Dietary supplementation of ruminant diets with an Aspergillus oryzae -amylase

Abstract

Research in the area of dietary enzyme supplements for ruminant diets has primarily focused on fibrolytic enzymes, while activities involved in the process of starch digestion have been largely ignored. Since starch represents a major component in diets fed to highly productive cattle, the use of enzymatic dietary supplements to manipulate starch digestion in the rumen may allow improved productivity. This review discusses current information on dietary supplementation of calf, dairy and beef cattle diets with an Aspergillus oryzae extract containing α-amylase activity. During starch hydrolysis, α-amylase randomly cleaves starch polymers to low molecular weight oligosaccharides and eventually produces maltotriose and maltose from amylose and α-limit dextrins, maltose and glucose from amylopectin. Through its hydrolytic action, supplemental α-amylase hypothetically increases the availability of starch hydrolysis products in the rumen consequently altering the ruminal fermentation process. Data from studies employing lactating dairy cows, steers or rumen-simulating continuous cultures suggest that supplemental α-amylase did not increase ruminal starch digestion but consistently increased butyrate and reduced propionate molar proportions in the rumen. The increase in ruminal butyrate was also reflected in higher blood β-hydroxybutyrate concentrations in both transition and lactating dairy cows. In addition, supplemental α-amylase enhanced ruminal epithelium growth in dairy calves, a tissue that preferentially uses butyrate as an energy source. Experiments with pure cultures of ruminal bacteria showed that supplemental α-amylase supported rapid growth of bacteria that cannot grow, or grow slowly, on starch such as Butyrivibrio fibrisolvens D1, Selenomonas ruminantium GA192 or Megasphaera elsdenii T81. In contrast, bacteria that grow rapidly on starch, such as Streptococcus bovis S1 or Butyrivibrio fibrisolvens 49, did not benefit from α-amylase supplementation. Animal performance studies showed higher weight gain, and longissimus muscle area, in finishing beef cattle fed supplemental α-amylase. Weight gain improvements were primarily mediated through increased dry matter intake, which may be a consequence of reduced ruminal propionate molar proportions reported in other studies. In lactating dairy cattle, supplemental α-amylase increased milk yield, reduced milk fat proportion without reducing milk fat yield and tended to improve milk protein yield when data from 45 commercial herds (approximately 8150 cows) were examined. Currently available data on effects of the Aspergillus oryzae α-amylase described here suggest that this enzyme supplement may improve animal productivity by modifying ruminal starch digestion without necessarily increasing starch digestion in the rumen.

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Animal Feed Science and Technology
145 (2008) 136–150
A
vailable online at www.sciencedirect.com
Dietary supplementation of ruminant diets
with an Aspergillus oryzae ␣-amylase夽
J.M. Tricarico a,∗, J.D. Johnstonb, K.A. Dawsona
aAlltech Biotechnology Inc., Nicholasville, KY 40356, USA
bRitchie Feed & Seed Inc., Ottawa, Ont. K1B 4V5, Canada
Accepted 27 April 2007
Abstract
Research in the area of dietary enzyme supplements for ruminant diets has primarily focused on
fibrolytic enzymes, while activities involved in the process of starch digestion have been largely
ignored. Since starch represents a major component in diets fed to highly productive cattle, the use
of enzymatic dietary supplements to manipulate starch digestion in the rumen may allow improved
productivity. This review discusses current information on dietary supplementation of calf, dairy
and beef cattle diets with an Aspergillus oryzae extract containing ␣-amylase activity. During starch
hydrolysis, ␣-amylase randomly cleaves starch polymers to low molecular weight oligosaccharides
and eventually produces maltotriose and maltose from amylose and ␣-limit dextrins, maltose and
glucose from amylopectin. Through its hydrolytic action, supplemental ␣-amylase hypothetically
increases the availability of starch hydrolysis products in the rumen consequently altering the ruminal
fermentation process. Data from studies employing lactating dairy cows, steers or rumen-simulating
continuous cultures suggest that supplemental ␣-amylase did not increase ruminal starch digestion
but consistently increased butyrate and reduced propionate molar proportions in the rumen. The
increase in ruminal butyrate was also reflected in higher blood ␤-hydroxybutyrate concentrations
in both transition and lactating dairy cows. In addition, supplemental ␣-amylase enhanced ruminal
Abbreviations: ADG, average daily gain; CP, crude protein; DHI, dairy herd improvement; DIM, days in milk;
DM, dry matter; DP, degree of polymerization; DU, dextrinizing unit; NDF, neutral detergent fibre; OM, organic
matter.
夽This paper is part of a special issue entitled “Enzymes, Direct Fed Microbials and Plant Extracts in Ruminant
Nutrition” guest edited by R. J. Wallace, D. Colombatto and P. H. Robinson.
∗Corresponding author at: 3031 Catnip Hill Pike, Nicholasville, KY 40356, USA. Tel.: +1 859 885 9613;
fax: +1 859 887 3233.
E-mail address: jtricarico@alltech.com (J.M. Tricarico).
0377-8401/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.anifeedsci.2007.04.017

