Nutrient Metabolism

Glucose-Based Oligosaccharides Exhibit Different In Vitro Fermentation
Patterns and Affect In Vivo Apparent Nutrient Digestibility and Microbial
Populations in Dogs

Elizabeth A. Flickinger, Bryan W. Wolf,* Keith A. Garleb,* JoMay Chow,* Gregory J. Leyer,*
Paul W. Johns* and George C. Fahey, Jr.

1

Division of Nutritional Sciences and Department of Animal Sciences, University of Illinois, Urbana, IL 61801,
and *Ross Products Division, Abbott Laboratories, Columbus, OH 43215

ABSTRACT

To evaluate the potential of indigestible oligosaccharides (OS) to serve as “dietary fiber-like” ingre-

dients, it is necessary to determine their extent of indigestibility. In vitro fermentation characteristics of two novel
OS,

␣-glucooligosaccharides (GOS) and a maltodextrin-like OS (MD), were compared to those of fructooligosac-

charides (FOS), gum arabic (GA), guar gum (GG) and guar hydrolysate (GH). Total short-chain fatty acid (SCFA)
production (

␮mol/g dry matter) as a result of MD fermentation was higher initially compared with GA (P ⬍ 0.01), but

GA was more extensively fermented at 24 h (P

⬍ 0.01). Total SCFA production for GOS was similar to that for FOS,

GG, GH and GA. In the second experiment, GOS and MD were added at 6% to an enteral formula control diet
(Control) and fed to ileal-cannulated dogs in a 3

⫻ 3 replicated Latin-square design. Ileal digestibility of glucose was

lower (P

⬍ 0.05) and carbohydrate (CHO) numerically lower (P ⫽ 0.08) for both GOS and MD compared with the

Control. Total tract digestibility of CHO and glucose was lower only for MD (P

⬍ 0.01) compared with the Control.

Total fecal weights were higher (P

⬍ 0.01) for both GOS and MD treatments. Fecal concentration of bifidobacteria

was numerically increased by GOS and MD supplementation (P

⫽ 0.13 and 0.23, respectively). Thus, GOS and MD

are indigestible yet fermentable OS, and may act as “dietary fiber-like” ingredients.

J. Nutr. 130: 1267–1273,

2000.

KEY WORDS:

●

oligosaccharides

●

fermentation

●

intestinal microbiota

●

short-chain fatty acids

●

dogs

Many oligosaccharides (OS)

2

are not hydrolyzed in the

small intestine but are fermented rapidly in the lower gastro-
intestinal tract of humans and nonruminant animals. Gibson
and Roberfroid (1995) used the term “prebiotics” for certain
nondigestible OS because they selectively stimulate favorable
endogenous bacterial populations when supplemented in the
diet at low levels. Both the fermentation process and the
proliferation of favorable bacteria caused by OS ingestion can
benefit the host animal. Oligosaccharides are fermented rap-
idly to yield short-chain fatty acids (SCFA), including bu-
tyrate, which is a fuel for colonocytes (Sakata 1987). Favorable
bacterial populations, such as bifidobacteria, can promote
health by inhibiting pathogenic bacteria such as Clostridium
perfringens and Escherichia coli (Araya-Kojima et al. 1995, Gib-
son and Wang 1994).

An

␣-glucooligosaccharide (GOS) was obtained through

enzymatic synthesis from sucrose and maltose to yield a
branched-chain (

␣-1,2, ␣-1,4 and ␣-1,6 linkages) glucose

polymer with an average degree of polymerization of 5. Mal-

todextrin-like glucose-based oligosaccharides (MD) were pro-
duced by heat and enzymatic treatment of cornstarch, creating
a random distribution of

␣- and ␤- (1,4), (1,6), (1,2) and (1,3)

linkages. It has an average molecular weight of 2000.

The objectives of this study were as follows: 1) to determine

the in vitro fermentation characteristics of GOS and MD in
reference to other fermentable oligosaccharides; 2) to deter-
mine the small intestinal digestibility of GOS and MD in
ileal-cannulated dogs; and 3) to determine the effects of GOS
and MD on fecal microbial populations in dogs.

MATERIALS AND METHODS

In vitro experiment

Substrates and donors.

Substrates used in this study were GOS

(Bioecolians, Solabia, Pantin Cedex, France), MD (Fibersol 2E, Ma-
tsutani Chemical Industry, Hyogo, Japan), fructooligosaccharides
(FOS) (NutraFlora, Golden Technologies, Westminister, CO), gum
arabic (GA), guar gum (GG) (TIC Gums, Belcamp, MD), and
hydrolyzed guar gum with 24,000 MW (GH) (Fiberon S, Dainippon,
Japan). Three healthy adult male human donors (average age 30 y;
average weight 78 kg) served as sources of fecal material from which
the inoculum was prepared. The donors consumed a “Western” diet
and no antibiotics in the 3 mo preceding the experiment.

Design.

Substrates were fermented in vitro for 24 h with fresh

human fecal microflora obtained from each of three donors. The
experiment was designed as a randomized complete block with the

1

To whom correspondence should be addressed.

