Published December 4, 2014
BOARD-INVITED REVIEW: Opportunities and challenges
in using exogenous enzymes to improve
nonruminant animal production
O. Adeola*1 and A. J. Cowieson†
*Department of Animal Sciences, Purdue University, West Lafayette, IN 47907;
and †Poultry Research Foundation, Veterinary Science Faculty, University of Sydney,
Camden, New South Wales 2570, Australia
ABSTRACT: Diets fed to nonruminant animals are
composed mainly of feed ingredients of plant origin. A
variety of antinutritional factors such as phytin, nonstarch polysaccharides, and protease inhibitors may be
present in these feed ingredients, which could limit nutrients that may be utilized by animals fed such diets.
The primary nutrient utilization-limiting effect of phytin arises from the binding of 6 phosphate groups, thus
making the P unavailable to the animal. The negative
charges allow for formation of insoluble phytin-metal
complexes with many divalent cations. Furthermore,
phytin and protein can form binary complexes through
electrostatic links of its charged phosphate groups with
either the free amino group on AA on proteins or via
formation of ternary complexes of phytin, Ca2+, and
protein. The form and extent of de novo formation of
binary and ternary complexes of phytin and protein
are likely to be important variables that influence the
effectiveness of nutrient hydrolysis in plant-based diets.
Nonstarch polysacharides reduce effective energy and
nutrient utilization by nonruminant animals because of
a lack of the enzymes needed for breaking down the
complex cell wall structure that encapsulate other nu-
trients. Enzymes are used in nonruminant animal production to promote growth and efficiency of nutrient
utilization and reduce nutrient excretion. The enzymes
used include those that target phytin and nonstarch
polysaccharides. Phytase improves growth and enhances P utilization, but positive effects on other nutrients
are not always observed. Nonstarch polysaccharidehydrolyzing enzymes are less consistent in their effects
on growth and nutrient utilization, although they show
promise and it is imperative to closely match both
types and amounts of nonstarch polysaccharides with
appropriate enzyme for beneficial effects. When used
together with phytase, nonstarch polysaccharide-hydrolyzing enzymes may increase the accessibility of phytase to phytin encapsulated in cell walls. The future of
enzymes in nonruminant animal production is promising and will likely include an understanding of the role
of enzyme supplementation in promoting health as well
as how enzymes may modulate gene functions. This
review is an attempt to summarize current thinking
in this area, provide some clarity in nomenclature and
mechanisms, and suggest opportunities for expanded
exploitation of this unique biotechnology.
Key words: energy, enzyme, fish, nutrient utilization, poultry, swine
©2011 American Society of Animal Science. All rights reserved.
J. Anim. Sci. 2011. 89:3189–3218
doi:10.2527/jas.2010-3715
INTRODUCTION
In the 1950s, pioneering scientists added amylases
and proteases to the diets of various farm animals and
observed benefits in productivity. Since then, the use of
exogenous feed enzymes has been one of the most widely studied and reported disciplines in animal science
1
Corresponding author: [email protected]
Received November 18, 2010.
Accepted April 18, 2011.
and has enjoyed tremendous growth worldwide in the
animal industry. Despite considerable commercial and
scientific success, new product development continues
unabated and literally hundreds of articles have been
published every year in the areas of enzyme biotechnology, animal nutrition, food science, and other related
disciplines. With this wealth of new information come
challenges, notably acute for the end user, as the array
of products and formulation strategies can be daunting.
This review summarizes current thinking in this area,
provides some clarity in nomenclature and mechanisms,
and suggests opportunities for continued and expanded
use of enzymes in nonruminant animal production.
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Adeola and Cowieson
OVERVIEW OF COMMERCIAL
FEED ENZYMES
Introduction
The global feed enzyme market is today worth in excess of $550 million US dollars and saves the global feed
market an estimated $3 to 5 billion per year. This market can be broadly divided into phytase (approximately
60%) and nonphytase (40%) enzyme segments. The
rapid growth of this market, notably in the last 5 to 10
yr, is associated primarily with the adoption of phytase
technology and the wider use of carbohydrases in cornbased diets, both ostensibly in response to increasing
input costs. Considerable investment in application research has led to several strategic developments in the
use of exogenous enzymes. Historically, phytases have
been used rather crudely as a cost-effective replacement
for sources of inorganic P, but recent evidence indicates that liberation of orthophosphate from inositol
phosphate (IP) may be of secondary importance to the
removal of reactive phytate from the diet. Strategic diet
modification is required to formulate in the absence
of phytate, and these changes extend to Ca, Na, AA,
and energy. Pivotally, it has also been demonstrated
recently that the magnitude of effect of exogenous xylanases depends on the nutritional value of the diet to
which they are added (Cowieson and Bedford, 2009),
precluding the possibility of full additivity between xylanase and phytase. The strategic use of phytases and
xylanases with appropriate diet modification, especially
with careful attention to the choice of feed ingredients,
can result in economic advantages for the end user, but
these advantages may be blunted or even lost if matrix
values (nutrient-equivalent values assigned to enzyme
products in least-cost formulation) are incorrectly assigned.
Carbohydrases
Broadly, carbohydrases include all enzymes that
catalyze a reduction in the molecular weight of polymeric carbohydrate, but more than 80% of the global
carbohydrase market is accounted for by 2 dominant
proteins, xylanase and glucanase. Xylanases (endo1,4-β-xylanase) carry the Enzyme Commission (EC)
identifier 3.2.1.8 and as such are in the family of hydrolase and in the subfamily glycosidase. Glucanases
are in the same family, carrying the identifier 3.2.1.6
[endo-1–3(4)-β-glucanase]. There is considerable obscurity in terminology regarding enzymes in general,
and this is notably acute for the carbohydrases. Endo1–3(4)-β-glucanases are often referred to as cellulases
or glycosidases, and these terms are misleading and, at
best, only partially correct. Cellulase does carry an EC
identifier (EC 3.2.1.4), but technically cellulase refers
not to a single activity but to a series of glycosidase activities, which depolymerize cellulose into glucose. Activities involved in this process are broadly: endo-1,4-
β-glucanases, cellobiohydrolases (1 and 2 depending on
whether they attack from the reducing or nonreducing
end), and β-glucosidases. Xylanases are often referred
to as pentosanases, nonstarch polysaccharide (NSP)degrading enzymes (nonstarch polysaccharidases),
hemicellulases, and so on, and again these distinctions
can be confusing and misleading. In addition to the
xylanases and glucanases, other commercially available
carbohydrases include (in order of decreasing importance) α-amylase, β-mannanase, α-galactosidase, and
pectinase (a broad term for a large family of pectindegrading enzymes, which may include, among others,
polygalacturonases, pectin transeliminases, and pectin
methyl esterases). Some commercial enzyme products
will also contain a variety of side activities, which are
often not well controlled or guaranteed and are variably
heat labile. However, it is important to note that virtually all feed-relevant carbohydrases are in the same
group of proteins, namely the hydrolase/glycosidase
family. Furthermore, virtually all are “endo” acting
(i.e., they hydrolyze carbohydrate polymers to generate
decreased molecular weight oligo- or polysaccharides
but generate virtually no free sugars).
Phytases
Phytases (myo-inositol hexakisphosphate phosphohydrolases) are a large family of hydrolases capable
of catalyzing the stepwise hydrolysis of myo-inositol
hexakisdihydrogen phosphate (phytic acid; IP6). Like
xylanases and glucanases, phytases are also in the hydrolase family, but rather than carrying the EC identifier 3.2.1 (glycosidase) they carry the identifier 3.1.3
(esterase/phosphatase). Feed-relevant phytases are
further divided into 2 subclasses dependent on which
phosphate on the myo-inositol nucleus they initiate catalysis (3- or 6-phytases). Kinetically, few commercial
phytases are capable of completely dephosphorylating
IP6 into myo-inositol and 6 free phosphates because
of the presence of a recalcitrant axially oriented phosphate at position 2 on the ring (Wyss et al., 1999).
However, mucosal or systemic phosphatases and some
microbial esterases are capable of removing this phosphate and generating free myo-inositol in the gastrointestinal tract of the animal providing the substrate
is soluble.
Other Enzymes
The feed enzyme market is dominated by phytases
and carbohydrases, and in the estimation of the authors, these activities account for at least 90% of the
global feed enzyme market. However, other enzymes,
which are occasionally supplied as part of a diet for
nonruminant animals, include subtilisin Ser protease
(EC 3.4.21.62), triacylglycerol lipase (EC 3.1.1.3), Nacetylmuramide glycanhydrolase, or lysozyme (EC
3.2.1.17), and very low quantities of various oxidore-
Exogenous enzymes in diets of nonruminants
ductases in some niche applications [e.g., laccase (EC
1.10.3.2) or hexose oxidase (EC 1.1.3.5)].
Enzyme Combinations
It has recently been reported that the beneficial effects of exogenous xylanase in poultry and swine diets
are inextricably linked to the amount of the undigested
fat, protein, and starch that leaves the ileum (Cowieson and Bedford, 2009). This observation rules out
full additivity between pronutrients (pronutrients being
compounds, which are added to feed to enhance the
digestibility of nutrients). An improvement in protein
and starch digestibility with added phytase reduces the
undigested fraction, which means that xylanase has less
fraction available as substrate. By definition then, only
the first added enzyme of choice can carry its full matrix when added to a diet, but subsequent additives
should have their matrices discounted to accommodate
the influence of the current incumbents. As theoretical
maximum ileal digestibility is 100%, enhancement of
digestibility constantly moves digestibility toward that
fixed asymptote, so opportunity for further improvement declines with each new added enzyme.
Enzyme Sources
Most commercially available feed enzymes are obtained from optimized fermentation systems relying
on the use of genetically modified bacteria or fungi.
These organisms have been engineered to overproduce
the protein of interest and will often deliver between 50
and 100 g of the active protein per liter of fermentation
broth, resulting in a product that contributes to a costeffective business model. Examples of production organisms include Trichoderma reesei, Aspergillus niger,
Escherichia coli, Bacillus licheniformis, and Pichia pastoris (note that this is by no means an exhaustive list).
After fermentation, a series of purification steps are
followed to ensure that no genetically modified recombinant DNA is detected in the final product and that
undesirable fermentation residues are removed. The active protein is then formulated for stability using compounds such as sorbitol and NaCl to improve storage
stability and general product characteristics. Thermal
tolerance may be improved by the use of various coating techniques, producing a granulated end product.
Feed enzymes are often offered to end users in a variety
of concentrations either as liquid products (often for
post-pellet application) or dry products.
Phytases, carbohydrases, and proteases will be naturally secreted by a range of bacteria and fungi to meet
their own metabolic requirements and these activities
are screened for useful characteristics appropriate to
feed application. Environmental extreme and diverse
habitats are explored to improve the likelihood of discovery of enzymes with inherently useful properties
(e.g., thermal vents for thermophilic bacteria that produce heat-tolerant enzymes or decaying fiber for cel-
3191
lulolytic proteins). More information on the industrial
production of enzymes and yeast expression systems
can be found in various reviews (e.g., Dhawan and
Kaur, 2007; Schuster and Schmoll, 2010).
EFFICACY OF CARBOHYDRASES
Exogenous enzymes that target NSP are used essentially to reduce inherent inefficiency of nutrient utilization and to combat the possible ill effects associated
with feeding diets that contain high concentrations of
NSP. Because NSP comprise many compounds with
very different structures, several enzymes have been
developed to target this diverse group of antinutrients.
Generally, poultry respond to a greater extent to supplementation of NSP-degrading enzymes than swine.
The following reviews more recent studies designed to
understand the various aspects of the modes of action,
as well as the potency of NSP-degrading enzymes in
poultry species and swine.
Tools to Assess the Effect of Carbohydrases
Enzyme effects on animal are assessed through
growth response, nutrient utilization, evaluation of
plasma composition, analysis of whole-body nutrient
utilization, and carcass quality. Effect of enzymes on
microbial activities in the gut or on the morphology
of the intestinal tract is elucidated using microbiological approaches such as plating techniques. These approaches are generally adequate for providing a gross
understanding of the effect of the enzymes but have
limitations. For example, growth performance does not
provide information about specific processes that are
enhanced or suppressed to result in enhanced BW gain.
Clearly, growth occurs as a result of many different processes in the animal. These processes, in turn, are controlled or fine-tuned by several biological or biochemical events, for which data on BW gain cannot provide
information.
However, molecular techniques can open new vistas
in our knowledge of the effect of enzymes and can elucidate pathways that explain the effect of the enzymes
in more precise ways. Some of the molecular techniques
useful to help in understanding the role of carbohydrases on make-up and changes in microbial population
in the digestive tract of animals include chromosomal
DNA of the total bacterial community in the gut (enterome method), microbial profiling using the percentage guanine plus cytosine (G+C), and superclusters.
The complex microbial ecosystem in the gut cannot
be accurately enumerated with the aid of microscope.
