Page 64 - World Aquaculture Magazine - March 2011

62 March 2011
The endothelial sealing of levan may have practical impor-
tance. Natural levans are serologically active and elicit an-
tibody production, but purified levan preparations are not
antigenic.
Sources of Levan
Levan is a diversely distributed component, particularly
in plants, yeasts, fungi and bacteria (Jang et al. 2002). Bac-
terial species, such as Zymomonas mobilis, Bacillus subtilis,
Bacillus polymyxa and Acetobacter xylinum produce extra-
cellular levan (Dina et al. 2007). Levans produced in grasses
(Dactylis glomerata, Poa secunda and Agropyron cristatum )
are present as storage carbohydrates in the stem and leaf
sheaths and are degraded in the latter stages of the growing
season to provide plants with carbohydrates for grain fill-
ing (Ploolock and Cairns 1991). Levan is also contained in
wheat and barley (Hordeum vulgare), fungi ( Aspergillus sy-
dawi and A. versicolor) and in trace amounts in yeasts (Han
1990). Levan is naturally present in various food products
and thus, is regularly consumed in very small amounts by
humans. However, it has been ignored as a functional food
ingredient until now. It has been argued that unlike inulin,
the high molecular weight and branched structure of levan
might cause it not be useful as a carbon source for animals
and humans (Marx et al. 2000).
Biosynthesis (Levansucrase)
The biosynthesis of levan requires the extracellular en-
zyme levansucrase (sucrose-6-fructosyltransferase), which
shows specificity for sucrose. Most methods for the bio-
synthesis of levan have been used enzymes from B.subtilis,
Aerobacter levanicum and S. salivarius. Those enzymes have
been extensively purified and the mode of action explored.
Levansucrase of B.subtitis is inducible and exocellular,
whereas that of A. levanicum is constitutive and endocellu-
lar (Dedonder 1966). It is uncertain whether levansucrase
is one enzyme or a complex of multiple enzymes that syn-
thesize the main ß (2→6) and the branch beta (2 →1) link-
ages. Chains of levan, like dextran and starch, grow in steps
by repeated transfer of a hexosyl group from the donor to
growing the acceptor molecule (Hehre 1955)
Application of Microbial Levan
Prebiotic effect of levan and its applications
Levan has numerous demonstrated uses in foods (Han
1990). Levan is non-toxic, non-mutagenic, odorless, taste-
less, and a soluble dietary fiber. Its hydrolysates help improve
gut function. Gibson and Roberfroid (1995) defined a prebi-
otic as a non-digestible and non-absorbable food ingredient
in the upper portion of the gastrointestinal tract that benefi-
cially affects the host by selectively stimulating the growth
and/or activity of one, or a limited number, of bacteria in
the colon, which can improve the host’s health. Therefore,
non-digestible carbohydrates, especially inulin oligosaccha-
rides, are classified as authentic prebiotics. Both inulin and
inulin oligosaccharides are utilized by Bifidobacterium sp. in
vivo (Gibson and Roberfroid 1995). Generally, fructo-oligo-
saccharides indicate β-2,1-d-fructans, with degrees of po-
lymerization varying between 2-20 (oligofructose) and 20-60
(inulin). Levan is composed of d-fructofuranosyl residues of
which molecular weights that reach several million Daltons,
with multiple branches.
The physiological effects of levan are dependent on its
size and linkage type and the fermentability of levan is,
therefore, an important issue. In vitro studies testing the
abilities of various genera to ferment levan and levan oli-
gosaccharides have been performed on pure cultures that
include Bifidobacterium adolescentis , B. longum, B. breve,
Lactobacillus plantarum and Pediococcus pentosaceus (Marx
et al. 2000). The in vitro fermentation properties of levan,
originating from Erwinia herbicola as the control, and levan
type exopolysaccharides originating from Lactobacillus san-
franciscensis, were studied using human feces as an inoculate
(Bello et al. 2001). An enrichment of Bifidobacterium sp. was
found with the levan-type exopolysaccharides, but not for
levan. The problem with this approach is that levan can be
hydrolyzed by gastric acids.
A possible explanation for the fermentation of levan
could be that its acid hydrolysis in the stomach produces
smaller sized levan or levan-oligosaccharides, which are sub-
sequently fully utilized by lumen bacteria. It is well known
that dietary fiber reaches the large intestine and is ferment-
ed by the colonic microflora, producing SCFAs, hydrogen,
carbon dioxide and biomass (Topping et al. 2001). This
fermentative process dominates human large bowel func-
tion and provides a means whereby energy is obtained from
the carbohydrates not digested in the small bowel through
the absorption of SCFAs. These SCFAs limit the growth of
harmful lumen bacteria and are an important energy source
for the host (Topping et al. 2001).
The ingestion of levanheptaose was shown to increase the
fecal counts of endogenous Bifidobacteria, without affecting
Lactobacillus sp. (Kang et al. 2000). The amount of butyrate
as well as β-fructosidase activity were increased, whereas to-
tal aerobes and pH were reduced in rats fed levanheptaose
diets as compared to those on a control diet. Although the
identity of the bacteria responsible for levan fermentation
remains unclear, the above concepts suggest that levan might
be degraded and then fully fermented by lumen bacteria in
the ceacum and colon.
Levan as an immunonutrient in aquaculture
High yields in aquaculture involve intensive management
systems, where antibiotics, drugs and chemicals are used to
prevent fish diseases caused by environmental stress and oth-
er factors. However, these are found to be effective only for a
short time, in addition to enhancing the risk of their bioac-
cumulation in the environment. On the other hand, the use
of immunostimulants in aqua-feed is considered to be safe
and effective against various pathogens. Immunostimulants
quickly activate non-specific defense mechanisms to protect
fish against pathogens (Siwicki 1994). Dietary manipulation
is the ideal approach to enhance the non-specific immunity
of fish along with good management practices. However,
limited studies have been undertaken to assess the influence
of dietary factors and the immune system. In recent years,
increasing consideration has been given to dietary immu-