2001 In vitro fermentation characteristics of native and processed cereal grains and potato

In vitro fermentation characteristics of native and processed cereal grains and

potato starch using ileal chyme from dogs

S. M. Murray*

, E. A. Flickinger*, A. R. Patil*, N. R. Merchen*, J. L. Brent, Jr.†,

and G. C. Fahey, Jr.*

*Department of Animal Sciences, University of Illinois, Urbana 61801 and †Department of Grain Science and

Industry, Kansas State University, Manhattan 66506

ABSTRACT:

Two in vitro experiments were con-

ducted to evaluate the ability of small intestinal bacte-
ria of dogs to ferment native and extruded cereal grains
and potato starch and cereal grain and potato flours.
Substrates included barley, corn, potato, rice, sorghum,
and wheat. In addition to testing native grains and
flours, extruded substrates also were tested. Substrates
were extruded at low temperatures (LT; 79 to 93

C) and

high temperatures (HT; 124 to 140

C) using a Wenger

extruder (model TX-52). Substrates varied widely in
concentrations of rapidly digestible starch (RDS),
slowly digestible starch (SDS), resistant starch (RS),
and total starch (TS). Extrusion of most substrates at
HT vs LT resulted in increased RDS and decreased RS
concentrations. Organic matter disappearance (OMD)

Key Words: Dogs, Extrusion, Fermentation, Small Intestine, Starch



J. Anim. Sci. 2001. 79:435–444

Introduction

Dogs, like most nonruminant animals with the excep-

tion of the horse, do not rely on fermentation as a major
strategy for digestion of nutrients supplied in their
diets. Although fermentation is primarily a colonic
event, studies (Ruseler-van Embden et al., 1992; Zen-
tek, 1995) suggest that a significant bacterial popula-
tion resides in the small intestine of dogs. This discov-
ery has prompted further investigation into determin-
ing both the beneficial and detrimental effects of these
bacteria and their relative importance to small intesti-
nal function. Several investigators (Batt et al., 1983;
Willard et al., 1994) have documented the overgrowth
of bacteria causing damage in the small intestine of
German shepherds. However, research also has docu-
mented the incidence of small intestinal bacterial over-
growth (SIBO) occurring in the small intestine of

Present address: The Iams Company, Lewisburg, OH 45338-0189.

Correspondence: E-mail: g-fahey@uiuc.edu.

Received March 15, 2000.
Accepted September 29, 2000.

435

values attributed to microbial fermentation for a 5-h
period were as high as 27% for native extruded sub-
strates (LT potato starch) and 39% for potato flour.
Average OMD was higher for cereal and potato flours
than for native extruded substrates (29.9 vs 25.4%).
Average molar percentages of short-chain fatty acids
produced from all substrates fermented for 5 h were
73, 14, and 13% (acetate, propionate, and butyrate, re-
spectively). Average lactate production for substrates
ranked as follows: flours

native and extruded cereal

grains and potato starch (0.33 and 0.18 mmol/g OM,
respectively). In vitro microbial fermentation of
starches by ileal bacteria can be substantial and is af-
fected by differences in starch source, fraction, and pro-
cessed form.

healthy, adult beagles (Batt et al., 1992) and healthy,
domestic short-hair cats (Johnston et al., 1993).

Most components of cereal grains and potato are di-

gested primarily via small intestinal enzymatic degra-
dation. It has not been well documented whether small
intestinal fermentation plays a significant role in their
degradation. Therefore, two in vitro experiments were
conducted to evaluate the fermentability of cereal
grains and potato (native and processed) as measured
by the disappearance of OM after 5 h and the production
of short-chain fatty acids and lactic acid by ileal mi-
croflora of dogs.

Experimental Procedures

All experiments were conducted under protocols ap-

proved by the Campus Animal Care Advisory Commit-
tee, University of Illinois, Urbana.

Small Intestinal Bacteria.

To verify the presence of a

bacterial population in the small intestine of the dog,
fresh ileal fluid was collected from five ileally cannu-
lated dogs with hound bloodlines, an average weight of
31

5 kg, and age of 3 yr

6 mo. Dogs, prior to sample

Murray et al.

436

collection, were surgically implanted with an ileal can-
nula. Ileal cannulation was conducted according to
Walker et al. (1994). Surgical and animal care proce-
dures for the experiment were conducted under a re-
search protocol approved by the Campus Animal Care
Advisory Committee, University of Illinois, Urbana.
Dogs were allowed a minimum of 2 wk of recovery before
collection of samples. The dogs were housed in 1.2-

3.1-

m clean floor pens in a temperature-controlled room. A
total of five ileal effluent samples were collected, one
(approximately 15 mL) from each individual dog over
a 15-min time period, via attachment of a Whirlpak
bag (Pioneer Container Corp., Cedarburg, WI). Samples
then were pooled, serially diluted with diluent (Bryant
and Burkey, 1953), and plated within 1 h of collection
to enumerate specific bacteria. Total anaerobes and aer-
obes were enumerated using a 40% ruminal fluid agar
according to Bryant et al. (1961) and Mackie et al.
(1978). Bifidobacterium species were enumerated using
a selective agar according to Munoa and Pares (1988).
Lactobacilli were cultured on Rogosa SL agar (Difco
Laboratories, Detroit, MI). Clostridium perfringens was
enumerated on a tryptose-sulfite-cycloserine (TSC)
agar with egg yolk (FDA, 1992). Counting precision of
the microbiota was maximized by inoculating drops of
seven different dilutions onto their respective plates.
After adsorption of the droplets, the plates were in-
verted and incubated either anaerobically or aerobically
at 38

C for 48 h. Colony counts were made after incuba-

tion to determine colony-forming units (cfu) per millili-
ter of ileal effluent. A colony-forming unit was defined as
a distinct colony measuring at least 1 mm in diameter.
Colony-forming units per milliliter of sample were cal-
culated as follows:

cfu/ml

(mean cfu)

(diluent dilution)

(mL of sample)

(mL in drop plated)

In Vitro Experiments.

