Protease enzyme and trypsin inhibitor: Novus’s results
Published:February 26, 2020
What should producers know when it comes to selecting a protease enzyme to combat trypsin inhibitor? Hear what Raquel Araujo said about this topic in the recent edition of IPPE 2020, in Atlanta, USA
A large number of commercial proteases have chymotrypsin type activity and oriented toward hydrophobic amide bonds. What type activity does your protease have?
Would it affect other supplemental enzymes such as amylase or phytase?
Thank you very much for the interesting questions! DP100 is a type of keratinase that belong to serine protease and it’s produced by Bacillus licheniformis PWD-1. Within the serine proteases, DP100 belongs to the subtilisin-like proteases group in which chymotrypsin belongs to. Data shows that the protease DP100 can be blended with other enzymes such as phytase, xylanase and NSPase without negative effects, if any, they will complement each other. The mode of action of DP100 is to hydrolyze dietary protein and it complements the activity of other enzymes that work on different feed substrates such as phytases, xylanases and other NSPases. The combination of different enzymes with DP100 has led to improvements in performance and gut health beyond the individual enzymes. It has also been reported that DP100 improves starch digestibility in sorghum-based diets by digesting the kafirin protein that protects the starch granules.
Mercedes Vazquez-Anon Thank you for the effort of your answer. Having chymotrypsin type activity provides broad based action on proteins by cleaving peptide linkages of hydrophobic amino acids which usually dominate most proteins. Kafirin is good example by having a very large amount of leucine. It nicely clears starch occlusion of all proamines in grain endosperm enabling supplemental amylase to function better. My concern is it’s potential at “injuring” other enzymes supplemented with it to reduce their activity. I don’t know if this happens, but it has always been a concern with supplemental proteases at-large.
Dear Dr Moran, thanks for your interest in the use of proteases in the presence of other exogenous enzymes. When we blend DP100 with phytase in feed, we don’t see losses of the phytase or DP100 in the feed, and when we feed the combination of the two enzymes we don’t see reduction in the response of the bird. This would indicate the protease is not degrading the exogenous phytase, if any we see benefits of including the two enzymes together. Hope this information is helpful.
Juxing Chen Dear Mrs.Chen, it is really so, I have the same experience. Protease doesn't destroy other added enzymes, incl.NSP-enzymes (also in liquid form, too). It is quite strange and it is difficult to explain because all enzymes are proteins, but it is really so. Thus, protease show no negative effect. Another question how big is positive effect. I think this is problem, and there is possible a lot of speculations. May be answer can give only commercial trial with broilers or layers.
Edwin T Moran Dear Mr.Edwin, yes it is. The best way is to mix enzymes cocktail and hold it some time during 30 centigrades in thermostat. We perform this test during a half year. Activity of all enzymes was approx.80% or normal level. But protease was not DP-100, it was from another producer.
Dr. Fiodor S. Marchenkov Dear DR. Fiodor S. Marchenkov, If I was you I would have no problem having a blend in dry format, but having it in Liquid I have seen this affect the recovery of the other enzymes and would strongly not recommend this. Rgds. Arne Korsbak.
If we think of a protease being added to the diet to compensate or destroy the anti-trypsin activity of soybean meal, would it be right to say that the net benefit from said protease would be dependent on the quality of the soybean meal (if properly deactivated there is less room for the protease to improve results) or also on the level of inclusion of soybean meal in the diet (if replaced by other protein sources)?
Luciano Andriguetto Hi Luciano, The effect of the protease is to improve amino acid digestibility of ingredients, in the presence or absence of trypsin inhibitor. The presence of trysin inhibitor in soybean meal reduces the capacity of the animal to digest protein, and this reduction is partially ameliorated by the protease. Hope this helps!
Luciano Andriguetto Protease is used to increase AA digestability (by 3-5% average) in the diet. It is not only SBM. The additional (side) effect is to deactivate tripsin inhibitors if SBM is not enough toasted/too low temperature. For "properly" toasted SBM the additional effect is not required. But protease-exogenous is still active and required for SBM and other proteins sources to increase the AA digestability.
Luciano Andriguetto YES, I agree to what you said the biggest advantage of using proteases is to destroy trypsin inhibitors, so whenever you get bad quality soya, you get good results; when you use proteases with good quality soya, we see less results with proteases.
Kapil Manwal It is not ONLY for SBM and trypsin inhibitor. Protease increase all AA/protein digestability from ALL protein sources. The "uplift" is not equall for each AA and each RM, of course.
Dr Piotr Stanislawski Uplift in AA digestibility is not increased if protein source already having better digestibility %. But still if you through light on this, will be very pleasureable. Protease also showing negative effect if used in higher dosage or unit values?
