name for the yellow garden pea from which “pea protein isolate” is extracted. Knowing the Latin name helps us in our search of PubMed, the US patent database on ways we can bypass those overpriced supplement stores.  The blog offers a nerdy overview of pea proteins, how certain preps may have good food chemistry properties, but may leave you deficient in certain amino acids.  You can have those creamy shakes and use the dregs to DIY veggie burgers.

Pea protein is complete “baby” plant protein

The cotyledons of Pisum sativum, in some obscure way, serve the same purpose for the germinating seedling as mammalian milk serves for the new baby. Both contain fat, carbohydrates, and complete proteins to provide for the new offspring until it can fend for itself. Recent interest in plant based diets have fueled the “pea protein vs. whey protein” debate. Is pea protein complete? Is whey protein complete? Whey is the watery part of milk that remains after the curds have coagulated in the process of cheese making. Lactalbumin, minerals, and traces of fat are what remain in the whey.      Whey proteins are quite incomplete compared to what comes out of a mammary gland to nourish the baby mammal.  By analogy, those pea protein powders that consumers add to beverages may be incomplete. Commercial pea protein isolates are quite incomplete compared to what one finds in a seed to nourish the developing pea embryo.  Do we DIYers really care when we can use the pea protein okara for other purposes?   

What is Pea Protein Isolate?

And Why bother? Peas are a member of the nitrogen fixing Leguminosae family which contain a significant portion of flatulent sugars such as raffinose, stachyose, and verbascose. Sure, we can get pea proteins by eating unprocessed peas.  As DIYers we can avoid offending  our office and gym mates.  A 2019 US patents uses lactic acid bacteria fermentation to remove the flatulence producing carbohydrates. A 2015 patent uses methods of selectively dissolving and precipitating pea proteins. These are just a few US patents protecting methods for isolating proteins we want from carbohydrates that we do not want. Dry versus wet extraction in relationship to those flatulent carbohydrates will be discussed later.   We humans have been using these lactic acid bacteria to metabolize the milk lactose that we often lose our ability to digest as we grow up.  Why not do the same with peas?  This patent is claiming to make pea yogurt before isolating the proteins.  If one is into probiotics, just make pea yogurt and skip the protein isolation part.  Another DIYer twist might be to add sour dough starter to milled peas.   (USPO, USPO, Bakerpedia)

Pea History

The garden pea Pisum sativum was domesticated in the near east in the Fertile Crescent about 11,000 years ago. Records suggest that Neanderthals were consuming wild peas about 46,000 years ago.

• Scientist were studying glutathione and non protein sulfur in peas back in 1926.
• In 1934 pea meal was being investigated as a means of boosting the protein content of wheat and rye bread.
• Efforts were underway in 1948 to separate constituents of Pisum sativum seeds. These peas were also as a source of muscle building growth in swine. Deficiency of Pisum sativum cotyledons in sulfur containing amino acids was a concern back in 1983. In the fully developed cotyledon, cysteine and methionine were present at 1.4 and 1.2 mol% of total NaOH extractable proteins. Changes in free and protein bound sulfur containing amino acids were found to fluctuate in the cotyledon as a function of days after flowering. A 54 residue peptide was sequenced in 1985. It contained no methionine but was found to be a significant source of cysteine. The same year an albumin was characterized and found to contain a large number of hydrophobic residues and only a few methiones and cysteines.   In wake of how easy it is total to sequence the genomes from which proteins are derived, this is ancient history.    (ThoughtCo, xPubMed , x,  x, x, PubMed , x , x, PubMed)

What is Pea Protein? 

 Pea protein includes four major classes of proteins: globulin, albumin, prolamin, and glutelin. The proportions of these proteins and how much they are disulfide cross linked may vary from one strain (genotype) to another.  As we shall see, glutelin of cereal grain fame is pretty close to legumin.  (PubMed, PubMed)

11S Globulin,

also known as cruciferin, is a storage protein found in many plants including Arabidopsis thaliana, thale crest, a crucifer.    We know a little bit about the biology of cruciferin in the thale crest.  This one gets sticky.  We have a huge family of related plant proteins.    

Albumin

Seven amino acid sequences are available for albumin in Pisum sativum in the UniProt database. Albumin 1A-F are 130 amino acid peptides that may be toxic to some insects by binding to proteins in their gut walls. This is only based on amino acid sequence similarity.  These small proteins have six cysteines involved in double bonds. Albumin 1 binds to globulin.   A PubMed reference suggests that albumin may be used as a foaming agent.  Okay DIYers, the relevant part of this is that these protein interactions may be dissolved by going raising the pH such that positively charged amino acids like lysine and arginine are uncharged.    (PubMed, UniProt )

Prolamin

is a cysteine rich 146 amino acid peptide in  the model legume Medicago truncatula . Cyseine is also found in rice prolambin.  