Two separate in vitro experi-

ments were conducted. Experiment 1 focused on the
fermentation characteristics of ileal microflora when
exposed to different forms (native, low- and high-tem-
perature extruded) of grains and potato starch. Experi-
ment 2 focused on the fermentation characteristics of
ileal microflora when exposed to different cereal grain
and potato flours. Both experiments involved the use
of ileal effluent as the source of inoculum.

Experiment 1

Substrates.

Substrates included five cereal grains

(barley, corn, rice, sorghum, and wheat), purchased
from local feed vendors, and one tuber starch (potato;
AVEBE, Holland). In addition to testing native forms,
substrates were modified using extrusion. Substrates
were ground through a 2-mm screen in a hammer mill
prior to extrusion. Substrates were extruded at low
temperatures (LT; 79 to 93

C) and high temperatures

(HT; 124 to 140

C) using a Wenger extruder (model

TX-52; Sabetha, KS), housed in the extrusion facility of
the Department of Grain Science and Industry, Kansas
State University, Manhattan. Native substrates were
not extruded. Temperatures were achieved by a combi-
nation of mechanical energy conversion into heat, sub-
sequent barrel heat transfer, and injection of steam
through the barrel jacket and monitored through indi-
vidual barrel head temperatures. Temperatures re-
ported are extruder discharge temperatures measured
at the die using a flush-mount transducer. End-product
temperature at the die was measured a minimum of
four times to ensure temperature stability before col-
lecting extruded substrate samples. Extruded sub-
strates were cut into 2.54-cm pieces to facilitate rapid
drying. The pieces were dried on a Wenger belt dryer
at 82.2

C for 30 min and air-dried overnight. Final

moisture content of the substrates was approximately
6%. Dried, extruded substrates then were ground
through a 2-mm screen in a Wiley mill in preparation
for chemical analyses as well as in vitro fermentation.

Chemical Analyses.

Substrates were analyzed for DM,

OM, ash, and Kjeldahl N according to AOAC (1984)
procedures. Total lipid content was determined by acid
hydrolysis followed by ether extraction according to
AACC (1983) and Budde (1952).

Starch fractions—rapidly digestible starch (RDS),

slowly digestible starch (SDS), and resistant starch
(RS)—of samples were determined using the methods
of Muir and O’Dea (1992, 1993). Corn, potato, and amy-
lomaize starches were used as standard substrates.
Standards were included as part of each fractionation
method to validate the efficacy of experimental con-
ditions.

Starch fractions were determined using a modifica-

tion of the Muir and O’Dea (1992, 1993) starch fraction-
ation technique. Approximately 0.1 g of substrate was
suspended in 1 mL of pepsin (Sigma Chemical Co., St.
Louis, MO) solution (1g/L; pH was adjusted to 2.0 with
HCl) and incubated for 30 min at 37

C. The solution

then was neutralized with 0.5 M NaOH (0.5 mL). Five
mL of 0.2 M sodium acetate (pH was adjusted to 5.0
with glacial acetic acid) and 1 mL of enzyme solution
containing 10 mg of α-amylase (Sigma Chemical Co.)
and 28 U of amyloglucosidase (Sigma Chemical Co.)
was dissolved in the sodium acetate buffer (pH 5.0) and
added. Samples were incubated at 37

C in a shaking

water bath for 2.5 and 15 h. After the appropriate incu-
bation time, samples were centrifuged at 3,000

g for

10 min and the supernate was removed. The pellet was
washed three times by resuspending the pellet with 1.5
mL of sodium acetate buffer (pH 5.0) and centrifuging
(10 min, 3,000

g). All supernates from washings were

pooled with the original supernate. Computation of the
concentration of this fraction allows for the prediction
of digestion that would occur in the small intestine (i.e.,
digestible starch). Samples analyzed for glucose at 2.5
and 15 h represent concentrations of RDS and SDS
present in the original substrate, respectively.

Ileal fermentation of starch by the dog

437

The washed pellet was lyophilized; this material was

considered the RS component of the sample. The pellet
was resuspended in 5 mL of DMSO (dimethyl sulfoxide)
and incubated in a boiling water bath for 30 min. Then,
20 mL of 0.15 N sodium acetate buffer (pH adjusted to
4.5 with glacial acetic acid) was added and incubated
in a boiling water bath for 20 min. Samples then were
autoclaved for 1 h at 15 psi and 121

C. Samples were

allowed to cool to room temperature before addition of
10 mL of amyloglucosidase solution containing 580 U
of amyloglucosidase dissolved in water. Samples were
incubated for 24 h at 55

C (with occasional vortexing),

then centrifuged for 10 min at 10,000 to 15,000

The supernate was removed for glucose analysis. After
starch digestion (hydrolysis), the released glucose was
measured by a glucose oxidase method (Glucose Test
Kit 510-A; Sigma Chemical Co.). Glucose concentration
was determined by reading the absorbance of individual
samples at 450 nm on a DU 640 spectrophotometer
(Beckman Instruments, Schaumburg, IL) and compar-
ing those values against a glucose standard curve. Amy-
lomaize (Opta Food Ingredients, Cambridge, MA), corn-
starch and potato starch (Sigma Chemical Co.) were
used as standard substrates. A complete database on
these standards is available, so these were included
with each assay as a quality control measure.

For all laboratory analyses, samples were analyzed

in duplicate, and analyses were repeated if a deviation
greater than 5% between duplicates occurred. Total
starch content of samples was determined using the
method of Thivend et al. (1972).

