Properties of Levan and Potential Medical Uses - American Chemical

Chapter 13

Properties of Levan and Potential Medical Uses

Downloaded by COLUMBIA UNIV on July 27, 2012 | http://pubs.acs.org Publication Date: June 22, 2006 | doi: 10.1021/bk-2006-0934.ch013

J. Combie Montana Polysaccharides Corporation, 1910-107 Lavington, Rock Hill, SC 29732

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 much smaller with the molecular weight generally under 10,000.

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


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 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.


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 preformed bonds, once excess moisture is removed, the levan regains its adhesive strength.

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


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 included either 1% or 5% levan. Blood cholesterol fell 17% or 41% 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).


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).

Conclusions 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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11. Saito, K . ; Kondo, K . ; Kojima, I.; Yokota, Α.; Tomita, F. Appl. Environ. Microbiol., 2000, 66, 2252-256. 12. Calazans, G . ; Lopes, C.; Lima, R.; de Franca, F. Biotechnol. Lett, 1997, 19, 19-21. 13. Ishihara,K. U . S. Patent 5,527,784, 1996. 14. Rolant, F.; Herscovici, B.; Wolman, M . Biochem. Exp.Biol.,1911, 13, 187-191. 15. Iizuka, M . ; Minamiura, N . ; Ogura, T. In Glycoenzymes. Ohnishi, M . , Ed.; Japan Scientific Societies Press: Tokyo, Japan, 2000; pp 241-258. 16. Yamamoto, Y . ; Takahashi, Y . ; Kawano, M . ; Iizuka, M . ; Matsumoto, T.; Sacki, S.; Yamaguchi, H . J. Nutr. Biochem. 1999, 10, 13-18. 17. Leibovici, J.; Stark, Y . ; Eldar, T.; Brudner, G.; Wolman, M . Recent Results Cancer Res. 1980, 75, 173-179. 18. Yoo, S-H.; Yoon, E . ; Cha, J.; Lee, H . Int. J. Biolog. Macromol.2004,34, 37-41. 19. Leibovici, J.; Stark, Y . ; Wolman, M . J. Exp. Pathol. 1983, 64, 239-244. 20. Rowland, I. In Fructan 2004; Arrieta, J., Ed.; Elfos Scientiae: Havana, Cuba, 2004;p120. 21. Leibovici, J.; Bleiberg, I.; Wolman, M . Proc. Soc. Exp. Biol. Med. 1975, 149, 348-350. 22. Papadimitriou, J.; Robertson, T.; Wolman, M . ; Walters, M . Pathology. 1978, 10, 235-241. 23. Sedgwick, Α.; Rutman, Α.; Sin, Y . ; Mackay, Α.; Willoughby, D. Br. J. Exp. Pathol. 1984, 65, 215-222. 24. Berman, Z.; Leibovici, J.; Wolman. Isr. J. Med. Sci. 1976, 12, 1294-1297. 25. Ritsema, T.; Smeekens, S. Curr. Opin. 2003, 6, 223-230. 26. Kang, S.; Park, S.; Lee, J.; T. J. Korean Soc. Food Sci. Nutr. 2000, 29, 3540. 27. Garegg, P.; Roberts, E . Patent WO9803184, 1998.


Properties of Levan and Potential Medical Uses - American Chemical

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