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J.M. Tricarico et al. / Animal Feed Science and Technology 145 (2008) 136–150 137
epithelium growth in dairy calves, a tissue that preferentially uses butyrate as an energy source.
Experiments with pure cultures of ruminal bacteria showed that supplemental ␣-amylase supported
rapid growth of bacteria that cannot grow, or grow slowly, on starch such as Butyrivibrio fibrisolvens
D1, Selenomonas ruminantium GA192 or Megasphaera elsdenii T81. In contrast, bacteria that grow
rapidly on starch, such as Streptococcus bovis S1 or Butyrivibrio fibrisolvens 49, did not benefit from
␣-amylase supplementation. Animal performance studies showed higher weight gain, and longissimus
muscle area, in finishing beef cattle fed supplemental ␣-amylase. Weight gain improvements were
primarily mediated through increased dry matter intake, which may be a consequence of reduced
ruminal propionate molar proportions reported in other studies. In lactating dairy cattle, supplemental
␣-amylase increased milk yield, reduced milk fat proportion without reducing milk fat yield and tended
to improve milk protein yield when data from 45 commercial herds (approximately 8150 cows) were
examined. Currently available data on effects of the Aspergillus oryzae ␣-amylase described here
suggest that this enzyme supplement may improve animal productivity by modifying ruminal starch
digestion without necessarily increasing starch digestion in the rumen.
© 2007 Elsevier B.V. All rights reserved.
Keywords: ␣-Amylase; Aspergillus oryzae; Ruminants
1. Introduction
Digestive processes in livestock and poultry are mediated through the action of enzymes.
This has generated interest in using exogenous enzyme preparations as dietary supple-
ments to improve animal productivity by researchers and practicing nutritionists. Successful
application of dietary enzyme supplement technology was first achieved with monogastric
animals and it is current practice in poultry and pig nutrition (Bedford and Schulze, 1998).
Nonetheless, the microbial fermentation process in the rumen has made adoption of dietary
enzyme technology more challenging in ruminants.
1.1. Dietary enzymes for ruminants
The vast majority of research in the area of exogenous enzyme supplements for rumi-
nants has focused on fibrolytic enzyme preparations and fiber digestion. Increased ruminal
fiber digestion may largely explain improvements in ruminant productivity resulting from
dietary supplementation with cell wall-degrading enzymes. Discussion of fibrolytic enzyme
supplements is outside the scope of this manuscript and the reader is referred to excellent
reviews on this subject by McAllister et al. (2001) and Beauchemin et al. (2003). The list of
ruminal effects implicated in the potential mode of action for exogenous enzymes proposed
in these reviews include direct hydrolysis, increased ruminal microbial attachment, stimu-
lation of ruminal microbial populations, and synergism with ruminal microbial enzymes.
Some of these factors may also have a critical role in the potential mode of action for
supplemental ␣-amylase.
1.2. Exogenous amylases in ruminant diets
Unlike cell wall-degrading enzymes, exogenous amylases have received little attention
by ruminant nutritionists. The general perception is that starch digestion by ruminants is

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extensive and does not generally limit production in the way that incomplete or slow fiber
digestion does. In addition, rapid digestion of excessive amounts of starch in the rumen may
lead to ruminal acidosis (Owens et al., 1998) representing a potential concern for inclusion
of exogenous amylases in ruminant diets. Consequently, supplementation with exogenous
amylases to increase ruminal starch digestion is not warranted. Nonetheless, we hypothe-
size that exogenous supplemental amylases could be employed to reduce the considerable
unexplained variation in ruminal starch degradation within dietary starch sources (Firkins
et al., 2001).
It is not surprising that the potential for amylase supplementation in ruminant diets has
been rarely studied and the absence of information on this topic is apparent in the literature.
Early studies included amylase in combination with other enzyme activities, none of which
were characterized (Burroughs et al., 1960). In more recent studies, amylase activities only
represented a minor component of primarily microbial (McGilliard and Stallings, 1998)or
predominantly fibrolytic preparations (McAllister et al., 1999; Hristov et al., 2000), while
other studies used undefined preparations (Chen et al., 1995) or thermostable ␣-amylase
from Bacillus licheniformis (Rojo-Rubio et al., 2001; Mora-Jaimes et al., 2002,Rojo et al.,
2005) with the specific objective of increasing starch digestion in sorghum.
The objective of this review is to present and discuss information collected over the last
four years on dietary supplementation of calf, dairy and beef cattle diets with an Aspergillus
oryzae extract primarily containing ␣-amylase activity.
2. Exogenous ␣-amylase from Aspergillus oryzae
2.1. Exogenous α-amylase characteristics
The dietary amylase supplement (AmaizeTM, Alltech Inc., Nicholasville, KY, USA)
discussed in this review is based on a powdered Aspergillus oryzae extract that contains
primarily ␣-amylase or 1,4-␣-d-glucan glucanohydrolase (EC 3.2.1.1) activity. One ␣-
amylase dextrinizing unit (DU) is defined as the quantity of enzyme required to dextrinize
soluble starch at the rate of 1 g/h at 30 ◦C and pH 4.8 according to the procedure described in
the Food Chemicals Codex (1996). Final ␣-amylase concentration in the Aspergillus oryzae-
based supplement is 600 DU/g. Protease, cellulase and xylanase actvities, determined with
hemoglobin, carboxymethyl-cellulase, and brichwood xylan as described by Tricarico et al.
(2005), are negligible in this preparation.
2.2. Effects of supplemental Aspergillus oryzae α-amylase on dairy and beef cattle
production
Dietary supplementation with the Aspergillus oryzae ␣-amylase preparation improved
dairy cattle performance (Table 1). A quadratic increase (P=0.02) in milk production was
initially reported by Tricarico et al. (2005) in lactating dairy cows fed a corn grain based
diet. These researchers reported a maximum milk yield at 240 DU/kg of dietary dry matter
(DM) with an increase of 1.5 kg/d over the non-supplemented control. DeFrain et al. (2005)
reported a higher (P=0.03) decrease in DM intake from week 2 to week 1 prepartum, but