Donors and Collection Methods.

Five purpose-bred,

mature (4 yr

2) ileally cannulated female dogs with

an average weight of 25

5 kg were given ad libitum

access twice daily to a commercially available diet (Dia-
mond Petfoods, Meta, MO ) containing approximately
21% CP and 12% fat for 14 d before collection of ileal
effluent. Dogs were housed in 1.2-

3.1-m clean floor

pens in a temperature-controlled room and were given
free access to water at all times. Fresh ileal fluid (ap-
proximately 15 mL) was collected from each dog for 15-
min intervals in a Whirlpak bag (Pioneer Container
Corp., Cedarburg, WI) until sufficient amounts needed
to inoculate all tubes were obtained. At the end of each
15-min period, bags were removed and replaced with
new ones. Bags containing samples were sealed imme-
diately after expressing excess air, placed inside a pre-
warmed insulated container (37

C), and transported to

the laboratory for processing.

Medium Composition and Substrate Fermentation.

The composition of the medium used to culture the ileal
microflora is listed in Table 1. All medium components
except the vitamin mixes were added before autoclav-
ing. The vitamin mixes were aseptically added after
they were filter-sterilized.

Upon arrival in the laboratory, fresh ileal samples

were immediately pooled under anaerobic conditions
and diluted 1:10 (vol/vol) in a 39

C anaerobic dilution

solution (Bryant and Burkey, 1953) by blending it for

Table 1.

Composition of microbiological medium used

in the in vitro experiments

Component

Amount

−

mL/L

−

Solution A

330.0

Solution B

330.0

Trace mineral solution

10.0

Water-soluble vitamin mix

20.0

Folate-biotin solution

5.0

Riboflavin solution

5.0

Hemin solution

5.0

Short-chain fatty acid mix

0.4

Resazurin

1.0

Distilled water

296.0

−

g/L

−

Yeast extract

0.5

Trypticase

0.5

4.0

Cysteine HCl

ⴢH

0.5

Composition (g/L): NaCl, 5.4; KH

, 2.7; CaCl

ⴢH

O, 0.16;

MgCl

ⴢ6H

O, 0.12; MnCl

ⴢ4H

O, 0.06; CoCl

ⴢ6H

O, 0.06; (NH

)

5.4.

Composition: K

HPO

, 2.7 g/L.

Composition (mg/L): EDTA (disodium salt), 500; FeSO

ⴢH

O, 200;

ZnSO

ⴢ7H

O, 10; MnCl

ⴢ4H

O, 3; H

, 30; CoCl

ⴢ6H

O, 20;

CuCl

ⴢH

O, 1; NiCl

ⴢ6H

O, 2; Na

MoO

ⴢ2H

O, 3.

Composition (mg/L): thiamin-HCl, 100; pantothenic acid, 100; nia-

cin, 100; pyridoxine, 100; p-aminobenzoic acid, 5; vitamin B

, 0.25.

Composition (mg/L): folic acid, 10; biotin, 2; NH

HCO

, 100.

Composition: riboflavin, 10 mg/L in 5 mM HEPES.

Composition: hemin, 500 mg/L in 10 mM NaOH.

Composition: 250 mL/L each of n-valerate, isovalerate, isobutyr-

ate, and

-α-methylbutyrate.

Resazurin, 1 g/L in distilled water.

10 s in a Waring blender. Blended, diluted ileal effluent
was filtered through four layers of cheesecloth and fil-
trate was sealed in 125-mL serum bottles under CO

Appropriate sample and blank tubes containing 26 mL
of medium and 300 mg of substrate were aseptically
inoculated with 4 mL of diluted ileal effluent. Tubes
were flushed with CO

and capped with stoppers

equipped with one-way gas release valves (Nalge Nunc
International, Rochester, NY). Blank tubes contained
4 mL of inoculum and 26 mL of medium but did not
contain any substrate.

Triplicate tubes were incubated in a forced-air oven

at 39

C with periodic mixing for the 5-h fermentation

time period. At the appropriate time, tubes were re-
moved from the incubator and processed immediately.
A 2-mL aliquot was removed from each tube for short-
chain fatty acid (SCFA) and lactate analyses. The re-
maining 28 mL was combined with 112 mL of 95%
ethanol and allowed to set for 1 h to precipitate the
soluble polysaccharide fractions. To recover unfer-
mented residues, samples were filtered through tared
Whatman 541 filter paper and washed sequentially
with 78% ethanol, 95% ethanol, and acetone. Samples
then were dried at 105

C, weighed, ashed in porcelain

crucibles (500

C), and weighed again to determine or-

ganic matter disappeaerance (OMD). In vitro OMD
(percentage) was calculated as (1

−

(OM residue

−

Murray et al.

438

blank)/ original OM)

100, where OM residue is the

OM recovered after 5 h of fermentation, OM blank is
the OM recovered in the corresponding blank after the
same fermentation times, and original OM is the OM
of the substrate placed in the tube.

The 2-mL aliquot of fluid removed from the sample

tubes for SCFA and lactate analyses was immediately
added to 0.5 mL of 25% metaphosphoric acid, precipi-
tated for 30 min, and centrifuged at 20,000

g for 20

min. The supernate was decanted and frozen at

−

in microfuge tubes. After freezing, the supernate was
thawed and centrifuged in microfuge tubes at 10,000

g for 10 min. Concentrations of acetate, propionate,
and butyrate were determined in the supernate using
a Hewlett-Packard 5890A Series II gas-liquid chroma-
tograph and a glass column (180 cm

4 mm i.d.) packed

with 10% SP-1200/1% H

on 80/100 mesh Chro-

mosorb WAW (Supleco, Bellefonte, PA). Short-chain
fatty acid concentrations also were corrected for by
blank tube production of SCFA. The supernates also
were analyzed for lactate concentration by the spectro-
photometric method described by Barker and Sum-
merson (1941).

