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Trypsin Inhibitor, the hidden enemy in Soyabean Meal

Published: June 24, 2021
By: Dr. Koushik De, Director-Technical Services, SCA Novus International
As global animal production has rapidly shifted towards reduced Antibiotic free, “Gut health” has become a popular expression and all-encompassing concept in the scientific community. The gastro-intestinal tract must provide a barrier function protecting against harmful environmental elements (e.g. toxins and pathogenic microbes), while simultaneously permitting appropriate nutrient absorption. Successful animal performance depends on the interplay between the intestine, microbiota, diet, and a multitude of environmental factors.
The shift to antibiotic free production or better gut health often results in the increase of soybean meal inclusion as there are limited in the number of efficacious protein sources that successfully reduce soybean meal content. Soybean meal is the most widely used major protein source in poultry production across the world. However, SBM contains various anti-nutritional factors that may affect intestinal homeostasis and impair nutrient utilization in poultry. The main anti-nutritional factors in SBM, are trypsin inhibitors (TI), oligosaccharides, such as raffinose and stachyose, and the antigen Glycinin, β-conglycinin and Lectins. Diets that include high levels of soybean meal contain proportionally higher anti-nutritional factors and may pose the risk of impaired performance.
Chen et al. (2016) analyzed the content of TI and Urease Activity (UA) in more than 1000 samples of SBM from all over the world and observed a high degree of variability in the reported ANF values, both within the same country and amongst different origins.
The levels of trypsin inhibitor (TI) of solvent-extracted soybean meal samples from different countries and world areas.
The elevated variability and its potential negative impact on performance highlights the importance of knowing the content of anti-nutritional factors in SBM for poultry formulations.
In this article, we will review mainly the role of Trypsin Inhibitors (TI) in broilers.
Why Should we care about TI?
Trypsin and chymotrypsin are important digestive enzymes that are secreted by the pancreas as the inactive enzyme precursors trypsinogen and chymotrypsinogen. Trypsin activates itself via positive feedback and converts chymotrypsinogen and other inactive enzymes into their active forms. As Tis are protein in nature and one of the most anti nutritional components of SBM, they compete to bind to trypsin therefore affecting the digestion process. They have been correlated with rapid feed passage and decrease in digestibility of broilers with a relevant economic impact. The analysis is still more expensive, complex and time consuming for TI, for this reason, other parameters are commercially used as indirect SBM quality indicators, such as Urease activity and Protein solubility.
There are two types of TI present in Soya, Kunitz TI which is larger molecule & Bowman-Birk TI which is smaller molecule. But soyabean seed contain around 14% more Bowman Birk TI than Kunitz TI.
Consequence of TI for Soya Bean and bird performance:
Excessive quantities of TI in feed will cause pancreatic hypertrophy leading to poor growth and decreased performance (Pacheco et al. 2014; García-Rebollar et al. 2016; Rada et al. 2017). This pancreatic hypertrophy is a compensatory modulation by the body to offset the effect of ingested trypsin inhibitors (Liener 1981; Waldroup et al. 1985).
Linear increase in pancreas size with increasing TI content in SB
TI also affect the nutritive value of SBM. Because of loss of endogenous protein there is reduced digestion which affects the nitrogen balance, gut viscosity resulting into reduced live weight and negative impact on feed efficiency. Palliyeguru et al. (2011) demonstrated dietary soya TI elicited an increased severity of sub-clinical necrotic enteritis. When amino acid digestibility is compromised, the ileal ingesta will have a relatively high content of undigested amino acids that pass into the large intestine and cecal tonsils, where microbial fermentation will occur. C. perfringens, a pathogenic agent of necrotic enteritis, needs specific amino acids and peptides for its proliferation (Nakamura et al., 1968). 
Using the Data from the simulation conducted by Havenstein et al.(2003) with “1957” broilers versus ‘2001” broiler it is possible to estimate the TI intake of the “1957” birds fed 1957 diets and compare it with the estimate of TI intake by the “2001” birds fed 2001 diets. 
Because of improvement of modern broilers in terms of average feed intake and body weight they consume more than three times TI than 1957 birds considering the same amount of TI in SBM.