2

Abbreviations used: CHO, carbohydrate; CP, crude protein; DM, dry matter;

FOS, fructooligosaccharide; GA, gum arabic; GG, guar gum; GH, hydrolyzed guar
gum; GOS;

␣-glucooligosaccharide; MD, maltodextrin-like glucose-based oligo-

saccharide; OM, organic matter; OS, oligosaccharide; SCFA, short-chain fatty
acids.

0022-3166/00 $3.00 © 2000 American Society for Nutritional Sciences.
Manuscript received 14 October 1999. Initial review completed 9 December 1999. Revision accepted 1 February 2000.

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three fecal donors serving as blocks. Treatments were allotted in a 6

⫻ 7 factorial arrangement with six substrates and seven incubation

lengths. Each block by treatment combination was assayed using
duplicate fermentation tubes. Duplicate tubes containing no substrate
also were fermented with each inoculum source and time point to
correct for SCFA not arising from the substrates.

Fermentation.

Aliquots (9 mL) of sterile anaerobic buffer (95%

CO

2

/5% H

2

, pH 6.8) were aseptically transferred into tubes contain-

ing 75 mg substrate. Buffer composition is presented in Table 1. To
maintain anaerobic conditions, the tubes were sealed with butyl
rubber stoppers in an anaerobic (95% CO

2

/5% H

2

) chamber. Sub-

strates were hydrated for

⬃2 h before incubation.

Feces from the three donors were collected in commode specimen

collection systems (Sage Products, Crystal Lake, IL). Immediately
after collection, feces were diluted (1:10 wt/v) with anaerobic fer-
mentation buffer. Substrate and blank tubes were then inoculated
aseptically with 1 mL diluted feces. Tubes were incubated at 37°C,
and 1.0 mL samples were collected at 0, 1.5, 3, 6, 11, 18 and 24 h.

Chemical analyses.

A 1-mL aliquot was removed for immediate

pH determination before centrifuging at 13,000

⫻ g at 22°C for 3

min. The supernatant was stored at

⫺70°C. Acetate, propionate,

butyrate and lactate concentrations in cell-free supernatant were
analyzed by ion exclusion chromatography using a Hewlett Packard
Model HP1090 Liquid Chromatograph equipped with an ION-300
ion exclusion column (30 cm

⫻ 7.8 mm i.d.) (Interaction Chemicals,

Mountain View, CA). The mobile phase consisted of 0.005 mol/L
H

2

SO

4

with a flow rate of 0.3 mL/min and 40°C column temperature.

Statistical analyses.

Data were analyzed as a randomized com-

plete block with fecal donor serving as the block. The model state-
ment included donor, substrate, time and substrate

⫻ time. All

analyses were performed according to the General Linear Models
(GLM) procedure of SAS (1994). Arithmetic means are reported

along with the

SEM

for all treatments. When treatment differences

were detected (P

⬍ 0.05), means were compared by the least signif-

icant difference method (Carmer and Swanson 1973).

In vivo experiment

Animals and diets.

Six purpose-bred adult female dogs (Butler

Farms USA, Clyde, NY) with hound bloodlines and an average
weight of 25.3

⫾ 4.6 kg and age of 3 ⫾ 1.5 y were surgically prepared

with ileal cannulas. Ileal cannulation was conducted according to
Walker et al. (1994). Dogs were housed individually in clean floor
pens (1.2

⫻ 3.1 m) in a temperature-controlled room at the animal

facility of the Edward R. Madigan Laboratory on the University of
Illinois campus. All dogs were allowed free access to water. The
surgical and animal care procedures were approved by the Campus
Laboratory Animal Care Advisory Committee, University of Illinois
at Urbana-Champaign.

Two OS treatments were tested against a control: Enteral Control,

Enteral Control

⫹ GOS (GOS), and Enteral Control ⫹ MD (MD).

An enteral diet was selected as the control because it provided very
highly digestible nutrients for the dogs, allowing a more exact test of
the less digestible OS. The OS were added at the 6% level [dry matter
(DM) basis] to the Control diet, such that they did not replace any
individual dietary component. Diets were reconstituted before feed-
ing by adding 235 g of Enteral Control powder to 835 mL H

2

O, or

249 g of the GOS or MD diets to 825 mL H

2

O.

The chemical composition of the experimental diets is reported in

Table 2. Dietary protein was provided as sodium caseinate, calcium
caseinate and soy protein isolate. Corn oil was used as the sole lipid
source, whereas corn syrup and sucrose comprised the carbohydrate
portion of the diet. On an energy basis, the enteral diets supplied

⬃14% of kJ as protein, 31.5% as fat and 54.5% as carbohydrate.

Experimental design.

