Microbial levan, an ideal prebiotic
and immunonutrient in aquaculture
S. K. Gupta, Pronob Das1*, S. K. Singh, M. S. Akhtar, D. K. Meena and S. C. Mandal
Aquaculture is one of the fastest growing agriculture
sectors in the world, providing food and nutritional secu-
rity to millions of people. However, disease outbreaks are a
constraint to aquaculture production, thereby affecting the
socio-economic status of people in many countries. For in-
stance, disease is now considered to be the limiting factor in
the shrimp farming industry. Disease control in the aquacul-
ture industry has been achieved using variou methods, in-
cluding traditional means, synthetic chemicals and antibiot-
ics. However, the use of such expensive chemotherapeutants
for controlling diseases has been widely criticized for their
negative effects, including accumulation of residues, devel-
opment of drug resistance, immunosuppression and reduced
consumer preference for products treated with antibiotics.
Traditional methods are ineffective against controlling new
diseases in large aquaculture systems. Therefore, alternative
methods need to be developed to maintain a healthy micro-
bial environment in aquaculture systems, thereby maintain-
ing the health of the cultured organisms. The use of prebiot-
ics, probiotics and immunonutrients is growing as means of
producing healthy organisms (Panigrahi and Azad 2007).
Prebiotics are non-digestible food ingredients that benefi-
cially affect the host by stimulating growth activity and/or
activity of a limited number of beneficial bacteria in the gas-
trointestinal tract (Gibson and Roberfroid 1995). According
to Panigrahi and Azad (2007), prebiotics are basically food
for probiotics. In the case of terrestrial animals, the use of
prebiotics has been progressing. Health-promoting bacteria
commonly augmented by prebiotics include those of the ge-
nus Lactobacillus and Bifidobacterium in terrestrial animals
(Manning and Gibson 2004). The use of prebiotics in aqua-
culture is a fairly recent development. Levan was reported
by Lippmann as early as 1881 and the name “Levulan” was
proposed for the compound. Microbial levan is used as a
prebiotic and immunonutrient in aquaculture.
The Chemical Structure of Microbial Levan
Levans are fructans, natural polymers of fructose. The
two main types of fructans are the levans with mostly ß
(2→6) linkages and the inulins with ß (2→1) linkages (Han
1990). Branched fructans with both types of linkages also
exist. Levan is the common name for a fructan in which
most fructose has ß (2→6) linkages. A more descriptive
name would be (2→6)-ß-D fructans. Lavans are homopoly-
saccharides composed of monomers of D-fructose attached
by ß (2→6) linkages that carry a D-glucosyl residue at the
end of the chain. They constitute a series of homologus oli-
gosaccharides and polysaccharides, which can be considered
derivatives of sucrose.
Although the structure of levan is represented by a
straight chain of ß (2→6) linkages, many bacterial levans
are branched through ß (2→1) bonds. The branch chains
are usually short and sometimes consist of one fructose resi-
due. In general, levans produced by different organisms have
similar structures. The difference may be a varying degree
of polymerization and branching of the repeating unit. Le-
vans are one of the few natural polymers in which the car-
bohydrate exists in the furanose form. This feature plays an
important role in the final confirmation of the molecules in
the solution.
Properties of Levan
Levan is a white crystalline powder having properties like
a strong adhesive. The particles are spherical shape, densely
packed, 75-200nm in diameter and do not swell in water,
which differs from other polysaccharides. The composition
and properties of levan depend upon the environment in
which the microorganisms are grown. The general properties
of levans resemble those of dextrans. Levans are levorota-
tory, amorphous or microcrystalline, of varying solubility in
cold water. They are very soluble in hot water and insoluble
in absolute ethyl alcohol. Levans are generally more soluble
than inulin, which is almost insoluble (<0.5 percent) in wa-
ter at room temperature. The high solubility of levan may
be a characteristic of the ß (2→6) linkage compared to the
ß (2→1) linkage. Branching may be only a support factor.
Levans are non-reducing, not hydrolyzed by yeast invertase
and amylase action, but very susceptible to hydrolysis by
acid. They are not colored by iodine, but hydrogen chloride
imparts a purple color that distinguishes them from other
polysaccharides not containing fructose. The molecular
weight and viscosity in aqueous solution increases sharply
when various salts are present, but it decreases with a small
increase in temperature (Kang and Cottrell 1979).
Certain biological properties of levan, such as tumor in-
hibition and stimulation and increase in cell permeability for
a cytotoxic agent (Leibovici and Stark 1984) have attracted
attention. These effects are partly caused by suppression of
a normal inflammatory response. Only levan with Molecular
weight 107 promotes infection; the effect is lost when poly-
mers degrade. Levan, given intravenously to mice, greatly
increases the virulence of intraperitonially injected bacteria.
This is partly caused by permeability and the prevention of
the escape of blood constituents into the peritoneal cavity.
World Aquaculture 61
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-
62 March 2011
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-
nostimulants or immunonutrients, which augment the im-
mune system of cultured aquatic animals. Immunomodula-
tion by dietary manipulation may, therefore, offer a novel
alternative to reduce the use of antibiotics and other drugs
in aquaculture, which in turn decreases the residual load of
those compounds in the food chain as well as in the aquatic
environment. Some researchers have reported increased fish
survival by using immunonutrients (Kawakami et al. 1998).