Statistical Analysis.

The General Linear Models pro-

cedures of SAS (SAS Inst. Inc., Cary, NC) were used to
analyze data from this experiment. The experimental
design was a 5

6 factorial arrangement of sub-

strates (six substrates with three treatments per sub-
strate;

potato

had

two

treatments).

Therefore,

substrate, treatment, and substrate

treatment were

used in the statistical model. Arithmetic means are
reported along with the SEM for all treatments. When
significant (P

0.05) differences were detected, individ-

ual means were compared with the least significant
difference (LSD) method of SAS.

Experiment 2

Substrates.

Substrates included six cereal grain and

potato flours (barley, corn, potato, rice, sorghum, and
wheat) purchased from a local vendor and prepared by
fine milling, sieving, and steam processing. Flours were
not extruded.

Chemical Analyses.

These were the same as those

described for Exp. 1.

Donors and Collection Methods.

The same number of

donors and methods of collection were used as in Exp. 1.

Medium and Substrate Fermentation.

Identical meth-

ods for medium preparation and in vitro fermentation
as reported for Exp. 1 were implemented.

Statistical Analysis.

Substrates were compared using

the General Linear Models procedures of SAS (SAS
Inst. Inc.) for a completely randomized design. Arithme-
tic means are reported along with the SEM for all treat-
ments. When significant (P

0.05) differences were

detected, individual means were compared with the
least significant difference method of SAS.

Results and Discussion

Substrates.

Substrates used in the present in vitro

experiments consisted of five cereal grains (barley, corn,
rice, sorghum, and wheat) and one tuber (potato starch),
either as native, ground materials; low- or high-temper-
ature extruded materials; or cereal grain and potato
flours. Three standard substrates (amylomaize, corn-
starch, and potato starch) were included as substrates
in the in vitro experiments.

Small Intestinal Bacteria.

Fermentation in most non-

ruminant species has been attributed to bacterial popu-
lations residing in the colon. However, bacteria popu-
late the small intestine of dogs as well (Ruseler-van
Embden et al., 1992). Over 25 different species of bacte-
ria were identified in ileal chyme of beagle dogs. Bacte-
ria included pathogenic species such as Clostridium
perfringens and Escherichia coli, and beneficial bacteria
such as Bifidobacteria and Lactobacilli. These research-
ers did not assess fermentation characteristics of the
small intestinal bacteria. In the present study, we found
the following colony-forming units per milliliter of ileal
effluent (approximately 15% DM) after isolation and
plating: total anaerobes, 4.2

; total aerobes, 7.1

; Escherichia coli, 1.3

; Clostridium perfringens,

1.7

; Bifidobacteria, 1.8

; and Lactobacillus,

3.3

. These concentrations were similar to values

reported by Ruseler-van Embden et al. (1992) and con-
firm that there is a substantial population of bacteria
that reside in the small intestine of dogs.

Experiment 1

Chemical Composition.

The chemical composition of

substrates is presented in Table 2. Dry matter concen-
trations were similar among sources, regardless of pro-
cessing condition, and ranged from 90% (LT potato
starch) to 94.9% (HT barley). Organic matter concentra-
tions differed by approximately 3 percentage units
among substrates. Crude protein content varied widely
among substrates, ranging from 0.2% (potato starch)
to 17.2% (barley). Concentrations of fat were highest,
on average, for corn substrates (4.6%) and lowest for
potato starch substrates (0.6%). In general, DM, OM,
and fat concentrations remained the same after extru-
sion processing. However, extrusion processing had an
influence on CP concentrations; as temperature in-
creased, CP concentrations decreased by approximately
2 percentage units for barley, rice, sorghum, and wheat
compared to their native counterparts. This reason for
this decrease is not clear.

Starch Fractions.

Concentrations of starch fractions

of substrates (expressed as a percentage of DM and as
a percentage of TS) are presented in Table 3. Starch in
cereal grains varied widely in concentrations of RDS,
SDS, and RS. Rapidly digestible starch concentrations
for native, LT, and HT treatments were highest for rice
(36.9%), LT potato starch (65.4%), and HT rice (76.1%),
respectively. Lowest values were 15.5% for wheat (na-

Ileal fermentation of starch by the dog

439

Table 2.

Chemical composition of native and extruded

cereal grains and potato starch (% DM basis)

Substrate

Fat

Barley

93.7

97.1

17.2

3.0

LT barley

93.6

97.2

15.7

3.6

HT barley

94.9

97.0

15.2

3.5

Corn

93.7

98.5

9.5

4.8

LT corn

94.0

98.5

9.5

3.7

HT corn

93.2

98.6

9.2

5.2

Potato starch

91.2

99.6

0.2

0.6

LT potato starch

90.0

99.6

0.6

1.0

HT potato starch

Rice

91.9

99.0

10.3

2.3

LT rice

94.3

99.0

9.9

2.4

HT rice

93.6

98.7

7.5

2.2

Sorghum

93.7

98.4

10.8

3.7

LT sorghum

94.1

98.4

10.2

3.4

HT sorghum

93.6

98.6

9.4

4.0

Wheat

93.0

97.7

15.0

3.0

LT wheat

93.6

97.9

13.8

3.2

HT wheat

94.2

98.0

14.0

3.3

low-temperature extrusion: barley, 83

C; corn, 83

C; potato

starch, 94

C; rice, 79

C; sorghum, 84

C; wheat, 86

high-temperature extrusion: barley, 135

C; corn, 140

C; rice,

124

C; sorghum, 145

C; wheat, 143

Not determined.

tive) and 30.3 and 47.8% for barley (LT and HT, respec-
tively). Concentrations of RDS in processed substrates,
expressed as a percentage of TS, were much higher (62

Table 3.

