Improved measurement tools for trypsin inhibitors
While trypsin inhibitors can reduce protein digestibility, traditional processing methods effectively mitigate inhibitors, resulting in high protein digestibility.
By Gregory L. Engelke, M.S., P.A.S., Cornerstone Resources LLC
and Emily Dustman, United Soybean Board
Livestock and feed producers agree that quality feed ingredients are key to animal growth and performance. Recently, discussions have centered around anti-nutritional factors (ANFs), like protease inhibitors (PIs), such as trypsin inhibitors (TIs) and their impact on intestinal homeostasis and nutrient utilization in animals. While TIs can reduce protein digestibility, traditional processing methods effectively mitigate inhibitors, resulting in high protein digestibility. Nonetheless, methods to directly quantify and better manage inhibitors in feed and food formulations are needed. Currently, few laboratories in the world measure them directly and variability between and among labs is high (Chen et al., 2019). Further, it has been suggested that the standard indirect methods are inadequate and that better methods are needed for greater accuracy, efficiency, and affordability (Liu, 2019). To enhance inhibitor management, United Soybean Board (USB) is partnering with the public and private sectors to develop quick and accurate methods to measure inhibitors commonly found in legume plants and in the germ of some cereal grains (Barth et al., 1993).
Plant Defense Mechanisms. Over the course of time, plants have evolved mechanisms to promote survivability and improve seed dispersal. One such defense mechanism is the production of compounds known as antinutrients or anti-nutritional factors (ANFs) (Dong et al., 2000). Anti-nutritional factors (ANFs) have important physiological functions like protein synthesis, plant growth regulation, and prevention of infections and infestations (Richardson, 1977, 1981; Ryan, 1973, 1979).
ANFs in Soybeans. Anti-nutritional factors (ANFs) are naturally found in a wide variety of plants like soybeans, and in general, do not positively contribute to animal nutrition though some research has reported benefits (Dong et al., 2000; Mohan et al., 2016). As such, further research is needed to determine if they should be preserved or eliminated (Mohan et al., 2016). In raw soybeans, ANFs have been associated with impairing the nutritional quality of protein by reducing enzyme activity (see Figures).Of particular focus for soybeans are two ANFs found within the protease inhibitor family, Kunitz trypsin inhibitors (KTIs) and Bowman-Birk inhibitors (BBIs). Kunitz trypsin inhibitors (KTIs) were first isolated from soybean meal through a crystallization process and were identified as globulin proteins with one or two disulfide bonds and a single reactive site (Kunitz, 1945, 1946; Onesti et al., 1991).
Bowman-Birk inhibitors (BBIs) are proteins with numerous disulfide bonds and two reactive sites (DiPietro and Liener, 1989; Mohan et al., 2016). Both protease inhibitors (PIs) are competitive inhibitors that interfere with enzyme active sites and render them unavailable to bind to and break down proteins into peptides and amino acids (AAs). Kunitz trypsin inhibitors specifically bind to and inhibit the enzyme trypsin, and weakly bind to and inhibit the enzyme chymotrypsin. Bowman-Birk inhibitors strongly bind to and inhibit both trypsin and chymotrypsin (Laskowski and Qasim, 2000; Clemente et al., 2011) (see Figure).
Several factors contribute to protease inhibitor activity (PIA), including soybean variety, geographic location of production, levels of insect infestation or damage, harvest timing and conditions, storage conditions and duration, and processing methodologies (Burns, 1987; Ibáñez et al., 2020).
There is substantial variation in PI levels between different soybean lines and agriculture biotechnology companies are exploring how to select the best production and nutrient characteristics.
Measuring PIs. The amount of active PIs in a soy product is generally evaluated by measuring overall trypsin inhibitor activity (TIA). The current standard method, approved by American Association of Cereal Chemists International (Method 22-4-.01; AACC, 1999), features mixing trypsin with a series of inhibitor levels and then adding substrate to start the colorimetric reaction (Liu, 2019). Previous studies have shown flaws with the method, particularly with using several inhibitor levels and the sequence of adding the substrate last. Liu (2019) reported the use of varying levels of dilution and volumes of a dilute sample extract and that the pH of the premix (the mixture of dilute sample extract and trypsin solution) ranged 3.3-3.6 for raw soy flour, and 3.2-6.7 for toasted soy. Within these premix pH ranges, the standard method of adding substrate last would give TIA values equal to or less than those measured by the same method. The last standard method was substantially improved by using a single sample extract level and by adding the enzyme last.
