Appl Microbiol Biotechnol (2003) 63:286–292
DOI 10.1007/s00253-003-1426-0
ORIGINAL PAPER
G. Tzortzis · A. J. Jay · M. L. A. Baillon ·
G. R. Gibson · R. A. Rastall
Synthesis of a-galactooligosaccharides with a-galactosidase
from
Lactobacillus reuteri
of canine origin
Received: 2 May 2003 / Revised: 24 July 2003 / Accepted: 26 July 2003 / Published online: 30 August 2003
Springer-Verlag 2003
Abstract Crude cell-free extracts from Lactobacillus
reuteri grown on cellobiose, maltose, lactose and raffi-
nose were assayed for glycosidic activities. When raffi-
nose was used as the carbon source, a-galactosidase was
produced, showing the highest yield at the beginning of
the stationary growth phase. A 64 kDa enzyme was
purified by ultra- and gel filtration, and characterized for
its hydrolytic and synthetic activity. Highest hydrolytic
activity was found at pH 5.0 at 50 C (K
M
0.55 mM, V
max
0.80 mmol min
1
mg
1
of protein). The crude cell-free
extract was further used in glycosyl transfer reactions to
synthesize oligosaccharides from melibiose and raffinose.
At a substrate concentration of 23% (w/v) oligosaccharide
mixtures were formed with main products being a
trisaccharide at 26% (w/w) yield from melibiose after
8 h and a tetrasaccharide at 18% (w/w) yield from
raffinose after 7 h. Methylation analysis revealed the
trisaccharide to be 6
0
a-galactosyl melibiose and the
tetrasaccharide to be stachyose. In both cases synthesis
ceased when hydrolysis of the substrate reached 50%.
Introduction
Most lactic acid bacteria are food-grade microorganisms,
generally regarded as safe, which can contribute towards
the taste, smell or preservation of food products (van
Geel-Schuten et al. 1999). Lactobacillus species have
been found in large numbers as part of the intestinal flora
of humans and other animals, where they are thought to
increase resistance to common intestinal disorders, espe-
cially those with a microbial pathogenesis e.g. gastroen-
teritis (Casas and Dobrogosz 2000). They can achieve this
by fortifying the normal microflora either through their
fermentation products or by the production of glycosi-
dases, which degrade carbohydrates, thereby supplying
energy for the growth of other bacteria (Sandine 1979).
Lactobacillus reuteri, until recently misclassified as
Lactobacillus fermentum, is hetero-fermentative and the
only Lactobacillus species thought to inhabit the gastro-
intestinal tract of all vertebrates and mammals (Casas and
Dobrogosz 2000). L. reuteri has been reported to be one
of the few lactobacilli isolated from the small and large
intestinal mucosae (Molin et al. 1993) and it has been
suggested that it produces cell surface proteins with
mucus-binding properties. It also produces anti-microbial
substances such as reuterin (El-Ziney and Debevere 1998)
and reutericin (Ganzle et al. 2000), which are active
against a range of Gram-positive and Gram-negative
pathogenic bacteria.
Until recently, oligosaccharides have been used in the
food industry as a source of energy or as sweeteners.
Nowadays knowledge of their biological function and
their role in cell-surface interactions has opened a new
field of glycotechnology. Apart from their traditional use,
oligosaccharides find new applications as immunostimu-
lating agents or prebiotic compounds able to modulate the
colonic microflora towards a healthy balance (Gibson and
Roberfroid 1995). This usually involves selectively
increasing the levels of bifidobacteria and lactobacilli at
the expense of less desirable bacteria (Fuller and Gibson
1998). a-Galactooligosaccharides as additives in func-
tional food are of great interest because their a-galacto-
sidic linkages are non-digestible by humans and
monogastric animals (Peterbauer and Richter 2001) and
therefore reach the colon intact, where few bacteria are
known to exhibit high a-galactosidase activity (Sakai et
G. Tzortzis · G. R. Gibson · R. A. Rastall (
)
)
School of Food Biosciences,
The University of Reading,
Whiteknights, PO Box 226, Reading, RG6 6AP, UK
e-mail: R.A.Rastall@reading.ac.uk
Tel.: +44-118-3786726
A. J. Jay
Institute of Food Research,
Colney Lane, Norwich, NR4 7UA, UK
M. L. A. Baillon
Waltham Centre for Pet Nutrition,
Waltham-on-the-Wolds, Melton Mowbray, Leicestershire,
LE14 4RT, UK