Starch fractions of native and extruded cereal grains

and potato starch (% DM basis)

Sample

% RDS

% SDS

% RS

Thivend

Barley

23.2 (45.0)

11.4 (22.0)

17.0 (33.0)

51.6

53.0

LT barley

30.3 (62.3)

13.5 (27.8)

4.8

(9.9)

48.6

46.7

HT barley

47.8 (82.1)

4.4

(7.6)

6.0 (10.3)

58.2

52.0

Corn

34.6 (47.5)

14.6 (20.1)

23.6 (32.4)

72.8

74.9

LT corn

54.2 (73.5)

13.1 (17.8)

6.4

(8.7)

73.7

70.9

HT corn

65.0 (87.6)

7.8 (10.5)

1.4

(1.9)

74.2

70.4

Potato starch

24.4 (28.0)

2.6

(3.0)

60.0 (68.9)

86.9

89.1

LT potato starch

65.4 (69.1)

27.0 (28.6)

2.2

(2.3)

94.6

92.6

HT potato starch

Rice

36.9 (45.6)

17.0 (21.1)

26.9 (33.3)

80.8

81.1

LT rice

57.7 (65.7)

22.9 (26.0)

7.3

(8.3)

87.9

75.3

HT rice

76.1 (90.1)

4.5

(5.3)

4.0

(4.7)

84.6

78.0

Sorghum

27.3 (36.8)

13.0 (17.5)

33.8 (45.6)

74.1

75.1

LT sorghum

49.1 (65.1)

10.9 (14.4)

15.4 (20.5)

75.4

76.6

HT sorghum

70.0 (90.3)

5.4

(7.0)

2.1

(2.7)

77.5

65.3

Wheat

15.5 (25.0)

33.6 (54.1)

13.0 (20.9)

62.1

65.5

LT wheat

54.6 (81.9)

6.0

(9.0)

6.1

(9.1)

66.7

76.6

HT wheat

65.5 (91.9)

5.2

(7.3)

0.6

(0.8)

71.3

66.0

Rapidly digestible starch.

Slowly digestible starch.

Resistant starch.

Total starch

RDS

SDS

RS.

Total starch as measured by the method of Thivend et al. (1972).

low-temperature extrusion: barley, 83

C; corn, 83

C; potato starch, 94

C; rice, 79

C; sorghum, 84

wheat, 86

high-temperature extrusion: barley, 135

C; corn, 140

C; rice, 124

C; sorghum, 145

C; wheat, 143

Not determined.

Values in parentheses are individual fractions expressed as a percentage of TS.

to 92%) than those in native substrates (25 to 48%).
Substrates extruded at HT contained considerably
higher concentrations of RDS (69 to 92%) than those
extruded at LT (62 to 82%).

Substrates contained moderate concentrations of

starch in the SDS form. The most notable exception
was the native wheat, which contained approximately
54% SDS when expressed as a percentage of TS.

Resistant starch concentrations were highest for na-

tive potato starch (60%) and lowest for HT wheat (0.6%).
As a percentage of TS, most native substrates contained
higher concentrations of RS (21 to 69%) than processed
substrates (1 to 21%). Slowly digestible starch and RS
fractions decreased substantially when substrates were
extruded at HT compared to their native counterparts.

Total starch of substrates obtained from the summa-

tion of fractions was compared to values determined
using the method of Thivend et al. (1972). Values ob-
tained using the two methods agreed closely for most
samples. However, a few samples (LT rice, HT rice, HT
sorghum, and LT wheat) did not agree as well. A possi-
ble explanation for the higher concentration of TS for
LT wheat using the method of Thivend et al. (1972)
may be the inclusion of sucrose in the measurement.
This method enzymatically converts sucrose into mono-
saccharides and allows for their recovery and measure-
ment in the supernate. The method of Muir and O’Dea
(1992, 1993) does not convert sucrose to monosaccha-
rides and, thus, it is not part of the starch component.

Murray et al.

440

Although not analyzed for sucrose content, LT wheat
may have contained a sufficient level of sucrose to result
in the lower TS value when analyzed using the method
of Muir and O’Dea (1992; 1993). The differences noted
for LT rice, HT rice, and HT sorghum are unclear, but
may be related to interference in enzyme activity by
the fiber (e.g., HT sorghum) or some other component
of these ingredients.

Establishment of Ileal Fermentation Times.

The 5-h

fermentation time used was based on several factors:
1) the total transit time of food residues through the
gastrointestinal tract of the dog is approximately 24 h
(Fahey et al., 1990); 2) we assumed that retention time
of ingested food particles is equal in the three major
segments of the gastrointestinal tract (stomach, small
intestine, and large intestine); and 3) the earliest expo-
sure of ingested food particles to small intestinal bacte-
ria would be approximately 8 h. Smeets-Peeters et al.
(1998) reported estimated times of 8 and 16 h for 90%
of liquids and solids (2-mm particles), respectively, to
be emptied from the small intestine of dogs after ingest-
ing a meat-based diet. To our knowledge, no documenta-
tion exists describing the time of first contact within
the small intestine between ingested food particles and
resident microflora. Therefore, 5 h was chosen as the
time chyme in the small intestine would potentially be
exposed to bacteria at significant enough levels to have
a measureable impact on ingested food particles. Also,
a pilot study conducted in our laboratory showed little
difference in values obtained at 5 vs 7.5 h.

Organic Matter Disappearance and Organic Acid Pro-

duction.

Table 4 reports the OMD of substrates and the

SCFA and lactate production data for Exp. 1. Substrate

treatment interactions for all fermentation character-

istics except lactate production were significant at P

0.05. Lactate production tended (P

0.09) to be different

among treatments (differences among mean values

three times the SEM are statistically different).