There are several polymers of carbohydrate and their
derivatives are being used as immunostimulants. Among
the most popular and effective immunostimulants is
β-glucan, found in the cell walls of plants, fungi and bac-
teria. Fructose-based polymers are also categorized as
the bioactive polysaccharides. Microbial levan is shown
to have immunostimulating properties. Dina et al. (2007)
studied the effects of dietary levan on the survival of Cy-
prinus carpio juveniles fed a feed containing levan at con-
centrations ranging from 0.1 to 1.0 percent. One hundred
percent survival was obtained with 0.5 percent levan in the
feed. Increasing the concentration to 1 percent probably
increased the antigenic load, leading to immunosuppres-
sion and thus, reducing the protection efficiency. This is
in agreement with Anderson (1992) who proposed that an
inadequate amount of immunostimulant will result in no
protection, whereas too much may cause immunosuppres-
sion. Levan activates the non-specific phagocytes, which is
important for reducing mortality in fish.
Recently, Gupta et al. (2008) reported that levan at 1.25
percent can be used as a dietary immunostimulant for
Labeo rohita juveniles. According to him, hemoglobin con-
tent and total leucocyte count were increased with dietary
supplementation of levan at 1 percent or more. An increas-
ing trend for total erythrocyte count was observed with in-
creasing levels of dietary levan. Lower levan-supplemented
groups showed a higher albumin/globulin ratio. As levan
supplementation was increased, there was a gradual increase
in serum lysozyme activity and respiratory burst activity
[nitroblue tetrazolium (NBT) assay] reduction values. The
highest lysozyme activity and NBT were observed in the diet
supplemented with 1.25 percent levan. No significant histo-
architectural changes were associated with dietary levan lev-
els. The relative survival percentage of juveniles after chal-
lenge with Aermonas hydrophila was the highest in the 1.25
percent fed group. These studies have been the only reports
submitted presenting data on levan application in aquacul-
ture, which should be explored in more detail with various
species of fish and shellfish.
Other applications of levan
In addition to the use of levan in foods, feeds, medicines,
and cosmetics, other rare uses of levan have been reported by
the chemical and biotech industries. Levan has been shown
to exert excellent cell proliferating, skin moisturizing and
skin irritation-alleviating effects as a blending component in
cosmetics. Levan derivatives, such as sulphated, phosphated
or acetylated levans, are asserted to be anti-AIDS agents.
In addition, levan is used as a coating material in a drug
delivery formulation. Levan has a number of industrial ap-
plications, such as a surfactant for household use and a gly-
col/levan aqueous two-phase system for the partitioning of
proteins. However, there are some limitations for the indus-
trial applications of levan as a result of its weak chemical
stability in solution and the complex process required for its
purification. Once those limitations are resolved, the market
for levan will gradually increase.
Future Prospects
To date, only a meager amount of information is avail-
able about the immunostimulatory efficacy of levan in the
diet. Further feeding trials are necessary before advocat-
ing the use of levan under immunosuppressive or stressful
conditions associated with intensive culture practices. The
immunostimulating effect of levan in L. rohita fingerlings
was found to be higher at the 1.25 percent dietary level but
the optimum was 0.5 percent for Cyprinus carpio juveniles.
However, the effect of a higher inclusion level on immunity
is not known and needs further study. The route of admin-
istration of levan may also affect immunity as reported in
several studies in the case of glucan. Hence, both route of
administration along with dosage may boost the immune
system. It appears that microbial levan is a potential immu-
nostimulant in aqua-feed but the dose depends on the spe-
cies and the type of management practices.
Conclusion
The research on prebiotics and immunonutrients is
mounting with the ever-increasing need for environmentally
friendly aquaculture. Even though some information on the
use of microbial levan as a prebiotic and immnonutrient has
been documented, it is limited to a few species. More precise
research information on the application of microbial levan
could provide an eye opening direction for betterment of
the aquaculture industry. In this article, we have tried to in-
corporate some valuable information on the present status
and future prospects of microbial levan and its use in aqua-
culture.
Notes
1
Central Institute of fisheries Education, Seven Bungalow, Versova,
Mumbai-61, India
*Corresponding author: pronobjaan80@gmail.com. Phone:
+919769733660, Fax: +912226361573
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(Continued on page 66)
World Aquaculture 63
 In the productive chain of Brazilian aquaculture, the feed
industry may be the link more capable of rapid growth. The
feed mills can increase production quickly enough to meet
the growing demand for feed and, because they are scattered
throughout the country, they can produce competitive feeds
with regional ingredients, despite being based on corn, soy-
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The main difficulties of the feed industry are the growing
gap between prices of agricultural commodities and byprod-
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don’t increase at the rate needed by the producers. Producers
of fish feed are experiencing tight profit margins and as are
farmers. Nevertheless, there is optimism, and many compa-
nies are setting up new factories in regions where production
is growing and where there is a supply of regional ingredients
that can make the feed cheaper and decrease freight costs.
According to Firetti and Sales (2007), the price of feed
has increased over the years in Brazil, from US$0.45∕ kg in
1996 to $0.52∕ kg in 2006, an increase of 15.81 percent. In
1999, the price of feed was higher, when a kilogram of feed
US$ 0.74. Sales et al. (2005) commented that since 2002,
national fish farming went through a restructuring process,
reflecting the worsening of the crisis and economic problems
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fish farmers from São Paulo, indicated that the crisis began
in 1999 when demand decreased, causing excess of fish on
the farms, and the production costs increased with rising
prices of the inputs (Scorvo et al. 2010).
Brazil has many opportunities, but also great challenges.
We need to make aquaculture profitable, employing many
people; but it needs to be sustainable and preserve the en-
vironment. With regard to nutrition, we need to develop re-
search to determine the real nutritional requirements of var-
ious species already farmed and the potential to be grown in
many different environments and systems.
Notes
1
Aquafeed Product Manager, Guabi Animal Nutrition Member of
the National Organizing Committee of World Aquaculture 2011.
2
Tacon, A.J.G. (personal communication) University of Las Pal-
mas, Gran Canaria, The Canary Islands, Spain
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