The effects of TIA are particularly strong in young animals. It has been shown that overcooking of soybean meal decreases digestibility of amino acids (Lee and Garlich, 1992; Parsons et al., 1992). The explanation for the decreased amino acid digestibility and reduced growth responses appear to be related to the Maillard reaction with cross-linking involved to a lesser extent.
Correlation of TI (AOAC) & indirect Parameters for SBM quality:
Currently, the analytical technique most commonly used to measure soybean meal quality is protein solubility, perhaps combined with the urease test. The urease test has been used for some time as a measure of soybean meal processing. Trypsin inhibitors (TI) and urease activity (UA) are the two most relevant quality measurements for soybean products as feed ingredients for animals. TI were reported to be correlated with UA, so feed processing plants use UA as an indicator of TI in soybean meal (SBM). Chen et.al (2019) conducted a study to determine the levels of TI and UA in 414 SBM samples from 19 different countries and to validate whether TI and UA are correlated. They found that TI were poorly correlated with UA in solvent extracted SBM samples, suggesting that UA should not be used as a surrogate indicator for TI content in soybean products.
 Araujo et al (2019) conducted similar study to determine the correlation of TI and KOH Protein Solubility.
How to deal with TI in SBM?
Soybean meal (SBM) is the most important source of dietary protein for poultry. Although TI is reduced by heat treatment, overheating has a negative impact on protein quality and amino acid digestibility. Exogenous Protease enzymes can improve digestibility of feedstuffs, lower feed costs and improve animal performance. Proteases improve animal performance and nutrient digestibility by decreasing digesta viscosity, improving endogenous enzyme activity and decreasing pancreas weight (Bedford and Classen, 1993; Bedford and Schulze, 1998; Erdaw et al., 2017a,b; Yan et al., 2017).
As mentioned earlier the determination in the laboratory of the TI content of SBM and its relationship with AA availability is tedious and time-consuming and provides inconsistent results. Also, the traditional processes of treating SBM can’t remove the anti-nutritional factors to a safe level. Therefore, use of exogenous protease is very effective in reducing the deleterious effect of TI in SBM. Liu et al., in 2013 conducted a study wherein they used a protease enzyme (Cibenza EP150) with different levels of TI and found that protease enzyme was able to destroy almost all trypsin inhibitors (both Bowman-Birk & Kunitz TI) present in soyabean meal (at 1:1 ratio) and destroy substancially even in higher concentración (2:1) of TI as well.
Wedekind et al., in 2020 showed that addition of exogenous protease (Cibenza EP150) in a diet containing FFS(with TI 8.15mg/g) improved the amino acid digestibility and at the same time reduce the pancreas weight also indicating a potential amelioration of the negative effect of TI from FFS.
Conclusion:
There are lot of scientific evidences on the negative effect of soybean trypsin inhibitors in chickens. They can not only adversely affect the productive performance of chickens but can also impair their intestinal health. The beneficial responses of protease are likely due to decreases in endogenous amino acid losses, but in vitro evidence also demonstrates the ability of protease to hydrolyze Bowman-Birk and Kunitz-trypsin inhibitor proteins. Thus, there might be both direct and indirect mechanisms whereby amino acid digestibility is improved with proteases and so is the bird’s performances.
Related topics
Authors:
Dr. Koushik De
Novus
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dan hofer
1 de noviembre de 2021
very good topic so would whole soybean roasted be trypsin free
Robert Swick
University of New England
7 de septiembre de 2021
Really good discussion. Certainly will be great to have such discussion face-to-face once again!
Dave Albin
Insta-Pro International
16 de agosto de 2021
Thank you for this article. A couple of issues must be raised, however. First, the correlations of urease activity and TI, measured as mg/g, may not be appropriate. Urease activity is a measure enzyme activity by assessing a rise in pH, while TI, in this case, is assessing the absolute amount of TI remaining in the meal. So, it's perhaps not surprising that a measurement of enzyme activity and a measurement of absolute amount of enzyme remaining would not correlate well, especially considering that these are two different molecules (urease vs. TI). It may be more appropriate to measure TI activity, vs. mg/g, and see how these values correlate with urease activity. Second, the use of proteases poses one major problem, namely that these enzymes could degrade other exogenous enzymes, such as carbohydrases or fiber-degrading enzymes, making them less effective.
Nelson Ruíz
Nelson Ruíz Nutrition LLC
29 de julio de 2021

