Download Enzymes and their effect on amino acid nutrition

Enzymes and their effect on amino acid nutrition
Mike Bedford and Carrie Walk, AB Vista Feed Ingredients, Marlborough, Wilts UK.
Whenever the application of enzymes to improve protein digestibility is discussed, one of
the immediate classes of enzymes that comes to mind is protease. However, this is not the only feed
enzyme that can directly influence protein digestibility. In some cases proteases may be completely
overshadowed by the effects of the other two main classes of enzymes used, namely phytases and
non-starch polysaccharideases (NSP’ases). A very brief explanatory review follows.
Scene setting – why is protein digestion compromised?
The digestion of protein is driven by the presence of endogenous proteases and in the case
of the monogastric this is a two stage process. The first phase is the gastric phase which is low pH
and exposes the proteins to pepsin. The second phase is the small intestinal phase, a neutral phase
where trypsin, chymotrypsin, elastase and several other exo-proteases are present to complete the
process of protein digestion. The secreted proteases are very effective in degrading dietary proteins
and as a result are potentially dangerous as they could digest the animal’s gastrointestinal (GI) tract
and the cells in which they are produced. However, this problem is averted since the enzymes are
secreted in an inactive form and only activated by pH or enzymes within the lumen . In addition, the
gut is protected by a layer of mucus which is relatively inert to proteolytic destruction. Generally this
system works well but protein digestion may be compromised for a number of reasons including:
1. Protease inhibitors within feed ingredients
2. Damage to intestinal structure and absorptive surface area
3. Rapid transit time through the gastrointestinal tract
4. Insufficient secretion of endogenous proteases
The latter includes impediments such as viscous non-starch polysaccharides (NSPs) which reduce the
turnover rate of all digestive enzymes, including proteases, and thus the net result is that there may
be insufficient proteases secreted to facilitate complete digestion. Young and diseased animals may
also be compromised in their ability to produce or secrete digestive enzymes in which case nutrient
digestion, particularly protein digestion, will be reduced. In many cases the animal is faced with one
or more of the above situations and thus protein digestion is often not optimal. Under such
circumstances, supplementation of the diet with enzymes which address one or more of the factors
compromising digestion would enable more complete protein digestion and presumably more
efficient growth.
Proteases
Augmentation of proteolytic activity in the GI tract clearly should be of advantage when
digestion is overwhelmed. The proteases used commercially to date tend to be serine proteases
isolated from Bacillus spp which maintain optimal activity in the alkaline region of the GI tract, in
some cases their optimum activity is as high as pH 9, which is much higher than any pH encountered
within the gut lumen. This suggests that they will be most active in the small intestine and likely not
active in the gastric phase at all. The data to date are not consistent and perhaps this relates to
variation in response which is governed by ingredients used, the presence of other enzymes and the
age of the animal. Each is discussed briefly below.
Recent work has shown significant improvements in amino acid digestibility when proteases
are employed but the concomitant improvement in performance is not always apparent (Angel et
al., 2011; Liu et al., 2013). Several authors have suggested that the benefit derives from the ability of
the exogenous proteases to target protease inhibitors and/or lectins. However, in the work of Liu et
al (2013), it was suggested that, given the pattern of effect on digestibility of specific amino acids,
the enzyme was probably preferentially targeting the cereal portion of the ration (Liu et al., 2013).
Variation in performance of proteases depending upon dietary formulation has also been observed
suggesting that the efficacy of a protease may be dependent upon the ingredients used in the ration
(Kocher et al., 2003). The adjustments made to a ration when employing a protease may therefore
need to take into account the ingredients used.
The benefit of a protease may also depend upon the presence of other enzymes, as it has
been shown that the benefit is lost when it is tested in the background of a xylanase and/or phytase
(Kalmendal, 2012; Sultan et al., 2011), although addition of a protease into a ration which already
contained a xylanase and an amylase was shown to improve apparent ileal amino acid digestibility
and AMEn in young broilers (Romero et al., 2013). The discussion regarding how matrices of
enzymes combine when different classes of enzymes are used in the same ration has led to much
debate but the general conclusion is that they are not additive (Cowieson and Bedford, 2009a) and is
discussed briefly below.
Regardless, the implementation of a protease in a ration requires formulation advice which
results in no loss in performance, and this has been the case in some (Angel et al., 2011; Yan et al.,
2012) but not all performance trials reported (Tempra, 2013). In the work of Yan et al (2012) it was
clear that the benefit of the protease was greater in the starter phase compared with the finisher
phase which suggested that the young animal may be more responsive to such products. If correct
then age specific recommendations would refine the protease offering considerably.
