Ingenieria En Industrias Alimentarias

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2000, p. 2682–2684
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Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Vol. 66, No. 6

Fermentation of Fructooligosaccharides by Lactic Acid
Bacteria and Bifidobacteria†
HANDAN KAPLAN

AND

ROBERT W. HUTKINS*

Department of Food Science and Technology, University of Nebraska–Lincoln, Lincoln,Nebraska 68583-0919
Received 17 December 1999/Accepted 21 March 2000

Lactic acid bacteria and bifidobacteria were screened of their ability to ferment fructooligosaccharides
(FOS) on MRS agar. Of 28 strains of lactic acid bacteria and bifidobacteria examined, 12 of 16 Lactobacillus
strains and 7 of 8 Bifidobacterium strains fermented FOS. Only strains that gave a positive reaction by the agarmethod reached high cell densities in broth containing FOS.
1-kestose (GF2), nystose (GF3), and 1F-fructofuranosyl nystose
(GF4). FOS have been self-affirmed by the manufacturers as
GRAS (generally recognized as safe) (19) and have been
added to infant formulas, yogurt, and other food products and
food supplements.
Despite the considerable commercial and research interests
in oligosaccharidesand probiotic bacteria, relatively little is
known about which strains actually metabolize these materials.
Because commercial oligosaccharide preparations often contain glucose, fructose, sucrose, or other fermentable sugars, it
has been difficult to establish that growth in FOS-containing
medium is due to actual utilization of FOS (10, 11, 22). In this
study, we incorporated a pure form ofFOS into MRS medium
in order to identify lactic acid bacteria and bifidobacteria capable of fermenting FOS.
A commercial FOS mixture containing three FOS species,
GF2 (32.0%), GF3 (53.6%), and GF4 (9.8%), was supplied by
The GTC Nutrition Company (Westminster, Colo.). The balance consisted of 2.3% glucose and fructose and 2.3% sucrose.
To prepare pure FOS, a 40% (wt/vol) FOS solution wasapplied to a column (30 by 5 cm) containing activated charcoal
(Sigma Chemical Co., St. Louis, Mo.). Glucose and fructose
were eluted by using distilled water, and sucrose was eluted by
the addition of 5% ethanol. Finally, FOS was eluted by using
15% ethanol, and the solution was lyophilized (FTS Systems,
Inc., Stone Ridge, N.Y.). The purity of the FOS and the concentration of each FOS moiety (GF2,GF3, and GF4) were
determined by high-pressure liquid chromatography (HPLC)
(Waters Corp., Milford, Mass.) using an Aminex HPX-42C
column (0.78 by 30 cm; Bio-Rad Laboratories, Hercules, Calif.) and an RI-410 detector (Waters). The column temperature
was kept constant at 85°C, and water was used as the mobile
phase at a flow rate of 0.6 ml/min.
An MRS-FOS agar medium was prepared by adding 2%(wt/vol) purified FOS to MRS agar (3) containing 0.05% Lcysteine, 1.5% agar, and 30 mg of bromcresol purple per liter.
The MRS basal medium (i.e., MRS without carbohydrate) was
autoclaved, and the FOS was filter sterilized and then added to
the tempered agar. A total of 28 strains were evaluated, including 6 strains used as commercial probiotics (Table 1). Each
strain was initially grown inMRS broth and then diluted and
spread onto the MRS-FOS agar plates to give approximately
25 to 50 colonies. The plates were incubated anaerobically for
24 h. Strains that fermented FOS (and produced acid end
products) grew as colonies surrounded by a yellow zone ( 3
mm) against a purple background. Nonfermenting colonies
produced smaller white colonies without a yellow zone. Thus,
althoughthe basal MRS medium was sufficiently rich to pro-

The number of food and other dietary products containing
live Bifidobacterium and Lactobacillus bacteria has increased
significantly in recent years (20); this is due, in part, to the
beneficial effects these probiotic organisms are believed to
confer. Although substantial research efforts are currently in
progress to investigate these claims,...