Among the native substrates, OMD was highest (P

0.05) for corn (14.5%) compared to potato starch, rice,

and sorghum (0, 1.5, and 4.0%, respectively). Potato
starch (27.0%) and wheat (25.0%) had the highest (P

0.05) OMD among LT extruded substrates. Barley,
corn, and sorghum had higher (P

0.05) OMD values

when extruded at HT vs LT. The greatest numeric in-
creases in OMD among treatments were observed for
native vs LT potato starch, for which an increase of 27
percentage units was noted as a result of extrusion.

Fermentation of native rice resulted in the greatest

0.05) acetate production (3.82 mmol/g OM) among

native substrates. Potato starch (4.56 mmol/g OM) was
higher (P

0.05) in acetate production than barley,

corn, and sorghum (3.67, 3.31, and 3.68 mmol/g OM,
respectively) extruded at LT. High-temperature wheat
(5.38 mmol/g OM) resulted in higher (P

0.05) acetate

production than all other substrates except HT rice.
Barley and wheat extruded at HT resulted in higher
(P

0.05) acetate values after 5 h of fermentation than

those extruded at LT. The largest increase in total ace-

tate produced among substrates was observed for na-
tive potato starch (threefold increase after extrusion
at LT).

The highest (P

0.05) propionate production for na-

tive substrates was noted for rice (0.92 mmol/g OM).
For the LT substrates, corn (0 mmol/g OM) was lower
(P

0.05) in propionate production than all other sub-

strates. Propionate production values for HT extruded
substrates were lowest (P

0.05) for HT corn (0 mmol/

g OM). Propionate production values for rice, sorghum,
and wheat substrates extruded at HT were higher (P

0.05) compared to their LT extruded counterparts.

Butyrate production for the native substrates was

greatest (P

0.05) for rice (0.71 mmol/g OM), intermedi-

ate for barley, corn, and sorghum (0.36, 0.42, and 0.36
mmol/g OM, respectively), and lowest (P

0.05) for

potato starch and wheat (0.07 and 0.16 mmol/g OM,
respectively). Butyrate production for LT extruded sub-
strates was higher (P

0.05) for LT rice (0.96 mmol/g

OM) than for LT barley, corn, and sorghum (0.60, 0.62,
and 0.63 mmol/g OM, respectively). High-temperature
extruded substrates had the highest (P

0.05) butyrate

values for HT wheat (1.25 mmol/g OM) and HT rice
(1.18 mmol/g OM). Comparison of LT and HT values
revealed that barley, rice, and wheat resulted in greater
(P

0.05) production of butyrate at HT than at LT.

Among native substrates, rice (5.45 mmol/g OM) had

the greatest (P

0.05) and potato starch (1.64 mmol/g

OM) the lowest total SCFA production values. Total
SCFA production was lowest (P

0.05) for LT corn

among all LT substrates. Data for HT substrates re-
vealed that the largest (P

0.05) total production of

SCFA was observed for HT wheat (8.13 mmol/g OM)
compared to all other substrates except HT rice. Com-
paring LT and HT data revealed that total SCFA pro-
duction was greater (P

0.05) for substrates extruded

at HT vs LT for wheat only.

Lactate production tended (P

0.09) to be different

among substrates. Native potato starch resulted in lit-
tle lactate production. Lactate production for LT sub-
strates was greatest (P

0.05) for LT corn (0.36 mmol/

g OM). Fermentation of barley and wheat extruded at
HT produced more (P

0.05) lactate compared to their

LT counterparts.

Experiment 2

Chemical Composition.

The chemical composition of

cereal grain and potato flours is presented in Table 5.
Dry matter concentrations were similar among flours,
with the exception of the potato flour, which was ap-
proximately 2 percentage units higher. Organic matter
concentrations were highest for corn, rice, sorghum,
and wheat, barley was intermediate, and potato was
lowest in OM content. Crude protein concentrations
were approximately 28% higher for barley and wheat
flours than for potato, rice, and sorghum and were ap-
proximately 123% higher than those for corn flour. Fat
concentrations were similar among flours except for

Ileal fermentation of starch by the dog

441

Table 4.

Organic matter disappearance (OMD) and acetate (ACE), propionate (PRO),

butyrate (BUTY), total short-chain fatty acid (SCFA), and lactate production

following 5 h of in vitro fermentation of native and extruded cereal

grains and potato starch with canine ileal fluid

(Exp. 1)

ACE,

PRO,

BUTY,

Total SCFA,

Lactate,

OMD,

mmol/g

Substrate

Barley

Native

10.0

2.66

0.59

0.36

3.61

0.07

12.5

3.67

0.98

0.60

5.25

0.14

25.0

4.27

0.85

0.96

6.08

0.27

Corn

Native

14.5

2.26

0.45

0.42

3.13

0.10

14.0

3.31

0.62

3.93

0.36

24.0

3.08

0.60

3.68

0.32

Potato starch

Native

1.46

0.11

0.07

1.64

0.01

27.0

4.56

1.42

0.88

6.86

0.16

Rice

Native

1.5

3.82

0.92

0.71

5.45

0.05

17.5

4.47

1.27

0.96

6.70

0.19

19.5

4.74

1.53

1.18

7.45

0.28

Sorghum

Native

4.0

2.67

0.67

0.36

3.70

0.05

9.0

3.68

1.30

0.63

5.61

0.19

21.5

4.13

1.61

0.73

6.47

0.26

Wheat

Native

9.5

2.17

0.52

0.16

2.85

0.04

25.0

4.19

1.15

0.94

6.28

0.21

24.5

5.38

1.50

1.25

8.13

0.37

SEM

1.9

0.24

0.08

0.06

0.36

0.04

The interaction of substrate

treatment was significant (P

0.05) for OMD, ACE, PRO, BUTY, total

SCFA, and (P

0.09) for lactate.

