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 (Figure 1; Figure 2—A, B). 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) (Figure 2—B).
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 2—B, C). 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:
- 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).
- 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).
- 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 1). 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 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 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.
FIGURE 2 –– A, B, C:
- 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.
- 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).
- 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.
TABLE 1: Indirect Tests for Protein Quality
American Association of Cereal Chemists (AACC). Approved methods of analysis, 11th Ed. Method 22-40.01. Measurement of trypsin inhibitor activity of soy products—spectrophotometric method. First approval Nov 7, 1973; Reapproved Nov 3, 1999. AACC International, St. Paul. doi: 10.1094/AACCIntMethod-22-40.01.
American Oil Chemists' Society (AOCS). 2011a. Urease Activity. Official Method Ba 9-58. Official Methods and recommended Practices of the AOCS, AOCS, 6th ed., Second Printing, Urbana, IL.
Araba, M., and N. M. Dale. 1990. Evaluation of Protein Solubility as an Indicator of Overprocessing Soybean Meal. Pou. Sci. 69:76-83. doi:10.3382/ps.0690076.
Barth, C.A., B. Lunding, M. Schumitz, and H. Hagemaistr, 1993. Soybean Trypsin Inhibitors Reduce Absorption of exogenous in Miniature Pigs. J. Nutr., 123, 2195-2200. doi: 10.1093/jn/123.12.2195.
Birk, Y., A. Gerler, S. Kkalef. 1963. A Pure Trypsin Inhibitor from Soya Bean. BioChem. J., 87: 281-284. doi: 10.1042/bj0870281
Birk, Y. 1961, Purification and Some Properties of a Highly Active Inhibitor of Trypsin and alpha-Chymotrypsin from Soybeans. Bichem. Biophy., Acta 54:378. doi: 10.1016/0006-3002(61)90387-0.
Burns, R.A. 1987. Protease Inhibitors in Processed Plant Foods. Journal of Food Protection. 50(2); 161-166. doi: 10.4315/0362-028X-50.2.161.
Chen, J., C. Atwell, K. Wedekind, J. Escobar, M. Vazquez-Anon. 2019. Distribution of Trypsin Inhibitor and Urease Activity in Soy Products in Different Countries and World Areas. Proc. IPPE - International Production & Processing Expo 2019. https://en.engormix.com/poultry-industry/articles/distribution-trypsin-inhibitor-urease-t43927.htm
Clemente A., G. Sonnante., and C. Domoney. 2011. Bowman-Birk Inhibitors from Legumes and Human Gastrointestinal Health: Current Status and Perspectives. Curr Protein Pept Sci. 12 (5): 358-73. doi: 10.2174/138920311796391133.
DiPietro, C.M. and I. E. Liener. 1989. Heat inactivation of the Kunitz and Bowman-Birk Soybean Protease Inhibitors. Journal of Agricultural and Food Chemistry 37 (1), 39-44. doi 10.1021/jf00085a010.
Dong, F.M., R. W. Hardy and D. A. Higgs. 2000. Antinutritional factors. In R. R. Stickney, Editor, The Encyclopedia of Aquaculture, John Wiley and Sons, New York, New York. P 45-50.
Erdaw, M.M. 2019. Natural Protease Inhibitors and Sulphur Containing Amino Acids. In: Nelson Pérez Guerra, Editor, Protease, Mechanisms, Function and Use. Nova Sciences Publishers, New York, New York. p. 233-268.
Francis, G, Harinder P.S. Makkar, Becker, K. 2001. Antinutritional factors present in plant-derived alternate fish feed ingredients and their effects in fish. Aquaculture 199(3-4): 197-227. doi.org/10.1016/S0044-8486(01)00526-9.
Ibáñez, M.A., C. de Blas, L. Cámara, and G. G. Mateos. 2020. Chemical composition, protein quality and nutritive value of commercial soybean meals produced from beans from different countries: A meta-analytical study. Animal Feed Science and Technology, 267: 114531. https://doi.org/10.1016/j.anifeedsci.2020.114531.