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J.M. Tricarico et al. / Animal Feed Science and Technology 145 (2008) 136–150 139
Table 1
Effects of supplemental ␣-amylase from Aspergillus oryzae on milk production and composition in lactating dairy
cattle (adapted from DeFrain et al., 2005; Tricarico et al., 2005)
Supplement
Control ␣-Amylase Change
DeFrain et al. (2005)a
Number of animals 12 12
␣-Amylase activity (DU/kg DM) 0 662
Milk yield (kg/d) d 1–70 43.6 44.2 +0.6
Milk yield (kg/d) d 1–21 17.8 17.8 −0.1
Milk fat (g/kg) d 1–21 47.8 41.8 −0.6
Tricarico et al. (2005)b
Number of animals 24 24
␣-Amylase activity (DU/kg DM) 0 240
Milk yield (kg/d)c29.2 30.7 +1.5
Milk fat (g/kg) 37.1 37.8 +0.7
aSupplemental ␣-amylase was fed from d −21 to 21. DM intake (kg/d) were 12.5 (control) and 11.9 (␣-amylase)
from d −21 to d 0, and 17.8(control) and 17.7 (␣-amylase) fromd1tod21.
bSupplemental ␣-amylase was fed at 0, 240, 480 and 720 DU/kg dietary DM. Only data from 0 and 240DU/kg
dietary DM are presented. DMI (kg/d) was 22.7 (control) and 22.9 (␣-amylase).
cQuadratic effect of ␣-amylase supplementation (P=0.02).
no differences in early lactation milk production up to 70 days in milk (DIM) in dairy cows
fed supplemental ␣-amylase at 662 DU/kg of dietary DM during the transition period (−21
to 21 DIM).
Dietary supplementation with the Aspergillus oryzae ␣-amylase preparation also
improved finishing beef cattle performance in two studies (Tricarico et al., 2007). In both
instances, the largest improvements in average daily gain (ADG) occurred during the ini-
tial 28 d on feed (Table 2), although overall carcass-adjusted ADG also increased in one
experiment. Increases in ADG were apparently a consequence of increased feed intake in
both instances and in one experiment the response was predominantly quadratic. Increased
DM intake as a result of ␣-amylase supplementation in finishing beef cattle, but not in
lactating dairy cattle, may be a function of different dietary and animal factors in these
two production systems. Interestingly, quadratic responses to ␣-amylase supplementation
occurred in lactating dairy and finishing beef cattle and are in general agreement with
the frequent occurrence of non-linear responses to exogenous enzyme supplementation
that have been reported (e.g.,Beauchemin et al., 2003). Other interesting observations
are that supplemental ␣-amylase increased intake and gain with both high moisture and
cracked corn in one experiment, but only with cottonseed hulls and not with alfalfa hay
in another. The lack of interaction between ␣-amylase supplementation and corn grain
processing suggests no increase in ruminal starch digestion in these studies and that our
initial hypothesis that supplemental ␣-amylase would be more efficacious in diets contain-
ing more resistant starch (cracked versus high moisture corn) was incorrect. Reasons for
the lack of effects of supplemental ␣-amylase in the alfalfa hay diet are unknown. How-
ever, positive responses to ␣-amylase supplementation reported by Tricarico et al. (2005)
in lactating cows and by DeFrain et al. (2005) in prepartum cows occurred with diets con-

