Proteases – a tool to improve soybean quality?
Dr. Vibe Glitsø, Senior Department Manager Feed Applications, Novozymes A/S, presented a series of in vitro studies carried out with the aim of better understanding the role of a protease to improve the quality of under and over-processed soybean meal (SBM). In under-processed SBM, where the endogenous protease system is impaired a protease was shown to degrade trypsin inhibitor (TI) and complement the endogenous protease system. Whereas, in over-processed materials, where the endogenous system is functional but protein solubility limits protein utilization, a protease was shown to improve solubility by complementing the endogenous system.
Workshop participants from western Europe had mixed views on the importance of under-processing, citing overcooking as more of an issue for their situation. In other regions, this view differed and the point was also made that users often do not have an alternative regarding SBM quality – they have to use what is supplied from the vendor. In this situation, the potential for a protease to stabilize the quality of soybean from batch to batch was seen as an interesting tool to improve uniformity of production.
Also debated was the occurrence and value of measuring trypsin inhibitor activity (TIA) in SBM given the role even marginally high levels of TI can have on feed passage rate and litter condition. Whilst useful it was seen by some to be an expensive and time consuming procedure, hence the reliance on urease as a measure of SBM processing despite its limitations. The ongoing development of NIR-based approaches to allow measurement of TIA routinely was seen as a novel and useful tool going forward to better assess the quality of SBM.
Formulating feeds with a protease
Dr. Adam Smith, Market Development Manager – Proteases, DSM EMEA, gave an overview of undigested protein at ileal level for different ingredients in broilers, and the approach DSM has taken to quantify the effect of RONOZYME® ProAct to reduce this. An overview of the data available for the effect of RONOZYME® ProAct on ileal digestibility of individual ingredients and the relative merit of different approaches for implementing such information into least cost feed formulation were discussed.
Dr. Smith commented: “DSM have invested more than €4 million in ileal amino acid digestibility studies over the last 3 years to provide a robust database describing the effect of RONOZYME® ProAct on the key ingredients used in poultry feed. This gives users of the product access to an unrivalled database of information to formulate feeds in confidence.”
Scrutinizing the database in more detail, participants in the workshop rightly highlighted the need for more understanding of the pattern of response to a protease. In particular, the relationship with protein structure/type and other interacting factors, such as NSP, were raised. Clarification on the currently equivocal position on the use of an AME matrix value with protease, more information on the potential of a protease to support greater use of by-products and the desire for similar data to those now available for broilers in laying hens were also discussed.
Are proteases beneficial for the environment?
Professor Ilias Kyriazakis demonstrated, by means of LCA studies, that the use of proteases in animal feed positively contribute to the environmental impact of animal production systems. They reduce the emission of greenhouse gas but more significantly lower acidification potential (through reduced ammonia emissions).
Prof Kyriazakis commented: “There is no single technology that will solve the environmental and food security issue we face as an industry. The effects of proteases we have seen are definitely contributing in the right direction.”
Workshop participants indicated that only in certain intensive livestock production regions do nitrogen emissions directly influence their business. However, all agreed that technologies that increase efficiency and allow reduction of absolute crude protein levels in diets contribute both to the environment as well as business profitability.
Questions which were debated but remained unanswered were:
- If and how much of a premium producers are willing to pay for technologies other than protease, that benefit the environment but do not create a direct cost saving as such.
- If and how information around technologies such as proteases and their environmental benefits need to be communicated further down the value chain to retailers and end consumers. Suggestions were made that proteases should be used as one of a range of technologies in complete feeding and/or production concepts around which clear messages can be formulated rather than as a single technology.
Can proteases play a role in enteric health?
Dr. ir. Pim Langhout, Director of Nutrition, DSM, started the workshop with a short review of the importance of litter quality, the link with poor enteric health and in turn protein. “There exists a paradox between decreased protein levels which are favorable for enteric health, but problematic for optimal growth in today’s modern broiler” commented Dr. Langhout. Evidence was subsequently provided from the scientific literature for a relationship between high protein levels in the diet and higher levels of detrimental bacteria such as E.coli and Clostridium. The possibility that a protease, as a way of improving protein utilization, could help avoid the negative effects of high protein in the diet was put to the group for discussion.