Holben et al. (2004) observed that only 0.1 to 1% of
bacteria detected by microbial enumeration can be
recovered by most media. Usually, nonculturable microbes or those that are proportionately smaller in the
community may end up not being identified. Total microbial community in any environment (including the
gut) can be assessed by fractionation of the total com-
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Adeola and Cowieson
munity DNA using the G+C fractionation technique
(Holben and Harris, 1995; Apajalahti et al., 2001). The
G+C microbial profiling technique is important for
rapid assessment of the shifts in microbial populations
in response to various stimuli. Apajalahti et al. (2001)
used the G+C technique for elucidating the effect of
feed source or feedstuffs on the difference in cecal microbial community of broilers. The G+C technique has
been combined with the use of denaturing gradient gel
electrophoresis to form a G+C denaturing gradient gel
electrophoresis technique (Holben et al., 2004), which
enables the identification of very diverse and complex
microbial population occurs in the digestive tract. The
approach is advantageous in that it enables the identification of the diversity of the whole microbial community instead of studying each genera or species separately
in response to dietary supplemental carbohydrases.
Observation on gene expression responses to carbohydrase supplementation is important for explaining
growth performance responses observed. A relevant example is demonstrated in the Yin et al. (2010) study,
in which greater starch digestion was found to be associated with greater AA absorption. This effect was
mediated via glucose absorption because glucose acts
as a signal molecule for pathway of AA transporters.
Absorbed glucose modulates the phosphorylation of
mammalian target of rapamycin, thus enhancing AA
absorption to meet the need for protein synthesis (Roos
et al., 2009). Observation of such interplays among
chemical pathways may offer insight into the drivers for
enhancement in nutrient utilization as a consequence
of carbohydrase supplementation. It may help explain
why amylase supplementation, for example, may also
enhance AA utilization. Currently, there is a paucity of
information on how exogenous enzymes alter gene expression in livestock and poultry. It is evident that the
future will see studies that are specifically designed to
elucidate the effect of carbohydrases on animal perfor-
mance, nutrient utilization, and gene expression, which
should elucidate the underlying mechanisms.
NSP-Degrading Enzymes and Growth
Performance Responses
Because a great variety of compounds make up NSP,
and an equally great variety of enzymes are used individually or in combination to target their antinutritive effects, it is generally difficult to make comparison
among studies with regard to the effect of NSP-degrading enzymes. There are differences associated with the
type of enzyme used individually or in combination,
the inclusion rates of the enzymes, the extent of reduction in nutrient density in the control diet, as well as
the type of cereal grain(s) being used. Tables 1 and 2
show some studies within the past 15 yr in swine, poultry species, and fish in which NSP-degrading enzymes
are used. By indicating the enzymes used as well as
the cereal grains used in the diet, it helps to identify
the sources of differences in performance responses observed.
Carbohydrases and Growth Performance
in Swine
There are generally no consistent effects of carbohydrase supplementation on the growth performance of
swine. There are reports of positive response to carbohydrase supplementation especially in diets in which
high-NSP cereal grains were used (Cadogan et al., 2003;
Barrera et al., 2004; Kiarie et al., 2007), whereas others
reported no improvement in BW gain in response to
the enzymes (Mavromichalis et al., 2000; Olukosi et al.,
2007a,c; Woyengo et al., 2008). Differences in response
to performance in these studies could be attributed to
differences in the type and quantity of cereal grains
used, the age of the animal, the extent of deficiency of
Table 1. Some studies in swine showing effect on exogenous nonstarch polysaccharide enzymes on growth performance
Reference
Stage of
growth
Barrera et al., 2004
Emiola et al., 2009
Growing
Finishing
He et al., 2010
Mavromichalis et al., 2000
Mavromichalis et al., 2000
Olukosi et al., 2007a
Olukosi et al., 2007c
Olukosi et al., 2007c
Weaning
Weaning
Finishing
Nursery
Nursery
Growingfinishing
Weaning
Weaning
Growing
Vahjen et al., 2007
Vahjen et al., 2007
Woyengo et al., 2008
Feedstuffs
Major enzyme
activity
Observation
Wheat
Corn, barley, wheat
distillers dried grains with
solubles
Corn, wheat, wheat bran
Wheat
Wheat
Corn, wheat, rye
Corn, wheat middlings
Wheat, wheat middlings
Xylanase
Multicarbohydrase
activities
15% improvement in daily BW gain
15% improvement in daily BW gain
Xylanase
Xylanase
Xylanase
Xylanase
Xylanase, amylase
Xylanase
20% improvement in daily BW gain
No effect on BW gain
Inconsistent effects on BW gain
No effects on BW gain
No effects on BW gain
No effects on BW gain
Wheat, wheat bran
Wheat, wheat bran
Wheat
Multiple carbohydrase
Xylanase
Xylanase
6% improvement in BW gain
7% improvement in BW gain
No effect on BW gain
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Exogenous enzymes in diets of nonruminants
Table 2. Some studies in poultry and fish showing effects of exogenous nonstarch polysaccharide enzymes on
growth performance
Major enzyme
activity
Reference
Species
Ingredient
Adeola and Bedford, 2004
Duck
Xylanase
Boguhn and Rodehutscord, 2010
Turkey
High- or low-viscosity
wheat
Wheat, barley, rye
Chauynarong et al., 2007
Pullet
Corn, wheat products
Five carbohydrase
activities
Cowieson and Ravindran, 2008a
Broiler
Corn
Farrell and Martin, 1998
Duck
Jaroni et al., 1999
Layer
Rice bran, wheat,
sorghum
Corn, oat, wheat
middlings
Xylanase, amylase, and
protease combination
Xylanase, amylase
Mathlouthi et al., 2002
Broiler
Corn- or rye-based
Xylanase and β-glucanase
Mathlouthi et al., 2003
Turkey
Wheat, barley
Xylanase, β-glucanase
Novak et al., 2007
Pullet
Corn and wheat
middlings
Xylanase, amylase, and
protease
Olukosi and Adeola, 2008
Broiler
Xylanase
Olukosi et al., 2007a
Broiler
Wheat and wheat
middlings
Wheat and rye
Olukosi et al., 2007b
Broiler
Corn
Olukosi et al., 2008b
Broiler
Corn and wheat
Roberts and Choct, 2006
Layer
Barley, wheat, triticale
Tahir et al., 2008
Broiler
Corn
Xylanase, amylase,
protease
Xylanase, amylase,
protease
Five carbohydrase
activities
Cellulase, hemicellulase,
pectinase
Timmler and Rodehutscord, 2001
Duck
Wheat, rye, triticale
Troche et al., 2007
Turkey
Corn, wheat
Ai et al., 2007
Japanese sea bass
Ogunkoya et al., 2006
Rainbow trout
Soybean meal, rapeseed
meal, peanut meal
Soybean meal
Farhangi and Carter, 2007
Rainbow trout
Lupin
limiting nutrient, and the extent to which the enzyme
increased digestible nutrient content. It is important
to note that improvement in nutrient digestibility does
not explain all the effects of carbohydrase supplementation on performance. This is demonstrated in Barrera
et al. (2004), in which xylanase supplementation to a
low-AA wheat-based diet only marginally improved
performance, whereas supplementation of crystalline
AA improved growth. In the same study, xylanase supplementation improved AA digestibility by an average
of 11%. It is likely that this observation is particularly
relevant to older swine, which are able to utilize fibrous
feedstuffs more efficiently. In younger swine, both the
limitations imposed by gut capacity, limiting nutrients,
as well as negative consequences of fibrous feedstuffs
Xylanase, β-glucanase
Xylanase, protease
Xylanase
Amylase, xylanase,
β-glucanase
Xylanase, amylase,
protease
Xylanase, glucanase,
pentosanase, cellulase
Xylanase, amylase,
glucanase, cellulase
Hemicellulases and
α-galactosidases
Observation
Up to 12% improvement
in BW gain
2% improvement in daily
BW gain
Greater ovary weight in
enzyme-supplemented
treatment
6% improvement in BW
gain
No effects on
performance
No effect on egg
production; positive
effect on egg weight
58% improvement in BW
gain
5% improvement in BW
gain
Enzymes alone had no
effect on growth but
improved feed conversion
No effect on BW gain
18% improvement in BW
gain
No effect on BW gain
No effect on BW gain
Effects differ with cereal
grains
Enzyme combination
improved BW gain by
9%
Inconsistent effects on
BW gain
No effects on growth
performance up to d 56
Specific growth rate
improved by 9%
No effect on growth rate
No effect on growth rate
could make carbohydrase supplementation an essential
dietary intervention.
Carbohydrases and Growth Performance
in Broilers
Mathlouthi et al. (2002) assessed the effect of enzyme
containing xylanase and β-glucanase activities in ryeor corn-based diets. Feeding the rye-based diet reduced
performance by 43% compared with the corn-based
diet, and enzyme supplementation at 560 and 2,800 IU
of xylanase and β-glucanase, respectively, restored performance to levels comparable with the corn-based diet.
In corn-soybean meal-based diets, Tahir et al. (2005)
observed that cellulase, hemicellulase, and their com-
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Adeola and Cowieson
bination increased BW gain without any effect on feed
intake in broilers but Cowieson and Ravindran (2008b)
observed both increased BW gain and feed intake in
response to supplementation with a mixture of xylanase, amylase, and protease (XAP). Similarly, Olukosi
et al. (2007a) reported a dose-related increase in BW
gain, feed intake, and feed efficiency in broilers receiving wheat and rye-based diets with xylanase supplementation. In other studies, there were no responses to
supplementation of carbohydrases (Olukosi and Adeola, 2008) or a mixture of carbohydrases and protease
(Olukosi et al., 2007b, 2008a,b). Part of the differences
observed in the various studies can be due to the extent
of nutrient density reduction in the control diets. It
seems that when the control diet is less nutrient dense
or the antinutritive effect is more expressed, there is a
greater growth performance improvement with enzyme
supplementation. Overall, these studies show potential
for carbohydrase use in broilers, but they also point out
the need to match nutrient need with potential capacity of supplemented enzyme to hydrolyze the cell wall
and release the needed nutrients.
Carbohydrases and Growth Performance
in Pullets and Laying Hens
In comparison with broilers, the effect of NSP-degrading enzymes was smaller for pullets (Karimi et al.,
2007), although the enzyme reduced digesta viscosity
to the same extent in the different chicks. Some studies reported no effects of NSP-degrading enzymes on
egg production (Jaroni et al., 1999; Hampson et al.,
2002), but at the same time, reported positive effects
on specific response criteria related to the quality of
eggs produced (Jaroni et al., 1999). Because of variations in treatments and enzyme activities used, it is difficult to make across-study comparisons for the effects
of the enzymes. Nevertheless, the results of the Roberts
and Choct (2006) study indicated that the influence of
NSP-degrading enzymes may be dependent on cereal
grain being used. But, even in the study, different NSPdegrading enzymes in different combinations and different dosage rates were used for different cereal grains.
The authors observed lighter shell color and reduced
albumen quality for barley- and wheat-based diets under the conditions in their study.
In pullets, addition of XAP to the corn, soybean
meal, or wheat middlings diets improved feed efficiency
in diets marginally deficient in ME, CP, or both (Novak
et al., 2007). The enzyme also reduced N excretion, but
its effect on energy and nutrient utilization was inconsistent. Similarly, Chauynarong et al. (2007) observed
reduced feed intake, a trend for improved BW gain,
and increased ovarian weight with a reduced-CP diet
supplemented with NSP-degrading enzyme containing
7 enzyme activities. The authors observed a carryover
effect of the enzyme supplementation that resulted in
better egg production even after the treatments were
discontinued. The indications from the studies are that
appropriate use of NSP-degrading enzymes may enhance growth performance of pullets, improve egg shell
quality in a nutritionally marginal diet, and reduce the
incidence of dirty eggs; however, some of the positive
effects may be inconsistent.
Carbohydrases and Growth Performance
in Turkeys
At least in the young turkey, supplementation of
diets based on barley, oats, and wheat with xylanase
and glucanase reduced the viscosity-induced reduction
in growth (Palander et al., 2005). Earlier, Mathlouthi
et al. (2003) showed that supplementation of NSPdegrading enzymes with xylanase and β-glucanase activities produced modest improvements in gain/feed of
turkey receiving wheat-, barley-, or wheat-based diets.
Boguhn and Rodehutscord (2010) also reported a small
increase (2%) in overall daily BW gain and G:F (3%)
in wheat-based diets. Troche et al. (2007), using a cornsoybean-based diet containing marginal amount of ME,
reported no effect of the XAP supplementation in their
study. The marginal increases in growth performance
in most of the studies indicate that the use of appropriate NSP-degrading enzyme may be beneficial in turkey
feeding, but because improvement in performance response is inconsistent, it is imperative to closely match
antinutrients with appropriate enzymes.
Carbohydrases and Growth Performance
in Ducks
Adeola and Bedford (2004), using either high- (46
mPa/s) or low-viscosity (6 mPa/s) wheat, observed a
depression in growth, especially in high-viscosity wheat,
and an amelioration of the growth depression, as well
as improvement in G:F when xylanase from Trichoderma longibrachiatum was added to the diet. However,
in an earlier study, supplementation of enzyme with
XAP and β-glucanase activities in diets with rice bran
did not improve growth performance, even though rice
bran depressed performance in some of the studies (Farrell and Martin, 1998). Others have observed improved
growth performance in ducks receiving NSP-degrading
enzymes (Hong et al., 2002; Adeola and Bedford, 2004).