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Table 2
Effects of supplemental ␣-amylase from Aspergillus oryzae on performance and carcass characteristics in finishing
beef cattle (adapted from Tricarico et al., 2007)
Supplement
ItemaControl ␣-Amylase Change
Exp.1b
Pens per treatment 6 6
␣-Amylase activity (DU/kg DM) 0 950
DM intake (kg/d)
d 0–28 7.53 7.84 +0.31
d 0 to end 7.61 7.93 +0.32
ADG (kg/d)
d 0–28 1.64 1.88 +0.24
Carcass-adjusted to end 1.30 1.42 +0.12
LM area (cm2) 82.72 88.96 +6.24
Yield grade 2.99 2.89 −0.10
Exp.2c
Pens per treatment 4 4
␣-Amylase activity (DU/kg DM) 0 580
DM intake (kg/d)
d 0–28 8.04 9.05 +1.01
d 0 to end 9.13 10.06 +0.93
ADG (kg/d)
d 0–28 1.98 2.30 +0.32
Carcass-adjusted to end 1.87 2.05 +0.18
LM area (cm2) 80.04 85.06 +5.02
Yield grade 2.29 2.05 −0.24
aUsed implants and ionophores (monensin sodium) in both experiments.
bExp. 1 used 120 crossbred steers (5 steers/pen) fed alfalfa hay or cottonseed hulls finishing diets for an average
152 d from d 0 to end (slaughter). Only data from cottonseed hulls diets are presented. Main effects of ␣-amylase
on: DMI d 0–28 (P=0.10) and d 0 to end (P=0.27); ADG d 0–28 (P=0.06) and carcass-adjusted to end (P=0.44);
LM area (P=0.02). Roughage ×␣-amylase interaction on: DMI d 0–28 (P=0.85) andd0toend(P=0.20); ADG d
0–28 (P=0.02) and carcass-adjusted to end (P=0.11); LM area (P=0.31).
cExp. 2 used 96 crossbred heifer (4 heifers/pen) fed cracked or high moisture corn finishing diets for an average
81dfromd0toend(slaughter). Combined data from cracked and high moisture corn diets are presented. Main
effects of ␣-amylase on: DMI d 0–28 (P=0.05) andd0toend(P=0.12); ADG d 0–28 (P=0.03) and carcass-adjusted
to end (P=0.04); LM area (P=0.12); yield grade (P=0.02). Quadratic effects of ␣-amylase on: DMI d 0–28 (P=0.06)
andd0toend(P=0.07); ADG d 0–28 (P=0.05) and carcass-adjusted to end (P=0.04); LM area (P=0.04); yield
grade (P=0.02).
taining alfalfa hay or haylage at 300 and 210 g/kg of total forage fed. In contrast, no effects
were reported in postpartum cows by DeFrain et al. (2005) or on ruminal fermentation by
Hristov et al. (2008) when ␣-amylase was included in diets containing alfalfa hay or haylage
at 450 and 620 g/kg of total forage fed. These observations suggest that a negative interac-
tion may exist between alfalfa and supplemental ␣-amylase and that additional research
is needed to examine potential interactions between exogenous ␣-amylase and dietary
ingredients.

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2.3. Effects of supplemental Aspergillus oryzae α-amylase on digestion
Dietary supplementation with the Aspergillus oryzae ␣-amylase preparation apparently
does not increase ruminal starch digestion. Ruminal in situ starch digestion did not increase
in steers or lactating dairy cows fed corn grain based diets containing ensiled forage
(Tricarico et al., 2005). Similarly, Hristov et al. (2008) reported no effects on ruminal
and total tract starch digestion in lactating dairy cows fed corn grain, barley grain, and
alfalfa and grass hay in a more recent study using the same exogenous enzyme preparation.
Finally, the possibility that supplemental ␣-amylase increases intestinal starch digestion
is not likely, since the enzyme is inactivated by gastric digestion (M.D. Abney and M.L.
Galyean, Texas Tech University, Lubbock, TX, personal communication).
Chen et al. (1995) used a commercial amylase and protease mixture, with the objective
of increasing starch digestion in dairy cattle fed sorghum grain, and reported no effects of
enzyme treatment on milk production or starch digestion, but observed higher DM, organic
matter (OM), crude protein (CP) and neutral detergent fibre (NDF) total tract digestibility
in enzyme-treated steam flaked sorghum grain. Unfortunately, a description of the specific
amylase present in the mixture, or its concentration, was not provided. Neither Tricarico
et al. (2005) nor Hristov et al. (2008) reported changes in ruminal NDF digestion with
supplemental ␣-amylase from Aspergillus oryzae. In contrast, supplementation with a ther-
mostable ␣-amylase from Bacillus licheniformis increased ruminal starch digestion both in
vitro (Rojo-Rubio et al., 2001) and in lambs (Mora-Jaimes et al., 2002; Rojo et al., 2005).
Comparison of our results with those reported by these researchers is complicated by the
use of different source microorganisms (Aspergillus oryzae versus Bacillus licheniformis),
enzyme assay conditions, unit definitions, diet composition (primarily corn grain versus
sorghum grain) and animal species used (i.e., bovines versus lambs).
2.4. Effects of supplemental Aspergillus oryzae α-amylase on ruminal fermentation
and metabolite concentrations
Dietary supplementation with the Aspergillus oryzae ␣-amylase preparation increased
acetate and butyrate and reduced propionate molar proportions (Table 3) in steers, lactating
dairy cows, and ruminal-simulating continuous cultures (Tricarico et al., 2005). Supplemen-
tal ␣-amylase also numerically increased ruminal butyrate molar proportions in prepartum
dairy cows (DeFrain et al., 2005). Consequently, ␣-amylase supplementation increased the
acetate to propionate ratio; an increase that is not indicative of increased ruminal starch
digestion. Benefits of increasing ruminal butyrate molar proportions relative to ruminant
productivity are not apparent. However, a review summarizing data from 20 studies reported
that ruminal concentrations of butyrate, followed by propionate, had the strongest positive
correlation with milk production (Seymour et al., 2005).
The increases in ruminal butyrate molar proportions were also accompanied by increases
in serum concentrations of ␤-hydroxybutyrate and non-esterified fatty acids (Table 4)in
prepartum (DeFrain et al., 2005) and lactating dairy cows (Tricarico et al., 2005). Con-
comitant increases in blood ␤-hydroxybutyrate, and decreases in blood glucose, have
been reported (Huhtanen et al., 1993; Miettinen and Huhtanen, 1996). Nonetheless, ␣-
amylase supplementation did not reduce serum glucose concentrations in lactating cows

