Hello, my name is Hannah Bailey, and today I will be talking about the new methodology developed to evaluate protein quality in processed human foods. This methodology is called the digestible indispensable amino acid score, or DIAAS as I will be referring to it for the duration of this presentation.

The DIAAS methodology was developed by the Food and Agriculture Organization of the United Nations (abbreviated FAO), and you can see their logo in the upper right-hand corner. Prior to DIAAS, the FAO had developed the protein digestibility corrected amino acid score, or abbreviated PDCAAS. The PDCAAS methodology has been used for over 25 years to evaluate the protein quality of human foods. However, many limitations have been identified, and therefore DIAAS was developed to replace PDCAAS. The main differences between these two methodologies are as follows: The PDCAAS methodology uses rat fecal digestibility, whereas DIAAS uses pig ileal digestibility values. This is important because we know amino acids are completely absorbed by the end of the small intestine. In addition, when analyzing protein in the feces, the value will be overestimated due to the effect of microbial protein. Also, the FAO has determined the pig as the most appropriate model when evaluating protein digestibility for humans. Secondly, PDCAAS uses a single crude protein digestibility value, whereas DIAAS utilizes the digestibility of each individual amino acid. This is important because amino acids differ in their digestibilities, and the requirements differ for each individual amino acid. Thirdly, PDCAAS uses one reference scoring pattern for children 2 to 5 years of age, whereas DIAAS uses three reference scoring patterns: one from birth to 6 months, 6 months to 3 years, and one for children older than 3 years, adolescents, and adults. Now although DIAAS has become more specific in its reference patterns, it still fails to recognize the different amino acid requirements for pregnant and lactating women, the elderly, and the malnourished population. Lastly, DIAAS no longer requires values greater than 100 to be truncated. This enables the identification of complementary proteins, which I will give an example of later on in this presentation, as well as the determination of DIAAS for mixed diets.

As I mentioned, the FAO has identified the pig as the most appropriate model when determining protein digestibility for human foods. So how do we determine DIAAS in the pig? First, a T-cannula is surgically inserted into the distal ileum of the pig. The pigs respond very well to this surgery and as you can see by the second picture, the pig is up and acting natural with no irritation around the surgical site. During experimental periods, we allow the pigs 5 days of adaptation to the diet and 2 days of collection. During collection days, the ileal digesta are collected for 9 hours each day and this is done by removing the caps and securing a 250 mL plastic bag to the cannula barrel via zip tie. The ileal digesta flowing into the bag are then collected. The bags are removed when filled with ileal digesta, or at least once every thirty minutes, and immediately stored at -20 degrees Celsius to prevent bacterial degradation of amino acid.

Now let’s move on to how we actually calculate DIAAS. First, we analyze the concentration of amino acids in the test ingredient. Then, as previously explained, we feed this test ingredient to a cannulated pig and collect the ileal digesta. This ileal digesta is then analyzed for amino acids to enable the calculation of standardized ileal digestibility, or SID, of amino acids in the test ingredient. These two steps complete the lab and animal work that is required for DIAAS. The rest of the steps in determining DIAAS are simply calculations. Next, the concentration of amino acids in the test ingredient is divided by the SID of the same amino acid in the test ingredient, resulting in the concentration of digestible indispensable amino acids in the test ingredient. The concentrations of digestible indispensable amino acids are then divided by one of three reference scoring patterns, resulting in the calculated digestible indispensable amino acid reference ratio. And the amino acid with the lower ratio is what determines the DIAAS for that test ingredient.

Now, to help make this calculation a little clearer, I will walk you through an example. For this example, we will determine DIAAS for wheat for persons older than 3 years. Wheat is a cereal grain, and we know that cereal grains are first limiting in lysine. So, for the sake of simplicity I will only be showing the calculation for lysine; however, it is important to note that all indispensable amino acids are required for this calculation. For the first step, the amino acid concentrations are analyzed in wheat and it is observed that the concentration of lysine is 33.4 mg per g of crude protein. Next, wheat is fed to a cannulated pig and the SID of lysine is determined to be 77%. The concentration of lysine in wheat is then multiplied by the SID of lysine, and this results in the concentration of digestible lysine in wheat to be 26 mg per g of crude protein. The concentration of digestible lysine is then divided by the lysine reference pattern determined by the FAO, for persons older than 3 years, which is 48 mg per g of crude protein. When these two values are divided, the resulting value is the digestible indispensable amino acid reference ration for lysine, which is 0.54. Now as I mentioned, we know lysine is the first limiting amino acid in wheat. Therefore, the DIAAS for wheat is 54%.