Glutelin = Legumin.

Glutin in cereal grains is composed of two fractions:  the gliadin and glutelin.  The gliadins are soluble in ethanol/water mixes.  The glutelins are not soluble in much of anything. The glutenin fraction is a disulfide cross-linked polymer of 500,000 to more than 10 million Da.   After reduction of disulfide bonds, the resulting glutelin subunits become more soluble.  Most research concerns gutelin as a storage protein in rice.  A rice glutelin was blasted against the Pisum sativum database.  The acidic subunit of Legumin J showed about 38% homology as did other legumin sequences.  .  ( PubMed,  x )

is a family of globular proteins of about 520 amino acids.  About four cysteines help maintain the globular structure by forming double bonds.   Legumins have acid and basic amino acids in their globular structure. (UniProt)  DIYers take note:  Pea legumin has some notable regions of homology with rice glutinin.  If we are crafty we can make pea proteins nice and gooey like high glutin rice.  

Lectin

Lectins are carbohydrate binding proteins.  A split green pea lectin tetramer has been isolated and shown to bind glucose and sucrose on the basis of hemagglutinating inhibition.  This protein was purified on a DEAE cellulose anion echange column.The UniProt sequence suggests that the tetramer is infact a dimer that has been post translationally cleaved into two subunits.

Okay DIYers, let’s forget the story about whether or not we have any sulfur containing amino acids in this protein.  We have two features for us to exploit:  (1) It is soluble meaning we can isolate it with water, the pH to be determined.  (2) It binds sugars that likely include starch.   Here we have our thickening agent.

Vicillin, convicillin, and lipoxygenase

These three proteins have been obtained with a pH 9 Pisum sativum using a pH 9 extraction.   This prep also yielded some legumin.   The authors used NaOH (Draino) rather than Ca(OH)2  (lime).  They lowered the pH to 4.5, chilled, and reextracted at pH 9.  The solution was precipitated a second time at pH 4.5.  This precipitate was resuspended at pH 3, 5, 7, and 8.  These authors used HCl (pool acid) to adjust the pH.   was Pure sunflower oil (15 mL) and 45 mL 1.0 g kg⁻¹ protein isolate suspension and  homogenized in a mechanical homogenizer.  We can use a blender.   pH 5 yielded the poorest emulsion.  So DIYers, best to stick to pH 7-8 water for your home made coffee creamer.    pH 5 final re suspensions made for the worst foaming agents.  In a simple sense, the authors bubbled air through the suspension to create a foam.  If we were making our own protein whipped topping, we’d probably want to add sweetener of some sort.  In case you are wondering, lipooxygenases catalyze the oxidation of fatty acids.  This could be a liability in making coffee creamer.

Chymotrypsin/trypsin inhibitors

A search of Pisum sativum seed  proteins yields many sequences of a chymotrypsin/trypsin inhibitor. If this/these inhibitors are not digested in the stomach, the digestion of pea proteins in the small intestine may be limited.  It is also hard to reconcile reports of amino acid deficiency in reports from the middle part of the last century to the post genome sequence database of today. Cysteine and methionine may always be below the expected 5% mole percent for the 20 amino acids. What does this mean for human nutrition? (x)

Alternative to whey protein (legumin = similar properties to casein)

or so a Wikipedia quote attributed to Gustus von Leibig claims. This  scientist lived from 1803-1873 A Blast of Pisum sativum legumin against bovine (Bos taurus) protein sequence database failed to reveal any similarities. The UniProt.org/ align algorithm was used to manually align bovine alpha casein and pea legalbumin A. There was no sequence homology. Even if the amino acid content is close, the structures can be expected to be totally different just because the sequence of those amino acids is different.(Wiki, NCBIprotein)

We DIYers have got to move past the supplement industry hype.  If we are lazy we can just make some pea milk and save the okara for veggie burgers.  This blog will hopefully provide some insights into the food chemistry if we want to really get into it.  Hopefully you  have some ideas about plant proteins.  Let’s look at ways that industry deals with something we’ve been eating for over ten thousand years.

How is Pea Protein Made? 

