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Chapter 13

Properties of Levan and Potential Medical Uses

J. Combie

Montana Polysaccharides Corporation, 1910-107 Lavington, Rock


Hill, SC 29732
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Publication Date: June 22, 2006 | doi: 10.1021/bk-2006-0934.ch013

Levan is an unusual polysaccharide that does not swell in


water and that has an uncommonly low intrinsic viscosity.
Animal studies have shown levan can lower blood cholesterol
and a derivative of levan will increase calcium absorption. As
a strong adhesive and a water soluble film former, levan has
the potential to make a temporary coating or bandage.

© 2006 American Chemical Society 263

In Polysaccharides for Drug Delivery and Pharmaceutical Applications; Marchessault, R., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 2006.
264

The term "levan" was introduced over 100 years ago to describe the
exopolysaccharide produced by a Bacillus when grown on sucrose (I). While
the glucose portion of the substrate is used as a microbial energy source, the
fructose units are linked together to build levan, a natural polymer of fructose.
β-D-fructo-furanosyl residues are connected by β-2,6 linkages. Branching is
accomplished through occasional β-2,1 bonds. The degree of branching varies
with the organism used in production but has been reported as high as 20%.
Several dozen bacteria are known to produce levan, including species of
Acetobacter, Aerobacter, Azotobacter, Bacillus, Corynebacterium, Erwinia,
Gluconobacter, Mycobacterium, Pseudomonas, Streptococcus, and Zymomonas
(1,2). The molecular weights of microbial levans are usually greater than 0.5
million and occasionally as high as 40 million (3). Levans made by plants are
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much smaller with the molecular weight generally under 10,000.


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Properties of Levan

Adhesive

One interesting property of levan is the adhesive strength. Although sugar


based materials are often sticky, the adhesive strength of levan is significantly
greater than that of most other natural polymers. For example, when
polysaccharides were applied to ten sets of bare aluminum adherends, cured for
10 days at 35 °C and then tested for tensile strength, levan had an average tensile
strength of 991 psi. Under the same conditions, dextran had only half the tensile
strength at 479 psi. Polysaccharides commonly used for thickening such as guar
gum and xanthan gum, had even lower adhesive strengths at 63 and 33 psi
respectively. Entanglement of the branches extending from the surface of levan
spheres contributes to the cohesive strength of levan. It should be noted that all
materials tested were diluted only with water. No formulation was done to
enhance adhesive strength or other properties such as flexibility, fatigue
resistance and shrinkage (4).

Levan is water soluble but does not swell in water. It has potential use in
bonding of tablets when dissolution is desired shortly after ingestion. If a more
gradual breakup of the tablet is desired, a more water resistant fructan would be
useful. Indeed, there is another fructan with low water solubility. This fructan,
inulin, is chemically identical to levan, but bonding through the 2 and 1 carbons
(as opposed to the 2 and 6 carbons of levan) results in a largely water insoluble
compound. However, the adhesive strength of inulin is only about one-tenth that
of levan.

Although there are numerous methods for decreasing the water solubility of
a material, the moieties responsible for the adhesive properties of levan are also

In Polysaccharides for Drug Delivery and Pharmaceutical Applications; Marchessault, R., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 2006.
265
responsible for the interaction with water. Numerous attempts were required to
solve the dilemma. Ultimately, a cross-linking procedure was found to be the
most successful. The formulation is being optimized and will be published in the
near future.

Like most polysaccharides, levan is very resistant to solvents. Coupons


bonded with levan were soaked in d-limonene, methylethylketone or toluene for
48 hrs. The solvent-soaked levan bond retained full adhesive strength (4). This
property is particularly useful when bonding materials that may be exposed to
solvents during production or cleaning.

Spherical Shape
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Publication Date: June 22, 2006 | doi: 10.1021/bk-2006-0934.ch013

Levan is one of the few polysaccharides in which the carbohydrate ring is in


the furanose form. With greater flexibility than the more common pyranose of
most polysaccharides, the 5-membered ring permits repeated folding (5,6). The
end result is a densely packed spherical structure. In the case of the present
levan, approximately 10,000 fructans are joined and crowded into the small
sphere. The sphere diameter ranges from 50 to 200 nm in diameter.