Dr Piotr Stanislawski You are correct and I agree with you, it was about topic and maybe I found it more useful and had found very good results when soya quality. Regards, Kapil
Dr. Vijay Swami as I know protease doesn't show negative results in higher dose. But the negative is that it doesn't show positive results if protein/AA in feed are high; simply not substrate/space to improve.
Dr Piotr Stanislawski Hi Piotr!!! Nice to hear from you again! What is problem? Tr-inhibitor? Simply drop off SBM and replace it by SFM, add a little bit lysine, proper enzymes mix (you know what is necessary to add:)) and everything will be ok! Including protease, of course. My factory apply proteases mix, too: alkaline, neutral and acid protease. Any case it is possible to improve AA digestibility.
Dr Piotr Stanislawski 100%. Everybody speak about activity, but nobody about substrate. Is it enough substrate quantity for enzyme in the feed? If not, what is reason to increase enzyme dosage? Are enzyme's matrix values also depending on substrate content? How can we calculate this? A lot of complicated problems for nutritionists.
Dr Piotr Stanislawski
I endorse your view, practically I had noticed little room for additional benefit of Protease regarding trypsin inhibitors if SBM is properly toasted.
kasaraneni suseela Keratinases hydrolyze more than just keratine. They are are serine proteases that hydrolyze a variety of dietary proteins and, in case of DP100, the trypsin inhibitors.
I am from shrimp farming industry. Most of the farmers are using fermented soya in the feed. Trypsin inhibitor is one of the anti nutritional factor. Can we use any protease enzyme to counter this and which product is suitable for this purpose? Thank you
Dr. RAVI KUMAR AMERNENI Not all proteases are effective in hydrolyzing trypsin inhibitors. Some of them have low specificity for TIs, others are severely inhibited by TIs and hence are not effective. Cibenza DP100 is highly effective in hydrolyzing soybean TIs.
Morning, hello there. Very interesting discussion, thanks for illustrating us. I have a question for Mercedes and Juxing; your answers, reflections, I assumed, are considering enzymes addition in dry form. What would be the effect if we would spray proteases + phytases + NSPases, let´s say more or less during a short period of time, or even at the same time over the pellet? Any experience? Many thanks! All the best to everyone.
Hi every one
Mercedes Vazquez - Anon
I wanted to know about flavoring compounds. Is there any product in the market for poultry, and what is the acceptance for sucrose octa acetate?
Sadaqat there are matrixes for NSP enzymes used by part of nutritionists/companies. In practice we use usually at least 2 enzymes: carbohydrase (xylanase and/or glucanase) and phytase. Then using matrix for phytase we add only ME uplift for cereals with carbohydrase ( eg.5-6% for wheat and/or 8% for barley).Other nutrients for carbohydrases are neglected.
Pirzado M Zakria Using "cocktail" of some enzymes there is not additive effect. As the general rule, NSP are calculated as 100% for energy, phytase 100% for minerals and protease 100% for protein/AA (I mean 100% as declared). Other effect of all enzymes are calculated partly or neglected (eg. AA for NSP or energy for phytase). This is not just simple adding. Each enzyme company have own research and recommendations. Pls ask your enzyme/premix supplier for details.
Pirzado M Zakria Also need to know which enzyme is contributing more in that cocktail (e.g.) if its NSPase base then you can consider ME, primarily, and ignore other factors. Do likewise for other enzymes.
Raquel Araujo, interesting and coherent information. However, I would like to ask a question about the nature of the protein, specifically that of soy. It is known that the gastric digestion of soy protein, produces a series of peptides with an action of importance for the health of the animal, as an antioxidant, of increasing the phagocytic capacity of macrophages, among others. Thus it can be deduced that the protein source goes beyond the simple supply of amino acids. It turns out that when we replace one protein source with another, we simply rely on the amino acid composition. And when we analyze the results, we usually associate the differences in animal performance, the possible differences in the digestibility of amino acids. This reality also has implications when we use amino acid supplementation, reducing the protein source. In this context, how is the use of proteases? I will be reasoning correctly.
A work published in 2017 (Santos et al., 2017) http://dx.doi.org/10.1016/j.aninu.2017.02.001 showed there is no additivity in the combination of phytase superdosing, xylanase and protease. Something to take into account when formulating diets and accept that having more enzymes in the formula does not mean they are going to work with the same efficiency. There is a nutrient limitation as key nutrients are digested first by the dominant enzyme leaving less space to work for other enzymes! So, it must to be accepted that working with enzymes does not mean 1+1+1 is 3!