Extracted from yellow pea

Companies that sell pea protein powders might not always tell the customer how they make it. The US Patent website is a good place to to investigate the manufacturing process of pea protein. One of the earlier patents was filed in 1977 by the A. E. Staley Manufacturing Company (Decatur, IL). This patent was intended for any vegetable protein flakes that could be soy, yellow field pea, or peanut. The oils and soluble carbohydrates are extracted with an aqueous ethanol solution. A 2014 patent was awarded to Frenchman Bernard Boursier for a method that uses pH to selectively isolate pea proteins from carbohydrates and other materials. A 2019 patent awarded to a group from Belgium claimed to remove the flatulence causing carbohydrates with a lactic acid bacterium fermentation step prior to milling. Bakerpedia simplifies the extraction process of sifting a dry flour to remove the starch particles to a water, sometimes alkali, extraction. ( x , x, x   , x )

Foods it’s included in

Bakerpedia claims that pea proteins are a good substitute for egg protein in baked goods. Research suggests that is can be a good emulsifying agent for making coffee creamers and mayonnaise.  Foaming agents can be used to make whipped cream. 

Just like making hominy

Those familiar with this art soak the dried corn kernels in an alkali solution before cooking in a process called nixtamalization.  The authors of this Wikipedia link claim that nixtimalization is needed to dissolute the hemicellulose.  It should also be noted that the alkali soak should be at a pH above the isoelectric point of lysine and arginine.  Nixtimalization is needed to get the corn kernels to swell in the cooking process.  Go to your local garden or hardware store and buy some lime.  A teaspoon should get 2-4 liters of water and corn to the right pH.  If in doubt, Ebay sells some really cheap pH paper.  This cheap pH comes in handy if you want to bring the pH back down after dissolving the pea proteins.

What is Pea Protein Isolate? 

Groan…. with nuances of the pH of water used for extraction and fractionation of the resulting “milk,”  pea protein isolate is often the equivalent of soy milk.  

Used by meat-alternative manufacturers 

A food industry market watch group reports several companies using pea protein in vegetarian burgers.  Concern about the health effects of genetically modified soy is fueling the demand for pea protein. Here is where the DIYer needs to pause and think about stuff.  Cellulose fiber might be good for holding the DIY veggie burger together.  The flatulent sugars raffinose, stachyose, and verbascose are maybe less desirable.  The goal would be to find a good brand of Greek yogurt that contains a microbiota that can take care of those unnecessary carbohydrates.  Save that pea milk okara!  ( x ) 

What are the Benefits of Pea Protein? 

Pea protein(s) in this section will be primarily discussed as a nutrient rather than individual proteins with may have some functional value in food physical chemistry, e.g. as emulsifying agents.  What is pea protein good for?

Food chemistry

It is also a humectant to keep foods moist, oil absorption agent for baked goods, and gelling agents. Soy proteins can act as emulsifying agents allowing oils to mix with water. Egg yolk is another such example in the making of mayo. Some pea proteins are foaming agents. Others are filming agents in foods such as meat, fish, soups, and sauces. Pea proteins are even making their way into coffee creamers. One has to wonder if a pea protein dietary supplement could have multiple uses in the kitchen and by the office coffee pot. It is beyond the scope of this article to get into the nitty gritty of food and industrial uses of each pea protein. It is now becoming possible to purify these proteins by biochemical techniques. (x, x , x)

As a supplement 

Pea protein may be trending as a nutritional supplement. Established Vegan may find this laughable.  Peas are food and have been for tens of thousands of years.   

Is pea protein complete?

Yes, yellow pea protein is complete. A central issue is how bio available those amino acids are. Bio availability of pea proteins was addressed in a patent awarded to the Innovative Protein Holdings LLC of  South Dakota. These authors added an alkaline proteolytic digestion step to some of the traditional methods of extracting pea proteins. (x)

What amino acids are in pea protein?

Innovative Protein Holdings analyzed essential amino acid composition of yellow pea protein using two of their methods and the methods of five of their competitors. They also listed the World Health and Food and Agriculture Organization standards (FAO/WHO) for older children, adults and young children up to three years of age.  FAO/WHO recommends 23 mg cysteine + methionine for every 1000mg of total protein for adults and children over three years of age.  Infants six months and younger require 27 mg per 1000mg total protein.    Innovative Proteins came up with a protocol that yielded 27 mg of methionine + cysteine per 1000mg total pea protein.    The lowest ranking competitor’s product had only 16 mg methionine + cysteine per 1000mg total protein.  

Here are all of the data presented in this patent:

• amino acid (>3yr –  <3yr,  lowest yield -IP yield)
• methionine + cysteine (23-27, 16-27), 
• tryptophan ( 6.6-8.6, 7-10.6),
• threonine (25-31, 31-41),
• isoleucine (30-32, 44-48),
• leucine (61-66, 78-84),
• phenylalanine + tyrosine (41-52, 80-106),
• valine (40-43, 47-50),
• histidine (16-20, 24-25).  