In the latter, the authors reported greater improvement
in growth response to enzyme supplementation with
diets containing high-viscosity wheat. Timmler and
Rodehutscord (2001), using enzymes containing glucanase, xylanase, and amylase activities in diets, in which
wheat, rye, and triticale comprised approximately 60%
of the diet, reported a decrease in digesta viscosity
but no consistent effect of enzyme supplementation on
growth performance of the ducks. The differences in responses to enzyme supplementation could be attributed
to the feedstuff used (e.g., less effect on the grains with
low soluble NSP concentration). There are not as many
studies in ducks as there are in broilers, whereas some
studies did not show improvement in performance from
Exogenous enzymes in diets of nonruminants
added carborhydrase, but most of the studies available
indicate positive response to enzyme supplementation
when the enzymes are selected to match the antinutrient of interest (e.g., intestinal high viscosity as an
antinutrient).
Carbohydrases and Growth Performance
of Fish
The search for alternatives to fish meal as protein
sources in aquaculture, as well as the need to reduce
potential water pollution, are some of the major drivers
for the use of exogenous enzymes in fish nutrition. The
use of plant protein sources such as soybean meal and
canola meal, among others, is limited by their increased
content of phytate and NSP. The use of exogenous enzymes can alleviate some of the negative effects of these
antinutrients. In some studies, carbohydrases improved
the growth rate of fish (Ai et al., 2007; Lin et al., 2007),
whereas there were reports of no effects on growth performance (Ogunkoya et al., 2006; Farhangi and Carter,
2007). Of course, these studies utilized different plant
protein feedstuffs, as well as different enzyme activities, highlighting the difficulty of cross-study comparison. Interestingly though, most of the studies reported
improved nutrient digestibility and reduced nutrient
excretion (Stone et al., 2003; Ogunkoya et al., 2006;
Farhangi and Carter, 2007; Lin et al., 2007). Those
studies indicate that there are possibilities of improved
nutrient utilization that are not accompanied by increased growth rate. In such instances, it is likely that
the control diets were not sufficiently limiting in nutrients for growth or that endogenous enzyme activities
in some of the feedstuffs masked the effect of the exogenous enzyme (Farhangi and Carter, 2007). Overall,
these studies indicate the inconsistencies in the effects
of carbohydrases to enhance feeding value of plant protein sources for fish.
Carbohydrases and Nutrient Utilization
in Swine
As with growth performance, reports of improvement
in nutrient utilization after carbohydrase supplementation are not universal. Improvements in DM (Li et al.,
1996; Nortey et al., 2007; Olukosi et al., 2007a), N (Yin
et al., 2000; Emiola et al., 2009; Reilly et al., 2010),
OM (Li et al., 1996), minerals (Olukosi et al., 2007c;
He et al., 2010), or energy digestibility (Yin et al., 2000;
Diebold et al., 2004; Olukosi et al., 2007c), and no effect on DM (Woyengo et al., 2008), CP (Nitrayová et
al., 2009), minerals (Olukosi et al., 2007c; Nortey et al.,
2008), or energy (Olukosi et al., 2007a,c) have been reported. Several studies also noted improved AA digestibility in carbohydrase-supplemented wheat- (Diebold
et al., 2004; Barrera et al., 2004; Vahjen et al., 2007)
and barley-based diets (Li et al., 1996) or diets containing wheat-DDGS (Emiola et al., 2009). Small improvement in AA digestibility is perhaps due to a reduc-
3195
tion in endogenous AA losses. However, observations
of increased digestibility of fiber or NSP fractions with
carbohydrase supplementation underscore the importance of release of inaccessible nutrients in enhancing
AA digestibility.
The relationship between content of NSP and improvement in AA digestibility after enzyme supplementation (Nortey et al., 2008) is illustrated in Figure 1.
Generally, there was no response to xylanase supplementation of wheat, whereas supplementation of the
same enzyme to different fractions of wheat showed responses that virtually follow the trend in the quantity
of NSP in these fractions. Wheat bran, which had the
greatest content of NSP, also had the greatest improvement in AA digestibility after xylanase supplementation. Consequently, the importance of enhanced digestibility after carbohydrase supplementation should be
considered in evaluating the role of carbohydrases in
enhancing nutrient utilization.
There is a dearth of information on the use of carbohydrases in sows. This is probably because of the
importance of fiber in the diet of sows. One study reported improved ileal and total tract DM and N utilization during lactation in corn-soybean meal diets supplemented with protease and xylanase (Souza et al., 2007).
More studies with sows are needed in this area.
Carbohydrases and Nutrient Utilization
in Poultry
In many poultry studies, carbohydrase supplementation has been shown to improve energy utilization in
corn-soybean meal diets (Meng et al., 2005; Leslie et
al., 2007; Rutherfurd et al., 2007; Cowieson and Ravindran, 2008a,b; Yang et al., 2010). Others noted no
improvement in energy utilization in response to carbohydrases (Olukosi et al., 2007b). In diets with cereal
grains containing greater quantity of NSP, carbohydrase supplementation also improved energy utilization
(Mathlouthi et al., 2003; Adeola and Bedford, 2004;
Adeola et al., 2007; MacLeod et al., 2008), except in 1
report (Adeola et al., 2008). The studies show that carbohydrases often improve energy value of diet or feed
ingredients containing increased concentration of NSP.
The differences in the effect of the enzymes on energy
of feedstuffs or diet may relate to the amount of substrate for the enzyme or availability of energy from the
ingredient itself, or both. As Figure 2 shows, it seems
that improvement in energy from cereal grains with
carbohydrase supplementation may be masked when
the energy value of the cereal grain is large. Similarly,
in Adeola et al. (2008) study, carbohydrases improved
ME in diets with reduced ME but not in diets with
greater ME. A similar effect is demonstrated in Figure
3, in which carbohydrase effect on improving apparent
ME (AME) became greater with a decrease in AME
of the control diet.
There are also reports of improvement in DM utilization (Leslie et al., 2007; Olukosi et al., 2007b; Cowi-
3196
Adeola and Cowieson
Figure 1. Relationship between total nonstarch polysaccharide (NSP) content and improvement in AA digestibility after xylanase supplementation of swine diets (adapted from Nortey et al., 2008). For total NSP, the y-axis unit is percentage total NSP, and the y-axis unit is percentage
improvement in AA.
eson and Ravindran, 2008b; Yang et al., 2010), fat
(Mathlouthi et al., 2002; Adeola and Bedford, 2004;
Boguhn and Rodehutscord, 2010), starch (Adeola and
Bedford, 2004; Meng and Slominski, 2005), and minerals (Olukosi et al., 2007b, 2008b) in response to carbohydrase supplementation. Others reported no effect
of carbohydrase supplementation on the utilization of
these nutrients (Adeola et al., 2008; Cowieson and Ravindran, 2008a; Olukosi et al., 2008b). The responses to
enzyme supplementation are feedstuff-, diet-, and enzyme-dependent. Generally, the feedstuffs with greater
amount of NSP, intuitively, respond to a greater extent
to carbohydrase supplementation.
Palander et al. (2005) observed an association between extent of digesta-viscosity reduction and im-
provement in N digestibility. Many others have also
reported improvement in N (Hong et al., 2002; Adeola
et al., 2008; Cowieson and Ravindran, 2008a,b; Yang
et al., 2010) and AA digestibility (Rutherfurd et al.,
2007; Cowieson and Ravindran, 2008a,b; Boguhn and
Rodehutscord, 2010) in response to carbohydrase supplementation. An interesting observation is that data
from 5 different studies with broilers (Rutherfurd et al.,
2007; Cowieson and Ravindran, 2008b), ducks (Hong
et al., 2002; Adeola et al., 2008), and turkeys (Boguhn
and Rodehutscord, 2010) showed that cysteine consistently had the greatest improvement in digestibility,
ranging from 4.7% in ducks (Adeola et al., 2008) to
22.9% in broilers (Rutherfurd et al., 2007). The AA
that most often had the greatest response to carbohy-
Figure 2. Relationship between ME of cereal grains and potential for carbohydrase to improve its energy value (adapted from Palander et al.,
2005). For ME, the y-axis unit is megajoules per kilogram, and the y-axis unit is percentage ME improvement by enzyme.
Exogenous enzymes in diets of nonruminants
3197
Figure 3. Dependence of carbohydrase effect on dietary ME (adapted from Zhou et al., 2009).
drase supplementation are, in descending order, Cys
> Ser > Thr > Gly > Val > Lys > Met. Cowieson
and Ravindran (2008b) made a similar observation and
speculated that this may be related to beneficial effect
of exogenous enzymes on reducing endogenous loss of
mucin. This assumption would be valid if it is assumed
that the main benefit of carbohydrase supplementation
is reduction of endogenous loss. However, Rutherfurd
et al. (2007) noted that reduction of endogenous loss
by carbohydrases is secondary to improvement in protein hydrolysis. In addition, it seems that Thr has a
greater effect on mucin production than Cys (Faure
et al., 2006). Clearly, there is need to understand why
specific AA respond to a greater extent and how this
can be used to increase the benefit from carbohydrase
supplementation.
COMBINATION OF ENZYMES
The use of multiple enzyme activities targets different antinutritive compounds in feedstuffs to obtain the
maximum benefit from the enzyme. Parkkonen et al.
(1997) observed that xylanase increases the permeability of the aleurone layer of wheat, which is the site of
phytic acid storage. Xylanase, by itself, will not target
phytic acid, but a combination of xylanase and phytase
may be mutually beneficial. By the same reasoning,
the use of multiple carbohydrase activities may produce greater benefit than each of the enzymes acting
individually (Juanpere et al., 2005; Meng et al., 2005;
Olukosi et al., 2007b). On the other hand, Saarelainen
et al. (1993) and Saleh et al. (2004) have observed that
the hydrolytic activity of carbohydrases may be limited or the carbohydrase itself may be digested in the
presence of protease. In view of this, it is important
to understand the optimum combination of enzymes
to use in animal diets. Beneficial interactions among
carbohydrases (Choct et al., 2004; Tahir et al., 2008)
with phytase (Diebold et al., 2004; Nortey et al., 2007;
Olukosi et al., 2007b) have been reported. There were
reports of no additional benefit of using enzyme combination than when enzymes were used individually (Wu
et al., 2004; Olukosi et al., 2007c) or of reduced improvement in nutrient utilization when enzymes were
combined when compared with the use of the enzymes
individually (Olukosi et al., 2007b). The beneficial effect of enzyme combination may be dependent on diet
composition (Meng and Slominski, 2005).
To maximize the efficacy of enzyme combinations, it
is essential to understand how the enzyme work together to hydrolyze their respective substrates. For example, because some xylanases target soluble and others
insoluble arabinoxylans, the most effective combination
is achieved when they are used together. Choct et al.
(2004) showed that the process of hydrolysis by xylanase is the breakdown of insoluble, and then soluble,
arabinoxylans. Xylanases with only an affinity for insoluble arabinoxylans give soluble hydrolytic products
and consequently increase digesta viscosity. Therefore,
effective reduction in viscosity is only attainable when
the enzyme has an affinity for both the soluble and
insoluble NSP. Tahir et al. (2008) paired hemicellulase,
cellulase, and pectinase or the 3 enzymes individually
and in combination. The observation indicated that the
hydrolysis of hemicelluloses is the rate-limiting step for
subsequent hydrolysis of other nutrients that may be
trapped in the cell. Therefore, the cellulase and pectinase pair was less effective in improving NSP hydrolysis.
Because the result obtained from enzyme combination
studies can be feedstuff specific, more studies that use
individual enzymes, as well different combinations of
enzymes, are needed to understand the optimum combination of enzymes.
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Adeola and Cowieson
Figure 4. Animal responses to control diet, control diet + enzyme 1, control diet + enzyme 2, and control diet + enzyme 1 + 2.
The potential benefit of enzyme combination can
usually be assessed by examining additivity or interaction in responses obtained when the enzymes are used
individually or in combination. In Figure 4, a typical
response showing step-wise improvement, as the enzymes were used individually and in combination, is
presented. In Figure 5, the response in the control diet
was set to 0 and the improvements in the enzyme-supplemented diet are expressed relative to response in the
control diet using 3 different scenarios. Although, in
all the instances, the response when the 2 enzymes are
used in combination seems greater than when the en-
zymes were used individually, only a statistical analysis
using a contrast of enzyme 1 plus enzyme 2 vs. enzyme
1 + 2 is able to indicate if there is additivity. In the
instance of additivity, it is expected that the sum of the
improvement in response to individual enzymes (i.e.,
the improvement from enzyme 1 plus the improvement
from enzyme 2, individually) will be the same with the
improvement in response when the enzymes are used
in combination (i.e., the improvement from enzymes
1 + 2) as expressed by nonsignificant P-value (Figure
5, scenario 1; 10 + 15 vs. 25). On the other hand, if
the contrast has a significant P-value, then there is no
Figure 5. Response, above control, to addition of enzyme for the determination of additivity or interaction of enzymes when used in combination.
