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Protease enzyme and trypsin inhibitor: Novus’s results

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Edwin T Moran Edwin T Moran
Animal Nutritionist
March 17, 2020

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?

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Mercedes Vazquez-Anon Mercedes Vazquez-Anon
Senior director of animal nutrition
Novus International Novus International
Missouri, United States
April 29, 2020

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.

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Edwin T Moran Edwin T Moran
Animal Nutritionist
April 30, 2020
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.
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Juxing Chen Juxing Chen
Structure
Novus International Novus International
Missouri, United States
May 1, 2020

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.

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May 4, 2020

Edwin T Moran There is no enzyme, so the mixture will not work?

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Dr. RAVI KUMAR AMERNENI Dr. RAVI KUMAR AMERNENI
Masters in Aquaculture
May 6, 2020
Mercedes Vazquez-Anon can you please share the DP 100 product details to my email. raviaqua@gmail.com. thank you
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June 5, 2020
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.
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June 24, 2020
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.
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Arne Korsbak Arne Korsbak
Marketing manager, Feed enzymes Europe
DSM DSM
June 25, 2020

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.

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June 27, 2020
Juxing Chen Do you have any results of that? And this will work same even in higher protease dosage?
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daniel severino daniel severino
Agriculturist
March 17, 2020

Thank you so much, this is a well detailed information.

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Carlos Vilchez Carlos Vilchez
Title: Ingeniero Zootecnista. Academic Degrees: Mg.Sc., M.S., Ph.D.
March 18, 2020
On the subject, for more information, read the article by Lee et al. (Poult Sci. 2018 Jun 1;97(6):2078-2085)
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Luciano Andriguetto Luciano Andriguetto
Owner of Ithaca Feed Solutions, Professor of Animal Nutrition at UFPr
April 30, 2020
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)?
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Mercedes Vazquez-Anon Mercedes Vazquez-Anon
Senior director of animal nutrition
Novus International Novus International
Missouri, United States
May 4, 2020
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!
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Dr Piotr Stanislawski Dr Piotr Stanislawski
Nutritionist
DSM DSM
May 6, 2020
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.
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Kapil Manwal Kapil Manwal
masters in animal nutrition
May 8, 2020

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.

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Dr Piotr Stanislawski Dr Piotr Stanislawski
Nutritionist
DSM DSM
May 9, 2020
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.
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 Dr. Vijay Swami Dr. Vijay Swami
Country Manager -SunHy Biology
May 13, 2020

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?

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Kapil Manwal Kapil Manwal
masters in animal nutrition
May 14, 2020

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

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Kapil Manwal Kapil Manwal
masters in animal nutrition
May 14, 2020

Dr. Vijay Swami
Good point what I had shared is my practical experience and yes, I am not scientist but an simple chicken man.

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Dr Piotr Stanislawski Dr Piotr Stanislawski
Nutritionist
DSM DSM
May 20, 2020
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.
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June 24, 2020
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.
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June 24, 2020
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.
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Youssef Attia Youssef Attia
Professor
June 25, 2020
Mercedes Vazquez-Anon very helpful, in addition the effect of protease will depend on age of broilers and diet quality
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Saif Ul Haq Saif Ul Haq
Animal Nutritionist
June 28, 2020
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.
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Shah Alam Shah Alam
Sales manager
July 14, 2020

Mercedes Vazquez-Anon
Have a nice day ! Can you tell me the name of trypsin inhibitors which are available in soybean?
Stay healthy.

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April 30, 2020
Thank you so much for being so informative
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April 30, 2020
keratinase is for hair hydrolysis , how does it help? what is the active for the enzyme?
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Novus International Novus International
Missouri, United States
May 5, 2020
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.
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May 3, 2020

Thank you for the kindness. But I needed enzymes for an experiment, but I have no way out since the borders are closed.

Does a diet based on processed soy and corn have no effect on broilers?

Mozambique
João Marime Maquena

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Dr. RAVI KUMAR AMERNENI Dr. RAVI KUMAR AMERNENI
Masters in Aquaculture
May 3, 2020

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

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Novus International Novus International
Missouri, United States
May 5, 2020
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.
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May 4, 2020

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.

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Mercedes Vazquez-Anon Mercedes Vazquez-Anon
Senior director of animal nutrition
Novus International Novus International
Missouri, United States
May 4, 2020
Rafael Duran Hi Rafael, our experience is with dry form of enzymes.
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May 6, 2020
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?
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May 6, 2020
Why is this difficult to construct matrix valves for xylanases and Nspase?
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Dr Piotr Stanislawski Dr Piotr Stanislawski
Nutritionist
DSM DSM
May 6, 2020
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.
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June 24, 2020
Dr Piotr Stanislawski thanks alot sir
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Pirzado M Zakria Pirzado M Zakria
Veterinary Doctor
May 7, 2020
How can we calculate matrix values when we use protease, phytase and NSPs in combination?
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Dr Piotr Stanislawski Dr Piotr Stanislawski
Nutritionist
DSM DSM
May 7, 2020

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.

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Haroon Mushtaq Haroon Mushtaq
PostDoc in Poultry Nutrition
June 8, 2020
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.
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May 7, 2020

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.

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Gemma González Ortiz Gemma González Ortiz
Research Manager
May 8, 2020
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!
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May 28, 2020
Gemma González Ortiz thanks
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June 2, 2020
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
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Guillaume Trepo Guillaume Trepo
Global Enzyme Lead, DSM Animal Nutrition & Health
DSM DSM
June 1, 2020

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

Best regards,

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Novus International Novus International
Missouri, United States
June 8, 2020
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.
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June 1, 2020

Can i reduce crude protein in diets when i used protease enzyme (cibenza d100) in corn soya diets

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June 1, 2020

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!

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June 1, 2020

Nasser H Odetallah thanks happy eid

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Novus International Novus International
Missouri, United States
June 1, 2020

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

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June 2, 2020

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.

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Mangalmurti Pathak Mangalmurti Pathak
MVSc & A.H. Animal Nutrition
June 9, 2020
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.
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June 18, 2020
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.
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Joshua Jendza Joshua Jendza
Animal Nutritionist
July 20, 2020
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.
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Arne Korsbak Arne Korsbak
Marketing manager, Feed enzymes Europe
DSM DSM
June 29, 2020

Dear Prof. Daulant Rehman Khan

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.

Rgds. Arne Korsbak

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