Participants in the workshop discussed wet litter occurrence and control measures in different regions. The fact that in EU countries high incidence of footpad lesions due often too wet litter result in penalties in the form of lower stocking density was particularly highlighted. Proteases were seen as a tool which helped with wet litter under practical conditions and could as part of an overall strategy help prevent such penalties. One participant commented ‘my vet called the nutritionist to find out what had changed due to the reduction in wet litter when we started to use a protease’. The suggestion was also made for more uniform pigmentation of broilers when a protease was used and this was linked to a positive enteric health status. The relationship between protein level and gender of birds was raised in the context of as hatched flocks. Whereas male birds can utilize higher levels of protein, female birds do not need this, thus resulting in wet litter. It was suggested that further research may investigate the role of a protease to overcome these problems.
Bird age and protease effects – what can we already conclude?
Professor Roselina Angel reviewed the literature on the effect of age on protein digestibility, endogenous enzyme secretion, endogenous flow and the implications this has for an exogenous protease. Early amino acid digestibility is influenced by variation in endogenous loss and lower endogenous enzyme secretions in the first 5 days in particular. When comparing amino acid digestibility between 5 and 21 days for specific ingredients, clear increases were seen with age for most. However, other ingredients, such as wheat, were shown to clearly buck this trend. Asked to summarize what this all would mean for the value of an exogenous protease with age Prof. Angel commented “the greatest potential impact of a protease will be in the young bird, but this will be partly diet-composition dependent.”
Workshop participants discussed the importance of age-related digestibility data in practical situations, why such effects were seen, and ways for reducing these effects like reducing time between hatch and first food to initiate endogenous enzyme production. It was concluded that generating an age related set of digestibility values for the major raw materials could be of value for the industry as a whole. Data for the effect of an exogenous protease at different ages could also be of interest to better understand protease value. Even without concrete data, it was suggested that, in practice, adjusting matrix values used with a protease by age makes sense, certainly in corn soy diets where age trends are more clear. The possibility that optimal dose of protease may vary with age also warrants more investigation.
Gut physiology and the use of exogenous proteases – what knowledge is needed to better optimise function?
Use of exogenous proteases is already accepted as an effective way to improve digestion of protein in feed ingredients. Although a study of the literature reveals a significant amount of data concerning endogenous enzyme systems, including proteases,the understanding of how exogenous proteases act and interact with these and the physiology of the gut remains limited. With this situation in mind participants of the workshop were asked to consider three questions:
Q1: What do we know about the interaction of exogenous and endogenous proteases and what do we need to know?
Scientific literature in this area remains limited. In order to optimise use of exogenous proteases, increasing our understanding is perceived as a priority for future research.
The main questions to be answered in such activities include:
- Are exogenous enzymes interacting with endogenous enzyme feedback mechanisms?
- Will exogenous: endogenous interactions result in an energy sparing effect?
- Are there any direct positive or negative effects of an exogenous protease on the gut wall
Q2: What tools are available to help us better understand gut physiology and interaction with enzyme activity?
Various innovative tools are available and already being used by DSM and collaborators to study gut physiology in relation to protein digestion. They give us the potential to better understand the effect of an exogenous protease on endogenous and exogenous protein digestion/recycling. Some of the methodologies already in use are given below but others, currently under-utilised in animal nutrition, need to be developed.
- Liquid chromatography - mass spectrometry (LC-MS) - to measure protein digestion in digesta at different levels in the gastrointestinal tract and with different ingredients/diets
- Use of stable isotopes - to monitor/follow endogenous losses
- Gene expression analysis - to measure changes in pancreatic activity/trypsin secretion
- Metabolic chambers to detect any energy sparing effects
Q3: How critical is intestinal transit time in determining the effect of an exogenous protease?