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Table 3
Effects of supplemental ␣-amylase from Aspergillus oryzae on ruminal butyrate molar proportions and the acetate
to propionate (A:P) ratio in lactating dairy cows, steers, continuous cultures (adapted from Tricarico et al., 2005)
and transition dairy cows (adapted from DeFrain et al., 2005)
Supplement
Control ␣-Amylase Change
Tricarico et al. (2005)a
Butyrate (mmol/mol)b
Lactating cows 129 141 +12
Steers 128 146 +18
Continuous cultures 178 193 +15
A:Pc
Lactating cows 2.85 3.15 +0.30
Steers 2.90 4.08 +1.18
Continuous cultures 1.68 1.75 +0.07
DeFrain et al. (2005)d
Butyrate (mmol/mol)e
Prepartum 91 100 +9
Postpartum 103 110 +7
A:Pf
Prepartum 3.53 3.53 0
Postpartum 2.99 2.75 −0.24
aSupplemental ␣-amylase was provided at: 0, 240, 480 and 720 DU/kg dietary DM to lactating cows (data from
0 and 240 DU/kg dietary DM are presented); 0, 360 and 720 DU/kg dietary DM to steers (averages for 1–11 h after
feeding from 0 and 360 DU/kg dietary DM are presented); 0 and 1200 DU/kg dietary DM to continuous cultures
(averages for 72–120 h are presented).
bSignificance for butyrate molar proportions: main effects of ␣-amylase supplementation in lactating cows
(P<0.05) and continuous cultures (P=0.03).
cSignificance for A:P: main effect of ␣-amylase supplementation in lactating cows (P<0.05) and ␣-amylase by
time interaction in steers (P=0.07).
dSupplemental ␣-amylase was provided at 0 and 662 DU/kg dietary DM from d −21 to 21 (prepartum and
postpartum averages are presented).
eSignificance for butyrate molar proportions: main effects of ␣-amylase supplementation prepartum (P=0.14).
fSignificance for A:P: ␣-amylase by day interaction prepartum (P<0.04; 3.60 vs. 3.33 d −21 and 3.46 vs. 3.74
d−7 for control and ␣-amylase, respectively).
at 240 DU/kg DM (Tricarico et al., 2005) and only tended to increase postpartum plasma
glucose concentrations at 662 DU/kg dietary DM (DeFrain et al., 2005). It is conceivable
that increased productivity in cattle may arise from effects of ␣-amylase supplementation
on ruminal fermentation and the concomitant changes in metabolite concentrations that
imply differences in nutrient metabolism in supplemented cattle.
2.5. Effects of supplemental Aspergillus oryzae α-amylase on ruminal development in
calves
Ruminal development is stimulated by microbial VFA production and especially by
butyrate and propionate (McLeod and Baldwin, 2000). Approximately 0.90 of ruminal
butyrate may be absorbed by rumen tissue providing energy for rumen wall thickening,