See Table 2 for a description of treatments.

Potato starch was not extruded at HT.

barley, which was approximately 2 percentage units
higher.

Grain flours commonly have a much finer consistency

than their native counterparts, mainly due to the pro-
cessing techniques used to prepare them. According to
Hoseney (1994), grain flours are made up primarily of
two components, protein and starch, whereas ground
grains can contain the pericarp, aleurone layers, and
germ portions of the grain, thus providing additional
components such as lipid, fiber, and ash. Except for
potato, DM content of flours was typically 2 to 3 percent-
age units lower than for ground grains. Again, except
for potato, the OM content of flours was 2 to 3 percent-
age units higher than that of ground grains. Crude

Table 5.

Chemical composition of cereal grain and

potato flours (% DM basis)

Substrate

Fat

Barley

91.9

97.2

11.9

4.4

Corn

90.1

99.4

5.6

3.2

Potato

93.5

95.5

9.8

1.6

Rice

90.8

99.4

8.2

2.8

Sorghum

90.9

99.5

9.2

2.6

Wheat

90.5

99.3

13.1

2.6

protein concentrations of flours differed widely com-
pared to their ground grain counterparts. The most
notable CP difference was the 49-fold difference be-
tween potato starch (0.2%) and potato flour (9.8%). Pro-
cessing of potato flour can include the skin of the potato,
which may contribute to the CP content of the flour.
Fat content of the flours differed slightly compared to
ground grains.

Starch Fractions.

Table 6 reports the starch fraction

concentrations contained in the flours. Flours varied
widely in the percentage of starch found in each of the
three fractions. Total starch content of the flours was
similar for corn, rice, and sorghum; these sources were
approximately 13% higher in starch than potato and
wheat and approximately 40% higher than barley,
which was lowest in total starch content. These starch
concentrations closely parallel values reported from de-
termination of TS using the method of Thivend et al.
(1972). Flours contained approximately 97% of their
total starch as RDS and SDS combined. Even potato
flour, which had been reported to contain high levels
of RS (Englyst et al., 1992), contained a majority of its
starch as RDS. This is perhaps the result of reorganiza-
tion of its starch moiety due to the steam processing
used to make the flour. However, sorghum and wheat

Murray et al.

442

Table 6.

Starch fractions of cereal grain and potato flours (% DM basis)

Starch fraction

Flour

RDS

SDS

Thivend

Barley

53.9 (91.7)

3.2

(5.4)

1.7 (2.9)

58.8

61.0

Corn

80.1 (91.4)

4.9

(5.6)

2.6 (3.0)

87.6

88.3

Potato

75.5 (93.2)

3.8

(4.7)

1.7 (2.1)

81.0

77.9

Rice

83.2 (95.7)

1.7

(2.0)

2.0 (2.3)

86.9

87.4

Sorghum

63.5 (70.8)

24.6 (27.4)

1.6 (1.8)

89.7

84.8

Wheat

54.7 (69.5)

21.6 (27.4)

2.4 (3.0)

78.7

78.1

Rapidly digestible starch.

Slowly digestible starch.

Resistant starch.

Total starch

RDS

SDS

RS.

Total starch as measured by the method of Thivend et al. (1972).

Values in parentheses are individual fractions expressed as a percentage of TS.

flours differed from all others by containing approxi-
mately 70% of their total starch in the RDS form and
27% in the SDS form. Starch fraction concentrations
for flours were much higher in RDS than were their
ground grain counterparts. With the exception of the
SDS content of sorghum and wheat flours, SDS and RS
concentrations were substantially lower for flours than
for ground grains.

Organic Matter Disappearance and Organic Acid Pro-

duction.

Table 7 presents the OMD of substrates and

the resultant SCFA and lactate production data. Be-
cause the flours were not extruded like their native
counterparts, there was no interaction of treatment in
the statistical model. Therefore, all substrate produc-
tion values resulted in an overall significance at P

0.05.

Organic matter disappearance was higher (P

0.05)

for potato flour (39.4%) than for all other flours. Wheat
was indigestible after 5 h of fermentation. Numerically,
acetate production ranked as follows: potato (3.38
mmol/g OM)

barley (2.73 mmol/g OM)

corn (1.68

mmol/g OM)

wheat (1.19 mmol/g OM)

rice (0.71

mmol/g OM)

sorghum (0.52 mmol/g OM). Propionate

production was highest (P

0.05) for potato (0.77 mmol/

g OM). Barley was intermediate (P

0.05) in propionate

Table 7.

Organic matter disappearance (OMD) and acetate (ACE), propionate (PRO),

butyrate (BUTY), total short-chain fatty acid (SCFA), and lactate production

following 5 h of in vitro fermentation of cereal grain and potato flours

with canine ileal fluid (Exp. 2)

ACE,

PRO,

BUTY,

Total SCFA,

Lactate,

OMD,

mmol/g

Substrate

Barley

30.2

2.73

0.35

0.40

3.48

0.49

Corn

25.1

1.68

0.11

0.09

1.88

0.23

Potato

39.4

3.38

0.77

0.57

4.72

0.56

Rice

23.1

0.71

0.05

0.18

0.94

0.39

Sorghum

17.7

0.52

0.10

0.13

0.75

0.26

Wheat

1.19

0.13

0.15

1.47

0.03

SEM

2.4

0.38

0.05

0.04

0.42

0.02

Organic matter disappearance and organic acid production values are different (P

0.05).

production (0.35 mmol/g OM) compared to all re-
maining flours. Butyrate production paralleled propio-
nate production. Statistically, potato (0.57 mmol/g OM),
when fermented, resulted in the highest (P

0.05) pro-

duction of butyrate compared to all other substrates.