Dogs were randomized in a replicated 3

⫻ 3 Latin-square design with 14-d periods. Dogs were offered 1000

mL of the reconstituted diets at 0800 and 2000 h daily to provide

⬃8.37 MJ metabolizable energy/d. The diet adaptation phase con-

sisted of d 1 through 10; d 11 through 14 were used for ileal and fecal
collections. Chromic oxide was used as a digestion marker. On d 6

TABLE 2

Chemical composition of diets fed to ileal cannulated dogs

1,2

Item

Diet

Control

GOS

MD

Dry matter, g/100 g (powder)

96.0

96.0

95.8

Dry matter, g/100 g (enteral solution)

21.9

23.2

23.1

g/100 g dry matter

Organic matter

96.7

96.8

96.9

Crude protein

15.6

15.0

15.2

Fat

14.7

14.9

15.1

Carbohydrate (by difference)

66.4

66.9

66.6

Glucose

28.9

29.1

26.8

1

Control, Enteral Control; GOS, Enteral Control

⫹ 6%

␣-glucooli-

gosaccharide; MD, Enteral Control

⫹ 6% maltodextrin-like glucose-

based oligosaccharide.

2

Protein: 84% caseinate and 16% soy protein isolate; lipid: corn oil;

nonstructural carbohydrate: 70% corn syrup and 30% sucrose. Vita-
mins (unit/L): retinyl palmitate, 2.65 mg; cholecalciferol, 5.3

␮g; dl-␣-

tocopheryl acetate, 32.3 mg; phylloquinone, 43

␮g; ascorbic acid C,

159 mg; folic acid, 424

␮g; thiamine, 1.6 mg; riboflavin, 1.9 mg; vitamin

B-6, 2.2 mg; vitamin B-12, 6.4 mg; niacin, 21.2 mg; choline, 318 mg;
biotin, 318

␮g; pantothenic acid, 10 mg. Minerals (unit/L): sodium, 846

mg; potassium, 1564 mg; chloride, 1312 mg; calcium, 530 mg; phos-
phorus, 530 mg; magnesium, 212 mg; iodine, 80

␮g; manganese, 2.7

mg; copper, 1.1 mg; zinc, 12.0 mg; iron, 9.6 mg; selenium, 38

␮g;

chromium, 53

␮g; molybdenum, 80 ␮g.

TABLE 1

Composition of buffer used for in vitro fermentation

1

Component

Amount, unit/L

mg

Na

2

CO

3

4000

Cysteine HCl

600

Trypticase

500

Yeast extract

500

(NH

4

)

2

SO

4

480

NaCl

480

K

2

HPO

4

292

KH

2

PO

4

292

MgSO

4

䡠 7H

2

O

100

CaCl

2

䡠 2H

2

O

64

mL

Hemin

1.25

Resazurin

1.0

Short-chain fatty acid mix

2

3.1

Trace mineral solution

3

10.0

Vitamin solution

4

10.0

1

Heat-tolerant portions of the buffer were heated (10 min in an

autoclave) to drive off O

2

and cooled in an anaerobic chamber (95%

CO

2

/5% H

2

) before aliquots were placed in fermentation tubes.

2

Composition (mL/L): acetate, 408; propionate, 144; butyrate, 72;

valerate, 24; isovalerate, 24; isobutyrate, 24; and 2-methylbutyrate, 24.

3

Composition (mg/L): Na

4

EDTA, 500; FeSO

4

䡠 7H

2

O, 200; ZnSO

4

䡠

7H

2

O, 10; MnCl

2

䡠 4H

2

O, 200; H

3

BO

4

, 20; CoCl

2

䡠 6H

2

O, 20; CuCl

2

䡠

2H

2

O, 1; NiCl

2

䡠 6H

2

O, 2; Na

2

MoO

4

䡠 2H

2

O, 3.

4

Composition (g/L of 5 mmol/L HEPES, pH 7.5): biotin, 0.025; folic

acid, 0.025; calcium-

D

-pantothenate, 0.200; nicotinamide, 0.200; ribo-

flavin, 0.200; thiamine-HCl, 0.200; pyridoxine-HCl, 0.200; para-amino-
benzoic acid, 0.025; cyanocobalamin, 0.025; 1,4-napthoquinone,
0.250.

FLICKINGER ET AL.

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through 14 of each period, dogs were dosed with 0.5 g chromic oxide
in a gelatin capsule at 0800 and 2000 h for a total of 1 g marker/d.

Sampling procedures.

During the collection phase, ileal effluent

and feces were collected for 4 d. Ileal effluent was collected 3 times
per day, with an interval of 4 h between collections. Individual ileal
collections were 1 h in duration. Sampling times on the remaining 3 d
were rotated 1 h from the previous day’s collection time. For example,
on d 1, sampling took place at 0800, 1200 and 1600 h; on d 2, samples
were collected at 0900, 1300 and 1700 h. Ileal samples were obtained
by attaching a Whirlpak bag (Pioneer Container, Cedarburg, WI) to
the cannula barrel and around the cannula hose clamp with a rubber
band. Before attachment of the bag, the interior of the cannula was
scraped clean with a spatula and initial digesta discarded. During
collection of ileal effluent, dogs were encouraged to move around
freely. The use of Elizabethan collars was necessary for some dogs to
deter them from pulling the collection bag from their cannula. Total
feces excreted during the collection phase of each period were col-
lected from the floor of the pen, weighed, composited and frozen at

⫺4°C.

Sample handling.