While this offers useful information about the overall presence of Protease Inhibitor Activity (PIA) in the sample, it gives no indication about which type of PI is responsible for the inhibitory activity. Distinguishing between KTI or BBI activity is important as the inhibitors have different antinutritional effects once they are ingested along with different degrees of affinity for the assay and thermal response.
In Europe, progress has been made as the Geneva-based International Organization for Standardization adapted an improved standard method in 2001 as ISO 14902 for the TIA assay, and reapproved it in 2012 (ISO, 2012). In the assay for activity of an enzyme inhibitor, there is a distinct sequence for adding an inhibitor (I), substrate (S), and enzyme (E). The IS +E sequence features mixing I with S first then adding the E (Liu, 2019). The ISO method incorporates elements of using a single dilution level as well as the IS+E sequence. At the same time, approved methodologies in the United States have remained unchanged.
PIs & Processing. Protease Inhibitors have been associated with reducing protein digestibility, but in general PIs are not a major issue in traditionally processed soybean meal (SBM) (solvent or extruded) due to temperature, pressure, and time thresholds during processing (Erdaw, 2019; DiPietro and Liener, 1989; Wright, 1981). During SBM production, most of the oil is removed through an extraction process. The resulting SBM is treated with moist heat, which denatures (or inactivates) specific proteins such as the PIs (Figure). The meal product is then dried and toasted to current market standards. Among the proteins denatured are inhibitors of pancreatic endopeptidases, trypsin, or chymotrypsin (Birk et et al., 1963; Liener, 1986). Kunitz trypsin inhibitors are considered the most important because they bind almost irreversibly to their targets (Kunitz, 1945).
Research denotes possible differences in the heat thresholds for KTIs and BBIs. Kunitz trypsin inhibitors have been described as “heat-labile” inhibitors, while BBIs have been referred to as “heat-stable” inhibitors (Tacon,1997; Francis et al., 2001). In most cases, descriptions of BBIs as “heat-stable” are derived from the early work of Birk (1961), and the heat inactivation mechanisms are not well understood. More research and consistent testing methods are necessary to validate the heat thresholds for KTIs and BBIs.
Indirect Measures. Traditional measures of protein quality are costly, so soybean processing plants typically use indirect methods to measure inhibitors / inhibitor activity. These methods provide an indication of overcooking (potential for reduced protein digestibility through production of Maillard products), or undercooking (residual PIA). Indirect tests commonly used include the following:
1. Urease Activity (UA) - Urease is a metalloenzyme present in soybeans that catalyzes the hydrolysis of urea to ammonia and CO2. Like PIs, the activity of urease is reduced when it is heated. The UA test measures the increase in pH as a result of the release of ammonia (AOCS, 2011a).
Protease inhibitors, like TIs, are not a major issue in traditionally processed SBM due to temperature, pressure, and time thresholds during processing.
2. Protein Solubility (PS) - Protein Solubility is used to evaluate heating processes. The solubility of soybean protein in potassium hydroxide (KOH) is inversely related to heat treatment (Araba and Dale, 1990).
3. Protein Dispersibility Index (PDI) - Protein Dispersibility Index (PDI) measures the solubility of protein in water, and it is another indicator of adequately heat processed SBM (Van Eys, 2015).
The above indirect tests are inadequate in a variety of ways (see Table). Each give slightly different results depending on the particle size of the sample used, what the laboratory’s standard procedures are, if they are properly adhered to, and what type of equipment is used and if the same equipment is used consistently in testing (Parsons et al. 1991; Ruiz, 1996). As a result, measurements for these indicators can have a high degree of intra- and inter-lab variability.