Except for sulfur containing amino acids cysteine and methionine, all six methods yielded complete essential amino acids for adults to nutrient demanding  six to 36 month old children. Pea protein has all of the essential amino acids. Depending on the method of isolation, it can meet WHO/FAO standards even for babies. The Innovative Proteins method has an impressive amount of leucine that might be of interest to some consumers. (Innovative Proteins Patent)

Essential vs. non-essential amino acids (brief description) Essential amino acids are ones that we cannot synthesize on our own.

Complete vs. incomplete amino acid profile was exemplified with excerpts from the Innovative Proteins patent.  What makes this Innovative Proteins method unique is that it goes or the total pea proteins in peptides rather than selectively isolating proteins that might have culinary appeal. (x)

Branched-chain amino acids are leucine, isoleucine, and valine. ( x )

Muscle Protein Synthesis 

• Protein = muscle building . Dietary protein stimulates muscle growth as well as supplying the raw materials. This is a nice review that offers insight on the timing of protein ingestion for optimal muscle protein synthesis. ( x )
  
• Muscle amino acid profile Muscle contracts by movement of myosin on actin filaments with regulation by other proteins. Let’s do a thought experiment and a keyword search on the sequence of human skeletal muscle myosin 1. Myosin 1 is a protein with 1939 amino acids. When we do a keyword search for cysteine using the single letter amino acid code “C” we find about 17 in the total 1939. This is less than 1%.   Myosin has about 55 methionines. Let us look at actin in this thought experiment. Actin is another major muscle protein. Actin is a giant polymer of small, globular protein with only 377 amino acids. Actin has 6 cysteines and 17 methionines. ( x , x )
  
• Research / evidence Dietary protein and essential amino acids may ameliorate muscle loss in conditions such as immobilization and aging. The amount of dietary protein needed to stimulate muscle protein synthesis appears to be greater in older men than younger men. One way to maximize muscle protein synthesis is to increase protein intake at breakfast and lunch in addition to the evening meal. ( x , x,   x )

Why Take Pea Protein? 

• Why eat peas anyway? Why not? We’ve been eating peas for carbohydrates and proteins for thousands of years. It is only in the past hundred years that we’ve recognized that plant protein can augment or substitute for animal protein. We have recently discovered ways of extracting pea proteins in ways that may afford the same food chemistry attributes as animal proteins. There may be other times when we just want to eat peas for the protein content and skip the carbohydrates.
  
  

  Absorption rate / satiety

One issue affecting absorption is digestion of the pea proteins into peptides. Pea globulins are readily digested but are deficient in sulfur containing amino acids. Pea albumins are a rich source of sulfur containing amino acids but are modest trypsin inhibitors. Trypsin is a proteolytic enzyme of the small intestine. Pea proteins were concluded to have a similar nutritional value to soy proteins as soy also has a trypsin inhibitor. A study from the Netherlands tested the direct administration of undigested protein directly to the duodenum on satiety. The duodenum is the first part of the small intestine that sees a meal as it exits the stomach. The authors reported that digestion of pea proteins is 93% complete in the stomach. Further, they found that whole pea proteins pumped into the duodenum elicit a greater satiety than pea proteins partially digested in the stomach. Whether pea proteins are taken for nutrition or satiety may depend on the formulation: (1) How much do the proteins aggregate with each other in the stomach and thus avoid digestion? (2) Have the soy proteins been subjected to an alkali digestion that could compensate for reduced trypsin digestion in the small intestine? A 1997 study addressed the issue of digestion of “heat treated” flour from peas grown in radioactive [14N]. The limited study of seven subjects indicated good digestion and absorption. Was the heat treatment needed to denature the chymotrypsin/trypsin inhibitor so that digestion could occur in the stomach?  Perhaps non heat treated trypsin inhibitors digest just fine in the stomach. Are trypsin inhibitors in seeds like pea and soy there to aid in dissemination by the animals that eat them? ( x , x , x , x)

Pea protein equivalent of soy okara?

It is hard to really relate these data to the creamy, water soluble pea protein powders we so dearly love in our beverages. If they are truly soluble and/or dispersible in water, will they survive our stomachs as whole proteins? Those of us who have made our own soy milk also know about the okara, the high fiber, insoluble residue.. Okara is not only a good addition to soups and such as that, it is also a rich source of cysteine rich,insoluble soy proteins that never make it into the soymilk. No research was found as to whether the soy proteins of okara are good appetite suppressants. Does the pea protein industry have an okara equivalent? Perhaps we will one day see a pea protein bar on our grocery shelves saying, “*contains appetite reducing, muscle building” sulfur rich amino acids.  Right now those bars only say “pea protein” so we do not know. … unless of course it is onn GMO! ( x )

Pea Protein vs. Whey Protein

Amino acid contents

The following data are from a study comparing pea protein vs. whey protein in high intensity functional training (HIFT) muscle development.   Free Paper.  They used True Nutrition, a pea protein isolate that the customer is instructed to add to a shake.  