Ileal samples were frozen at

⫺4°C in their

individual bags. At the end of the experiment, all ileal effluent
samples were composited for each dog for each period, and then
refrozen at

⫺4°C. Ileal effluent then was freeze-dried in a Tri-Philizer

MP microprocessor-controlled lyophilizer (FTS Systems, Stone
Ridge, NY). Feces were dried at 55°C in a forced-air oven. After
drying, both feces and ileal samples were ground through a 2-mm
screen in a Wiley mill (model 4, Thomas Scientific, Swedesboro, NJ).
Diets were analyzed in the unhydrated powdered form.

Freshly voided feces were collected within 15 min of defecation;

individual aliquots were immediately transferred to preweighed
Carey-Blair transport media containers (Meridian Diagnostics, Cin-
cinnati, OH) for subsequent bacterial enumeration. Feces were scored
for each dog during each period according to the following system: 1

⫽ hard, dry pellets, small, hard mass; 2 ⫽ hard, formed, dry stool,

remains firm and soft; 3

⫽ soft, formed, moist, softer than stool that

retains shape; 4

⫽ soft, unformed, stool assumes shape of container,

pudding-like; 5

⫽ watery; liquid that can be poured.

Chemical analyses.

Diets, feces and ileal effluent were analyzed

for DM, organic matter (OM) and ash using AOAC (1984) methods.
Crude protein (CP) was calculated from Kjeldahl N values (AOAC
1984). Total lipid content was determined by acid hydrolysis followed
by ether extraction according to the American Association of Cereal
Chemists (1983) and Budde (1952). Chromium was analyzed accord-
ing to Williams et al. (1962) using an atomic absorption spectropho-
tometer (Model 2380, Perkin-Elmer, Norwalk, CT). Carbohydrate
(CHO) content was calculated as the difference between OM con-
tent and the sum of crude protein and lipid contents. Glucose content
was determined by monosaccharide analysis according to Bourquin et
al. (1990). Monosaccharides were hydrolyzed with H

2

SO

4

and quan-

tified by anion exchange HPLC with pulsed electrochemical detec-
tion. Briefly, 50

␮L of each neutralized, hydrolyzed sample was in-

jected into an HPLC fitted with a Dionex Carbo-Pac PA-1 column
(250

⫻ 4 mm) (Dionex, Sunnyvale, CA). Individual monosacchar-

ides were eluted with degassed water (1.0 mL/min, ambient temper-
ature) and 300 mmol/L NaOH was added postcolumn. For all labo-
ratory analyses, samples were analyzed in duplicate, and analyses were
repeated if a deviation

⬎ 5% between duplicates occurred.

Total anaerobes, total aerobes, bifidobacteria, lactobacilli and

bacteroides spp. were determined by serial dilution of fecal samples in
anaerobic diluent (Bryant and Burkey 1953) before inoculation onto
respective petri dishes of sterile agar. Total anaerobe and total aerobe
agars were prepared according to Bryant and Robinson (1961) and
Mackie et al. (1978). The selective medium for bifidobacteria (BIM-
25) was prepared using reinforced clostridial agar (BBL Microbiology
Systems, Cockeyville, MD) according to the method described by
Mun˜oa and Pares (1988). Lactobacilli were cultured on Rogosa SL
agar (Difco Laboratories, Detroit, MI). Bacteroides spp. were cultured
on a trypticase yeast extract glucose agar listed in Table 3. Plates were
incubated anaerobically (95% CO

2

/5% H

2

) at 38°C. Colony forming

units were defined as being distinct colonies measuring at least 1 mm
in diameter.

Calculations.

Dry matter (g/d) recovered as ileal effluent or

excreted as feces was calculated by dividing the Cr intake (mg/d) by
ileal or fecal Cr concentrations (mg Cr/g ileal effluent or feces),
respectively. Ileal and fecal nutrient flows were calculated by multi-
plying the DM flow by the concentration of the nutrient in the ileal
or fecal DM. Ileal and total tract nutrient digestibilities were calcu-
lated as nutrient intake (g/d) minus the ileal or fecal nutrient flow
(output, g/d), divided by nutrient intake (g/d).

Statistical analysis.

Data were analyzed by the General Linear

Models procedure of SAS (1994). The experimental design was a
replicated 3

⫻ 3 Latin square. Six sequences of the diets (one

sequence per dog) were used (ABC, CAB, BCA, ACB, BAC, CBA),
where A was Enteral Control, B was Enteral Control

⫹ GOS, and C

was Enteral Control

⫹ MD. Model sums of squares were separated

into treatment, period and animal effects. When significant (P

⬍ 0.05) differences were detected, treatment means were compared

by the least significant difference method of SAS (Carmer and
Swanson 1973).

RESULTS AND DISCUSSION

In vitro experiment

pH values.

In general, pH decreased as time of fermenta-

tion increased (Fig. 1). All substrates had similar pH values
(6.9) at 0 h. Fructooligosaccharide fermentation resulted in
the most rapid decline in pH and the lowest pH values (P

⬍ 0.05) at 3 and 6 h. Conversely, GA had the slowest decline

in pH and the highest pH values (P

⬍ 0.05) at 1.5, 3, 6 and

11 h of fermentation. The pH values for GOS, GH, GG and
MD generally were intermediate to that of FOS and GA
between 0 and 6 h. However, at 24 h, GOS had a numerically
lower pH (P

⫽ 0.18) than FOS and a lower pH (P ⬍ 0.05)

than all other substrates. The pH decline for MD was rapid
from 0 to 3 h, but then was relatively stable. At 24 h, the

TABLE 3

Composition of agar used to enumerate Bacteroides spp.