Additionally, these indirect measures are insufficient for informing animal nutritionists and processors of the presence of KTIs and BBIs, critical to overall assessment of PIs and their effects on soybean protein and amino acid quality, digestibility, and animal performance. Improved methods should also deliver low inter- and intra-lab measurement variability, be rapid, efficient, cost-effective, and adaptable to existing infrastructure. Test accuracy, simplicity, and affordability will aid processing plants in plant line management in producing high-quality SBM.
New Measures for Quality. Soybean meal has excellent nutritional qualities, and as a result of SBM inclusion in animal diets, evaluation of quality indicators has become an essential component in the application and utilization of raw materials.
A firm understanding of ingredient characteristics becomes critical and includes aspects like nutrient composition, nutrient density, nutrient availability, and the presence of less desirable chemical compounds that may interfere with nutrient availability or directly affect the performance of the animal (advantageously or adversely).
Understanding these ingredient characteristics will directly rely on accurate methods to quantify them.
The presence of PIs in raw and improperly treated soybeans is undesirable, and values for PIs have become highly sought-after as a quality indicator, but the nutritive value of SBM should not rely on current measurements of PIs until more accurate methods to analyze them exist.
Until then, there will be great uncertainty for processors and producers in understanding why variation exists and in making decisions regarding optimal heating conditions for SBM, threshold levels for PIs in animal diets, and appropriate enzyme supplementation.
Arguably, PIs are the most important group of ANFs and are the key to unlocking a more comprehensive understanding of protein quality.
The United Soybean Board (USB) has identified this critical gap and is funding work with government and industry to develop a quick, accurate, and cost-effective method to measure PIs that will provide this very critical key.
When soybean meal is properly heat-treated, inhibitors like TIs are deactivated and do not interfere with protein digestion. Steps 1 – 3 demonstrate the process involved in the digestion and absorption of nutrients from properly heat-treated soybean meal.
A. When an animal ingests protein, the pancreas releases trypsinogen. Trypsinogen enters the small intestine and is cleaved/activated into trypsin, an enzyme that aids in protein digestion. Protein binds to the active site of trypsin, forming an enzyme-substrate complex. This complex breaks down (digests) the protein into peptides and amino acids (products), making them available for absorption in the bloodstream.
B. Improperly treated/under-processed soybean meal (SBM) may reduce enzyme activity due to the natural presence of trypsin inhibitors (TIs) in the soybeans. Trypsin inhibitors compete with protein (substrate) by binding to the active site of trypsin (enzyme). The resulting complex is unable to bind protein, and the protein is not broken down/digested (no reaction; reduced protein digestibility).
C. Protease inhibitors, like TIs, are not a major issue in traditionally processed SBM due to temperature, pressure, and time thresholds during processing. During SBM production, most of the oil is removed through an extraction process. The resulting SBM is heat-treated which denatures (inactivates) specific proteins like TIs. These inactivated inhibitors are no longer able to bind to the active site of trypsin (enzyme), and protein (substrate) can bind once again and digestion proceeds.
New research shows health-challenged pigs perform better with higher lysine to energy ratios
Increasing ratio of lysine to metabolizable energy helps pigs fight health challenges.
“In today’s swine industry, disease pressure is a reality many producers have to navigate. Exploring nutritional strategies to help mitigate the negative growth performance associated with health challenges such as PRRS, is of value to the producer,” says Jessica Jasper, lead author.
Pigs facing a health challenge such as Porcine Reproductive and Respiratory Syndrome virus (PRRSV) show a significant loss of performance. In 2019, Schweer et al. showed that increasing the ratio of lysine to metabolizable energy to 110 to 120% of the National Research Council (NRC) recommendations improved both performance and feed efficiency in pigs challenged by PRRSV. Building on that work, Jasper and co-workers recently validated this and compared different ways of increasing that ratio, and monitored pig performance.
Swine nutritionists generally formulate feed based on energy, amino acids, and the ratio of grams of standardized ileal digestible lysine per Mcal metabolizable energy (SID Lys:ME). In diets composed of corn and soybean meal (SBM), the SBM is the primary source of amino acids, including lysine, which is the first-limiting amino acid. It may be that sick pigs eat less overall, thus reducing both their dietary energy intake and their amino acid intake. In this study, the ratio was changed to 120% in two different ways – by increasing the amount of SID Lys (numerator) or by reducing the ME (denominator).