ISO. (2012) International Organization for Standardization, standard 14902:2001. Animal feedingstuffs—Determination of trypsin inhibitor activity of soya products. Approved October 2001; Reapproved August 2012. Geneva, Switzerland.
Kunitz, M., 1945, Crystallization of a Trypsin Inhibitor from Soybean, Science, 101, 668; Gen. Physiol., 29, 149.
Kunitz, M. 1946. Crystalline Soybean Trypsin Inhibitor. J Gen Physiol. 29(3):149-154.
Laskowski, M. and M. A. Qasim. 2000. What can the structures of enzyme-inhibitor complexes tell us about the structures of enzyme substrate complexes? Biochimica Biophysica Acta. 1477 (1-2): 324-37. doi 10.1016/S0167-4838(99)00284-8.
Liener, L.E. 1986. Trypsin Inhibitors: Concerns for Human Nutrition or Not? J. Nutr. 116(5):920-923. doi: 10.1093/jn/116.5.920.
Liu, K. 2019. Soybean Trypsin Inhibitor Assay: Further Improvement of the Standard Method Approved and Reapproved by American Oil Chemists’ Society and American Association of Cereal Chemists International. J. Am. Oil Chemists Soc. 96(6). https://doi.org/10.1002/aocs.12205.
Mohan, V.R., P. S. Tresina, and E. D. Daffodil. 2016. Antinutritional Factors in Legume Seeds: Characteristics and Determination. Oxford: Academic Press. Encyclopedia of Food and Health, 211-220.
Onesti, S., P. Brick, D. M. Blow. 1991. Crystal structure of a Kunitz-type Trypsin Inhibitor from Erythrina coffra Seeds. Journal of Molecular Biology, 217(1), 153-176. doi:10.1016/0022-2836(91)90618-G
Parsons, C. M., K. Hashimoto, K. J. Wedekind, and D. H. Baker. 1991. Soybean protein solubility in potassium hydroxide: an in vitro test of in vivo protein quality. J. Anim. Sci. 69:2918-2924. doi: 10.2527/1991.6972918x.
Richardson, M. 1977. The Protease Inhibitors of Plants and Microbial Organisms. Phytochemistry, 16(2):159-169. https://doi.org/10.1016/S0031-9422(00)86777-1.
Richardson, M. 1981. Protein Inhibitors of Enzymes, Food Chem., 6(3):235-253. 10.1016/0308-8146(81)90012-1.
Ruiz, N., 1996. Avances en la estandarización de la técnica de la solubilidad de la proteína en KOH. Pages 1-8. In: Memorias del Seminario Internacional Nutrición Integral Aviar de Cara al Siglo XXI, Bogotá, Colombia.
Ryan, C. A. 1973. Proteolytic Enzyme and Their Inhibitors in Plants, Ann. Rev. Plant Physiol., 24:173-196. doi: /10.1146/annurev.pp.24.060173.001133.
Ryan, C. A. 1979. Proteinase Inhibitors, in: “Herbivores: Their Interaction with Secondary Plant Metabolites,” Rosenthal, G. A., D.H. Janzen, and S.W. Applebaum.. Academic Press, New York, New York.
Tacon, A.G.J.. 1997. Fishmeal replacers: Review of antinutrients within oilseeds and pulses - A limiting factor for the aquafeed Green Revolution? Zaragoza : CIHEAM Cahiers Options Méditerranéennes; n. 22. https://om.ciheam.org/article.php?IDPDF=97605920
Van Eys, J.E.. 2015. Manual of Quality Analysis for Soybean Products in the Feed Industry. 2nd Edition. U. S. Soybean Export Council. St Louis, MO. https://ussec.org/wp-content/uploads/2012/09/Manual-of-Quality-Analyses-2nd-edition.pdf
Wright, K. N. 1981. Soybean meal processing and quality control. Journal of the American Oil Chemists' Society. 58 (3): 294-300. https://link.springer.com/article/10.1007/BF02582362