Component

Amount, unit/L

g

Trypticase

10.0

Yeast extract

5.0

Agar

20.0

mL

Potassium phosphate buffer, 1 mol/L

100.0

TYG salt solution

1

40.0

CaCl

2

solution, 8 g/L

1.0

FeSO

4

, 40 g/L

1.0

Resazurin, 25 g/L

4.0

Hemin solution

2

10.0

Double-distilled H

2

O

828.3

After boiling, saturate with CO

2

, autoclave, cool to 60°C and add:

mL

Vitamin K solution, 40 g/L

1.0

Vitamin B-12 solution, 5.0 g/L

1.0

Gentamicin, filter sterilized, 50 g/L

4.0

Cysteine solution, free base, 0.3 kg/L

1.7

Glucose solution, 0.25 g/L

8.0

1

Composition (g/L): NaHCO

3

, 10.0 g; MgSO

4

䡠 7H

2

O, 0.5 g; NaCl,

2.0 g; double-distilled H

2

O, 1000 mL.

2

Composition: 500 mg hemin dissolved in 10 mL of 1 mol/L NaOH,

then diluted to 500 mL final volume with double-distilled H

2

O.

DIGESTIBILITY OF GLUCOSE-BASED OLIGOSACCHARIDES

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highest (P

⬍ 0.05) pH value was noted for MD (6.31) com-

pared with all other substrates. Overall, the pH declines for
GOS, FOS, GH and GG were not different (P

⫽ 0.35) from

those of MD and GA.

Organic acid production.

Organic acid (acetate

⫹ propi-

onate

⫹ butyrate ⫹ lactate) concentrations increased over

time (Table 4). Fructooligosaccharides resulted in the most
rapid accumulation of organic acids, achieving 93% of total
organic acid production after only 6 h. However, by 11 h, FOS,
GOS, GH and GG had similar concentrations of organic
acids. Gum arabic resulted in the lowest (P

⬍ 0.05) organic

acid concentrations at 11 h, and MD was intermediate. After
24 h of fermentation, GOS resulted in the numerically highest
(P

⫽ 0.15) organic acid concentration, whereas MD had the

lowest (P

⬍ 0.05). Fermentation of GA, GG, GH and FOS

resulted in intermediate organic acid concentrations, which
were not significantly different from values obtained for GOS.

After 6 or 11 h of fermentation, GOS and FOS resulted in

the highest (P

⬍ 0.05) concentrations of acetate. Hydrolyzed

guar gum, GG and MD had intermediate concentrations of
acetate. Gum arabic resulted in the lowest (P

⬍ 0.05) con-

centrations of acetate at 6 and 11 h. At 6 h, propionate
concentration was numerically lowest (P

⫽ 0.11) for GA

compared with all other substrates. At 11 h, propionate con-
centration was highest (P

⬍ 0.05) for GG, and remained

lowest (P

⬍ 0.05) for GA. Fructooligosaccharide and GOS

were intermediate in propionate concentration after 11 h of
fermentation. Butyrate concentrations at 6 or 11 h were nu-
merically highest (P

⫽ 0.23) for FOS and were lowest (P

⬍ 0.05) for GA. At 6 and 11 h, GOS, GH and GG had

intermediate concentrations of butyrate.

In contrast to the 11-h observation, after 24 h of fermen-

tation, the concentration of acetate for GA was greater than
FOS (P

⬍ 0.05) and numerically greater than GOS (P ⫽ 0.10)

FIGURE 1

The pH values of substrates after different times of in

vitro fermentation. Values are means, n

⫽ 6. Asterisks indicate a sig-

nificant difference from all other means at that time, P

⬍ 0.05. FOS,

fructooliogsaccharide; GOS,

␣-glucooligosaccharide; GH, hydrolyzed

guar gum; GG, guar gum; MD, maltodextrin-like glucose-based oligo-
saccharide; GA, gum arabic.

TABLE 4

Acetate, propionate, butyrate and total organic acid accumulation after different hours of in vitro fermentation of substrates