Intestinal transit time is affected by many parameters and is known to be a critical determinant of the effect of exogenous enzymes. The presence of the enzyme may also change the residence time of digesta in the different parts of the gut. In the case of proteases, as for other enzymes, more detail on this interaction is needed. When adding an exogenous protease the relationship could be further complicated due to their ability to degrade anti-protease factors known to increase passage rate of digesta. An attempt to try and understand better the dynamics of adding an exogenous protease on the transit time of digesta and vice versa is needed and should be a topic for future research.
Measuring amino acid digestibility with exogenous proteases in individual feed ingredients?
There are various methodologies currently available for measuring amino acid digestibility in broilers (Apparent, Standardized and True ileal amino acid digestibility). Each has its own advantages and disadvantages. The generation of digestibility values for individual ingredients, as currently being done by DSM and research collaborators is seen as critical for demonstraiting value of a protease. Which of the available techniques is the most appropriate and whether improvements/changes are needed for this purpose has so far been of little consideration. Participants of this workshop were therefore asked two questions:
Q1: What is the best available method for evaluating the effect of an exogenous protease on amino acid digestibiity in ingredients?
Standardized ileal amino acid digestibility (SIAAD) methodology (despite various technical limitations particuarly associated with use of N free and semi synthetic diets) was considered the most approporaite current tool for routine assessment of an exogenous protease on digestibility. The reasoning was mainly practical (in commercial feed formulation this is the most widely used methodology for evaluating digestibiity of amino acids and for specifying requirements). The importance of other techniques to improve understanding should not be ignored. For example, the use of the regression/TIAAD method, for ingredients used in feed at low inclusion levels. Such an aproach could allow more realistic evaluation of the effect of endogenous loss than the high ingredient content of semi synthetic diets used in SIAAD methods.
Q2: How could such methodologies be further modified to better evaluate the effect of an exogenous protease?
Standardisation of existing methodologies between institutes was seen as the most important factor needed to allow better evaluation of the effect of a protease. The impact of small but maybe important differences in intepretations of the SIAAD method need to be evaluated. Already good initiatives within the scientific community are running to address this issue. Other important issues and topics for future development were raised:
- Assessment of digestibility in the young chick (7d) aswell as the normal age choosen for ileal digestibility methods (16-24d) to help better understand the protease age interaction
- Validation of a protease on digestibility of whole diets to check if data from individual ingredients is additive or not
- Use of meta-analysis to better understand effects of protease
- NIR technology to use digestibiity data to create predictive tools
- Evaluation of digestibility of other ingredients such as calcium, phosphorus fat etc. to more fully understand protease effects
The role of proteases in gut health
It is well documented that high levels of undigestible protein in the gut can have an adverse effect on the gut microflora and may trigger proliferation of pathogens such as Clostridia perfringens. Given that use of an exogenous protease can improve the digestibility of protein in the gut and also allows use of lower protein diets in an efficient way it makes sense that an exogenous protease will have a possitive effect on gut health. Participants of this workshop were therefore invited to debate the following question:
Q1: What are the potential benefits of an exogenous protease on microbial challenge?
Current understanding of this complex area is rather limited in comparision to other feed addtives. First studies have been able to show reduced E.coli numbers in the hindgut when a protease was added to feed (in combination with a carbohydrase), reduced clostridial counts, benefits on performance during a necrotic enteritis challenge and improved performance (alone and in combination with a carbohydrase) during and after use of a live vaccination for coccidiosis. It therefore seems that proteases may have a possitive role to play during a microbial challenge.
Beneficial effects of a protease on litter quality are also frequently seen and although often explained by the effect of lower nitrogen diets and associated reduction in water intake/excretion the benefit typically seems greater than what can be attributed to these factors alone. An effect of gut health could be one explanantion for this difference.
Additional points were also highlighted as warranting further investigation/research if the effect of a protease on gut health is to be better understood and the value captured by industry:
- Interaction between mucin secretion and protease use – can a protease digest mucin to prevent an increase in viscosity and accumulation of undigested protein in the hindgut?
- Is there a direct relationship between an exogenous protease and gut integrity (e.g. villi structure and integrity of tight junctions)?
- Use of mild necrotic enteritis challenge models with and without a protease
- Use of proteases as integral part of a coccidiosis vaccination programme