Hello, my name is John K.  Mathai. I am a student at the University of Illinois pursuing a PhD in
Nutritional Sciences under Dr. Hans H. Stein. Today, I’m going to present on protein quality and
evaluation basics, a current hot topic in human nutrition, and what we here at the U of I in the
Stein lab are working on in that field through a presentation I’ve titled Protein Quality:
Transitions in Food Protein Evaluation.

I’ll begin the presentation with a brief history of protein evaluation. You’ll notice I have the
logo of the Food and Agriculture Organization of the United Nations here, not only because I will be
citing them heavily, but also because they have proven to be  leaders in spearheading transitions in
protein evaluation methodologies. And then I have our university logo and a drawing of a pig,
because after discussing these trends, I’ll describe the work that we are doing here with pigs to
further those means.

I think first it’s important to establish what we mean when we say protein quality. A quote from an
FAO publication sums it up best: “Nutritive value of a protein depends on its capacity to provide
nitrogen and amino acids in adequate amounts to meet the requirements of an organism”. Essentially,
a protein’s value is based upon its ability to provide essential amino acids. I think everyone can
agree on this, however, when discussing protein values in humans there are certain challenges that
must be overcome. Unlike in animals, there are certain ethical, technical, and even economical
barriers that prove difficult to overcome. So, as a result, and in line with a lot of other human
biological research, the rat proved an accessible and easy alternative.
Using the rat, the first mainstream method for evaluating protein was determining the Protein
Efficiency Ratio. This method was in use as early as 1919. The ratio is simply calculated by taking
the rat’s gain in body mass over a certain time and dividing that by their protein intake. One could
then take this ratio and multiply that ratio by a Reasonable Daily Intake of said protein to
determine a Protein Rating. Now, there are a couple of caveats to this approach. First and foremost,
this method uses the rat’s growth as a marker, which means that the rat’s amino acid requirements
are determining the protein rating. Secondly, this method did not account for the maintenance
requirement of the animal. Therefore, individual protein scores could not be appropriately compared
to each other. For example, a protein with a PER of 2.0 is not necessarily twice as good a protein
source as a protein with a score of 1.0. Acknowledging these flaws, an expert consultation
conference in 1980 determined that the PER needed to be replaced.
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At that conference, it was determined that certain concepts needed to be addressed. There needed to
be some accounting for the actual digestibility of a protein, and to address the unavailability of
certain amino acids. There also needed to be a way to standardize amino acid analytical chemistry
methodology. Ultimately, it was decided there needed to be a move towards digestibility of amino
acids, as opposed to the protein as a whole. And finally, there needed to be a greater understanding
of human amino acid requirements.

As a result of these desired changes, an expert consultation was held in 1989. Several things came
out of this meeting. Standardized methodologies for amino acid analysis were determined. A reference
pattern of amino acid requirements for preschool children was established for future scoring
systems. And interestingly, fecal digestibility of nitrogen was decided upon as the correction
pattern for scoring systems. This is interesting because ileal digestibility was at the time
understood to be a better estimator for protein digestibility. This is primarily for two reasons:
one, absorption of amino acids ends at the ileum; and two, the microbiota in the large intestine
generate nitrogen and therefore alter the nitrogenous composition of the feces. However, fecal
digestibility was settled upon because methodologies to determine ileal digestibility were just not
where they needed to be at the time.

But finally, the major outcome of this meeting was that the Protein Digestibility Corrected Amino
Acid Scoring, or PDCAAS for short, was established as the successor to PER.
PDCAAS is calculating by taking the mg of the first limiting amino acid in 1 g of test protein over
the mg of the same amino acid in the reference protein and then multiplying that value by the
digestibility of fecal nitrogen. This system was a major improvement over PER. For one, it factored
in digestibility, and two it was based on human requirements for amino acids. As a result, the
PDCAAS became the commonly accepted way to evaluate proteins for over 20 years.

However, while acknowledging the strengths of PDCAAS, there were still concerns with how the values
were determined. For one, the score was based on the concentration of one amino acid, and was, as we
mentioned before, corrected by fecal nitrogen digestibility. Second, the fecal digestibility was
determined in rats, a model that was becoming increasingly questioned as to its appropriateness as a
model for human nutrition. Therefore, the pig was proposed as a superior model when compared with
rats. And finally, all PDCAAS values were truncated at 1.00 or 100%, under the assumption that
excess protein would only be excreted. However, this idea assumes that said protein source is the
only source in the diet, and doesn’t factor in how amino acids from different protein sources may
complement each other.