1

Item

Time

FOS

2

GOS

2

GH

2

GG

2

MD

2

GA

2

SEM

3

LSD

4

h

mmol/g/substrate

Acetate

1.5

0.70

0.88

0.66

0.37

0.68

0.06

0.302

0.891

3

2.66

1.96

1.17

0.99

1.52

0.12

6

4.35

3.40

2.05

2.15

2.37

0.19

11

4.33

4.66

3.41

3.22

2.97

0.90

18

4.40

5.22

3.75

3.55

3.78

3.56

24

4.37

5.08

3.68

3.57

4.18

5.34

Propionate

1.5

0.11

0.14

0.13

0.11

0.12

0.01

0.272

0.803

3

0.35

0.33

0.29

0.30

0.25

0.01

6

0.99

0.73

0.72

0.83

0.48

0.08

11

1.17

1.31

1.81

1.94

0.68

0.47

18

1.35

1.78

2.00

2.26

1.10

1.61

24

1.47

2.03

2.03

2.23

2.03

2.35

Butyrate

1.5

0.18

0.22

0.17

0.08

0.17

0.01

0.193

0.570

3

0.59

0.40

0.39

0.26

0.32

0.03

6

1.44

0.62

0.70

0.59

0.36

0.02

11

1.75

1.00

1.33

1.40

0.44

0.05

18

1.77

1.39

1.54

1.48

0.47

0.19

24

1.82

1.39

1.58

1.45

0.54

0.39

Total SCFA

2,5

1.5

1.08

1.37

0.99

0.56

1.02

0.06

0.410

1.211

3

3.97

2.77

1.89

1.55

2.09

0.16

6

7.12

4.75

3.46

3.56

3.20

0.30

11

7.26

6.97

6.54

6.56

4.08

1.42

18

7.51

8.38

7.28

7.29

5.34

5.37

24

7.66

8.49

7.28

7.25

6.01

8.08

1

Values are means of six tubes (3 donors in duplicate).

2

FOS, fructooliogsaccharide; GOS,

␣-glucooligosaccharide; GH, hydrolyzed guar gum; GG, guar gum; MD, maltodextrin-like glucose-based

oligosaccharide; GA, gum arabic; SCFA, short-chain fatty acids.

3

Pooled

SEM

for each short-chain fatty acid.

4

Least significant difference between any two mean values in the same row (P

⬍ 0.05).

5

Acetate

⫹ propionate ⫹ butyrate ⫹ lactate.

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(Table 4). The concentration of acetate produced by GOS
remained greater (P

⬍ 0.05) than that of MD, which was

similar to the values for GG, GH and FOS. After 24 h of
incubation, propionate concentration was numerically lowest
(P

⫽ 0.15) for FOS (Table 4). Butyrate accumulation was

greater (P

⬍ 0.05) for FOS, GOS, GH and GG, than for MD

and GA (Table 4) after 18 and 24 h of incubation. The
highest butyrate-producing substrates resulted in

⬃5–35 times

greater values compared with the lowest butyrate-producing
substrates after 11–24 h of fermentation. Relatively little lac-
tate accumulated, and none was detected after 6 h of fermen-
tation (data not shown). Fructooligosaccharides had the high-
est (P

⬍ 0.05) concentrations of lactate at 3 and 6 h of

fermentation. No lactate accumulated as a result of the fer-
mentation of GG or GA.

The more rapid pH decline and organic acid production as

a result of FOS and GOS fermentation indicated that these
two OS were fermented more rapidly than the other substrates
tested in this study. Maltodextrin-like oligosaccharides re-
sulted in the lowest in vitro production of organic acids and
the highest pH at 24 h, indicating some resistance to fermen-
tation. Short-chain fatty acid profiles, in addition to concen-
trations, varied among substrates. Butyrate production was
highest and similar for FOS, GOS, GH and GG. Butyrate is a
major fuel for colonocytes (Roediger 1980) and has been
demonstrated to stimulate epithelial cell proliferation (Sakata
1987). Therefore, substrates that produce high levels of bu-
tyrate, such as FOS, GOS, GG and GH, may provide similar
beneficial effects when included in diets. The rapid in vitro
accumulation of butyrate with FOS fermentation at 6 h indi-
cates that FOS would serve as an excellent source of butyrate
in vivo. On the other hand, the slower rate of fermentation of
GA and MD indicates that they would perhaps provide a
source of fermentable CHO to the more distal part of the large
intestine. A blend of rapidly and slowly fermented carbohy-
drates should result in production of SCFA throughout the
large intestine.

In vivo experiment

Chemical composition.

The chemical composition of the

diets is reported in Table 2. Dry matter, OM, CP, fat, glucose
and CHO content did not differ among diets.

Nutrient intake and apparent digestibility.

Intakes of

DM, OM and CP did not differ (Table 5). Fat intake was lower
(P

⬍ 0.05) for the Control treatment than for either the GOS

or MD treatments due to lower DM intakes. Dogs consuming
either the GOS- or MD-containing diets had greater (P

⬍ 0.05) glucose and numerically greater (P ⫽ 0.09) CHO

intakes compared with the Control diet. This was expected
because GOS and MD were added at 6% “on top” of the
control diet.

Ileal digestibility of DM, OM, CP and fat did not differ

among treatments. Although the overall model was not sig-
nificant (P

⫽ 0.12), ileal digestibility of glucose was signifi-

cantly different (P

⬍ 0.05) due to diet. Ileal glucose digest-

ibility values were 4.6 and 4.8 percentage units lower for both
the GOS- and the MD-containing treatments, respectively,
compared with the Control diet. Diet tended (P

⫽ 0.08) to

reduce ileal digestibility of CHO (5.5 and 4.3 percentage units
lower) compared with the Control diet. These data indicate
that the supplemental OS, GOS and MD resisted hydrolytic
digestion and passed intact into the large intestine. These
results are supported by data of Valette et al. (1993) who
demonstrated that GOS was

⬃20% digested by germfree rats.