Exogenous enzymes in diets of nonruminants
additivity between the enzymes and it might be that
1 enzyme synergizes (Figure 5, scenario 2; 10 + 15 vs.
30) or hinders (Figure 5, scenario 3; 10 + 15 vs. 20) the
activity of the other.
Although this relationship is simple in principle and
has been used in some studies (Diebold et al., 2004;
Cowieson and Adeola, 2005; Olukosi et al., 2007c),
there are potential complications. In some instances,
additivity is reported for the effects of the enzyme for
a particular response and not for another (Olukosi et
al., 2007b). The issue, then, is in deciding which of the
conclusions is valid. Another complication is the possibility of obtaining values or digestible nutrient content
that is meaningless. An example of this is depicted in
Figure 6, where digestibilities in the control, enzyme 1,
enzyme 2, and enzymes 1 + 2 were 75, 85, 95, and 95%,
respectively. The improvements in responses above the
control diet are 10, 20, and 25 percentage points for enzymes 1, 2, and 1 + 2, respectively. Statistically, the addition of the individual enzymes vs. their combination
(30 vs. 25) may not be different. However, if additivity
is assumed and this is used to compute digestibility of
nutrients when the enzymes are used in combination,
the digestibility value will be 105% for the combination
of the 2 enzymes. Clearly, this will yield values for a
quantity of digestible nutrients that is greater than the
total quantity of nutrients available in the diet or ingredient. Consequently, it is important to evaluate the
values obtained when assessing additivity in enzyme
activities used in combination. As our understanding
of enzyme activities in liberating nutrients improves,
other measures to assess interactions among enzymes
when used together may emerge. For now, using statistical evaluation to assess how enzymes work together
will be a vital tool for a gross evaluation of benefits to
enzymes combination.
3199
OVERVIEW OF THE MODES
OF ACTIONS OF CARBOHYDRASES
Impact on NSP
The main reason for the use of carbohydrases is to
hydrolyze complex carbohydrates that nonruminant
animals are unable to hydrolyze by themselves. Some
of these compounds are present as part of the cell wall,
thus shielding substrates from contact with the digestive enzymes, or as part of cell content where their
presence may interfere with digestion and absorption
by their chemical nature. Nitrayová et al. (2009) reported improved ileal disappearance of NSP in diets
containing 96% rye for weanling swine; there was a
740% improvement in disappearance for xylose and
a 144% improvement in disappearance for total NSP
when xylanase was added at the rate of up to 200 mg/
kg. These data indicate that NSP removal is one of
the critical roles of carbohydrases when added to diets containing NSP. Nevertheless, the release of these
oligomers is not necessarily the greatest benefit of the
use of carbohydrases, especially when these oligomers
are still indigestible. It seems that reduction in digesta
viscosity may be one of the most important benefits
of carbohydrase supplementation (Vahjen et al., 2007).
Several studies have shown that xylanase attacks the
arabinoxylan backbone, causing a decrease in the degree of polymerization (Bengtsson et al., 1992; Courtin
and Delcour, 2002; Hu et al., 2008) and thus liberate
oligomers. The importance of this hydrolysis is that
the direct link between digesta viscosity and animal
performance has been demonstrated in several studies (Żyła et al., 1999; Dänicke et al., 2000; Zhang et
al., 2000a). Adeola and Bedford (2004) demonstrated
that 1 of the modes of action of carbohydrases is their
Figure 6. Assessing additivity in the effects of enzymes used individually and in combination.
3200
Adeola and Cowieson
ability to reduce NSP-induced digesta viscosity. Highviscosity wheat responded more (in terms of improved
nutrient utilization) than low-viscosity wheat when the
diets were supplemented with xylanase. On the basis of
these observations, with specific reference to NSP disappearance, reduction of viscosity seems to be the most
important mode of action of carbohydrases. Hydrolysis
of these NSP certainly produces other benefits, as will
be discussed.
Impact on Energy
Energy digestibility in swine generally decreases with
increased fiber intake (Stanogias and Pearcet, 1985;
Nortey et al., 2008). Explanations for reduced energy
digestibility could be increased endogenous energy loss,
reduced digestibility of energy yielding fraction because
of impaired nutrient absorption, reduced contact of
substrates and digestive enzymes, reduced proportion
of energy-yielding fractions in high-fiber feedstuffs, or
reduction in feed intake because of bulkiness of highfiber diets combined with inherent stomach capacity of
the animal. Others may include decreased digestive enzyme quantity and activities. Johnson and Gee (1986)
observed that feeding of high-NSP diet to rats reduced
DNA and protein contents of the brush border. This reduction could be a result of increased cell turnover rate
engendered by increased cell proliferation. Therefore,
if carbohydrases hydrolyze the NSP fractions, these effects could be reversed and energy utilization should
improve. Indeed, there have been observations of increased quantity of mono- and oligosaccharides in the
ileum after the use of cellulase or xylanase (van der
Meulen et al., 2001) and β-glucanase (Li et al., 1996)
or multiactivity carbohydrases (Kiarie et al., 2007). It
seems that one of the ways by which carbohydrases
improve energy utilization is by shifting production of
VFA and absorption of energy-yielding monosaccharides to proximal intestine. This is supported by observations of decreased net disappearance of nutrients
in the large intestine of swine receiving β-glucanasesupplemented diets (Li et al., 1996). The shift in nutrient utilization to the more proximal intestine would
decrease host-microbe competition for nutrients, ensure
availability of nutrients where absorption efficiency is
greater, reduce fermentative loss, and contribute to
overall improved efficiency of energy utilization. The
effect of digesta viscosity is more pronounced in poultry
than in swine. However, reduction in digesta viscosity is
usually far greater than improvement in energy utilization. In Macleod et al. (2008), a small increase (<5%)
in ME of naked oats was associated with a substantial
decrease (>250%) in jejunal digesta viscosity. Similarly,
in Adeola and Bedford (2004), the decrease in jejunal
viscosity (70%) was associated with only a small increase in ME (4%).
Because NSP may reduce the capacity for absorption
by reducing enzyme accessibility to substrate, it is reasonable that there are observations of increased digest-
ibility of energy-yielding nutrients after carbohydrase
supplementation. For example, Adeola and Bedford
(2004) reported improved starch and fat digestibility
in wheat after xylanase supplementation. Tervilä-Wilo
et al. (1996), Juanpere et al. (2005), and Vahjen et
al. (2007) also noted improved fat and starch digestibility in response to supplementation of xylanase and
β-glucanase. The improvement in fat digestibility is
especially noteworthy because NSP are known to increase hydrolysis of bile salts (Mathlouthi et al., 2002)
and hence reduce fat utilization. Meng and Slominski
(2005) suggested that hydrolysis of encapsulating cell
walls may be responsible for enhanced energy utilization in a corn diet but that disruption of cell matrix
resulting in a release of structural protein may be responsible for improved energy utilization in a soybean
meal-based diet.
Impact on N and AA
Nitrogen digestibility reduction by NDF and ADF
has been ascribed to increased loss of endogenous and
microbial N, low availability of N in the fibers themselves, or increased excretion of N trapped in the fibers or the digesta (Stanogias and Pearcet, 1985). The
increased N loss with feeding of fibrous feedstuffs resulted from endogenous (59%) and exogenous N (41%)
sources. Loss of pancreatic enzymes and bile, as well as
sloughed mucosa, will result in endogenous N and AA
losses (Schulze et al., 1995). Consequently, reduction
of the endogenous and exogenous losses and increased
hydrolysis of dietary protein are 2 possible modes of
action of carbohydrases in improving N and AA utilization. However, observations regarding carbohydrases
reducing endogenous protein or AA have been inconsistent. Yin et al. (2000) reported a modest decrease in endogenous AA loss after carbohydrase supplementation,
whereas Rutherfurd et al. (2007) did not observe such
effects. These may be diet-, ingredient-, or enzyme-specific (i.e., presence or absence of certain antinutrients,
difference enzymes, and others). More observations on
how the enzymes affect losses of N and AA in different
ingredients will be instructive.
As shown for the effects of carbohydrase on other nutrients, it is possible that carbohydrases act to improve
N and AA utilization indirectly by increasing the access
to protein for digestive proteases. Tahir et al. (2008)
noted that combination of enzymes were only effective
in the presence of hemicellulases, which is capable of
cleaving the resistant galacturonic acid and rhamnose
bonds. This was corroborated by the observation that,
for soybean meal, there was a strong correlation between the amount of galacturonic acid (product of the
breakdown of rhamnogalacturonans in soybean meal)
and CP digestibility, indicating that the hydrolysis of
pectic substances in the cell wall enhanced protein digestibility. Therefore, failure to observe increased N or
AA utilization with the use of carbohydrases in some
studies may be because of intact cell walls, especial-
Exogenous enzymes in diets of nonruminants
ly when the carbohydrases do not target the specific
bonds shielding the nutrient. Another indirect effect
of carbohydrases on N and AA utilization was recently
demonstrated in the Yin et al. (2010) study, in which
carbohydrases enhanced starch digestibility indirectly
and improved N and AA digestibility and absorption.
The authors observed that starches with increased
amylose:amylopectin are more resistant to digestion
and have decreased digestibility, especially at the proximal part of the small intestine, in the overall length of
the intestine, and the decreased digestibility is closely
associated with decreased AA digestibility and plasma
AA concentration. These effects were mediated through
amelioration of digestive and absorptive issues relating to increased digesta viscosity that could be caused
by increased amylose concentration. However, it is also
likely that the poor absorption of glucose (in increased
amylose:amylopectin diets) reduced efficiency of AA
transport because glucose is important in regulating
the pathways for AA transporters and protein synthesis (Roos et al., 2009). Reduced glucose concentration
ultimately affects phosphorylation of the pathway that
drives protein synthesis, which ultimately reduces AA
absorption.
Impact on Minerals
As with other nutrients, Nortey et al. (2008) observed
reduced digestibility of minerals in diets, in which different by-products of wheat milling were added to a
wheat-soybean meal basal diet. In several studies, supplementation of xylanase (Nortey et al., 2007) or xylanase, amylase, and protease (Olukosi et al., 2007b)
resulted in improved P digestibility. One possible explanation is provided by an observation of the relationship between phytic acid and NSP in plants. In cereal
grains and legumes, most of the P is bound in phytic
acid. Frølich (1990) indicated phytic acid and NSP are
both found in the aleurone layer of wheat. In many
cereal grains, or fractions thereof (e.g., wheat bran,
arabinoxylans, and other NSP) are sequestered with
phytic acid. Consequently, when carbohydrases hydrolyze their substrates, phytic acid and other minerals
may be exposed to digestive enzymes. As Parkkonen et
al. (1997) observed, carbohydrases may increase permeability of the aleurone layer and consequently increase
the release of otherwise unavailable minerals. Therefore, the increase in mineral availability as a result of
carbohydrase supplementation is an indirect response
to the carbohydrase effect.
Impact on Gut Health
Increased digesta viscosity encourages slower diffusion rate, accumulation of particulate matter for
microbial adhesion, and greater flow of solids rather
than liquid. These factors encourage slower shedding
of microorganism and encourage the proliferation of
harmful microorganisms (Meyer et al., 1986; Vahjen et
3201
al., 1998). In newly weaned swine receiving diets that
promote increased digesta viscosity, McDonald et al.
(2001) observed increased shedding of enterotoxigenic
E. coli. Poorer gut health in high-NSP diets may also
arise from alteration of the morphology of the digestive
surfaces in animals receiving a high-NSP diet. Johnson and Gee (1986) observed that rats consuming gelforming NSP in their diets have adaptive growth of the
small intestine, leading to greater mucosa proliferation,
broader villi base, and heavier ceca weight. Similar observations have been made in swine and poultry (McDonald et al., 2001; Mathlouthi et al., 2002). Teirlynck
et al. (2009) observed greater evidence and markers of
gut damage, apoptosis, increased mounting of immune
defense, and microbial invasion of intestinal tissues in
broilers receiving wheat-rye diets compared with those
on corn-based diets. The observation by Langhout et
al. (2000) that the negative effects of NSP were less
pronounced in germ-free birds indicates that microorganisms play a critical role in mediating the negative
effects of NSP.
Carbohydrase supplementation reverses these negative effects by increasing the proportion of lactic and
organic acids (Högberg and Lindberg, 2004; Kiarie et
al., 2007), reducing ammonia production (Kiarie et al.,
2007), and increasing VFA concentration (Hübener et
al., 2002), which is indicative of hydrolysis fragmentation of NSP and supporting growth of beneficial bacteria. Increased proportions of lactic acid promote gut
health by suppressing the growth of presumptive pathogens (Pluske et al., 2001). Hillman et al. (1995) showed
that certain strains of Lactobacillus inhibit the growth
of coliforms such as pathogenic E. coli. Increased colonization of the gut with Lactobacilli has been associated with xylanase supplementation of a wheat diet
and reduction in digesta viscosity (Vahjen et al., 1998).
