Hello, my name is Hannah Bailey, and I’m a PhD student working under Dr. Hans Stein at the University of Illinois. Today I will be reviewing current data from our lab for digestible indispensable amino acid score, or DIAAS as I will be referring to it for the duration of this presentation, that have been determined for human foods. The DIAAS methodology was developed by the Food and Agriculture Organization of the United Nations, or abbreviated FAO, and you can see their logo in the bottom left-hand corner. Prior to DIAAS, the FAO had developed the protein digestibility corrected amino acid score, or abbreviated PDCAAS. In 2013, DIAAS was announced as the successor of PDCAAS with three new human amino acid reference patterns developed and the recognition of the growing pigs as the more appropriate model for humans compared with the rat. The main differences between the PDCAAS and DIAAS methodologies are as follows. The PDCAAS methodology uses rat fecal digestibility values, whereas DIAAS uses pig ileal digestibility values. This is important because, as 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 a superior model to the rat 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 requirements differ for each individual amino acid. Thirdly, 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. Lastly, 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. As I mentioned, the FAO has identified the pig as the most appropriate model when determining protein digestibility for human foods. To determine DIAAS in the pig, we first have to determine the standardized ileal digestibility (or SID) of amino acids for a particular ingredient. We do this by first surgically inserting a T-cannula into the distal ileum of the pig. The T-cannula is shown in the image on the left, and these can vary in size depending on the weight of the pig. The pig responds very well to this surgery and as you can see by the picture in the middle, the pig is up and acting natural with no irritation around the surgical site. During experimental periods, we allow the pig 5 days of adaptation to the diet and 2 days of collection. During the collection days, 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, and the ileal digesta flowing into the bag are 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 acids. Now let’s move on to how we actually calculate DIAAS. First, we analyze the concentration of amino acids in the test ingredient in mg/g of crude protein. Then, as I previously explained, we feed this test ingredient to a cannulated pig and collect the ileal digesta for 2 days. This ileal digesta is then analyzed for amino acids to enable the calculation of SID of amino acids in the test ingredient. These two steps complete the lab and animal work that is required for DIAAS, and the rest of the steps in determining DIAAS are simply calculations. Next, the concentrations of amino acids in the test ingredient are 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 in mg/g of crude protein. The concentrations of digestible indispensable amino acids are then divided by one of the three reference scoring patterns, resulting in the calculation of digestible indispensable amino acid reference ratio. And the amino acid with the lowest ratio—the first limiting amino acid in the test ingredient when compared to the human amino acid requirements—is what determines the DIAAS for the test ingredient. 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 75 and 99. And if a food item has a DIAAS less than 75, there can be no claim made about its protein quality. 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. 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, our lab has determined DIAAS for a number of cereal grains, plant proteins, and dairy proteins. In addition, we have determined DIAAS in different cuts of pork and beef that have undergone different processing methods. Dr. Moughan’s lab in New Zealand has determined DIAAS for one specific muscle cut of beef that underwent different processing methods. So, it is very important to analyze different processing methods that animal proteins, or any food for that matter, may undergo because humans rarely eat raw meat, or raw beans, or even raw nuts. 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 in orange, and cereal grains are in gray. 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 and may be able to complement food items with lower quality protein. Plant proteins, for example soy and peas, generally are observed having DIAAS between 75 and 99. 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. It is important that when we are determining DIAAS for different food items that these items are analyzed in the form that humans commonly consume them in. This is because processing, specifically heat processing, can denature and aggregate proteins, making them unavailable for digestion, absorption, or metabolism in the body. In the diagram on the left adapted by Patel and colleagues in 2015, we see a native whey protein that is folded in its quaternary form. When heat is added, these proteins start to unfold or denature around 70 to 90 degrees Celsius. Denaturation may actually be beneficial to the protein’s bioavailability because unfolding will increase the surface area of the protein, giving more space for enzymes to act on and digest the proteins. However, if even more heat is added and the protein reaches temperatures greater than 100 degrees Celsius, they will start to refold and aggregate into forms that enzymes cannot digest or that the body cannot absorb and utilize. The extent of protein denaturation and aggregation largely depends on the protein type and the cooking temperature and duration. The last few years, our lab has focused on determining DIAAS in a number of food items consumed by humans. Today, I will be talking about three specific experiments that we conducted. Experiment 1 was conducted to determine the PDCAAS and DIAAS of pistachio nuts and to also assess the effect of roasting on the protein quality of pistachio nuts. For experiments 2 and 3, we focused on meat and determined the DIAAS in a number of pork and beef products. We also wanted to assess the effect processing has on meat products. Jumping into experiment 