“True Nutrition’s Pea Protein Plus is sourced from high-quality raw ingredients, using only vegan and non-GMO peas. Pea Protein Plus is processed using cold-water microfiltration, and provides 24g of vegan protein to help support muscle-building!

• Uses: Supports Muscle Building: Provides 24g of protein to help support muscle building!
• Mix-ability: Shake or stir thoroughly for best results.
• Taste: Mild earthy taste that goes best with chocolate, vanilla, mocha, and caramel flavors!
• Notes:Vegan, Dairy-free, Soy-free, Gluten-free

Directions  Mix 1 Serving (About 1 Scoop) in 8-12floz of water, juice, or milk. Stir or shake, and enjoy!”

This is a not so expensive product from China.  The cold filtering part tells us that they are probably leaving behind some insoluble sulfur containing amino acids. This product is not that deficient.   There was not significant difference between whey and pea supplements in this study.  A DIYer would wonder if the dregs of this products are fed to Chinese hogs and chickens to bulk them up.  Make your own and use the dregs in your veggie burger!

Leucine distribution

is of interest to those interested in increasing muscle mass. Leucine  signals the cell to ramp up the synthesis of new proteins and inhibits the catabolism of existing proteins. Leucine signals the cell to ramp up energy metabolism to support this protein synthesis.    Leucine is a precursor to α-ketoisocaproate (α-KIC) and β-hydroxy-β-methylbutyrate (HMB) in skeletal muscle. The mechanisms of HMB in promoting athletic performance have been reviewed elsewhere. The bottom line is that pea protein is almost identical to whey protein in its leucine content. ( PubMed )

How to Use Pea Protein ?

This one seems to be a gimmick of those companies catering to body builders.  For those DIYers practiced in the art of making soy milk, making pea milk is with in the realm of our skill sets.  We can bypass these supplement stores.  What we must never do is throw away the pea milk okara.  By analogy this is a rich source of sulfur containing amino acids that we will need to build muscle mass.

Novozymes and your own pea protein digest

The alkali extraction and the brilliant Innovative Proteins patent that gives us an incredible extraction of cysteine and methionine has been covered.  This patent never quite names the alkali proteases as trypsin and chymotypsin.  The DIYer can buy these enzymes as porcine preparations, but we will not even go to this site because it is neither cheap nor vegan.  Novozymes sells bacterial versions of these enzymes:  Formea® TL 1200 BG Trypsin-like bacterial protease, granulate and Formea® CTL 600 BG Chymotrypsin-like bacterial protease, granulate.  Novozymes has a lot of interesting things on their site that suggest the wrong use of enzymes may create off flavors.  They don’t seem to be selling directly to consumers of of now.

Possible Dangers of Pea Protein 

• Flatulence  is a side effect if the polysaccharides are not properly removed.   The authors that claimed that poorly digested pea protein entering the duodenum may act as an appetite suppressant.  If it does not get digested in the small intestine either, it may cause flatulence in the colon.  Novozymes starch solutions are already in place for corn and wheat industrial markets.  A DIYer may dream of buying some starch/fiber digesting enzymes to maximize the protein protein yield and minimize the intestinal gas of those DIY veggie burgers.    Forth coming in this blog will be the use of lactic acid bacteria to digest these carbohydrates.
• Allergies  Those with peanut allergies may be sensitive to proteins in lentil and pea proteins. When one considers specific proteins, those with lentil allergies may also be allergic to Pisum sativum convicilin (63 kDa), as well as vicilin (44 kDa) and one of its proteolytic fragments (32kDa). Smaller proteolytic fragments might not be as problematic. ( x )
  
• How to potentially avoid side effects Avoid eating peas if you are allergic to peanuts, lentils, or other legumes you may wish to avoid pea protein.

Keep checking back.  I’ve sent a few inquiries to Novozymes.  I’ve used their enzymes in the past or other things.  They may never be ready  to market to individual consumers.  Pea protein could be an awesome substitute for the soy that many shy away from due to fear of the foreign proteins in commercial soy genomes. Contact me if you’d like to be kept in the loop.

Post note.  I alkali treated some milled yellow peas, adjusted the pH back to neutral.  This was fermented for three days with Teraganix EM-1 probiotics.  After cooking, this material had a strong cheese flavor.  Next time I swill sift out the larger fiber particles.