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Table 4
Effects of supplemental ␣-amylase from Aspergillus oryzae on circulating ␤-hydroxybutyrate (BHBA), non-
esterified fatty acids (NEFA) and glucose in lactating (adapted from Tricarico et al., 2005) and transition dairy
cows (adapted from DeFrain et al., 2005)
Supplement
Control ␣-Amylase Change
Tricarico et al. (2005)a
BHBA (␮mol/l) 434 492 +58
NEFA (␮Eq/l) 160 192 +32
Glucose (mg/l) 468 474 +6
DeFrain et al. (2005)b
BHBA (␮mol/l)
Prepartum 376 603 +227
Postpartum 895 990 +95
NEFA (␮Eq/l)
Prepartum 115 373 +258
Postpartum 535 471 -64
Glucose (mg/l)
Prepartum 689 715 +26
Postpartum 640 693 +53
aSupplemental ␣-amylase was provided at 0, 240, 480 and 720 DU/kg dietary DM to lactating cows. Metabolite
concentrations in serum are presented for 0 and 240 DU/kg dietary DM. Main effects of ␣-amylase supplementation
for BHBA and NEFA (P<0.05).
bSupplemental ␣-amylase was provided at 0 and 662 DU/kg dietary DM from d −21 to 21. Metabolite concen-
trations in plasma are presented as averages for prepartum and postpartum. Effects of ␣-amylase supplementation
for BHBA and NEFA prepartum (P<0.01) and glucose postpartum (P=0.08).
and papillae and capillary development (Weigand et al., 1975). Therefore, supplementation
with a butyrate enhancing additive may be beneficial to ruminal epithelium development.
This hypothesis was examined with neonatal dairy calves by providing supplemental ␣-
amylase from Aspergillus oryzae in the first 200 g of calf starter consumed daily at 0, 6,762
or 13,524 DU/d in an initial study and 0, 4,710 or 9,420 DU/d in a second study. Dietary
supplementation with the ␣-amylase preparation enhanced ruminal epithelium development
in both studies, as evidenced by increased papillae length and width (A.J. Heinrichs, Penn
State University, State College, PA, USA, unpublished), suggesting that a dose of supple-
mental ␣-amylase that is adequate to improve ruminal epithelium development in calves is
between about 7000 and 9000 DU/d.
3. Aspergillus oryzae ␣-amylase mode of action
3.1. Ruminal starch hydrolysis
Starch is composed of an insoluble linear polymer of glucose bound by ␣-1,4 linkages
(amylose) and a highly branched polymer with ␣-1,6 bonds at each branch point (amy-
lopectin). The process of starch digestion involves ␣-amylase, which cleaves internal ␣-1,4
linkages of the polymer backbone randomly and releases low molecular weight oligosaccha-

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rides (maltodextrins), and isoamylase, which cleaves the ␣-1,6 linkages of the amylopectin
branch points. Glucoamylase and ␤-amylase cleave glucose and maltose from amylase non-
reducing ends. Although ruminal bacteria, protozoa and fungi are all involved in ruminal
starch digestion, the contributions of protozoa and fungi are not clearly defined.
Examination of the amylolytic activity of the predominant starch-digesting ruminal bac-
teria, and their hydrolysis products, can give some insight into potential modes of action
for supplemental ␣-amylase in the rumen. The ruminal bacteria with the highest capacity
for starch digestion are Ruminobacter amylophilus and Streptococcus bovis, followed by
Prevotella ruminicola and some Butyrivibrio fibrisolvens strains like 49 and A38 (Cotta,
1988). All of these ruminal bacteria produced mixed oligosaccharides as a result of amylose
digestion in the laboratory (Cotta, 1988). Butyrivibrio fibrisolvens 49, Ruminobacter amy-
lophilus H18, Streptococcus bovis JB1 and Prevotella ruminicola 23 produced primarily
maltose through maltotetraose. Butyrivibrio fibrisolvens A38 also produced maltopentaose
and maltohexaose, and Prevotella ruminicola B14 also produced maltoheptaose. In addi-
tion, prolonged exposure to the enzyme decreased the larger, and concurrently increased
the smaller, oligosaccharide products from amylose hydrolysis. This pattern of hydrolysis
is consistent with production of endo-acting enzymes by all studied bacteria whose activity
is similar to that of ␣-amylase. Genes encoding ␣-amylase activity have since been cloned
from Streptococcus bovis (Clark et al., 1992; Cotta and Whitehead, 1993) and Butyrivibrio
fibrisolvens (Rumbak et al., 1991) providing further support for this hypothesis. Therefore,
it is likely that starch in the rumen is hydrolyzed to a variety of products ranging from
glucose to maltoheptaose that could be used as growth substrates by a variety of ruminal
microorganisms.
3.2. Effects of α-amylase supplementation on growth of ruminal bacteria
Data from our studies suggest that supplemental ␣-amylase from Aspergillus oryzae does
not increase ruminal starch digestion, but shifts ruminal fermentation to a higher molar pro-
portion of butyrate and acetate at the expense of propionate, presumably by modifying
microbial metabolism or microbial populations in the rumen. Our hypothesis is that supple-
mental Aspergillus oryzae ␣-amylase produces maltodextrins that provide substrate, and a
competitive advantage, to non-amylolytic organisms that produce butyrate and acetate from
starch hydrolysis products.
A series of experiments were conducted to examine effects of ␣-amylase supplemen-
tation on growth of representative strains of ruminal bacteria on starch. Pure cultures of
Butyrivibrio fibrisolvens strains D1, 49 and A38, Streptococcus bovis S1, Megasphaera
elsdenii T81 and Selenomonas ruminantium GA192 were grown anaerobically at 37 ◦C
on medium 10 containing soluble potato starch (1.0 g/l) as the sole carbohydrate source.
Enzyme treatment was applied immediately prior to bacterial inoculation by adding 0.1 ml
of a solution to provide a final concentration of 0.06 DU/ml. Control cultures received 0.1 ml
of a solution prepared with fermentation solubles (enzyme carrier). Microbial growth was
estimated in each culture by measuring optical density at 600 nm over time. Each experi-
ment consisted of either two or three replicates per treatment. As expected, Streptococcus
bovis S1 and Butyrivibrio fibrisolvens 49 grew rapidly on starch-containing medium and
␣-amylase supplementation had no effects on their growth (Fig. 1). Butyrivibrio fibrisol-

