Exogenous protease is known to increase the digestibility of protein and amino acids when added to the animal feed. But there are more benefits to be gained from this enzyme.
A protease (Ronozyme ProAct) with a substrate protein chain (dark red) bound. The close-up shows how charge/ proton transfer among the three residues in the classical catalytic triad (red, blue, yellow) activates the nucleophile (yellow) to enable cleavage of the substrate.
The motivation for the use of exogenous protease in non-ruminant nutrition is typically the reduction of feed cost with a constant animal performance. But also secondary effects can be seen, including environmental benefits, litter management, enteric resilience, uniformity and microbial stability. This is the result of the increased amount of fundamental knowledge gained on protease over the last few decades. In addition, the availability of protease (only since the last 5-10 years) as a mono-component has increased the knowledge on the effectiveness of this enzyme. In this article, we delve a bit deeper in the ‘extra-proteinaceous’ effects.
The effect of exogenous protease on ileal amino acid digestibility is significant and moderately consistent and has been recently reviewed by Cowieson and Roos (2014). At the same time, there has been increasing interest over the past few years in ‘extra-proteinaceous’ effects of protease such as influences on enteric resilience, litter quality, interactions with non-protein nutrient digestibility e.g. fat or starch, carcass yields and so on. Exogenous enzymes, including protease, play an important role in improving environmental sustainability of pig and poultry operations via reduced N emissions for example. The origin of the promotional effect of protease on gut health is not clear
but may be a combination of several interacting factors. Such factors may include a reduction in putrefaction in the distal digestive tract, reduced viscosity of lumen contents, altered flow of NSP in the intestine, improved retention of calcium and phosphorus and enhanced availability of amino acids for mucin synthesis amongst others.
Figure 1 - Effect of exogenous protease on the thickness of the adherent mucus layer (AML;_m) in 3 intestinal segments in broilers. The effect of protease was statistically significant in each segment (adapted from Peek et al., 2009).
Role in gut health
The ‘extra-proteinaceous’ effects also include the reduction of bacterial load for example. Mynott et al. (1991) demonstrated that oral delivery of ‘an enterically-protected protease preparation was able to substantially reduce enterotoxigenic Eschericia coli (ETEC) attachment to the intestinal mucosa of rabbits. Protease supplementation also resulted in a significant reduction in colony-forming units per centimetre of intestine and a significant reduction in diarrhoea and diarrhoea-related death. A study from 2009 showed that protease addition to a corn/wheat/soy-based diet alleviated the negative effects of coccidial (E. acervulina, E. maxima, E. tenella) infection in broilers, significantly increasing weight gain equivalent to uninfected birds. Furthermore, birds that received the diet supplemented with exogenous protease had a significantly thicker adherent mucus layer in the duodenum, jejunum and caecum compared with birds that received the un-supplemented control diet (Figure 1). In another trial (Cowieson et al. (2015)), the effect of a mono-component exogenous protease on immune competence and jejunal health of broilers fed either a SBM-free diet (based on corn, canola meal and DDGS) or a SBM-based diet (standard corn/soy) was studied. Bird performance was more obviously improved in the diet based on SBM compared with the diet based on canola meal though ileal N digestibility was improved equivalently in both diets. Furthermore, protease application resulted in upregulation of Claudin1 and in various amino acid transporter proteins in the jejunum of broilers on day 21, an effect suggestive of increases in the integrity of tight junctions in the intestine and absorptive capacity per se.
Effect on fat digestibility
Feed enzymes have a wide influence beyond their target/focal nutrients. Proteases often increase the digestibility of non-protein nutrients (such as fat). Cowieson et al. (2015) noted that protease addition to a diet based on corn and soybean meal increased ileal digestible energy from 3077 kcal/kg to 3154 kcal/kg and apparent metabolisable energy from 3130 kcal/kg to 3261 kcal/kg. Kalmendal and Tauson (2012) reported that supplementation of a wheat-based diet for broilers with exogenous protease significantly increased the ileal digestibility of starch from 93 to 96%, of fat from 89 to 91% and AME from 13.68 to 14.16 MJ/kg. Yuan and Wang (2010) noted that elevated taurine concentrations in the jejunum of broilers inhibited intestinal development by increasing total bile acid concentrations, an observation that was recently confirmed (Huang et al., 2014). It is therefore possible that exogenous protease enhances fat digestibility by disrupting the feed nutrient matrix and reduces bile acid synthesis, secretion and intestinal taurine concentration which in turn promotes intestinal development.
Meta-analysis of 50 broiler trials
Application of protease often results in performance responses that are beyond what could be explained by the sum of the effect on amino acid digestibility. To further study this, a meta-analyis was carried out, covering more than 50 independent broiler trials including more than 300 ‘control’ diets, each fed with, and without, a mono-component exogenous protease (Cowieson et al., unpublished), several statistically significant factors emerged that are particularly associated with performance benefits. These factors include (+/− indicates whether this factor promotes or demotes a favourable effect of protease on weight gain and FCR): added fat/oil (+), amino acid balance (+/− depending on the individual amino acids and their ratios), presence of full-fat soybean meal (+), the dose of enzyme used (the higher the dose, the higher the response), limestone inclusion (−) and the balance of amino acids relative to energy (+/− depending on the individual amino acid). Additional added fat for example promoted the beneficial effect of exogenous protease on weight gain and FCR in broilers. Another remarkable result, taken from the meta-study is that diets with lower limestone inclusion concentrations are more responsive to exogenous protease than those with higher limestone levels. It was also shown that protease responses are linked to a variety of amino acid concentrations and ratios to each other and to energy.
It is clear from the published literature currently available exogenous proteases are effective tools to increase amino acid digestibility in non-ruminants. Furthermore, substantial beneficial effects on animal performance have been recorded that cannot be fully explained by the sum of the effect on amino acid digestibility, responses that point to ‘extra proteinaceous’ effects of protease. As the understanding of the wide range of effects of protease grows, it is expected that the market for this unique enzyme will also expand and will lead to novel application options and multifactorial solutions to ongoing production concerns.
This article has been edited from the original paper ‘Toward optimal value creation through the application of exogenous mono-components protease in the diets of non-ruminants’, published in Animal Feed Science and Technology (in press, 2016).