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Animal Feed Science and Technology
128 (2006) 96–107
Degree of starch access: An enzymatic method to
determine starch degradation potential of
corn grain and corn silage
H.M. Blasel, P.C. Hoffman
∗
, R.D. Shaver
Department of Dairy Science, University of Wisconsin-Madison, 266 Animal Science,
Madison, WI 53706, USA
Received 27 April 2005; received in revised form 19 August 2005; accepted 27 August 2005
Abstract
Starch supplied from corn grain or corn silage is an important source of dietary energy for lactating
dairy cows and other ruminants, but few laboratory methods are available to determine starch digestion
potential. A laboratory method, degree of starch gelatinization (DSG), commonly used by the food
industry to assess relative differences in starch characteristics of human foods was modified for
application to corn grain and corn silage. The modified assay, degree of starch access (DSA, g/kg
starch), was used to evaluate starch recovery by enzymatic hydrolysis in gelatinized undried, unground
corn grains and corn silages, which differed in particle size, dry matter content and endosperm type.
Effects of particle size (370, 500, 640, 1100, 3140 and 4000
m) of corn grains, which are known
to influence starch digestion in ruminants, were evaluated. For each 100
m increase in particle
size, DSA decreased (P < 0.001) 26.8 g/kg starch. In high-moisture corn grain, for each 10 g/kg fresh
matter increase in DM content, DSA decreased 20.0 g/kg starch (
r
2
= 0.76). In corn grain of differing
endosperm vitreousness (0 g/100 g
versus
100 g/100 g endosperm) DSA values were approximately
200 g/kg starch higher for corn grain with no vitreous endosperm as compared to corn grain with
highly vitreous (100 g/100 g) endosperm. For corn silage, DSA was positively correlated to latent
starch and starch retained on screens < 2.38 mm. While no comparisons between DSA and
in vivo
Abbreviations:
DSA, degree of starch access; DSG, degree of starch gelatinization; FM, fresh matter; PAF,
processing adjustment factors
∗
Corresponding author. Tel.: +1 715 387 2523; fax: +1 715 387 1723.
E-mail address:
pchoffma@facstaff.wisc.edu (P.C. Hoffman).
0377-8401/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.anifeedsci.2005.08.018
H.M. Blasel et al. / Animal Feed Science and Technology 128 (2006) 96–107
97
starch digestibility exist, the DSA assay may be useful as an index for evaluating differences in starch
digestion potential of corn and corn silage fed to ruminants.
© 2005 Elsevier B.V. All rights reserved.
Keywords:
Corn grain; Starch digestion; Vitreousness
1. Introduction
Starch supplied from corn and corn silage is an important source of dietary energy
for lactating dairy cows and other ruminants. However, various sources of corn starch have
highly variable ruminal and total tract digestibilities (
Ørskov, 1986; Theurer, 1986
). Factors,
such as particle size (
Remond et al., 2004
), partial gelatinization by heat processing (
Callison
et al., 2001
), conservation method (
Oba and Allen, 2003
) and type of corn endosperm
(
Correa et al., 2002
), can influence ruminal and or total tract digestion of corn starch by
lactating dairy cows. In an attempt to address some of these sources of variation,
NRC (2001)
suggested empirical processing adjustment factors (PAF) to adjust non-fiber carbohydrate
digestion coefficients for high-starch feeds. However, there is no system to measure or
estimate PAF on feedstuffs, and so, it is a subjective value that can be difficult to apply in
practice.
The food processing industry (
Varriano-Marston et al., 1980; Marconi et al., 2004
)
employs a relatively simple laboratory method entitled degree of cook or degree of starch
gelatinization (DSG) to assess physiochemical properties of high-starch human foods that
relate to their digestibility. This laboratory approach, to determine DSG, is quantifiable and
can be represented by the formula:
DSG (g
/
kg starch)
=
[recovered starch : cooked]
total starch
×
1000
The advantage of using a system, such as DSG, rather than PAF to adjust non-fiber carbohy-
drate digestion coefficients is that DSG is a defined and quantifiable laboratory procedure,
while PAF is subjective and empirical.
The objective of this study was to evaluate whether a methodology, such as DSG
could serve as an index to evaluate relative starch digestion potentials for corn or corn
silage.
2. Materials and methods
2.1. Pre-trial
Upon reviewing DSG methods (
Chiang and Johnson, 1977
,
Varriano-Marston et al.,
1980
,
Marconi et al., 2004
), we determined that changes in the assay would be required to
adapt DSG for livestock feeds as the DSG procedure was principally designed to measure
the effect of cooking on degree of starch gelatinization. Thus, DSG is typically determined
 98
H.M. Blasel et al. / Animal Feed Science and Technology 128 (2006) 96–107
on finely ground material (i.e., 1 mm grind), which eliminates effects of physical form
on DSG. Because physical form and DM content of starch are of critical importance in
ruminants (
Oba and Allen, 2003; Remond et al., 2004
), the volume of the assay was increased
to accommodate a larger sample, which is required to obtain a representative sample of
feeds that are not ground nor dried. Because corn silage and high-moisture corn grain are
fermented products with pH values often less than 5.0 and amylase or amyloglucosidase
used in the DSG procedure are pH specific (
McCleary et al., 1997
), additional buffer control
within the assay was required.