Furthermore, Tsuji and Gordon (1998) reported that MD

disappearance was only 10% after four consecutive in vitro
incubations with salivary

␣-amylase, gastric juice, pancreatic

␣-amylase and reconstituted intestinal mucosa, which simu-

lated hydrolytic digestion.

Total tract digestibilities of DM and fat did not differ

among treatments. Crude protein digestibility was lower (P

⬍ 0.05) for dogs consuming the GOS and MD treatments.

The lower values for total tract digestibility of the GOS and
MD diets may be attributed to an increased fecal excretion of
microbial protein. Wolf et al. (1998) proposed that as the
percentage of fermentable carbohydrate increases in the diet,
the amount of microbial mass increases in the feces. This
would result in an increase in fecal N excretion and an
apparent decrease in crude protein digestibility. Organic mat-
ter, carbohydrate and glucose digestibilities were lower (P

⬍ 0.05) for the MD treatment compared with either the

Enteral Control or the GOS treatments. This may indicate
that MD was not completely fermented in the large intestine
as suggested by our in vitro study. Incomplete fermentation
also was reported by Tsuji and Gordon (1998), who recovered
38% of orally dosed MD in the feces of adult rats. There was
a trend for carbohydrate and glucose digestibility to be slightly
lower for the GOS diet compared with the Control. This
suggests that GOS was extensively fermented. These findings
agree with those of Djouzi et al. (1995) who demonstrated that
GOS was fermented extensively by human intestinal bacterial
strains in vitro and in vivo by trixenic rats associated with
Bacteroides thetaiotaomicron, Bifidobacterium breve and Clostrid-
ium butyricum.

Fecal weight, fecal score, and body weight changes.

There were no differences among treatments in body weight
changes of dogs (data not shown). Fecal weights for dogs
consuming the GOS diet were greater (P

⬍ 0.05) than those

TABLE 5

Nutrient intakes and apparent digestibility data for ileal-

cannulated dogs fed diets supplemented with

␣-

glucooligosaccharide (GOS) or maltodextrin-like glucose-

based oligosaccharide (MD)

Item

Diet

SEM

Control

GOS

MD

Intake, g/d

Dry matter

447

473

464

6.2

Organic matter

433

458

449

5.9

Crude protein

70

71

71

0.8

Fat

66

a

70

b

70

b

1.4

Carbohydrate

297

316

308

4.0

Glucose

211

a

250

b

241

b

3.2

Ileal digestion, %

Dry matter

90.6

87.0

89.3

2.02

Organic matter

92.3

88.7

90.5

1.75

Crude protein

80.8

82.0

87.0

4.07

Fat

96.1

95.2

96.6

0.83

Carbohydrate

94.2

88.7

89.9

1.53

Glucose

97.0

b

92.4

a

92.2

a

1.16

Total tract digestion, %

Dry matter

96.4

95.8

94.2

0.33

Organic matter

97.5

b

96.8

b

95.3

a

0.29

Crude protein

92.3

c

90.1

b

87.9

a

0.60

Fat

95.8

96.0

95.9

0.32

Carbohydrate

99.2

b

98.5

b

96.8

a

0.32

Glucose

99.9

b

99.8

b

98.0

a

0.30

a,b,c

Means (n

⫽ 6) in a row not sharing a superscript letter differ (P

⬍ 0.05).

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of the Control on an as-is basis and tended (P

⫽ 0.07) to be

greater on a DM basis (Table 6). Fecal weight, on both an as-is
and DM basis, was greater (P

⬍ 0.05) for dogs consuming the

MD treatment compared with the Control. On a DM basis,
the fecal weight for MD was greater (P

⬍ 0.05) than that of

GOS. These data, along with the high 24-h pH value for MD
after 24 h of in vitro fermentation, indicate that it is not
completely fermented by the colonic microflora of dogs or
humans. Increased fecal weight is a common outcome of
dietary fiber consumption. Depending on its individual char-
acteristics, dietary fiber can increase fecal bulk by increasing
bacterial cell mass, undegraded fiber residue, fecal water or a
combination of these effects (Fahey et al. 1990, Roberfroid
1993, Schneeman, 1987). Fecal scores were higher (P

⬍ 0.05)

for dogs consuming the GOS and MD treatments compared
with the Control, indicating a higher fecal moisture content.
Overall, the fecal scores were relatively high. The loosely
formed stools were caused in part by consumption of an enteral
formula. Increased fecal moisture content is a common effect
attributed to dietary fiber. Fecal water content can be in-
creased by the physical water-holding properties of fibers, and
possibly by the osmotic action of SCFA produced by fermen-
tation (Roberfroid 1993, Schneeman 1987).

Fecal bacterial concentrations.

There were no significant

(P

⬎ 0.05) differences in bacterial concentrations among

treatments (Table 7). However, trends were apparent. The
concentrations of total anaerobes were increased slightly by
the addition of GOS or MD to the basal diet. Bifidobacteria
concentrations also tended to increase when GOS or MD were
added to the diet (P

⫽ 0.13 and 0.23, respectively). Bacte-

roides concentrations were numerically lower (P

⫽ 0.10) for

the MD diet compared with the Control. These results agree
with data collected by Kohmoto et al. (1988), who reported
that dietary isomaltooligosaccharides (13.5 g/d for 14 d) in-
creased fecal bifidobacteria levels (P

⬍ 0.05) in healthy adult

males.