Therefore, in 2011, another FAO Expert Consultation was held to evaluate PDCAAS and determine
potential alternatives. At this meeting it was decided that individual amino acids needed to start
being considered as individual nutrients. Ileal digestibility was proposed as the best currently
available method for determining amino acid digestibility. Additionally, three new amino acid
reference patterns were developed: one for birth to 6 months, one for 6 months to 3 years, and one
for 3 years and above. And most importantly, the digestible indispensable amino acid scoring, DIAAS
for short, was established as the successor to PDCAAS.

DIAAS are calculated by taking the mg of digestible indispensable amino acids in 1 g of the protein
divided by the mg of the same indispensable amino acid in 1g of the reference protein and then
multiplying this value by 100. DIAAS is an improvement over PDCAAS because it corrects for the ileal
digestibility of each amino acid – meaning that each amino acid is corrected by its own specific
digestibility at the ileum. Additionally, DIAAS scores are not truncated at 100 percent. This is
important because it allows for an understanding of how various proteins can play complementary
roles to each other in the diet. It also can potentially eliminate any bias that may occur from
truncation.

Now, as the DIAAS value has only recently been adopted as the preferred means for protein
evaluation, PDCAAS will still be used until a sufficient database of DIAAS values have been
established. It is in the development of this data base that the Stein lab comes in. But before we
get into some of our current and planned research, it might be valuable to go through a brief
overview of how we here at the Stein lab determine digestible indispensable amino acid scores.

It begins with the determination of ileal digestibility of amino acids in pigs. We go about this in
the same manner as we do to determine apparent and standardized ileal digestibility in amino acids
for feed ingredients. We begin by inserting a cannula, as shown here on the right, into the terminal
ileum of a pig.

Here we see a photo of Dr. Stein and a former student performing the surgery on a pig. The middle
shows the final product immediately after surgery, and the rightmost picture is of a pig a couple
weeks post-surgery. You can see here the surgical incisions are nicely healed, the cannula is tight
and flush to the body, and the pig is behaving normally. So, by using these cannulated pigs to
collect ileal effluent samples, we can determine the standardized ileal digestibility of amino acids
in various products. 

So here we have some ileal digestibility data on soy protein isolate determined in our lab. Fairly
high amino acid digestibility all around. Just to note, SAA refers to methionine and cysteine, and
here AAA refers to only phenylalanine and tyrosine.

Using the digestibility values we just looked at, we are able to calculate the actual concentration
of digestible amino acids in mg/g of crude protein, by multiplying the digestibility of an amino
acid by its concentration in the actual ingredient.

Then, by dividing our concentrations of digestible amino acids by the values in our reference
tables, again based on human requirements, we can calculate digestible indispensable amino acid
reference ratios.

So, here we have the DIAAS reference ratios for each of the indispensable amino acids in soy protein
isolate. We see here that the lowest value is for sulfur amino acids. So, by taking this value and
multiplying it by 100 we have the DIAAS score for SPI: 98%, as determined by its first limiting
amino acid or in this case, acids, the sulfur amino acids. So this was just an example from some of
our data.

But we have also done some work in other food products. Here we have some data for various cereal
grains used as food sources throughout the world. We can see here that as far as amino acid quality,
for the most part, grains are not a good protein source. In light of this data, we had the questions
as to how the DIAAS system represents the protein quality of various plant and animal proteins.

So here we have some brand new data comparing animal and plant proteins. On the X axis, going from
the left we have whey protein isolate, whey protein concentrate, milk protein concentrate, skim milk
powder, pea protein isolate, soy protein isolate, soy flour, and wheat. The first limiting amino
acid for each of these ingredients is listed in the table to the right. From this data we can see
that it appears that, by and large, the sources of animal protein (on the left hand side of the
table) have greater amino acid quality when compared with the plant sources (the sources on the
right side of the table). Now this is just a brief look at this data, but you can see more on this
data in a future publication, and perhaps, a future podcast.

Currently we have plans to do more DIAAS work. Particularly we have begun work on an experiment to
determine the effects that cooking may have on the DIAAS values in pork roast. We also are planning
on being a part of a large collaborative work to determine the DIAAS values in 100 different food
proteins in order to further develop the DIAAS database. We also see some potential opportunities
for the future. We see opportunities in understanding how different ingredients can come together to
provide amino acids in complete diets. And also, we see a need for the further development of amino
acid reference patterns for humans at different stages of life.

I hope this presentation was helpful in understanding where protein evaluation is today and also in
giving you a snapshot of some of the projects we are working on here at the Stein lab! I’d like to
thank you for your attention and to let you know that if you enjoyed this presentation, or wanted to
find more information, visit us at nutrition.ansci.illinois.edu.