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J.M. Tricarico et al. / Animal Feed Science and Technology 145 (2008) 136–150 145
Fig. 1. Growth (optical density at 660 nm) on soluble potato starch (1 g/l) in the absence (empty square) or presence
(solid square) of supplemental ␣-amylase (0.06 DU/ml) by (A) Butyrivibrio fibrisolvens D1; (B) Selenomonas
ruminantium GA192; (C) Megasphaera elsdenii T81; (D) Streptococcus bovis S1; (E) Butyrivibrio fibrisolvens
49; and (F) Butyrivibrio fibrisolvens A38.
vens A38 grows equally well on maltose and starch (Cotta, 1988) and grew more slowly in
the presence of supplemental ␣-amylase in this experiment. Conversely, Butyrivibrio fibri-
solvens D1, Selenomonas ruminantium GA192 and Megasphaera elsdenii T81 only grew
poorly or not at all on starch. However, these non-amylolytic species grew rapidly when
supplemental ␣-amylase was included in the starch-containing medium (Fig. 1).
Effects of ␣-amylase supplementation on growth of Butyrivibrio fibrisolvens D1 were
further examined using commercial maltodextrin products (Maltrin®, Grain Processing
Corporation, Muscatine, IA, USA) with various degrees of polymerization (DP) as carbo-
hydrate source (Table 5). Microbial growth conditions and monitoring were as described

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146 J.M. Tricarico et al. / Animal Feed Science and Technology 145 (2008) 136–150
Table 5
Saccharide composition (g/kg) of commercial maltodextrins (Maltrin®, Grain Processing Corporation, Muscatine,
IA, USA) used as a carbohydrate source for in vitro growth of Butyrivibrio fibrisolvens D1 in the absence and
presence (0.06 DU/ml) of ␣-amylase from Aspergillus oryzae
Maltrin®product code
M440 M500 M550 M580 M600
Dextrose equivalencea510151820
Average theoretical MWb3600 1800 1200 1000 900
Average degree of polymerizationc221 111 74 62 58
Composition (g/kg DM basis)
Glucose (DP1) 3 8 13 16 23
Maltose (DP2) 9 29 48 58 74
Maltotriose (DP3) 14 44 67 78 91
Maltotetraose (DP4) 14 38 55 61 68
Maltopentaose (DP5) 13 34 47 54 63
Maltohexaose (DP6) 18 57 84 102 119
Maltoheptaose (DP7) 24 68 91 102 100
>Maltoheptaose (>DP7) 905 722 595 529 462
aDextrose equivalence (DE) is a quantitative measure of the degree of starch polymer hydrolysis (DE of starch = 0
and DE of dextrose or glucose = 100).
bMW: molecular weight.
cDegree of polymerization (DP) refers to the number of glucose units joined in the molecule and is presented.
above. Growth of Butyrivibrio fibrisolvens D1 was similar in the presence or absence of
supplemental ␣-amylase with maltodextrins providing an average DP of 11.1 or less (Fig. 2).
However, ␣-amylase supplementation increased Butyrivibrio fibrisolvens D1 growth with
a maltodextrin providing an average DP of 22.1. These results confirm that Butyrivibrio
fibrisolvens D1 can grow efficiently on low DP maltodextrins and that supplemental ␣-
Fig. 2. Growth (optical density at 660 nm) of Butyrivibrio fibrisolvens D1 after incubation for 15 h on soluble potato
starch (1 g/l) or oligosaccharides (maltodextrins) of varying average degree of polymerization in the absence (light
grey) or presence (dark gray) of supplemental ␣-amylase (0.06 DU/ml) from Aspergillus oryzae. Means within a
substrate with different superscripts differ (P<0.05).