Membrane Protection

In plants, fructans serve as carbohydrate storage compounds. Evidence


suggests they may also provide plant cells with enhanced drought tolerance and
freeze resistance. Studies to elucidate the mechanism of these properties have
begun to reveal the interaction between membrane components and the fructans.
Vereyken showed that both an intermediate sized levan (DP 125) and a low
molecular weight inulin protected the membrane barrier more effectively than
dextran during dehydration-rehydration cycles. It appears that levan is inserted
in the headgroup region between lipid layers. Experiments were done in vitro
using unilamellar vesicles of l-palmitoyl-2-oleoyl-5«-glycero-3-phosphocholine
(7,8). Similar studies have not been done on high molecular weight levans.

Low Intrinsic Viscosity

Despite a high molecular weight, levan has an exceptionally low intrinsic


viscosity, resulting from the compact spherical shape. Levan produced in this
laboratory by an unidentified species of Bacillus has a measured intrinsic
viscosity of 0.14 dl/gm. Compare this with the intrinsic viscosity of dextran at
about 1 dl/gm or that of polysaccharides typically used as thickeners which are
frequently over 10 dl/gm (5). The low viscosity facilitates application of levan
as an easily spread paint or as an aerosol, not subject to clogging of the nozzle.
When dry, a levan coating is hard, although brittle as reflected by the glass
transition temperature of 123 °C.

In Polysaccharides for Drug Delivery and Pharmaceutical Applications; Marchessault, R., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 2006.
266

Film Formation

Levan can be readily formed into a film. Not allowing the film to
completely dry by adding a small amount of plasticizer will keep the film
flexible. Mixing levan with another polysaccharide, curdlan, will also result in a
flexible film. Perhaps more unexpected, was the result of mixing levan with the
clay, montmorillonite. One part of montmorillonite was mixed with either 2 or 5
parts of levan. A flexible film was formed. The surprise was that the film was
water resistant. These properties suggest potential application as a flexible
coating or bandage. For a natural material, levan is quite heat stable with a
melting point of 225 °C. Although autoclave moisture will interfere with pre-
formed bonds, once excess moisture is removed, the levan regains its adhesive
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strength.
Publication Date: June 22, 2006 | doi: 10.1021/bk-2006-0934.ch013

Medical Applications of Levan

Calcium Absorption

Ingestion of certain sugar alcohols, oligosaccharides and polysaccharides is


known to enhance calcium absorption (9). Cyclic disaccharide derivatives of
levan or inulin, difructose anhydrides (DFA) have been the subject of recent
studies in Tomita's laboratory. Four difructose anhydrides have been identified.
One of them, D F A IV, can be made by growing certain species of Arthrobacter
and Pseudomonas on levan. D F A IV has about half the sweetness of sucrose
and a melting point of 177-178 °C, sufficient stability to permit use in many food
applications. It is not digested or absorbed from the intestine of the rat. Rats fed
D F A IV absorbed significantly more calcium than control animals (10). The
absorption was mainly in the small intestine and it was suggested these DFAs
have potential in preventing osteoporosis (11).

Lowering Cholesterol

Perhaps the most interesting health-related property of levan is its ability to


lower cholesterol levels. Several drugs are currently marketed for lowering
blood cholesterol but for some people, there is the possibility of adverse side
effects. Water soluble dietary fibers are often used as antihyperlipidemics but
the high viscosity of these vegetable gums makes them difficult to ingest a
sufficient amount. Levan overcomes both of these problems. Studies indicate
levan is a safe material (12,13,14) and the low viscosity simplifies formulation
for easy consumption.