Once a food item has an established DIAAS, a nutrition claim may be made about the food item’s protein quality. The FAO has established two protein claims that can be added to a food item’s label based on its determined DIAAS. If the DIAAS is greater than or equal to 100, the food item can be claimed as an excellent source of protein for that specific age group. For a food item to be claimed as a good source of protein for a specific age group, its DIAAS needs to be between 99 and 75. And if a food item has a DIAAS less than 75, there can be no claim made about its protein quality. For example, wheat had a DIAAS of 54, therefore, no claim can be made about the protein quality of wheat. These DIAAS ranges are important because a DIAAS greater than or equal to 100 indicates the potential of that protein to complement lower quality proteins, such as wheat. And I will give an example of this later on in the presentation.

Since the establishment of DIAAS in 2013, many proteins have been analyzed and assigned a DIAAS. For example, DIAAS has been determined for a number of cereal grains, plant proteins, and dairy proteins. In addition, DIAAS was determined for one muscle cut of beef that underwent a few different processing methods. However, pork has yet to analyzed for DIAAS, and it is very important to analyze different processing methods that animal proteins may undergo, because humans rarely eat raw meat.

I mentioned that since 2013 a number of proteins have been assigned a DIAAS, and I will give a few examples of those here for persons older than 3 years. The animal proteins are shown in blue, the plant proteins are shown in orange, and cereal grains are in gray. The animal proteins, for example milk, whey, and beef, generally have DIAAS greater than 100. And if you recall, a DIAAS greater than 100 means these proteins are of excellent quality. Plant proteins, for example soy and peas, generally are observed having DIAAS between 99 and 75. That means these proteins generally are good sources of protein. Now, the cereal grains, for example oats, rice, and corn, generally have DIAAS less than 75 and, therefore, need to be complemented by a higher quality protein in order to supply adequate amounts of amino acids to humans.

The Stein Monogastric Laboratory recently determined DIAAS for beef and pork products. We thought these animal proteins were important because pork is the most widely consumed meat in the world and beef is the third most widely consumed meat in the world. For the experiment, we wanted to test the hypotheses that both pork and beef products have DIAAS greater than 100, and that processing will increase the DIAAS of these products.

For the first experiment, we analyzed three cuts of pork: the pork belly, pork ham, and pork loin. Within each cut, we analyzed three different processing methods. For the pork belly we analyzed it in its raw form; as a cured and smoked product, but still uncooked; and as a cured, smoked, and fully cooked product. For the pork ham, we analyzed it in its uncured form, cured with celery salt, and cured with Prague powder. In addition, all of these pork hams were fully cooked to approximately 73 to 74 degrees Celsius. For the pork loins, we wanted to evaluate the effect that temperature may have on protein quality, so analyzed loins were cooked to 63 degrees Celsius, 68 degrees Celsius, and 72 degrees Celsius.

Moving on to the results of the first experiment. For the sake of time, I am only going to show the DIAAS results determined for persons older than 3 years, but please note that the results for children 6 months to 3 years follow the same trends, and the DIAAS for the age group birth to 6 months was not determined because this group does not commonly eat meat products. For the pork bellies, the smoked-cooked belly had the greatest DIAAS of 142, while raw belly had a DIAAS of 119 and smoked belly had a DIAAS of 117. For the pork hams, the ham cured with celery salt (or the alternatively cured ham) had the greatest DIAAS of 133. However, I want to point out that the conventionally cured ham with Prague powder and the alternatively cured ham had numerically greater DIAAS than the uncured ham, suggesting that curing may increase the protein quality of pork hams. For the pork loins heated to different temperatures, the loin heated to 63 degrees Celsius had the greatest DIAAS of 133, whereas the DIAAS of the 68- and 72-degrees loins did not differ. The amino acid in least concentration when compared with the amino acid requirements of persons older than 3 years, and that ultimately determined the DIAAS for all these products, was valine.