Therefore, the basic concept of DSG was redefined as degree of starch access (DSA) as:
DSG (g
/
kg starch)
=
[recovered starch : undried
/
unground]
×
1000
total starch
The term DSG was renamed DSA to define the assay in terms of the degree of enzyme
access to starch after gelatinization of undried and unground feeds.
2.2. Recovered starch procedure
H
2
O/986 ml H
2
O
and brought to pH 6.5 using 2N NaOH was added, flasks were fit with a loose fitting
rubber stopper or beakers were covered, placed on a heating unit, swirled frequently and
heated for approximately 25 min to 90
◦
C. Samples were removed from heat, 100 ml of
distilled water added and when temperatures reached 80
◦
C, 3 ml of a heat stable amylase
(Sigma A-3306) working solution containing 7200 units/ml was added. Sample solutions
were continuously stirred and allowed to cool to 50
◦
C. All stirring procedures were per-
formed on magnetic stir plates. When samples reached 50
◦
C, 100 ml of pH 4.2 acetate
buffer (
Ehrman, 1996
) and 50 ml of amyloglucosidase (Sigma A-3042) working solution
containing 60 units/ml were added. Sample flasks were stirred for 60 min, at which point
6 ml of hydrolysate was transferred via pipette into a 10 ml test tube containing 0.5 ml
of a solution containing 50 g of trichloroacetic acid mixed with 200 ml of distilled water,
which terminated the enzymatic reaction. Samples were stabilized by adding a 3.5 ml of pH
6.5 phosphate buffer (
Ehrman, 1996
), vortexed and free glucose determined with a YSI-
2000 (YSI Incorporated, Yellow Springs, OH, USA). Starch, expressed as recovered starch
g/kg DM, from glucose determination was calculated by the equations of
Ehrman (1996)
with recovered starch adjusted based on recovery potential of the reference corn starch.
Mean recovery potential for pure corn starch was 950 g/kg DM with a S.D. of 9.0 g/kg
DM.
The procedure developed to determine recovered starch on undried and unground corn-
based feed used 20 g of undried and unground corn silage (approximately 7 g of DM)
transferred to a 1000 ml Berzelius beaker. Alternatively, 4 g of dry or high-moisture corn
grain (2.8–3.6 g of DM), was weighed and transferred to a 1000 ml Erlenmeyer flask. A
reference sample of 2.5 g corn starch (Sigma, S-4126, St. Louis, MO, USA) was included
with each assay run to measure starch recovery potential and also used to correct recovered
starch determinations for the test samples (
Ehrman, 1996
). A 150 ml volume of distilled
water was added to each flask or beaker and swirled vigorously to ensure sample disper-
sion. A 200 ml volume of phosphate buffer containing 14.0 g NaH
2
PO
4
·
H.M. Blasel et al. / Animal Feed Science and Technology 128 (2006) 96–107
99
2.3. Total starch
Samples of corn-based feeds were dried for 48 h at 55
◦
C in a forced air oven, weighed,
determined for initial DM and ground through a Udy mill (Udy Corp., Boulder, CO, USA) fit
with a 1 mm screen. Final DM was determined by drying 1 g of 1 mm grind sample at 105
◦
C
for 3 h. Total starch was determined by methods of
Ehrman (1996)
, where gelatinization
is aided by sodium hydroxide and final glucose concentration is determined with an YSI-
2700 fit with a dextrose detection probe.
2.4. Corn grain and corn silage
m and bottom pan)
using U.S. Standard sieves (E.H. Sargent Co., Chicago, IL, USA) and mean particle size
was calculated according to
ASAE (1968)
.
To evaluate effects of corn grain DM content on DSA, 18 samples of high-moisture
shelled corn grain were obtained from routine submissions to the Marshfield Soil and Forage
Analysis Laboratory (Marshfield, WI, USA). Samples were evaluated for DM, DSA and
particle size as described previously. To evaluate effects of corn endosperm type on DSA, 17
dried corn samples of varying endosperm types were ground through a Willey mill fit with
an 8 mm screen and analyzed for DM, DSA and particle size as described previously. Corn
grain vitreousness was determined by manual dissection of kernels (
Dombrink-Kurtzman
and Bietz, 1993
) using the kernel selection and preparation scheme of
Correa et al. (2002)
.
Kernel vitreousness was chosen as the criterion to compare with DSA values, because
corn grains with a greater proportion of vitreous endosperm have been shown to have
decreased ruminal starch degradation (
Correa et al., 2002; Philippeau and Michalet-Doreau,
1997
).