In addition, xylose (possible product of exogenous and
endogenous carbohydrase activity) has been shown to
be important in preferentially enhancing the growth of
beneficial bifidobacteria (He et al., 2010). There were
reports of improvement in the health of poultry as a
result of carbohydrase supplementation (Fernandez et
al., 2000; Hampson et al., 2002). In layers, Hampson et
al. (2002) observed a reduced excretion of Brachyspira
intermedia in hens receiving 265 mg/kg of an enzyme
product containing xylanase and protease activities,
which are indicative of infection reduction as a result of
improved intestinal microbial population.
There are observations of reduced relative weight of
the digestive tract and associated organs, as well as
improved villi length and reduced digesta transit rate,
with carbohydrase supplementation (Choct et al., 2004;
Hopwood et al., 2004; Sieo et al., 2005). Reduced relative weight of the intestine is indicative of less cell
proliferation and thus less gut nutrient maintenance
requirement. Mathlouthi et al. (2002) reported improved gut morphology with xylanase supplementation
of a rye-based diet. It is also argued that carbohydrase
supplementation favors the digestion of nutrients at the
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Adeola and Cowieson
more proximal portion of the digestive tract (Mathlouthi et al., 2002), thus promoting less host-microbe competition and enhancing growth of the animal. Consequently, there is a basis for improved gut and overall
health of the animal as a result of carbohydrase supplementation.
MATRIX VALUES FOR ENZYMES
To optimize enzyme supplementation, reduce waste
in enzyme use, and obtain optimum responses, efforts
have been made to derive matrix values for energy and
specific nutrients in enzyme-supplemented diets. This
is based on the premise that enzymes release nutrients
that are otherwise unavailable, and thus, it is possible
to make allowance for that in feed formulation. Zhou et
al. (2009) demonstrated the need for making allowances
for contribution of energy from carbohydrases in diets
using decreasing amount of AME with XAP supplementation. The greatest response to XAP was observed at
the least energy concentration, indicating that enzyme
supplementation is more beneficial when ME is suboptimal. Francesch and Geraert (2009) observed that reduction in energy (−65 or −85 kcal/kg) and CP (−1.5
or −3.0%) during the starter phase did not depress
performance but reduced available P (−0.15 percentage
points) and Ca (−0.12 percentage points), and enzyme
supplementation alleviated reduction in performance
that was induced by the nutrient reduction. Cowieson
and Ravindran (2008a,b) noted that supplementation
of a nutritionally marginal diet with XAP restored performance to that of a nutritionally adequate diet. The
authors suggested that the enzyme had a net effect
equal to that of 2.7 g/kg of lipid, 0.1 g/kg of dl-Met,
and 0.15 g/kg of lysine∙HCl.
Specifically designed studies can be used to determine the matrix values for enzymes. However, proprietary predictive models are used to estimate matrix
values for carbohydrases (Cowieson et al., 2006a,b).
Although these predictive models may be straightforward when using individual enzymes, complexities arise
when enzymes are used in combination, especially because the relationship among enzymes can be anything
but additive. Inappropriate use of matrix values can
lead to masking of the effect of enzymes. For example,
in Troche et al. (2007), a 140-kcal matrix value used
for corn in feed formulation was suggested to be the
reason why the carbohydrase did not produce an improvement in performance. If the matrix value used is
too small, the enzyme effect is masked, whereas if the
matrix value used is too large, the diet remains nutritionally inadequate. The matrix value of enzyme will
vary depending on ingredients that are put together
because the amount of nutrient or energy availability as
a consequence of enzyme supplementation will depend
on the structure of the feedstuff itself (i.e., fiber matrix
and others). Understanding the use and limitations of
matrix values to carbohydrases is essential. When used
appropriately, the benefit from enzyme supplementa-
tion is maximized; thus, the ME or digestible nutrient
content of the diet may be strategically reduced, but at
the same time, optimum production levels can be maintained. On the other hand, inappropriate use of matrix
values for enzymes will result in depressed performance
because inadequacy of nutrients will lead to wastage of
resources.
CURRENT APPROACHES
TO OPTIMIZE ENZYMIC ACTIVITY
OF CARBOHYDRASES
To make enzyme application more economical, there
continues to be interest in improving extant enzymes.
In the past, the attention was on producing enzymes
that survive longer in the digestive tract by making
them less susceptible to proteolysis during transit in
the digestive tract. New approaches optimize the activity of the next generation of enzymes and are designed
to increase their aggressiveness toward the substrate of
interest or increase their versatility for various methods
of feed processing.
One method that is being used to optimize hydrolytic
activity of new generation enzymes is heterologous expression. He et al. (2010) described a new-generation
Trichoderma reesei xylanase that was previously used
only in industrial applications. The crude enzyme has
many side activities including cellulase and protease,
which may hamper the activity of the xylanase itself
over time. Specific expression of the gene for the fungus
in P. pastoris allows a near-total elimination of harmful
side activities and, thus, enhances its xylanase activity.
Enzymes can lose up to 90% of their activity when exposed to temperatures greater than 90°C during pelleting of diets (Silversides and Bedford, 1999). One of the
approaches currently being used to enhance thermostability is the application of a coating for the enzyme.
A protective coat, usually a lipid, is used to protect
the enzyme during feed processing. Characteristics of
a good coat are its stability during feed processing and
its ease of dissolution during digestion. A good coat
must be strong enough to resist the high temperature
and pressure of pelleting processes but must also be
malleable enough as to be easily digested in the lower
temperature and moisture conditions of more proximal
part of the digestive tract, especially for phytase (as
phytase must be active in the gastric phase of digestion
when phytate is most soluble). Other approaches are to
genetically manipulate the AA structure of the enzyme
product so it is more inherently thermostable or to discover intrinsically thermostable enzymes (Bedford and
Cowieson, 2009).
PROTEASES
Proteases have been added to poultry and swine diets
routinely for many years as part of enzyme admixtures
containing xylanases, pectinases, glucanases, amylases,
and other activities (Simbaya et al., 1996; Cowieson
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Exogenous enzymes in diets of nonruminants
and Adeola, 2005; Cowieson and Ravindran, 2008b).
However, though an integral part of such products, the
relevance of the presence of this activity has not attracted a great deal of attention. In recent years, proteases have grown in profile, there are currently several
stand-alone proteases available, and new mechanisms
of action have been proposed.
Early research on the usefulness of proteases as
supplemental feed enzymes is equivocal. For example,
Caine et al. (1997) treated soybean meal with Bacillus
subtilis in an attempt to reduce the adverse effects of
proteinaceous antinutrients in soy when fed to weaner
piglets. However, the authors observed a protease-induced decrease in AA digestibility from 68.7 to 63.9%.
Ghazi et al. (1997) and Rooke et al. (1998) supplemented broiler and piglet diets respectively with either
an acid fungal (Aspergillus) or alkaline bacterial (Bacillus) protease. In both studies, the acid fungal protease
proved effective in improving BW gain and feed conversion, whereas the bacterial protease resulted in depressed growth and poor feed conversion. These studies
indicate that different proteases with different inherent
characteristics may elicit divergent responses in vivo.
That different proteases result in dramatically different
responses in both swine and poultry may be related
to compatibility with endogenous proteases or perhaps
modification of soy protein such as is observed in the
manufacture of tofu. For example, Blazek (2008) observed that some proteases are capable of coagulating
soy protein and the extent of this reaction is dependent
both on the characteristics of the soy protein and the
nature of the protease. Gelation of soy protein, as could
occur in situ in the gut of poultry and swine, may be
one explanation for some of the variable responses that
have been observed in the literature. These effects have
been previously reported where treatment of soy protein with 3 different proteases resulted in substantial,
though transient, gelation of the protein (Hrckova et
al., 2002). Interestingly, incubation with the different
proteases resulted in different quantities and types of
free AA production, with 1 protease producing mainly
His (30%), Leu (24%), and Tyr (19%) and another Arg
(22%), Leu (11%), and Phe (13%). The importance of
these product profiles is not clear, but generation of
free AA may interact with feed intake or absorption
or both. Further study is warranted to elucidate these
mechanisms and interactions between soy protein and
supplemental protease.
Contrary to some observed negative responses to
exogenous protease, there have been several reports
where beneficial effects were reported. Mahagna et al.
(1995) found positive effects of protease (and amylase)
supplementation of sorghum-based diets for broiler
chicks, and this was associated with a reduction in
chymotrypsin secretion by the pancreas. This apparent feedback mechanism may explain why feed conversion efficiency was improved because synthesis of endogenous protein is energetically expensive for animals.
Of interest is that Mahagna et al. (1995) observed no
effect of supplemental enzymes on digestibility coefficients, indicating that the mechanisms involved in feed
efficiency improvement may be “net.” Odetallah et al.
(2003) observed improved performance of broiler starters when a corn/soy-based diet was supplemented with
a keratinase from B. licheniformis. However, these authors reported results from 3 individual broiler growth
trials and benefits were not consistent in each study, a
factor perhaps linked to the AA or CP concentrations
(or both) in the control diets. Furthermore, the beneficial effects of protease did not persist to market weight,
an observation that was later confirmed (Odetallah et
al., 2005). O’Doherty and Forde (1999) found that supplementation of barley/wheat/soy-based diets for swine
with a neutral protease resulted in an improvement in
feed efficiency.
There is potential for protease in the diets of swine
and chickens. However, interactions with the digestive
architecture of the host, with other supplemental enzymes and with dietary proteins, require further investigation.
PHYTASE
Introduction
Phytase dephosphorylates insoluble phytic acid in
grains and oilseeds into orthophosphate and inositol
phosphates. In broad terms, phytases are classified as
3- and 6- phytase on the basis of the site on the phytic
acid molecule of initial dephosphorylation. Generally,
3-phytases are of microbial origin (EC 3.1.3.8) and
commence hydrolysis at the carbon 3 atom of the inositol ring, whereas 6-phytases are of plant origin (EC
3.1.3.26) and commence phosphate cleavage at the
carbon 6 atom of the inositol ring (Dvoráková, 1998).
There is no single enzyme that is capable of fully dephosphorylating phytic acid; therefore, a combination
of phytase and nonspecific phosphatases are involved
in the process (Maenz, 2001). Phytate (myo-inositol
hexakisdihydrogen phosphate or IP6), the substrate for
phytase, was first identified over 150 yr ago (Hartig,
1855). Phytate is found in most vegetable feed ingredients at concentrations from 5 to 25 g/kg, contributing between 1.4 and 7 g/kg of phytate-P. A common
misconception is that nonruminant animals lack endogenous phytase and this is the reason for poor phytate
disappearance. In fact, most nonruminant animals possess very effective phytase/phosphatase activity in the
intestinal mucosa, blood, and liver and can readily dephosphorylate phytate into inositol and free phosphate
for systemic distribution (Oshima et al., 1964; Birge
and Avioli, 1981; Maenz and Classen, 1998). Oshima et
al. (1964) presented data that showed a rapid increase
in IP6 concentration in the blood of chickens in the first
3 wk posthatch, indicating that chicks can absorb IP6
and that IP6 may have an important metabolic function
for maturing erythrocytes. Moore and Veum (1983)
demonstrated that not only is IP6 digestible but that
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Adeola and Cowieson
the digestibility can be artificially inflated by depriving
the growing animal of available P. Germ-free rats were
able to hydrolyze as much phytate as their conventional
counterparts (Miyazawa et al., 1996), indicating that
the intestinal microflora does not play an important
role in phytate hydrolysis.
McCollum and Hart (1908) were among the first authors to report phytase activity in animal tissues and
this has been confirmed in virtually every decade since
then. An important issue with phytate digestion is,
therefore, not a lack of compatible endogenous enzymes
but poor substrate solubility in the small intestine, and
this hinges on luminal cation (notably, Ca) concentrations. Schlemmer et al. (2001) assessed the solubility
of various IP esters in the small and large intestine of
swine. Interestingly, the solubility of IP6, IP5, IP4, IP3,
and IP2 in the small intestine of swine (pH 6.6) was 2,
7, 8, 31, and 75%, respectively. Corresponding values
for the large intestine (pH 6.2) were 2, 3, 0, 6, and 24%
(Schlemmer et al., 2001). However, it is also important
to note that the relative concentrations of these esters
may be decreased in the luminal contents, as some are
only present transiently because phytate is dephosphorylated (Wyss et al., 1999). Persson et al. (1998) also
noted that, in addition to the increased solubility of
the lower IP esters, the complexes formed with minerals were proportionately much weaker, indicating that
to encourage the persistence of solubility of both the
minerals and IP esters at higher pH, gastric reduction
to at least IP3 is necessary.