NSP EnzymesThe ability of an NSP’ase to enhance protein digestibility is well documented
(Bedford et al., 1998; Danicke et al., 1997; Silva et al., 1997) and is driven by one of three
mechanisms – reduced intestinal viscosity, direct endosperm cell wall puncturing and thus exposure
of contents to digestion, and production of fermentable oligosaccharides. The viscosity mechanism is
more relevant for small grain cereals such as wheat and barley, but its effect cannot be discarded for
maize based diets, particularly with reference to fat digestibility. The likelihood that the third
mechanism is actually responsible for increased cell wall puncturing has recently been discussed
(Masey O'Neill et al., 2012; Singh, 2012). In essence it is suggested that the NSP’ase provides
oligosaccharides which are fermented to volatile fatty acids (VFAs) in the large intestine in such
concentrations that they trigger an entero-hormonal response which results in delayed gastric
emptying (Goodlad et al., 1987) and duodenal transit rates (Park et al., 2013). This encourages more
complete gastric and proximal small intestinal digestion of the whole diet, which fits with the data in
the literature. Indeed when NSP’ases are used the digestibility of all amino acids in the undigested
fraction is improved by approximately 15-17% (Cowieson and Bedford, 2009a; Cowieson and
Bedford, 2009b), suggesting the enzyme is not just targeting the cereal fraction. If NSP’ases were
specifically targeting the cell walls of the cereal fraction then it would be expected that the benefit
would be greatest for those amino acids which are dominant in the cereal and this clearly is not the
case. One consequence of this proposed general tenet is that the goal for NSP’ase enzymes should
be to provide the correct oligosaccharides to propel the gastric/duodenal response. Thus the search
for more and more ancillary NSP degrading enzymes to “digest” the fibre fraction is likely a strategy
which will ultimately fail to improve performance as the complete dissolution of cereal cell walls is
clearly not responsible for the response to NSP’ases in monogastrics. Indeed the fact the effect of
an NSP’ase is is generalised across the whole diet lends credence to the oligosaccharide mechanism
whilst challenging that of the cell wall mechanism.
Phytases
A great deal of literature has focussed on the topic of amino acid digestibility and the effect
of phytases, with many authors showing significant benefits (Agbede et al., 2010; Augspurger and
Baker, 2004; Cadogan et al., 2009; Onyango et al., 2004; Onyango et al., 2005; Pirgozliev et al., 2011;
Ravindran et al., 1998; Ravindran et al., 1999; Ravindran et al., 2001; Selle et al., 2003). However,
several papers have shown no benefit of phytase on this parameter (Agbede et al., 2010; Augspurger
and Baker, 2004; Boling-Frankenbach et al., 2001; Peter and Baker, 2001; Peter et al., 2000),
although Boling-Frankenbach et al (2001) did suggest the effects of phytase were ingredient specific.
The current understanding is that phytate negatively interferes with gastric digestion and the
concomitant GIT response, i.e. increased pepsin and HCl and mucin production, is responsible for the
apparent reduction in digestibility of amino acids (Cowieson et al., 2004; Cowieson et al., 2006;
Cowieson et al., 2009; Cowieson et al., 2011; Peter and Baker, 2001; Peter et al., 2000). Thus the
application of a phytase under the right circumstances will reduce endogenous inputs for protein
digestion which results in an improvement in digestibility of those amino acids which predominate in
the endogenous losses, specifically cysteine, aspartate, threonine, glycine and serine (Cowieson et
al., 2004; Cowieson et al., 2006; Peter and Baker, 2001; Peter et al., 2000; Selle et al., 2012). The fact
that phytate varies in its ability to interfere with gastric digestion depending upon the identity of the
dietary protein investigated may explain some of the inconsistency in the literature (Kies, 2006).
Generally the use of a phytase can be associated with a reduced endogenous input rather than an
improvement in digestibility of the diet per se, and as a result the amino acid matrices applied to the
enzyme should reflect this fact. Thus lysine and methionine should not feature as significant amino
acids in the matrix of any phytase.
Combinations of enzymes
This topic has been dealt with elsewhere in great detail but the basic tenet is that the
phytase will reduce endogenous losses as a result of its ability to facilitate gastric and subsequent
small intestinal digestion (Cowieson and Bedford, 2009a; Cowieson and Bedford, 2009b). Specific
amino acids are spared more so than others when a phytase is employed, such as those involved in
mucin and endogenous secretions. If an NSP’ase is then utilised it will recover approximately ~15%
of the remaining undigested fraction, which will clearly include the endogenous inputs. Thus when a
phytase is used in addition to an NSP’ase the amino acid matrix of the NSP’ase needs to be adjusted
downwards to take into account the reduction in the undigested fraction of those amino acids
recovered by the phytase. The tertiary combination with a protease now needs to take into account
that the combination of both the phytase and NSP’ase has reduced endogenous losses and
recovered 15% of the “undigested” fraction, the latter probably being the most accessible amino
acids which escaped digestion in the absence of the NSP’ase. Application of a matrix for amino acids
for a protease derived from diets which did not contain a phytase or a xylanase therefore needs to
be considered with caution. In fact, Sultan et al (2011) suggest that the combination of more than
two enzyme classes from phytase, protease and xylanase, did not result in any further improvement
in N digestibility. Each enzyme alone or in combination with one other tended to improve N
digestibility, but addition of the third to a combination of the two resulted in no further
improvement. Users need to be aware of such limitations when applying feed enzymes as the values
on paper look attractive, but the biological value is less attractive and may result in significant losses
in performance as the enzymes fail to deliver their matrices in an additive manner.
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