Dear Dr. Koushik De. I find interesting your discussion on trypsin inhibitors and their role as antinutritional factors in broiler nutrition. It would be nice however, if you add a list of all the literature cited and provide credit where credit is due. Havenstein et al. (2003) didn't discuss trypsin inhibitors in their comparison of the 1957 and 2001 genetics, the slide that you have in your paper comes from my discussion in 2012 at the Arkansas Nutrition Conference [Ruiz, N., 2012. New insights on the urease activity range for soybean meal: a worldwide opportunity for the poultry industry. 2012 Arkansas Nutrition Conference Proceedings]. In my opinion, and of course, it is only my opinion, trypsin inhibitors are not "the hidden enemy" in soybean meal. Since the 1940's they started to be clearly identified by Kunitz (1945), Bowman (1944), and Birk (1961). And between 1969 and 1974 Kakade et al. developed the necessary analytical chemistry for their quantification. A considerable amount of the work on trypsin inhibitors has been done in soybeans and their products such as soybean meal.

I would say that rather than hidden, trypsin inhibitors in soy have been ignored for a variety of reasons. One important reason why during decades the industry didn't refer directly to trypsin inhibitors in soybean meal was the high correlation between urease activity and the inhibitors although it was not explicitly recognized. For decades the range of adequacy measured with urease activity was 0.05 to 0.20 pH units, and millions of tons of excellent quality SBM was produced and utilized in animal nutrition worldwide including broilers. This was true until the 1990's when the rapid feed passage syndrome showed up in broiler farms, particularly out of the United States [Kouwenhoven, B., R.M. Dwars, J.F.M. Smeets. 1992. Wet litter and high feed conversion, a new problem in broilers. Pages 558-561. Vol. 1. In: Proceedings XIX World’s Poultry Congress. WPSA. Amsterdam.] The rapid feed passage syndrome was the result of the dramatic increase in feed intake of broilers which accompanied with the predominance of soybean meal worldwide as the number one source of digestible amino acids exceeded the tolerance of broilers to trypsin inhibitors in the diet.
In 2005 [Ruiz, N., and F. de Belalcázar. 2005. Field observation: Trypsin inhibitors in soybean meal are correlated with outbreaks of feed passage in broilers. Poultry Science 84(Suppl. 1): 70.] we presented field data that indicated that commercial soybean meal displaying urease activity values within the range of adequacy were clearly involved in rapid feed passage in broilers older than 21 days of age. But in 2005 we measured simultaneously trypsin inhibitors and urease activity in lots of soybean meal that resulted in excellent performance of broilers as well as in lots that generated rapid feed passage. It became clear that the old range of adequacy had changed, and by 2012 at the Arkansas Nutrition Conference we proposed a new range: 0.000-0.050 pH units. Using the ISO 14902:2001 method to determine trypsin inhibitors we correlated this new proposed range for urease with a 1.65 - 2.35 mg/g range of adequacy for trypsin inhibitors. Since 2005 we also proposed the importance of measuring trypsin inhibitors as QC parameter in soybean meal and full-fat soybeans.