Total SCFA production was highest (P

0.05) for

both potato (4.72 mmol/g OM) and barley (3.48 mmol/
g OM). On average, these substrates produced approxi-
mately fivefold greater total SCFA in comparison to the
four remaining flours. Lactate production was rela-
tively high for all flours after 5 h of fermentation, except
for wheat flour. Statistically, lactate production was
highest (P

0.05) for potato (0.56 mmol/ g OM) and

lowest for wheat flour (0.03 mmol/g OM).

An interesting observation from this experiment is

that OMD and total SCFA production were low for rice
flour and high for potato flour. This is in direct contrast
to results of Exp. 1, in which the highest SCFA produc-
tion of all substrates tested was noted for native rice
and the lowest for native potato starch. This latter ob-
servation is corroborated by in vivo nutrient digestibil-
ity data (Murray et al., 1999). Preparation of the flour
fraction of grains and tubers can affect not only their
chemical composition but also their susceptibility to
fermentation. This perhaps is evidenced more strongly

Ileal fermentation of starch by the dog

443

in an in vitro system, as was used here, rather than
in one using colonic microflora, in which the time of
fermentation would be increased beyond 5 h. In some
cases, processing increases susceptibility of a substrate
to digestion/fermentation, whereas in other instances,
the opposite occurs. More information needs to be col-
lected on this topic on a substrate-by-substrate basis.

Our data show that small intestinal bacteria ferment

native and processed grains and potato starch differ-
ently. Evidence that raw starches are less digested than
cooked starches, as well as the negative side-effects of
their consumption (e.g., intestinal cramps, flatulence,
diarrhea), have been documented for decades (Thorpe,
1913; Langworthy and Deuel, 1920; Beazell et al.,
1939). Examination of our data on native substrates
and their concentrations of RS reconfirm findings of
early investigators reporting potential problems associ-
ated with feeding of unprocessed starch to animals.
McCay (1949) demonstrated the onset of diarrhea in
dogs fed too much starch caused by overflow of carbohy-
drate into the colon and subsequent fermentation by
bacteria. Further evidence supporting McCay’s claim
is revealed when comparing both low- and high-temper-
ature extruded substrates and the observed increase in
concentrations of RDS. The higher concentrations of
RDS imply that these starches would be utilized to a
greater extent in the small intestine and not pass into
the colon.

Cereal grain and potato flours represent substrates

with a chemical composition different from that of their
native counterparts. Flours are derived from cereals
through a series of grinding, milling, steam processing,
and fine-sieving processes (Hoseney, 1994). In our ex-
periment, flours were not extruded. Data in Table 6
illustrate why flours are highly digestible and have an
excellent correlation with increases in glycemic re-
sponses (Crapo et al., 1980). All flours contained ap-
proximately 70% of their TS in the form of RDS, making
them highly digestible products.

A common trait among all substrates was the fact

that all contained some level of SDS and RS. These
starch fractions, especially RS, would not be digested
in the small intestine and would, therefore, serve as
potential substrates for fermentation, either within the
ileum or large bowel.

In our experiments, it was apparent that microflora of

the small intestine were capable of fermenting starchy
substrates and producing SCFA and lactate. Organic
matter disappearance by small intestinal bacteria was
reasonably high. Flours (except wheat) displayed size-
able disappearance values as well. The average molar
percentage of SCFA for all substrates fermented in Exp.
1 and 2 was 75, 12, and 13% for acetate, propionate, and
butyrate, respectively. The slight elevation in butyrate
production was due mostly to the flours (13 molar %,
on average). Using grain and potato flours in the diet
may be an advantage to the dog because elevated buty-
rate production has been coupled with stimulation of
epithelial cell growth in the jejunum (Sakata, 1987).

Lactate production by ileal microflora did not follow

any consistent pattern but was influenced by substrate
source and processed form. Banta et al. (1979) indicated
that the concentration of lactate in the intestine of dogs
was influenced by the type of diet consumed (cereal- vs
protein-based). In addition, dogs produce substantial
quantities of lactate in their stomach and small intes-
tine. Lactate can be utilized for glucose synthesis by
the liver. Small intestinal fermentation might provide
the animal with an additional glucogenic substrate.

Organic matter disappearance and organic acid pro-

duction were the response criteria used to evaluate
treatment efficacy. In our opinion, the most accurate
measure of fermentative events would be organic acid
production measured during small intestinal fermenta-
tion. The molar percentages of SCFA obtained in our
study correlated well with previously published values
for nonruminants (Dukes, 1989; Smeets-Peters, 1998).
Our OMD values seem to be reasonable, but this is a
less precise measure of fermentative activity than is
SCFA and lactate production. Disappearance values
sometimes do not always agree with the organic acid
production results, as was noted for the wheat flour
treatment in Exp. 2, for which the OMD percentage
was 0 yet 1.47 mmol of SCFA/g OM was produced.

In conclusion, fermentation of high-starch substrates

is capable of occurring in the terminal ileum of dogs. In
addition, substrate source and processed form influence
the extent to which starchy substrates are fermented
by small intestinal bacteria.

Implications

Extrusion of cereal grains and potato at both low and

high temperatures alters the concentrations of rapidly
digestible starch, slowly digestible starch, and resistant
starch, in comparison to their native counterparts. In
addition, an ileal microbial population capable of fer-
menting starchy ingredients representing different
sources, fractions, and processed forms resides in the
small intestine of dogs and can make a contribution to
overall starch utilization. This bacterial population is
significant in determining the portion of starch and its
fractions that will be fermented for potential benefit to
overall colonic health. Understanding these differences
in utilization among various starch sources and their
processed forms will allow for more accurate inclusion
of cereal grains as part of companion animal diets.

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Citations

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