Ishihara was among the first to establish the value of high molecular weight
levan as a hypocholesterolemic agent. He found that high molecular weight

In Polysaccharides for Drug Delivery and Pharmaceutical Applications; Marchessault, R., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 2006.
267

levan or a partial hydrolysate of levan could effectively lower blood cholesterol


and aorta lipid deposits in rabbits. Triglycerides and the amount of adipose
tissue were lowered significantly in rats on diets including levan or levan
hydrolysates (13). These studies were later confirmed by Iizuka and colleagues
(15). Taking these findings to the next step, Ishihara also claimed levan or levan
derivatives were an effective antiobesity agent. The low viscosity of the
solutions facilitated consumption of sufficient levan to be effective (preferably
100 mg levan or hydrolysate per kg of body weight). Animal studies showed no
acute or chronic toxicity (13).

Work in Yamaguchi's lab used a high molecular weight (ca. 2,000,000)


levan in a systematic testing done in rats. The animals were fed diets which
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included either 1% or 5% levan. Blood cholesterol fell 17% or 41%


Publication Date: June 22, 2006 | doi: 10.1021/bk-2006-0934.ch013

respectively. Neither triacylglycerol nor glucose was affected by the dietary


levan. Total sterol excreted in the feces of levan-fed rats was approximately
double that excreted by control animals. In vitro testing indicated levan was not
fermented by the selected bifidobacteria. It is possible that the mechanism by
which the cholesterol lowering effect is accomplished may differ between high
molecular weight levan and the low molecular weight fructooligosaccharides.
One possible mechanism is that the levan binds or entraps sterols in the intestine,
interfering with their reabsorption (16).

Additional Applications

Levan has long been known to be an antitumor agent (12, 17, 18). Multiple
mechanisms have been attributed to this activity. The host immune response is
modulated, there is a direct inhibitory effect on tumor cells and levan augments
the activity of other antitumor compounds (17,19). Administration of fructans
has been shown to reduce the incidence of carcinogen-induced pre-cancerous
lesions in rats (20).

Additional findings related to the immunomodulatory effect of levan,


include the fact that levan can delay the rejection of skin grafts (21) and reduce
the number of macrophages attaching to subcutaneously implanted foreign
bodies (22). Also, levan has been shown to decrease the accumulation of
polymorphonuclear leucocytes in an experimentally induced inflammatory lesion
(23). In actively sensitized animals, levan markedly reduced the incidence and
severity of allergic encephalomyelitis in guinea pigs (24).

Oligosaccharides have been found useful as prebiotics, metabolized by the


beneficial bifidobacteria and lactobacilli in the large intestine (25). Few studies
have examined the value of high molecular weight levan. However, it is known
that large levans can be fermented by these beneficial bacteria but not by the
undesirable Clostridium perfringens and E. coli (26).

In Polysaccharides for Drug Delivery and Pharmaceutical Applications; Marchessault, R., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 2006.
268

Like dextran, levan can be used to create two phase liquid systems, of
potential value in purification of biological materials (5).

Garegg et. al. tested high molecular weight levan derivatives and found a
number of potential applications. For example, in an assay for inhibition of
smooth muscle cell proliferation, the activity of the levan sulfate was one log
greater than for the commercial heparin used for comparison. They also found
levan sulfate effective in reducing virus growth in an in vitro test. Phosphated
levan caused water and certain solvents to gel. Suggested uses for this gelled
form of levan were in pharmaceuticals and as a fat substitute (27).
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Conclusions
Publication Date: June 22, 2006 | doi: 10.1021/bk-2006-0934.ch013

Levan is a spherical polymer of fructose with branches extending from the


surface. Entanglement of these branches contributes to the cohesive strength.
Unlike polysaccharides used as thickeners, levan does not swell in water and has
an intrinsic viscosity of only 0.14 dl/gm. Like some other polysaccharide-based
compounds, levan can enhance calcium absorption and host immune responses.
The mechanism by which levan effectively lowers blood cholesterol in animals
has not been fully elucidated. Additional preliminary findings suggest a variety
of medical applications for levan in the future.

Acknowledgement

The author gratefully acknowledges the support of the Strategic


Environmental Research and Development Program (SERDP) and the U.S.
Environmental Protection Agency for funding work reported here.

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In Polysaccharides for Drug Delivery and Pharmaceutical Applications; Marchessault, R., et al.;
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