Yes, trypsin inhibitors is a QC parameter for both soybean meal and full-fat soybeans. Solubility in KOH is the parameter to measure if overprocessing has occurred in SOYBEAN MEAL and it is highly correlated to amino acid digestibility in soybean meal. We have reported that KOH protein solubility is not a QC parameter for full-fat soybeans [Ruiz, N., and C.M. Parsons. 2016. The absence of correlation between protein solubility and digestible amino acids in full-fat soybeans emphasizes focusing on trypsin inhibitors reduction. International Poultry Scientific Forum, Abstract T195 (p. 58); Poult. Sci. 95(E-Suppl. 1):251.]. Soybean meal and full-fat soybeans are two different ingredients each with distinctly quality control parameters [80th Minnesota Nutrition Conference, p. 169-175, 2019].

As far as how to deal with trypsin inhibitors in the feed formulation of broilers there are several alternatives beyond proteases. Nutritionists and formulators under commercial conditions have to think in overall cost, and formulating to minimize trypsin inhibitors in a broiler finisher diet is expensive because limiting soybean meal, the economic number one provider of digestible amino acids as already said, would result in higher price per metric ton of feed. As suggested by Dr. Marchenkov there are alternative ingredients such as sunflower meal that compete against soybean meal when a maximum spec for trypsin inhibitors in the formula is in play. Canola is another one. Per our experience, 0.58-0.60 mg of TI/g of FEED in a broiler finisher feed is roughly the upper limit.

Another way to effectively control trypsin inhibitors in broiler feeds is the utilization of full-fat soybeans (FFSB) which occur in several countries out of the United States. Given the fact that FFSB in practice is a no-commodity because is processed by the end user, then it is possible to decrease trypsin inhibitors in FFSB well below the trypsin inhibitor content of commercial soybean meal (a commodity). Soybean meal cannot reach by the solvent extraction process values below 1.60-1.65 mg TI/g without being overprocessed, while FFSB, PROPERLY PROCESSED may reach values as low as 1.0 mg TI/g (measured with the ISO 14902:2001 method). Consequently, by generating FFSB of superior quality than commercial soybean meal as far as trypsin inhibitors are concerned the only limitations are cost and inventory. In our experience FFSB competes excellently with soybean meal, reduces cost per MT of feed and allows to control trypsin inhibitors below the 0.58-0.60 mg TI/g of FEED. Nelson Ruiz Nutrition, LLC, Suwanee, GA USA.

Oketch Elijah Ogola
Chungnam National University
30 de junio de 2021
"The shift to antibiotic free production or better gut health often results in the increase of soybean meal inclusion as there are limited in the number of efficacious protein sources that successfully reduce soybean meal content"..... Could you please elaborate a bit more on how shift to AGP-free production has led to use of more SBM?
Dr. Fiodor S. Marchenkov
Kronos -Agro private company
29 de junio de 2021
Excellent article! Everybody knows about protein advantages of soya bean meal, but not enough about disadvantages of soya bean meal.
Dr.Ashwin Mahurkar
2 de septiembre de 2022
Dear Nelson What are the culprit parameters or factors affecting TI during Soya processing particularly in extruder & expeller method ?And how to control it ?
Yakub Ibrahim
Albarka APS Poultry
18 de septiembre de 2021

Very nice and educative topic, I learned a lot.
Thank you to all the contributors.

Dr. Rama Prasad Chakraborty
27 de julio de 2021
Trypsin inhibitor so looming large to become a threat to use SBM in bulk albeit the same ingredient is the rich source of protein and a fair source of both L Lysine and DL Methionine. Urease activity in SBM doesn't reveal much significance in poultry feeds but it restricts the rampant use of SBM along with Urea in cattle feed. The measure to reduce TI activity in SBM in poultry feeds like heat treatment or any chemical exposure requires more exploration in near future in order to ensure more growth in poultry to meet out true marketable demand. It is to reiterate that any other source as source of protein to poultry feed other than SBM is deoiled mustard cake or rape cake though they too have had their limiting factors prior using in greater scale.
Dr Kibiike David
14 de julio de 2021
Am looking for testing unit to test the presence of trypsin inhibitor in roasted soy.
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