Dear Gemma González Ortiz, yes your are right but we can get 1+1+1=3, if we supplement enzymes with their best, and the enzymes best is to provide enough room(substrate) to enzymes. Moreover, enzymes are meant to be cost saving with sustaining performance of birds. i suggest we should follow the bell shaped curve for enzymes. reducing the nutrient specs of feed, supplement the enzymes you will notice the 1+1+1 is 3 effect. Thanks
Thanks all for the good discussion. I would like to confirm a few points mentioned above:
Indeed exogenous Proteases do contribute to mitigate variability in quality from SBM (and protein sources) by improving aa digestibility and degrading Anti Nutritional Factors like Trypsin inhibitors and as well as lectin. True that the benefits are all the more important that digestibility of the amino acids of the raw material is suboptimal. As also mentioned previously, exogenous Proteases also contribute positively to Gastrointestinal Functionality by reducing the risk of development of enteric pathogens.
Furthermore, exogenous proteases have a proven compatibility and even clear additivity with other feed enzymes such as Phytases, Xylanases & Amylases. In a recent collaboration with Massey University, New Zealand, we at DSM have also observed synergistic effect on a.a. digestibility between DSM ProAct and DSM HiPhos: A.J.Cowieson, J.O.B.Sorbara, G.Pappenberger, M.R.Abdollah, V.Ravindran. Toward standardized amino acid matrices for exogenous phytase and protease in corn–soybean meal–based diets for broilers. Poultry Science. Available online 25 March 2020. https://doi.org/10.1016/j.psj.2019.12.071
We at Novus observed a similar synergistic behavior of proteases and phytases. We ran a 42d experiment to evaluate the effects of supplementation of CIBENZA® PHYTAVERSE® G10 in combination with a protease CIBENZA® DP100 in broiler chicks. It was evident that supplementing with both phytase and protease with their full nutrient values led to improved performance beyond that achieved with either enzyme supplemented separately.
Amin Nahal In general, yes. However, as a nutritionist, the focus needs to be on the digestibility of Amino Acids (AA) and energy value (ME) of the protease in use. It will depend on many factors mainly related to the quality of SBM used in the diets and the formulation (AA/ME ratio!) and many other factors. I will recommend you reach out to one of the R&D team members at Novus International, and they will be very happy to help you. You can also start with the Novus representative/distributor in your Region/Country!
Amin Nahal Yes, you can reduce crude protein levels in the diet when you use protease in corn soya diets, but the matrix will depend on many factors including quality of soybean meal
Guillaume Trepo, message of interest for understanding the matter at hand. However, with regard to the compatibility of the action of the protease with the different carbohydrates, exactly one of the ones that has the greatest action on the soy fiber, mannanase, was missing. The relevance of this detail is related to the fact that the fiber soybean, admittedly, has a negative effect on the swine microbiota. I believe that this enzyme may also be compatible with protease.
It is likely that addition of protease enzyme in feed with the reduction of protein in the feed could help in better balance between essential and non essential amino acids there by reducing the endogenous losses.
In one of my experment, we observed that the protease has no significant improvement in the amino acids digestiblities in corn, SBM and Wheat based diet. It was an independent university research. There is a lot of discussion about the matrix values of the enzymes which are advocated by the producers. In fact, it need to be critically evaluted by the feed mill nutritionists and try to establish their own matrix for each product they wanted to use under their own raw material and production conditions.
Daulat Rehman Khan I have a similar experience. Often times non-protease products such as phytase will have a greater and more consistent uplift in amino acid digestibility than proteases. In these situations it is likely more about reduction in endogenous losses, or breakdown of complexes (phytate interactions for example).
I do believe in the POTENTIAL for proteases, but not in any commercially available product. For me this comes down to target substrate. Phytase has a well defined substrate, that is largely indigestible in the absence of exogenous enzymes. Protein on the other hand has generally high digestibility. Some individual amino acids are digested with over 95% efficiency. And this high digestibility is due to an abundance of endogenously produced proteases.
Therefore, if we are to push protein digestibilty higher we need to develop a protease that targets resistant substrates, which are not consistent. Unfortunately, all commercial proteases of which I'm aware are not designed for animal feed, but adapted from proteases developed for other applications. They are generally non-specific, and will as a result be more likely to spare the need for endogenous acitivity, than to act as a supplement to it.
Furthermore, when proteases are used with other enyzmes, I've seen data suggesting that the first substrate for these added proteases are *THE OTHER* added enzymes (Phytase, carbohydrases, etc.), thus reducing the impact of these other enzymes insead of enhancing the response.
Please make sure the protease you are using is low pH gut stable. From my experience there are many so called proteases av. in the market but some of them is not pH stable. As the protease will first start function in small intestine it has to pass the stomach. From the nature point of view Proteases have the best working condition at pH 8 to 10. Producing Feed Proteases need to take this into consideration and create them gut stable and lowering the optimum Ph. A different thing could be your diet formulation has been too concentrated and the birds didn't need this higher digestibility and thereby didn't make use of it. Running a trial with protease you need to be around 15 % below breeder recommendation to see the effect, that is my experience.