P
As mentioned before, phytate is found in virtually
all seeds at concentrations from around 5 g/kg up to
well over 20 g/kg and, therefore, typical pig, poultry,
and fish diets will contain between 8 and 12 g of phytic
acid/kg (Selle et al., 2003b). By weight, phytic acid is
282 g of P/kg, but because of a poor solubility of phytate in the small intestine, swine and poultry do not
retain this P efficiently. Microbial phytases, when added to the animal diet, are able to hydrolyze the ester
bond between carbon 3 (in the case of 3-phytases) or
carbon 6 (in the case of 6-phytases) and the associated
phosphate group, liberating the phosphate for the animal. After hydrolysis at the favored site, the phytases
then move sequentially round the inositol ring, liberating additional phosphate groups until kinetics, environmental conditions, substrate solubility, or some other
mechanism prevents further activity. Importantly, the
phosphate located on carbon 2 in the ring is axially (as
opposed to equatorially) oriented and as such is recalcitrant. This means that in most instances, endogenous
phytases will not degrade phytic acid to inositol and
free phosphate, though the mucosal phosphatases may
generate inositol at the small intestinal level.
In a standard diet, there may be 10 g/kg of phytic
acid (2.8 g of phytate P/kg) and up to 60% of this may
be hydrolyzed by a microbial phytase, if appropriately
dosed, and absorbed by the terminal ileum. This release
varies and will be more immediately useful for metabolism if the phosphate is released proximal to the ileum
(as phosphate transporters become increasingly scarce
distal to the jejunum). Thus, the P effect of microbial
phytase will realize improvements in digestible P of up
to 0.17 percentage units depending on dietary phytate
concentration, the source of phytate, animal age and
species, mineral concentrations in the diet, vitamin D
concentration, phytase source, and phytase dosing.
Extraphosphoric Effects
Phytate is a ubiquitous, reactive, and extremely influential compound with physiological effects far beyond digestible P dilution. In feed, phytate is usually
present as an unreactive salt of Mg and K (sometimes
Ca is also implicated) known as phytin (Lott et al.,
2000). Once ingested and exposed to acidic conditions,
H+ ions readily replace the divalent cations and phytate
is solubilized as a partially protonated moiety. Because
the pKa values on 6 of the 12 ionizable orthophosphate
groups are too small to be fully protonated at pH of
between 2 and 3 (Costello et al., 1976), phytate retains
a net negative charge, even at the low pH encountered
in the gastric phase of digestion. The interaction between phytate and protein is not entirely clear, but it
may be that phytate, at intermediate concentrations
(10 IP6 g/kg and 200 g of protein/kg), alters the thermodynamics of water such that protein:protein aggregation is encouraged, reducing the solubility of dietary
protein. At greater concentrations of IP6 (>20 g/kg and
200 g of protein/kg), a rapid electrostatic attraction
between basic AA residues of dietary protein, which
are positively charged at gastric pH (pH below their
isoelectric point) and phytate occurs, pulling protein
back into solution (Mothes et al., 1990; Cowieson et
al., 2009). This process is partially dependent on the
nature of the dietary protein and the concentration of
phytate ingested. For example, some evidence suggests
that it is a critical phytate:protein (perhaps around
0.05:1; i.e., 10 g of phytic acid and 200 g of protein)
that is needed to minimize the solubility of the protein (Mothes et al., 1990). This ratio is marginally less
than that of a typical corn-soy diet, which suggests that
before use of phytase, the industry has been unwittingly dealing with an unrecognized problem of compromised and variable gastric digestion. Moreover, this
raises the possibility of generating negative responses
to xylanase, if fed in the absence of phytase, because
solubilization of previously encapsulated phytate may
push the phytate:protein further into the critical zone.
Regardless of this cautionary note, it is the reduction
in protein solubility that is responsible for the compromising effect of phytate on Na, Ca, AA, and energy
digestion because hypersecretion of HCl, mucin, pepsin,
bile, and NaHCO3 can be the result, increasing energy,
specific AA, and Na flow into the lumen, with the latter interfering with active transport (Cowieson et al.,
Exogenous enzymes in diets of nonruminants
2004; Cowieson and Ravindran, 2007; Liu et al., 2008).
Furthermore, though phytate:protein complexes will be
partially recovered by the time the feed reaches the
terminal ileum (because of changing pH and secretory
accommodation), intact phytate (i.e., fully phosphorylated), which reaches the duodenum, will rapidly scavenge divalent cations, especially Ca, forming insoluble
precipitates, which effectively remove these valuable
ions from the digestive milieu. Thus, the ingestion of
modest concentrations of phytate (<1% of the diet) has
a profound impact on endogenous processes and the
solubility and digestibility of dietary nutrients.
The nutrients that are most detrimentally influenced
by the ingestion of phytate are the AA Gly, Ser, Thr,
and Pro and the minerals Ca, Zn, Fe, Na, and Mg
(Cowieson and Ravindran, 2007; Peter et al., 2009;
Schlegel et al., 2009). There are also energetic effects,
both direct via improved solubility and digestibility of
dietary nutrients (e.g., release of metal cofactors for
endogenous enzymes or inositol liberation) and indirect via reduced endogenous loss, or net effects delivered via reduced synthesis of endogenous protein (gut
or support organ mass, enzymes, or mucin; Pirgozliev
et al., 2007; Cowieson et al., 2008; Onyango et al.,
2009). The obvious solution to these antinutritional
effects of phytate is to dephosphorylate IP6 as quickly
as possible in the proximal gastrointestinal tract to
lower molecular weight esters of IP. This approach will
release P from IP6, causing a commensurate decrease
in IP6 concentrations, and so reduce net antinutritive
effect as the lower esters are less antinutritional (Lutrell, 1993; Persson et al., 1998). Further, as the lower
esters of IP have a disproportionately reduced capacity
to chelate divalent cations, the solubility of such esters
in the small intestine will be improved, allowing access to these by the endogenous phytase/phosphatase
array. Thus, the primary responsibility for exogenous
microbial phytase is not so much to dephosphorylate
IP6 into inositol and free phosphate but to minimize
the entry of IP6, IP5, IP4, and IP3 into the duodenum.
The presence of phytate in the gut has a range of extraphosphoric consequences that extend to mineral,
AA, vitamin, and energy metabolism and ultimately
to animal viability, health, and efficiency.
AA
The effect of phytase on ileal AA digestibility have
been reported in at least 20 peer-reviewed articles in
both swine and poultry (Kornegay et al., 1996, 1999;
Sebastian et al., 1997; Namkung and Leeson, 1999; Ravindran et al., 1999, 2000, 2006; Zhang et al., 1999;
Camden et al., 2001; Selle et al., 2003a, 2009; Dilger
et al., 2004; Onyango et al., 2005; Agbede et al., 2009;
Cadogan et al., 2009). The effect of phytase on ileal
AA digestibility in these reports has ranged from no effect to fairly substantial effects (6 to 7% improvement
over the appropriate control), and these discrepancies
make interpretation difficult. One of the more conten-
3205
tious suggestions as to why some studies have observed
no effect of phytase and others substantive effects is
the choice of indigestible marker used in the work (Selle
and Ravindran, 2007). It may be coincidence, but it
seems that in studies where TiO2 or AIA are used as
markers, there is a notable improvement in ileal AA
digestibility with phytase, whereas in studies where
Cr2O3 was used, no effects are apparent. It is also clear
that benefits are more commonly reported for poultry
as opposed to swine (Cowieson and Bedford, 2009). A
common observation in all these studies is that typically Cys, Thr, Ser, Pro, and Gly respond to phytase,
whereas Met, Arg, Glu, and Lys respond much less. In a
review that presented a perspective that supplemental
dietary microbial phytase does not improve AA utilization, Adeola and Sands (2003) summarized literature
on AA digestibility and protein utilization responses
to phytase. The efficacy of phytase in improving protein utilization has presented conflicting information
with some investigators observing small improvements
in AA digestibility and protein utilization. Although
a limited pool of data exists on small increases in apparent AA digestibility in swine and poultry literature,
these seldom translated into improved growth performance when the effect of enhanced phytin-P utilization is factored out. Conversely, there are also data on
a lack of response in AA utilization (both pre- and
postabsorptive) to microbial phytase supplementation
(Adeola and Sands, 2003). More recently, data from
studies showing a lack of ileal AA digestibility response
to phytase supplementation in poultry and swine have
been reported (Liao et al., 2005; Onyango et al., 2005;
Woyengo et al., 2008; Sands et al., 2009).
Minerals
Nutrient requirements of swine and poultry were set
in the presence of phytate and so, inadvertently, accommodated some of the negative consequences of the
ingestion of phytate. One example of this is the relatively high concentrations of Na and Ca that are often
fed to modern broilers. Of course, not all the Na and
Ca requirements of the birds are dictated by phytate,
but perhaps 30% of the Ca requirement and 20 to 30%
of the Na requirement are. It is no coincidence that
Na is capable of disrupting phytate:protein complexes
as described by the following equation: percent protein bound to IP6 = −14.53ln(millimolar Na) + 86.113;
R2 = 0.9957 (De Rham and Jost, 1979; Mothes et al.,
1990), and consequently, it is likely that prephytase Na
requirements inadvertently took this mechanism into
account.
The interaction between Na and microbial phytase
is not well understood. The first paper to indicate that
there may be an effect of phytate and phytase on Na
was Cowieson et al. (2004) where large (>100%) increases in endogenous Na loss were reported in birds
that received a pure phytic acid solution by oral gavage.
This increased loss of Na was partially ameliorated by
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Adeola and Cowieson
the addition of phytase to the phytic acid solution immediately before oral gavage. The mechanism proposed
by Cowieson et al. (2004) was that phytate induced Na
influx into the small intestinal lumen as NaHCO3 in response to the hypersecretion of HCl in the gastric gut.
Clearly, this physiological response would alter intestinal electrolyte balance and may compromise, among
other processes, Na-K-ATPase active transport. These
effects were explored further by Ravindran et al. (2006)
where ileal Na digestibility coefficients were improved
by the addition of phytase to corn-based diets when
these diets were fed to broilers. In the study (Ravindran et al., 2006), ileal Na digestibility was enhanced
from −0.51 with no phytase to −0.18 with 1,000 FTU
of an E. coli 6-phytase/kg of diet. Subsequent to these
studies was a report by Ravindran et al. (2008) where
4 corn/soy-based diets with graded Na concentrations
from 1.5 to 5.2 g/kg were fed to broiler chickens with
and without phytase. The addition of Na was as NaHCO3, and it resulted in an increase in dietary electrolyte balance (DEB) from 150 to 375 mEq/kg. Nevertheless, it was observed that the diets with the greatest
DEB and Na concentrations were the least responsive
to microbial phytase, with improvements in, for example, ileal AA digestibility in the low Na diet of approximately 3.6% and in the high Na diet of 0%. These data
indicate that either DEB or dietary Na concentration
per se are implicit in the magnitude of response to microbial phytase in poultry. A further contribution to
the elucidation of this mechanism is Cowieson et al.
(2011) where the effect of phytate, phytase, and Na on
ileal endogenous flow was reported. The antinutritive
effects of phytate were confirmed as were the ameliorative effects of microbial phytase. However, several interactions were detected between Na (0.15 or 0.25%) such
that greater Na concentrations were associated with a
reduced antinutritive effect of phytate and a less obvi-
ous effect of phytase. As mentioned previously, it has
been shown that Na is capable of partially disrupting
the detrimental effect phytate has on protein solubility, and consequently greater dietary Na concentrations
may reduce the antinutritive effect of phytate and reduce nutrient digestibility response to phytase. These
effects were complex and AA specific. The reader is
referred to Cowieson et al. (2011) for a full description
of the results and a more comprehensive discussion of
the implications.
In terms of the role of Ca in phytate and phytase
nutrition, it is of importance that 1 phytate molecule
(28% P) is capable of complexing with up to 5 molecules of Ca (Selle et al., 2009), which is implicit in the
equation proposed by Nelson (1984):
Ca (%) = 0.6 + (phytate − P% × 1.1).
Thus, in a phytate-free diet, the associated Ca requirement is only around 0.6%. However, it is also important to consider that not all esters of phytate are
similarly malignant and it is not necessary to reduce
all phytate to myo-inositol and free phosphate to remove the antinutritive effects. Indeed, Luttrell (1993)
showed clearly that there is a disproportionate decrease
in the capacity of phytate to bind Ca as phosphates are
sequentially lost, which is shown by the equation Y =
5.1164X2 − 15.889X + 11.004; R2 = 1, where Y is the
relative Ca binding affinity as a percentage of IP6 and
X is the degree of phosphorylation. The lower IP3 has
approximately 11% of the binding affinity of IP6. Thus,
the equation proposed by Nelson (1984) could be more
accurately written as
Ca (%) = 0.6 + (IP6% × 1.1) + (IP5% × 0.65)
+ (IP4% × 0.32) + (IP3% × 0.11).
Figure 7. Theoretical relationship between P release from phytate and associated Ca value showing disproportionate extra­phosphoric effect
with initial destruction of the higher esters (Cowieson, 2010). Color version available in the online PDF.
Exogenous enzymes in diets of nonruminants
The above equation considers that removal of IP6
and IP5 is the primary objective rather than complete
dephosphorylation of all esters of phytate, which will
liberate more P but have no associated extraphosphoric
consequence (Figure 7).