Four hundred pigs were used, all 19-21 days old at the start. They were randomly divided into two identical barns, and on day one all the pigs in one barn were vaccinated with a live modified PRRSV vaccine (Ingelvac MLV, Boehringer Ingelheim), while those in the other barn were not. All pigs in both barns were fed the same nursery diet.
After 42 days, they were further subdivided into grower pens and fed the same corn-soybean meal diet for the next 14 days.
At this point and within vaccine status, pigs were allotted to one of three diets:
• Control - 2.69 g SID Lys:ME, or 100% Lys:ME based on NRC recommendations
• High Lysine (HL) - 3.23 g SID Lys:ME, including soybean meal and synthetic amino acids
• Low Energy (LE) - 3.22 g SID Lys:ME, dietary ME content was reduced by including sand.
Both the HL and LE diets contained 120% of the requirements for 35 to 75 kilogram (kg) pigs, and the total amounts of calcium, phosphorus, and ratios of SID threonine (Thr), tryptophan (Trp), methionine (Met), isoleucine (Ile), and valine (Val) to SID Lys were similar in all diets. Crude protein (CP) levels and soybean meal (SBM) inclusion rates were similar in both the control and LE diets, but the HL diet had an increased level of soybean meal (26.5%, as compared to 19.4% (control) and 22.0% (LE)), which probably increased intake of multiple amino acids, not only lysine. On day 56 post-weaning, all pigs in both barns were challenged with PRRSV (day post-inoculation [dpi] 0), and fed these diets for 42 days.
The pigs were then allowed unrestricted access to their assigned diet and water for the next 42 days, and their health and growth monitored weekly.
By dpi 7, all pigs were confirmed viremic and positive for PRRS. All pigs become naturally, and unintentionally, infected with porcine circovirus 2 (PCV2) between dpi 7 and 14.
Due to the co-infection, the vaccinated barn experienced 11 mortalities (5.6%) and the unvaccinated barn experienced 22 mortalities (11.3%). Mortality did not differ across dietary treatments. All pigs were placed on water amoxicillin, to decrease the risk of additional opportunistic pathogens.
During the first seven 7 dpi on test diets, the non-vaccinated LE pigs had greater average daily gain (ADG) and feed efficiency. However, vaccinated pigs on the LE and HL diets had only slightly higher ADG and increased average daily feed intakes (ADFI) compared to the control pigs.
The final body weight of pigs fed the HL and LE diets were 6.9 kg and 4.2 kg heavier, respectively, when compared to the control pigs. While the final body weight of pigs fed the HL and LE diets were 5.4 kg and 3.2 kg more, respectively, than the control pigs in the non-vaccinated barn.
Over the entire challenge period (dpi 0-42; Table 1), ADFI increased by 19.8% (20% and 17% in vaccinated and non-vaccinated pigs, respectively) for pigs on the LE diet, but the HL treatment was not statistically different from the control. This shows that immune-stimulated pigs can voluntarily adjust their feed intake and will attempt to eat to their energy needs, regardless of the energy density of the feed. Irrespective of vaccine, these data agree with earlier work that increasing the SID Lys:ME ratio to 120% in growing pigs facing a viral challenge significantly mitigated the usual negative growth performance associated with PRRS.
The increased ADFI in the LE diets could explain the improved ADG. While the use of sand to dilute the energy is not a practical commercial strategy, dietary fiber could be a more practical approach. However, the degree to which fiber augments satiety and impedes appetite will need to be examined further.
Experiencing a performance-limiting disease during the growth phase is almost inevitable in the swine industry. Increasing the ratio of SID Lys:ME is a viable strategy for improving performance during disease challenges, regardless of how that increase is achieved.
Soybean meal delivers more energy than previously thought
New research suggests that soybean meal provides 105-125% of the net energy of corn, when used as a feed ingredient for growing pigs.