For this first experiment with the pork products, we were able to conclude that based on the DIAAS, all of these products can be considered excellent quality protein sources for the specific age group. In addition, the amino acid in least concentration when compared with the human amino acid requirements was valine for all pork products. For processing, we can conclude that overheating may reduce DIAAS, as we observed in the pork loins. In addition, there may be a positive effect of curing and moderate heating on DIAAS, as observed with the pork hams and loins, respectively.

Moving on to the second experiment, we analyzed eight different beef products. Again, we looked at three types or cuts of beef, ready-to-eat products, ground beef, and ribeye roasts. Within each cut we evaluated different processing methods. For the ready-to-eat products, we evaluated salami, bologna, and beef jerky. For the ground beef, we evaluated it in the raw form and fully cooked form. For the ribeye roasts, they were heated to different internal temperatures, similar to how the pork loins were heated. We evaluated the ribeyes heated to 56 degrees Celsius, 64 degrees Celsius, and 72 degrees Celsius.

Now, moving onto the results. Again, I will only show results for DIAAS calculated for persons older than 3 years, but we saw similar trends for DIAAS calculated for children 6 months to 3 years.  Looking at the ready-to-eat products, bologna had the greatest DIAAS; however, salami and beef jerky also had a DIAAS well above 100. We observed something different with the ground beef. The cooked ground beef had a DIAAS less than 100 and the raw ground beef had a very high DIAAS of 121. Because the beef was ground prior to heating, the surface area of the product increased, causing greater heat damage to the proteins and resulting in the significant decrease in DIAAS for cooked ground beef compared to raw ground beef. For the ribeye roasts, the ribeye heated to 64 degrees Celsius had the greatest DIAAS—and if you recall, the pork loin heated to 63 degrees Celsius also had the greatest DIAAS among the heated loins, suggesting that moderate heating of intact meat may increase the protein quality of those products. The amino acid in least concentration when compared with the amino acid requirements for persons older than 3 years varied slightly among products. Leucine was the amino acid in least concentration for bologna and the two ground beefs, sulfur amino acids were in least concentration for beef jerky, and valine was the amino acid in least concentration when compared with human amino acid requirements for the remaining products.

For this second experiment, we can conclude that based on the DIAAS, these beef products are generally excellent sources of protein. And the amino acids in least concentration when compared with human amino acid requirements are leucine, valine, and sulfur amino acids. For processing, there was a negative effect of grinding and overheating on DIAAS. In contrast, there was a positive effect of curing, drying, and moderate heating on DIAAS.

Moving on to protein complementation. If you recall, I mentioned that food items with a DIAAS greater than or equal to 100 indicates the potential of that food item to complement a lower quality protein. This concept is especially important for developing countries that rely heavily on cereal grains, which supply a low-quality protein, or an unbalanced amino acid pattern.

I am going to give an example of how two proteins can complement each other. This bar graph will show the digestible indispensable amino acid reference ratios for all amino acids for cooked ground beef and polished white rice. If you recall, the cooked ground beef evaluated in the second experiment had a DIAAS of 99, and polished white rice has been previously evaluated and assigned a DIAAS of 64. Rice is a cereal grain, so we know it is first limiting in lysine. In contrast, beef has a greater concentration of lysine resulting in a reference ratio well above 1 or 100 for lysine. However, the cooked ground beef has low concentrations of leucine and valine. But the polished white rice has a leucine and valine reference ratio above 1 or 100. Therefore, when we combine these products together, the amino acid patterns will complement each other and result in a DIAAS greater than 1 or 100 for this mixed meal. Now it is important to point out that DIAAS does not take into consideration quantity. So, if a human consumes a spoonful of this mixture, it is most likely not meeting 100% of their amino acid requirements. However, DIAAS does show that this food combination supplies a balanced amino acid pattern.

Overall, we can conclude that pork and beef products generally have DIAAS greater than 100, meaning they are excellent quality protein sources; that curing and moderate heating of pork and beef may increase the protein quality of the end product; and that pork and beef may be used to complement lower quality proteins to ultimately have a mixed meal that is balanced in indispensable amino acids.

With that, I would like to thank you for listening to this podcast, and if you would like to know more about this topic, or know more about nutrition in general, I encourage you to visit our website at nutrition.ansci.illinois.edu.