1, many types of nuts are roasted or processed prior to consumption. However, the protein quality of only a few nuts have been evaluated and the effect of roasting on protein quality had only been evaluated in one type of nut. The chart on the right shows the nuts that have an established PDCAAS or DIAAS and the animal model that they were determined in. The top four nuts—almonds, brazil nuts, cashews, and peanuts—were all evaluated in the rat model and have PDCAAS values of 60 to 70, with the exception of cashews that have a PDCAAS of 90. Peanuts in their roasted form also have a DIAAS of 43. Pistachio nuts, shown on the bottom of the chart, were evaluated in our lab using the pig model, where we assessed the effect of roasting. One batch of pistachio nuts were in their raw form and the other batch was roasted to 115 degrees Celsius, which took approximately 30 minutes. The SID of amino acids was determined in the cannulated pig, and then the DIAAS was calculated. The graph on the left shows the SID of a few indispensable amino acids, with the orange bar representing raw pistachios and the blue bar representing roasted pistachios. As you can see, the SID of all amino acids shown was greater in raw pistachio nuts than in roasted pistachio nuts. For PDCAAS and DIAAS shown in the graph on the right, roasted pistachio nuts had a PDCAAS of 81, which was actually greater than raw pistachio nuts that had a PDCAAS of 73. And the first limiting amino acid that determined the PDCAAS was threonine in both nuts. For DIAAS, we see the opposite. Raw pistachio nuts had a DIAAS of 86, which was greater than roasted pistachio nuts that had a DIAAS of 83. And the first limiting amino acid that determined the DIAAS was lysine for both nuts. So in summary, based on the DIAAS for pistachio nuts, they can be considered good quality protein sources. And the first limiting amino acid that determined PDCAAS was threonine, and lysine determined the DIAAS for both pistachio nuts. For processing, we observed that roasting reduced the SID of amino acids and, therefore, reduced DIAAS and protein quality. Controversially, PDCAAS was not reduced by roasting and was unable to account for the decreased amino acid digestibility due to heat damage. For experiments 2 and 3, we evaluated the protein quality of pork and beef. These animal proteins are important because in 2017, pork was the most widely consumed meat in the world, and beef was the third most widely consumed meat in the world. The experiment we conducted was 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 pork, we analyzed three different cuts: 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 is 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 for pork. For the sake of time, I am only going to show 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 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 these hams. For the pork loins heated to different temperatures, the loin heated to 63 degrees Celsius had the greatest DIAAS of 139, whereas the DIAAS for the 68- and 72-degrees Celsius 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 pork products, was valine. For this experiment with the pork products, we were able to conclude that based on the DIAAS, all of these pork 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 beef experiment, we analyzed 8 different beef products. Again, we look 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 its 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 on to 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, and no DIAAS was calculated for infants from birth to 6 months. 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 these 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 salami and all ribeye roasts. For this experiment we can conclude, that based on DIAAS, these beef products are generally excellent sources of protein. And the amino acids in least concentration when compared with the human amino acid requirements are leucine, valine, and sulfur amino acids. Based on 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 low quality protein or an unbalanced amino acid pattern. This slide gives an example of how two proteins can complement each other. The bar graph shows the digestible indispensable amino acid reference ratios for amino acids for cooked ground beef and polished white rice. If you recall, the cooked ground beef evaluated in the beef 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, which is the bar in grey with a red star above it. In contrast, beef has a greater concentration of lysine (grey bar with red star) resulting in a reference ratio well above 1 or 100 for lysine depicted by the red line across the bar graph. However, the cooked ground beef has low concentrations of leucine and valine, which are depicted by the light brown and dark brown bars, respectively, with the yellow star above it. But looking at the polished white rice, the reference ratio of leucine and valine are above 1 or 100. Therefore, when we combine these two 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 generally, animal proteins have DIAAS greater than 100, plant proteins have DIAAS between 70 and 100, and cereal grains have DIAAS between 30 and 70. Curing and moderate heating of pork and beef may increase the protein quality of the end product, while roasting nuts may decrease amino acid digestibility and therefore decrease DIAAS. The animal proteins, such as pork and beef, may be used to complement lower quality proteins to ultimately have a mixed meal that is balanced in indispensable amino acids. However, more work is needed in this area. There should be further investigation of the effect that processing has on the protein quality of human foods. We need to continue to expand the DIAAS database for individual human foods using the growing pig. And we need to move forward in determining the additivity of DIAAS in order to calculate the protein quality for mixed meals instead of just individual food items. With that, I would like to thank you for listening to this presentation, and if you would like to know more about this topic, or know more about nutrition in general, I would encourage you to visit our website at nutrition.ansci.illinois.edu. Thank you!