To evaluate the utility of DSA in a heterogeneous feed, 44 corn silage samples were
obtained from routine submissions to the Marshfield Soil and Forage Analysis Laboratory
(Marshfield, WI, USA). Corn silages were analyzed for DM and DSA as previously
described with a 20 g of undried and unground samples used for the DSA determination.
Corn silage samples were ranked by DSA with every other (
n
= 22) sample selected for
subsequent particle size determination. Pre-screening a larger population (
n
= 44) of corn
silages and subsequent re-selection (
n
= 22) assured a robust set of corn silage samples for
evaluation. Particle size of starch in corn silages was determined by drying approximately
200 g of each corn silage sample for 48 h at 55
◦
C in a forced air oven. Dried corn silage
samples were placed into a vertical shaker (W.S. Tyler Incorporated, Mentor, OH, USA)
with nominal square apertures of 13.20, 9.50, 6.70, 4.75, 3.35, 2.36, 1.18, 0.60 and 0.30 mm
and shaken for 20 min. After shaking material remaining on each screen was weighed,
To evaluate the DSA assay, samples of corn grain and corn silage were prepared to
evaluate assay sensitivity to particle size, DM content, corn endosperm type and utility in
a heterogeneous feed (i.e., corn silage). To evaluate potential influences of particle size on
DSA, a single lot of dry shelled corn grain was ground through a Wiley mill (Arthur A.
Thomas Co., Philadelphia, PA, USA) fit with 1, 2, 4 or 8 mm screens. Additionally, two
samples of dry shelled corn grain were prepared by grinding through a Wiley mill without
a screen or left unground (whole dry shelled corn grain). All ground and whole corn grain
samples were dry sieved (sieve apertures: 4000, 2000, 841, 420, 177
100
H.M. Blasel et al. / Animal Feed Science and Technology 128 (2006) 96–107
ground through an Udy mill fit with a 1 mm screen and analyzed for residual DM by drying
for 3 h in a 105
◦
C oven and starch by the methods of
Ehrman (1996)
. Mean particle size of
starch was calculated as described by
ANSI (1993)
by using the amount of starch remaining
on each screen and the nominal dimension of the square aperture instead of the diagonal
dimension.
Recovered starch and total starch and the corresponding DSA were determined in qua-
druplicate for corn grains of differing endosperm type, particle size (dry corn grains) and
high-moisture corn grains. Duplicate evaluations of recovered and total starch and the cor-
responding DSA were conducted for corn silage. Mean DSA was calculated and expressed
as g/kg starch for all samples. The effect of particle size on DSA for dry corn grain was
evaluated using ANOVA with particle size as the independent variable. The effect of corn
DM content on DSA in high-moisture corn was evaluated using the GLM procedure of
SAS
(1999)
with DSA covariately adjusted for particle size with DM set as a random independent
variable. For corns of differing endosperm type, DSA values were adjusted for unavoidable
particle size differences created due to differences in grinding characteristics of corn grain
samples using a slope coefficient developed from the particle size evaluation and particle
size adjusted DSA values were compared to kernel vitreousness using the REG procedures
of
SAS (1999)
exploring first-, second- and third-order relationships. For corn silage cor-
relations between DSA and starch particle size, DM and total starch were explored using
the CORR procedures of
SAS (1999)
.
3. Results
3.1. Corn grain
Particle size influenced (P < 0.0001) DSA and ranged from 1068 to 15 g/kg starch for
corn grains ranging from 370 to 4000
m corn grain is
not possible because recovered starch should never be higher than total starch, indicating
that the value observed was affected by some error associated with total and recovered
starch determinations. A DSA value near 1000 g/kg starch for 370
m corn, is reasonable
and was expected because recovered starch and total starch were determined on the same
corn grain after grinding through a 1 mm screen. This suggests that recovered starch and
total starch procedures, although different, have similar starch hydrolysis potential. When
dry shelled corn grain samples were ground through screens of larger diameter (>370
m)
m), recovered starch decreased as proportion of total starch resulting
in declining DSA values. While we did not attempt to invoke linear particle size differences
in the corn grain samples, we did explore (
SAS, 1999
) first- and second-order relationships
of the data and observed that particle size effects on DSA were linear (
r
2
= 0.98). For each
100
m increase in particle size, DSA was reduced to 26.8 g/kg starch. Consistent with
observations of
Ehrman (1996)
, where enzymatic hydrolysis of starch to glucose in corn
decreased as particle size increased. The DSA value observed for >4000
m (whole) corn
grain was 15 g/kg starch indicating that the seed coat of dry shelled corn grain is a barrier to
enzymatic hydrolysis of intact kernel starch. Based on these observations, the DSA assay
m, respectively, indicating DSA is sensitive to corn
grain particle size of corn (
Fig. 1
). The DSA of 1068 g/kg starch for 370
or unground (>4000
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