By Lisa M. Balbes, Ph.D., SmithBucklin
Quantifying the effects of soybean meal (SBM) as a feed ingredient on swine growth performance is crucial to maximize farmer profit. As processing methods have improved over time, SBM has become a high quality and consistent source of highly digestible amino acids (AAs), but SBM also provides energy. The NRC (2012) lists the digestible (DE) and metabolizable energy (ME) content of SBM at 105 and 97%, respectively, that of corn. But when expressed on a net energy (NE) basis, SBM’s NE value is only 78% that of corn – but recent field data and studies have called that value into question.
For a long time, swine nutritionists have been trying to determine the best way to quantify the energy values of various feed ingredients. Traditionally, DE or ME values have been used. However recently, there has been greater implementation of the NE system in swine diets, which more closely correlates with growth performance. However, direct or indirect calorimetry to measure DE and ME is very labor-intensive and requires expensive, specialized equipment. Therefore, some have suggested a more practical approach , feeding pigs increasing amounts of SBM and using the differences in caloric efficiency to estimate the energy content of SBM relative to the known energy of corn.
Did you know that current research suggests that soybean meal provides growing pigs with more energy than previously thought? Did you know that current research suggests that soybean meal provides growing pigs with more energy than previously thought?
Two experiments reported in a recent paper4 indicate that SBM actually delivers between 105% and 125% of corn energy, which is significantly higher than the traditional published value in the NRC. This new value was determined by feeding swine diets with increasing amounts of SBM but balanced for essential amino acids, and observing changes in feed efficiency and caloric efficiency (CE, the amount of calories needed to produce a pound of weight gain). If there is no change in feed-to-gain ratio (F/G) or caloric efficiency (CE) as SBM levels increase, then the designated energy value used for SBM is accurate. However, if the F/G and CE change, the energy value of SBM is incorrect. Specifically, it F/G and CE improve, then there is likely more energy in SBM than initially estimated.
Henrique Cemin and colleagues at the Department of Animal Sciences and Industry at Kansas State University in collaboration with nutritionists at JBS USA, Greely CO, set out to measure the net energy value of today’s SBM. In the first experiment, 2,233 pigs were fed diets containing 21, 27, 33, or 39% SBM for 21 days, with 23 replicates of each treatment. The amount of corn in the diet was kept the same, but the inclusion rate of feed-grade amino acids was varied. In the second experiment, a total of 3,796 pigs were used, and they were fed diets with 17.5, 22, 26.5, 31, 35.5, or 40% SBM. In each case, pigs were weighed and feed disappearance was measured weekly, then used to calculate average daily gain (ADG), average daily feed intake (ADFI), feed-to-gain ratio (F/G), and caloric efficiency (CE).
The results were intriguing. In experiment 1, there was a tendency (linear, P = 0.092) for a decrease in ADFI as SBM increased. There was also a tendency (P = 0.090) for a quadratic response for ADG, with a decrease in ADG observed with 39% SBM inclusion. Both F/G and CE improved with increasing SBM content, indicating that its NRC (2012) energy value was underestimated. Using a value for corn energy of 2,672 kcal/kg NE, the NE of SBM was calculated to be 2,816 kcal/kg, or 105% of corn energy.
Experiment 2 showed a linear decrease in ADFI as SBM increased, and tendencies for ADG to decrease, but again, like experiment 1, F/G and CE improved. Using the data from the second experiment, the energy value of SBM was calculated to be 125% of corn energy, or 3,332 kcal/kg NE. Taken together, these results suggest that increasing the amount of SBM in swine feed improves both F/G and CE. It also indicates that the energy value of SBM is considerably higher than the current literature standard value, and probably in the range of 105 – 125% of corn, or between 2,816 and 3,332 kcal/kg.
There is the possibility that the beneficial results of SBM were derived not simply from energy content, but partially from a specific component of the SBM, such as isoflavones, which have been shown to have anti-inflammatory, anti-oxidant and anti-viral properties.
Furthermore, changes in carcass composition were not evaluated in these studies. Regardless of the reason for the increased energy estimate, nutritionists should take this new data into consideration when formulating swine diets.