Total anaerobes and total aerobes indicate general fermen-

tative activity, whereas bifidobacteria and lactobacilli are in-
dicators of a more remedial, beneficial bacterial population.
Increased concentrations of bifidobacteria and lactobacilli
have been associated with decreased fecal concentrations of
potentially pathogenic bacteria (Araya-Kojima et al. 1995,
Gibson and Wang, 1994) and decreased levels of carcinogenic
and putrefactive compounds (Hara et al. 1994, Mitsuoka 1982,

Terada et al. 1992). Although these compounds were not
measured in this study, the bifidogenic effect of GOS and MD
also may reduce fecal putrefactive compounds.

Bacteroides are the predominant colonic bacterial genera,

comprising

⬃30% of the total culturable microflora (Macfar-

lane and Macfarlane 1995). Most bacteroides are considered
neither beneficial nor detrimental to host health. They readily
utilize resistant starch as well as nonstarch polysaccharides
(Hudson and Marsh, 1995). In our experiment, GOS and MD
led to a numerical increase in fecal concentrations of bacte-
roides.

Ileal-cannulated dogs were chosen as the animal model for

this study for the many similarities they share with humans.
Both dogs and humans are omnivorous monogastrics. Like
humans, dogs have numerous species of endogenous bacteria
in their lower gastrointestinal tract (Balish et al. 1977, Davis
et al. 1977) that contribute to significant amounts of colonic
fermentation (Banta et al. 1979). The microflora of both dogs
and humans contains bacteroides, bifidobacteria and lactoba-
cilli as predominant species (Davis et al. 1977, Gibson and
Roberfroid 1995). Dogs have been employed in numerous
studies pertaining to human nutrition (Diez et al. 1997 and
1998, Willard et al. 1994). The ileal-cannulated dog model
has been widely used to evaluate ileal digestibility of nutrients
in many types of ingredients (Muir et al. 1996; Murray et al.
1997 and 1998, Zuo et al. 1996). Therefore, the ileal-cannu-
lated dog is an appropriate model to utilize in evaluating
oligosaccharides with potential human applications.

The in vitro and in vivo studies provided complementary

data regarding the fermentation of the novel OS, GOS and
MD. In vitro, the fermentation of GOS resulted in rapid
production of high concentrations of SCFA, whereas MD
fermentation resulted in a more gradual and overall lower
SCFA production. In vivo, GOS and MD resisted hydrolytic
digestion and were present at the terminal ileum.

␣-Gluco-

oligosaccharide appears to be extensively fermented because
very little was recovered in feces. However, MD was fermented
only partially, resulting in significantly lower total tract CHO
and glucose digestibility values.

In conclusion, both GOS and MD appear to be indigestible

in the small intestine, supplying CHO to the large intestine for
bacterial fermentation. Both in vitro and in vivo digestibility
data suggest that GOS was fermented more extensively than
MD. Dietary supplementation of these OS at the 6% level in
an enteral formula did not greatly alter the digestibility of
macronutrients. Both GOS and MD increased the volume of

TABLE 7

Fecal bacterial concentrations for cannulated dogs fed diets

supplemented with

␣-glucooligosaccharide (GOS) or

maltodextrin-like glucose-based oligosaccharide (MD)

1

Item

Diet

SEM

Control

GOS

MD

CFU log

10

/g fecal DM

2

Total anaerobes

10.82

10.93

11.09

0.249

Total aerobes

9.13

9.53

9.25

0.285

Bifidobacteria

9.01

9.67

9.52

0.251

Lactobacilli

7.84

8.37

7.74

0.521

Bacteroides

9.89

9.29

8.58

0.405

1

Values are means, n

⫽ 6.

2

CFU, colony-forming unit; DM, dry matter.

TABLE 6

Fecal characteristics of ileal-cannulated dogs fed diets

supplemented with

␣-glucooligosaccharide (GOS) or

maltodextrin-like glucose-based oligosaccharide (MD)

Item

Diet

SEM

Control

GOS

MD

Feces, as-is, g/d

35.0

a

64.8

b

79.4

b

6.74

Feces, DM

1

basis, g/d

16.0

a

20.0

a

26.7

b

1.38

Fecal score

2

3.4

a

4.1

b

4.4

c

0.10

1

DM, dry matter.

2

Scores based on the following scale: 1

⫽ hard, dry pellets; 2

⫽ hard, formed, dry stool, remains firm and soft; 3 ⫽ soft, formed,
moist, softer than stool that retains shape; 4

⫽ soft, unformed, stool

assumes shape of container, pudding-like; 5

⫽ watery, liquid that can

be poured.

a,b,c

Means (n

⫽ 6) in a row not sharing a superscript letter differ (P

⬍ 0.05).

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feces excreted and the moisture content of the feces. These OS
also tended to increase fecal concentrations of beneficial bac-
teria, including bifidobacteria. These findings indicate that
GOS and MD are indigestible in the upper intestinal tract.
However, these OS may serve as fermentable dietary fiber-like
substrates and positively affect gastrointestinal tract health.

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