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J.M. Tricarico et al. / Animal Feed Science and Technology 145 (2008) 136–150 147
amylase activity can provide these hydrolysis products from starch or maltodextrins with a
higher DP.
3.3. A comprehensive hypothesis
Maltodextrins produced from hydrolysis of native starch can be used by a wide variety of
ruminal bacteria including amylolytic and non-amylolytic species. Our studies, and those by
Cotta (1992), showed that although Selenomonas ruminantium and Megasphaera elsdenii
grow poorly on starch, both are able to grow rapidly on maltodextrins. Similarly, cellodex-
trins produced by cellulolytic bacteria can be used by non-cellulolytic species (Russell,
1985) and xylooligosaccharides from xylan hydrolysis can be used by non-xylanolytic
species (Cotta, 1993). These observations suggest that cross-feeding mechanisms are a
general feature of the ruminal microbial ecosystem and those microorganisms that utilize
hydrolysis products from other species will contribute to ruminal fermentation (Van Soest,
1982).
The hypothetical mode of action proposed for the specific Aspergillus oryzae ␣-amylase
preparation may be applicable for fibrolytic exogenous enzymes as well. The comprehensive
hypothesis would be that exogenous enzymes hydrolyze complex carbohydrates (i.e., starch,
cellulose and xylans) into oligosaccharides (i.e., malto-, cello- and xylo-oligosaccharides)
that support cross-feeding in the rumen. The oligosaccharide cross-feeding hypothesis is
compatible with reports of improved total tract digestibility (Rode et al., 1999), pre-ingestive
(Hristov et al., 1998) and ruminal effects (Yang et al., 1999), feed-enzyme specificity
(Colombatto et al., 2003a), structural changes rendering the polymers more amenable to
degradation (Nsereko et al., 2000), increased bacterial attachment (Wang et al., 2001), stim-
ulation of ruminal microbial populations (Nsereko et al., 2002), and synergism between
exogenous and endogenous ruminal enzymes (Morgavi et al., 2000).
The oligosaccharide cross-feeding hypothesis is also attractive because it is consistent
with most of the controversial features described for exogenous enzymes in the literature.
First, increased reducing sugar concentrations resulting from exogenous enzyme supple-
mentation in the absence of ruminal microbes have been described in some instances
(Hristov et al., 1998), although it is not an absolute requirement and cannot fully explain
responses to enzyme supplementation (Beauchemin et al., 2004). Production of oligosaccha-
rides is a function of enzyme activity that does not necessarily result in increased reducing
sugar concentrations. Second, ruminal digestion is generally considered a first order kinetic
process with substrate availability, rather than enzyme concentration, as the limiting fac-
tor (Weimer, 1998). Production of oligosaccharides from polymers by exogenous enzyme
action would effectively increase substrate availability by exposing new sites for hydrolytic
attack in the polymer by polymer-degrading bacteria, and by exposing the oligosaccharides
for cross-feeding by microbes that would not normally have access to it. This increase in
substrate availability would also explain the increase in the initial rate of digestion reported
for fibrolytic enzyme supplements (Wallace et al., 2001; Colombatto et al., 2003b). Third,
low enzyme doses may be beneficial while high enzyme doses that increase overall ruminal
enzymatic activity are not required to obtain improvements in digestion and performance
(Beauchemin et al., 2004). Fourth, the cross-feeding mechanisms resulting from oligosac-
charide production by exogenous enzyme action may explain the increased production of

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propionate from fiber digestion or acetate and butyrate from starch digestion. This concept
agrees with the classic example of two-species microbial interaction for propionate pro-
duction from cellulose; whereas Fibrobacter succinogenes produces cellulose fragments
and succinate from cellulose that are in turn converted to acetate, propionate and carbon
dioxide by Selenomonas ruminantium (Van Soest, 1982). Finally, and most importantly,
the oligosaccharide cross-feeding hypothesis is consistent with the frequent occurrence of
quadratic responses to enzyme supplementation. Low exogenous enzyme concentrations
would not produce enough oligosaccharides for effective cross-feeding to occur while high
enzyme doses, or prolonged exposure to enzymes, would extensively hydrolyze polymers to
di- and mono-saccharides thereby failing to support an effective cross-feeding mechanism.
4. Conclusions
Dietary supplemental ␣-amylase from Aspergillus oryzae may improve ruminant
productivity by modifying ruminal starch digestion without necessarily increasing starch
digestion in the rumen. The proposed hypothetical mode of action for ␣-amylase involves
production of oligosaccharides from amylose and amylopectin that can be used by amy-
lolytic and non-amylolytic bacteria in cross-feeding mechanisms that modify the resulting
products of fermentation in the rumen. The hypothesis of oligosaccharide cross-feeding
is also consistent with various observations associated with exogenous fibrolytic enzymes
that have been reported.
Acknowledgements
The authors acknowledge the contributions to the collaborative research efforts of the
following individuals: A.M. Gehman, A.J. Heinrichs, C.M. Jones, S.I. Kehoe, J.M. DeFrain,
A.R. Hippen, K.F. Kalscheur, K.C. Hanson, D.L. Harmon, K.R. McLeod, S.M. Speight,
G.A. Harrison, A.E. Kozenski, L. Johnston, M.D. Abney, J.D. Rivera, J.J. Cranston, N.A.
Elam, J.F. Gleghorn, J.T. Richeson, and M.L. Galyean.
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