New generation bacterial phytases have a very specific affinity for IP6 and IP5, and because they resist proteolytic digestion more than their fungal counterparts,
they are very aggressive phytate removers (Greiner and
Farouk, 2007; Cowieson et al., 2008). Indeed, Wyss et
al. (1999) presented information showing that E. coli
phytase would have removed all available (assuming it
is not encapsulated in cell wall material) IP6 and IP5
and most of the IP4 found in a typical corn-soy diet by
the time 0.13% P (a conventional P matrix for some
of the new bacterial phytases at around 500 FTU/kg)
is liberated. It is important to recognize that the current methods for assessing phytase activity focus on
the product formed (i.e., P, and not on the substrate
removed). Fungal and bacterial phytase at 500 FTU
will deliver the same amount of P under the conditions of assay, but the bacterial phytase, because of its
lack of affinity for IP4 and lower esters, delivers this P
through removal of far more phytate than its fungal
counterpart. This means that the liberated P comes
disproportionately from the greater molecular weight
esters when bacterial phytases are considered, making
the removal of the antinutrient (IP6 and IP5) at least as
important in the responses observed as the liberation
of the P per se. Not only that, but because very small
concentrations of the higher esters survive to reach the
duodenum, the aftermentioned equations indicate that
the new generation phytases may more appropriately
be assigned Ca matrices of 0.2% or even greater, increasing both remarkable commercial opportunity and
increased negative consequence of misuse.
Energy
The energy effect of phytase ranges from 0 to perhaps
120 kcal/kg. This effect is both the most economically
significant in terms of diet cost reduction but is also,
unfortunately, the most variable (Selle and Ravindran,
2007). For example, Scott et al. (2001) observed that
phytase increased ME by around 100 kcal/kg in cornand wheat-based diets for laying hens, whereas Liebert
et al. (2005) reported no effect of phytase in corn-based
diets for layers. In broilers, Selle and Ravindran (2007)
summarized 12 individual studies where the effect of
phytase on ME was reported and concluded that the
mean response is around 85 kcal/kg at an average phytase inclusion of 662 FTU/kg. However, this response
varied from almost 170 kcal/kg (Driver et al., 2006) to
−20 kcal/kg (Selle et al., 2003b) and, counterintuitively,
was not correlated with phytase dose. In fact, in 1 trial,
Driver et al. (2006) found that 24,000 FTU/kg of an A.
niger phytase improved AME by 170 kcal/kg, whereas
the same phytase dosed at just 750 FTU/kg had the
same effect in the study of Shirley and Edwards (2003).
3207
Thus, the energy effect of microbial phytase is difficult
to predict and may depend on the nature of the phytate
in the diet [i.e., its solubility/reactivity, which is different as noted by Leske and Coon (1999)], the divalent
cation concentration, the ingredients used, and many
other interactive factors. Of interest is that there is a
weak (r2 = 0.34), but significant, relationship between
the magnitude of response to phytase and the ME of
the unsupplemented control diet. This has been found
previously with phytase in terms of effects on ileal AA
digestibility (Cowieson, 2010) and indicates that one
reason for the variance in response to phytase for extraphosphoric nutrients may be the inherent digestibility
of these nutrients before phytase intervention. Several
mechanisms by which phytase may improve ME have
been proffered, including improved solubility of dietary
nutrients/reduction in insolubility of other complexes
(Matyka et al., 1990), improved solubility of cofactors
(e.g., Ca or Zn) for digestive enzymes (Selle and Ravindran, 2007), reduced endogenous energy flow (Cowieson
and Ravindran, 2007; Cowieson et al., 2008), improved
capacity for active transport of nutrients from the gut
associated with Na (Cowieson et al., 2004; Liu et al.,
2008), reduced integrity of fibrous complexes (in which
phytate may be implicit), and direct inhibition of digestive enzymes (or their activation) by phytate. These
various mechanisms have been partially proven at one
time or another, but no generally accepted mechanism
currently exists to explain the effect of phytase on energy digestibility.
Efficacy in Poultry and Swine
The beneficial effects of phytase in the diets of poultry and swine have been reported in thousands of
peer-reviewed publications, and this has been recently
and comprehensively reviewed by Selle and Ravindran
(2007) and Cowieson et al. (2009). It is beyond the
scope of this article to give a detailed review and to
do so would result in considerable repetition, but in
the interest of providing the reader with a reasonably
exhaustive conceptual framework, the following section
will briefly summarize the salient points.
Microbial phytases have been used commercially in
poultry and swine nutrition since the early 1990s but
have been assessed experimentally since (at least) the
1960s or 1970s. For example, Nelson and colleagues
conducted several studies using phytase (Nelson et al.,
1968, 1971). However, the paper that is generally credited as pivotal between experimental and commercial
use of phytase is Simons et al. (1990), where A. niger
phytase was found to improve the digestibility of P in
broilers and swine. Since 1990, the beneficial effect of
phytase on P digestibility has been confirmed and then
confirmed again in broilers (Kornegay et al., 1996),
swine (Adeola et al., 2004), turkeys (Ravindran et al.,
1995), laying hens (Gordon and Roland, 1998), ducks
(Orban et al., 1999; Adeola, 2010), and even some minor species such as quail (Sacakli et al., 2006). The im-
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provement in P digestibility with phytase is associated
with hydrolysis of phytate in the crop, proventriculus,
and gizzard, liberating P, which would otherwise be
rendered partially insoluble in the proximal small intestine and so recalcitrant to digestion. Typically, phytases used at standard commercial doses of 500 to 750
FTU/kg will release between 0.05 and 0.15% digestible
P, and this effect is accommodated in poultry and pig
diets by removal of inorganic P sources. Of importance
is that the concentration of phytate-P in the diet must
be known because in certain circumstances (e.g., use of
low phytate grains or inclusion of animal by-products
or both), the phytate concentration in the diet is too
small to consistently support bullish P matrix values.
It is, however, obvious from the literature that phytases
consistently improve the digestibility of P in diets that
contain low digestible P concentrations for swine and
poultry. Less clear, however, is the effect of phytase
on Ca, energy, AA, and other minerals (as described
before). Less intuitive factors that influence the usefulness of phytase in pig and poultry diets include interactions with other feed additives such as zinc oxide
(Augspurger et al., 2004), organic acids (Brenes et al.,
2003), vitamin D (Snow et al., 2004), pellet binders,
formaldehyde (Park et al., 1999), and other enzymes
(Cowieson, 2010). Further complexity is introduced by
phytases being offered in various product forms, including liquids, granulates, and coated, and with various
concentrations, particle sizes, stabilities, and thermotolerances.
A broad conclusion is that microbial phytases are
a well-accepted partial replacement for inorganic P in
the diets of poultry and swine. However, the extraphosphoric effects are more variable, and although of
some importance economically, ecologically, and scientifically, they remain poorly understood.
Efficacy in Fish
To the knowledge of the authors, the first report of
the use of phytase in aquaculture is Cain and Garling
(1995) where phytase was used to pretreat soybean
meal before feeding salmon to reduce the excretion of P
from hatcheries. This contrasts with phytase use in the
diets of swine and poultry dating at least back to the
1960s (Warden and Schaible, 1962). The relatively slow
uptake of phytase in aquaculture and a lag in scientific
investigation is likely due to a lack of compatibility
between the characteristics of commercially available
phytases and the requirements of aquaculture, as well
as the relative size of the fish-feed market (compared
with that of poultry and swine). For example, the diets
of farmed fish are typically extruded at high temperatures, which introduces technical difficulty in phytase
application and also fish diets typically contain animal
protein and so can be relatively low in phytate content.
Further, the optimum temperature of microbial phytases is typically 30 to 40°C, temperatures well above
the body temperature of many fish species, reducing
the relative activity of phytases [i.e., fish reared in
warm water (20 to 25°C) will likely have an improved
response to phytase compared with fish reared in cool
water (10 to 15°C)]. Nevertheless, several reports have
shown that phytase improves phytate-P availability
(Cain and Garling, 1995; Schaefer and Koppe, 1995;
Yu and Wang, 2000; Rodehutscord and Pfeffer, 2005;
Dalsgaard et al., 2009) in fish, reducing P pollution
(Jackson et al., 1996; Storebakken et al., 1998; Furuya
et al., 2001).
In terms of the extraphosphoric effects of phytase,
the experience of its use in fish nutrition reflects that
of its use in poultry and swine (i.e., equivocal). Forster
et al. (1999) found that up to 4,500 FTU/kg of an A.
niger phytase had no effect on energy digestibility in
rainbow trout (87.4 vs. 87.6%), despite phytate digestibility increasing from 7% to greater than 45%. Debnath et al. (2005) found that up to 2,000 FTU/kg of A.
niger phytase improved energy and protein retention in
Pangasius fed soy-based diets. Vielma et al. (2002) reported that dephytinization of soy protein concentrate
using a wet incubation process with an A. niger phytase improved P retention from 32 to 72%, N retention
from 35 to 43%, and Ca retention from 9 to 147% (fish
can absorb Ca from water via their gills). Several other
papers have shown positive or no effect of phytase on
energy, N, Ca, and Zn retention by fish (Liebert and
Portz, 2005, 2007; Biswas et al., 2007).
There seems to be only one report that shows the effect of phytase on AA digestibility in fish. Cheng and
Hardy (2003) added 200 to 1,000 FTU/kg of A. niger
phytase to rainbow trout diets based on casein, raw
soybeans, gelatin, dextrin, and fish oil. The digestibility of DM and CP was maximized at 800 FTU/kg,
and the same dose improved phytate digestibility from
19.6 to 93.7%. Amino acid digestibility was 97.4% in
the control group, and so there was little scope for further improvement with phytase as shown by Cowieson
and Bedford (2009). Nevertheless, 800 FTU/kg of phytase improved AA digestibility by an average of 1.1%,
ranging from 0.6% for Arg to 2.2% for Cys, a pattern
reminiscent of the typical response in poultry and swine
(Selle and Ravindran, 2007; Cowieson, 2010). Because
the diets of fish often contain greater concentrations of
readily digestible protein, it may be that the benefits
associated with the effects of phytase on AA digestibility are muted compared with other species.
It can be concluded that the usefulness of phytase in
the diets of fish is an emerging area that warrants further research. The appropriateness of currently available phytases may be questioned because their temperature profile and thermotolerance are not optimal
for aquaculture, especially for cold-water fish species.
The development of low-temperature active phytases
would be an advantage in the feeding of cold-water species and may improve the consistency of the benefit of
microbial phytase. Nevertheless, supplemental phytase
has been found to be relatively consistently advanta-
Exogenous enzymes in diets of nonruminants
geous to improve the retention of P by fish. For a more
detailed review of this topic the reader is directed to
Cao et al. (2007).
Supradosing
Supradosing is defined by the present authors as
≥2,500 FTU/kg of feed. Because conventional doses
are 500 to 1,000 FTU, this definition is considered to
be a magnitude above what most nutritionists would
use. After the pioneering work by Nelson et al. (1968,
1971), interest in phytase lagged notably until the late
1980s when commercialization of phytase as a feed additive took place in 1991. More recently, as phytase has
generally enjoyed incumbent status in the vast majority of pig and poultry diets, interest in phytase, and
supradosing specifically, has gained traction. Shirley
and Edwards (2003) supplemented corn-based diets
with up to 12,000 FTU/kg and observed a quadratic
increase in phytate-P disappearance with increasing log
phytase dose from approximately 42% at 93 FTU/kg
to almost 95% at 12,000 FTU. Although, similar responses in AME and N retention were observed, up to
12,000 FTU, the response to increased dose was very
much muted compared with that of phytate disappearance. Whereas Shirley and Edwards (2003) may have
been the first to report the effectiveness of particularly
large doses of phytase, there were a few previous papers where doses of up to and including 25,000 FTU/
kg were used (Simons et al., 1990; Huyghebaert et al.,
1992; Zhang et al., 2000b) and where continued advantages of these larger doses were observed. Such effects
have also been confirmed quite recently by Augspurger
and Baker (2004), Cowieson et al. (2006a), Brana et al.
(2006), Pirgozliev et al. (2007), Nyannor and Adeola
(2008), and Nyannor et al. (2007), all of which involved
studies where doses up to or beyond 10,000 FTU/kg
were used. Although these reports clearly show advantages of using unconventionally large doses of phytase,
there was no clear understanding of the mechanism.
Indeed, Shirley and Edwards (2003) indicated that it is
important to elucidate the mechanisms because there
are opportunities for the use of large doses of phytase.
There may be 3 main mechanisms whereby using large
doses of phytase may elicit beneficial effects: 1) more
liberated P or restoration of P/Ca proportionate release (if the animal remains below P requirement at
smaller doses or disproportionate Ca release at smaller
doses has disrupted Ca:P, larger phytase doses may
restore the digestible Ca:digestible P ratio); 2) less
residual phytate [i.e., destruction of the antinutritive
effect and increased generation of more (persistently)
soluble lower esters]; and 3) generation of myo-inositol
with vitamin-like/lipotropic effects. Others include a
possible interference by nonphytase side-activities [only
relevant for some products (e.g., fungal or “solid-state
produced” phytases)] and minor side-activities of other
enzymes being magnified at high inclusion rates.
3209
The diets of most nonruminant animals contain between 2 and 4 g of phytate-P/kg, and so notionally,
this is the maximum increase in digestible phosphate
possible by the use of phytase. However, phytate-P is
not entirely indigestible in the absence of phytase, and
largely depending on dietary Ca concentrations, phytate-P digestibility, in the absence of phytase, will be in
the range of 20 to 30%. Thus, P digestibility responses
to increasing phytase inclusion are likely to be maximized between 1.5 and 3 g/kg depending on the dose
of phytase and the concentration of dietary phytate.
Importantly, improvements in digestible P associated
with large doses of phytase would only be expected
to improve performance if P is limiting as a nutrient.
In the vast majority of the “super-dose” phytase trials mentioned before, digestible P concentrations in
the negative control diets were not unusually small and
achieving requirement should not have taken more than
750 FTU/kg. The question then is why liberation of P
beyond the requirement of the animal for digestible P
would continue to improve performance response criteria. This question does not have a satisfactory answer,
and it is more reasonable to conclude that liberation of
P in excess of requirement is not the principal mechanism by which high doses of phytase improve performance. Angel and Applegate (2002) have shown that
use of phytase in diets sufficient in available P results in
an increase in soluble P in the manure, mainly through
urinary excretion, confirming such a tenet that excess
P is not directly of benefit. However, an area of obscurity does persist, and that is asynchronicity in P and
Ca “release” by phytase.
Currently, most phytase products have a 1:1 relationship between Ca and P release values (e.g., 0.1% Ca and
0.1% P regardless of dose employed and both increase
in synchrony following a log-linear curve). However, as
clearly demonstrated by Wyss et al. (1999) and Greiner
and Farouk (2007), bacterial phytase preferentially target the higher molecular weight esters of IP and destroy
proportionately more IP6 and IP5 than IP4 and IP3 per
unit of P release in the initial reactive phase. Luttrell
(1993) and Persson et al. (1998) have demonstrated
that IP6 or IP5 have a much greater chelation capacity
for Ca than IP4 or IP3. Indeed, IP3 has only 10% of the
chelation capacity of IP6, not 50% as stoichiometry may
indicate. Thus, the release of the first 0.1% digestible P
from phytate through the use of 500 FTU/kg is likely
to be associated with destruction of the more malignant
esters and, consequently, a disproportionately large response for Ca. The use of 500 FTU may release Ca to
P in the ratio of greater than 2:1 rather than 1:1 that is
the assumed ratio when matrix values are applied. As a
result, the use of larger doses of phytase may continue
P release in a more linear fashion, whereas Ca is closer
to the asymptote. This could explain why large doses
of phytase give performance responses beyond associated positive controls or intuitive nutrient requirement
levels or both. Thus, dislocation of phytase matrices for
3210
Adeola and Cowieson
P from other nutrients may be justified (as illustrated
in Figure 2, page 229 of Cowieson et al., 2011). In fact,
Shirley and Edwards (2003) show clearly that phytateP disappearance may be linearly influenced by phytase
dose, whereas improvements in N retention and AME
are logarithmically correlated with dose. However,
when the effect of phytase on feed intake is considered,
these effects are diminished, indicating that phytase effects are bimodal, with an initial improvement in digestibility and a second stage improvement in digestible
nutrient intake.
Phytate is generally recognized as an antinutrient beyond its effect on digestible P dilution, and therefore
its removal, especially its complete and rapid removal,
from the diets of nonruminant animals will likely have
several beneficial effects. These effects are likely to be
particularly notable for the AA Thr, Cys, Gly, Ser, and
Pro, for the minerals Ca, Na, Zn, and Fe, and to some
extent for energy. Thus, the use of high doses of phytase may improve performance because of a combination of these beneficial effects.
Perhaps one wild card in phytase and phytate nutrition is the relevance of myo-inositol in phytase response. Myo-inostiol is widely distributed in animal
and vegetable tissues (Clements and Darnell, 1980) and
is found in poultry and swine diets between 1.4 and 6.8
g/kg. In these diets, it may be found in its free form
and as myo-inostiol-containing phospholipids but will
mainly be present as a component of myo-inostiol hexaphosphate (IP6), and it is less likely to be an immediate
participant in metabolic process. The first article to
show growth-promoting effects of myo-inostiol in chickens was Hegsted et al. (1941), and Dam (1944) suggested that myo-inostiol assists in ameliorating the detrimental effects of tocopherol deficiency in chicks. Few
contemporary articles have presented the effect of myoinostiol on the performance, physiology, or nutrition of
poultry or swine. However, from the classical work in
the 1940s/1950s, it seems reasonable that an increase
in myo-inostiol concentrations by direct addition, diet
modification (e.g., more animal/fish by-products), or
phytase supplementation may have a growth-promoting effect. Furthermore, because of the lipotropic effect
of myo-inostiol, it is possible that supradosing phytase
may have an indirect protective effect. When microbial phytases are added to diets, phytate is partially
dephosphorylated in the gastric phase. In the small intestine, the residual esters with lower molecular weight
are either absorbed or precipitate with cations, and are
subsequently excreted from the animal. The extent of
absorption of these lower esters is dictated by both the
degree of phosphorylation and ambient cation concentration (Nahapetian and Young, 1980; Schlemmer et
al., 2001). If the lower esters are absorbed, they will
be further dephosphorylated in the mucosa, blood, and
liver, and yet lower esters, free phosphate, and myoinostiol are rendered available for systemic distribution
and secondary metabolic processes such as the assembly of secondary messengers via phosphorylation with
endogenous kinases (Verbsky et al., 2002). In a recent
study, synthesis of myo-inostiol by phytase addition
was inferred by Karadas et al. (2010) where the addition of 12,500 FTU/kg of a bacterial phytase resulted
in increases in vitamin E and A and coenzyme Q10 in
the liver of chickens. Thus, it seems likely that although
exogenous phytase is not capable of generating myoinostiol in the lumen, it may present the mucosa with
soluble IP esters that may be absorbed and further
dephosphorylated systemically. Myo-inostiol genesis in
the liver or elsewhere in the body may play an important role in the growth-promoting effects of high doses
of phytase.
FORMULATING WITH FEED ENZYMES
To maximize the value of enzymes and to mitigate
risk of misuse, appropriate modifications to diet nutrient density are required and should include a matrix
for P, Ca, Na, AA, and energy. In the interests of establishing a frame of reference, it is suggested that the
release values for the new third-generation (engineered
E. coli as opposed to wild-type E. coli) phytases (500
FTU/kg) could be 0.13 to 0.14% P, 0.17 to 0.20% Ca,
0.02 to 0.05% Na, 1 to 6% improvement in digestibility
of AA (1% for methionine, 6% for cysteine, with others
following an established pattern; Cowieson, 2010), and
approximately 40 kcal/kg for ME. The use of carbohydrases usually allows the displacement of between 30
and 100 kcal of ME/kg (often achieved via the removal
of lipids). Failure to account for these effects (e.g., application of P matrices but no associated Ca value)
can lead to water retention issues, perhaps mediated
via renal damage associated with inappropriately high
circulating Ca concentrations.
In many ways nutrition research has, at least for the
past few decades, primarily been to keep up with changing genetic potential offered to nutritionists by the animal breeding companies. Thus, nutrition research has
been reactive rather than proactive in nature where the
new breeds arrive on the market and nutritionists must
respond with (usually modest) changes to nutrient requirements. Phytase, particularly the third-generation
bacterial (E. coli) phytases, are engineered in such a
way that they are extremely aggressive (Greiner and
Farouk, 2007), not only at liberating orthophosphate
from the inositol ring of phytate but also of removing,
almost completely, the antinutritive effects of phytate
in the process (Cowieson et al., 2004, 2008). Whereas
the liberated P can be quantified with relative ease, either via retention and digestibility or through titration
against a conventional phosphate source with known
availability, the extraphosphoric effects of phytases are
much more difficult to contend with. These extraphosphoric effects must be considered as potentially valuable effects of the use of phytase, but at the same time
are a considerable threat to productivity, litter/foot
pad quality, and bone strength if not appropriately accounted for in diet formulation.
Exogenous enzymes in diets of nonruminants
3211
Figure 8. Effect of fat removal from a corn/soy-based broiler diet on ileal AA digestibility [the negative control (NC) is expressed as percentage change from the positive control (PC); Cowieson et al., 2010].
Conventionally, the energetic benefits conferred by
exogenous enzymes are captured by a reduction of fat
concentration in the diet. However, it is important to
note that enzymes are not necessarily a suitable direct
replacement for fats and oils because extracaloric effects of lipids will not be delivered through the use of
enzyme technology. Examples of extracaloric benefits of
fat include pellet quality, essential fatty acids, fat-soluble vitamins (A, D, E, and K), balancing gastric emptying with protein and carbohydrate digestion, mill efficiency (energy use and throughput), and perhaps even
heat increment. Clearly, xylanases and phytases are not
direct replacements for these important effects, so the
removal of fat to accommodate the energy matrices of
enzymes should be done with care. In a recent study
(Figure 8; Cowieson et al., 2010), the removal of 2%
soy oil from a corn/soy-based broiler diet resulted in a
decrease (approximately 3%) in ileal AA digestibility at
d 21. Interestingly, these effects were not observed by
d 42 (change from positive control to negative control
approximately 0.4%; Figure 8), and furthermore, not
all AA were similarly influenced. This observation supports a previous report in piglets (Li and Sauer, 1994)
where the removal of canola oil resulted in a reduction
in AA digestibility. Presumably, these effects are mediated by changes in gastric emptying, which is driven in
part by dietary fat concentrations [Stacher et al., 1990;
Gentilcore et al., 2006; i.e., low fat diets may reduce
residency time of feed in the proventriculus/gizzard,
or even residency of food in the intestinal tract per
se (Mateos et al., 1982)]. It is interesting that the AA
most detrimentally influenced by the removal of added
fat are those AA that have been shown to be released
last from the sequence of endogenous proteolytic mechanisms (Low, 1980). Thus, the removal of fat/oil to
accommodate the ME advantages that enzymes confer
may be unwise in young animals because this strategy
may inadvertently compromise ileal AA digestibility,
especially for Thr, which tends to be the last dietary
AA to be exposed to exopeptidase activity. A further
consequence of reduced gastric residence time is that
the efficacy of a phytase will also be compromised because the stomach or proventriculus/gizzard is thought
to be most relevant for phytase activity. Thus, a dietary
modification made to profit from the energy-sparing
benefit observed when a xylanase is used may result
not only in direct losses in AA and starch digestibility,
but also in phytate hydrolysis with the ensuing further
losses in mineral, energy, and AA benefits, which were
attributed to phytate hydrolysis.
CONCLUSIONS
Current global feed enzyme use in diets of swine and
poultry is substantially greater than originally anticipated. Appropriate use of exogenous enzymes in feeds
requires strategic reductions in dietary energy and
nutrient content, as well as careful choice of feed ingredients to capture economic benefits of the various
enzymes. The efficacy of enzymes will vary depending on ingredients because nutrient and energy release
caused by enzyme supplementation will depend on the
structure of the feedstuff itself. It is important to continue the effort to understand the use and limitations
of matrix values of enzymes, which, if inappropriately
applied, will result in depressed performance because
of inadequacy of diets or will lead to wastage of resources. Because enzymes such as phytase hydrolyze
up to 60% of dietary phytate, there is considerable
room for the development of strategies that will further improve hydrolysis of phytate-P. The concept of
supradosing of phytase discussed earlier is promising.
Furthermore, there is a need to clarify and accurately
define the P requirements of swine and poultry and to
3212
Adeola and Cowieson
develop appropriate terminology to express these requirements uniformly. In this regard, a move away from
the widely used but nebulous available P to digestible
P is warranted. For Ca and P, therefore, standardized
total tract digestible Ca and P are recommended for
use in swine, but standardized ileal digestible Ca and P
are recommended in poultry. These will be more meaningful for a variety of reasons, including additivity in
diet formulation, efficiency of utilization, and accounting adequately for excretion.
The nonruminant feed enzyme market that includes
phytases, carbohydrases, and proteases has generated a
lot of interest in recent years. It is incontrovertible that
phytase is consistently efficacious in releasing orthophosphate from IP6 contained in plant-based feeds for
swine and poultry. Carbohydrases on the other hand
are equivocal based on several factors discussed in this
paper. Regardless, the use of exogenous enzymes in diets of nonruminants continues to be promising for a
variety of reasons that hinge on sustainability, economics, and the environment. Future research will no doubt
increase our understanding of feed enzymes and how
they can be more beneficially used to further improve
the efficiency of nonruminant animal production. A key
objective of future study should be to improve the consistency of observed responses to nonphytase enzymes,
which has been a persistent challenge for nutritionists
over the past 2 decades.
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