박범식*, 김철호*,**, 장은경**, 장기효***, 송기방*, 이상기*
*한국생명공학연구원, **(주)리얼바이오텍, ***경희대학교 동서의학대학원
Buem-Seek Park*, Chul-Ho Kim*,**, Eun-Kyung Jang**, Ki-Hyo Jang*** , Ki-Bang Song* and Sang-Ki Rhee*
* Korea Research Institute of Bioscience and Biotechnology (KRIBB), ** RealBioTech Co., Ltd., Bioventure Center, KRIBB, *** Department of Medical Nutrition, Graduate School of East-West Medical Science, Kyung Hee University
The fructose homopolymer, levan, is found in plants and especially in bioproducts of microorganisms. Plant levans, graminans and phleins, have shorter residues (varies from 10 to approximately 200 fructose residues) than microbial levans of which molecular weights are up to several million daltons with multiple branches. Microbial levans are produced extracellularly from sucrose- and raffinose-based substrates by levansucrase (sucrose 6-fructosyltransferase, EC 22.214.171.124) from a wide range of taxa such as bacteria, yeasts, and fungi (Han, 1990; Hendry and Wallace, 1993). The production and utilization of levan in the industrial field have been strictly limited, and only a few papers have reported the production of levan using fermentation techniques. Recently, great interest in this fructan has been renewed to discover applications for levan as a new industrial gum in the fields of cosmetics, foods like dietary fiber and pharmaceutical goods.
2. Biotechnological Production of Levan
Microbial production of levan requires fermentation and handling of highly viscous solutions. The conditions for producing levan by growing cultures of bacteria vary according to the microorganisms used, but yields of levan production are fairly low, due to the utilization of sucrose as energy source, formation of by-products, low level of levansucrase production, and presence of levanase activity in bacteria. Theoretically, yield of levan production by the levansucrase is 50% when sucrose is used as the substrate. In addition, the recovery process of levan from the fermentation broth is often very difficult due to the high viscosity of levan. Routinely, the yields of levan production are less than 58% of the theoretical yield. The levan can be also formed in vitro by bacterial levansucrase, sucrose serves as fructosyl donor and the released glucose inhibits the levan formation. The inhibitory action is influenced by competition with the glucose moiety of sucrose for the enzyme activity.
3. Applications of Levan
Microbial levan itself displays a direct effect on tumor cells which is related to a modification in the cell membrane including changes in cell permeability (Calazans et al., 2000), as well as radioprotective and antibacterial activities (Vina et al., 1998). Moreover, levan derivatives were suggested for medical applications as inhibitors of smooth muscle cell proliferation, as recipients in making tablets and as agents to transit water into gel. Levan derivatives such as sulfated, phosphated and acetylated levans were developed as anti-AIDS agents (Clarke et al., 1997).
The water-soluble polymers such as cellulose derivatives, pectin and carrageenan play key roles in the formulation of solid, liquid, semisolid and even controlled release of dosage forms (Guo et al., 1998). The viscosity of levan varies with its DP and degree of branching (Numbers of side fructose chains which are attached to one fructose unit in the main fructose chain.). In this respect, levan is applicable in pharmaceutical formulations for various purposes. Low-molecular weight, less branched levan usually give the low viscosity. This type of levan is used as a tablet binder in the immediate-release dosage forms, and levans with medium- and high-viscosity grades are used in the controlled-release matrix formations. Levan also has a great potential as a substitute for blood expanders (Gamal et al., 1974).
Microbial levans were introduced in plants to promote the agronomic performances of plants in the temperature zone and their natural storage capacities (Vijn and Smeekens, 1999). The transgenic tobacco plants expressing levansucrase genes from B. subtilis (Pilon-Smits et al., 1995) or Z. mobilis (Park et al., 1999) showed an increased tolerance against drought and cold stresses. The transgenic plants accumulating fructan were suggested as novel nutritional feed for ruminants (Biggs and Hancock, 1998). Recently, the microbial levan produced enzymatically was also developed as an animal feed (Rhee et al., 2000). Microbial levan could be used as a soil conditioner improving germination of various seeds (Gamal et al., 1974).
Novel applications of levan have been suggested in various aspects, particularly in food (Han, 1990; Suzuki and Chatterton, 1993). Levan acted as a prebiotic changing the intestinal microflora and this is offering beneficial effects when present in the human diet. Levan and its partially hydrolyzed products were fermented by intestinal bacteria including bifidobacteria and Lactobacillus species (Marx et al., 2000). Nowadays, it appears to be the night moment for the application of levan into dairy industry since L. reuteri produces levan-type EPS. EPS-producing lactic acid bacteria, including the genera of Streptococcus, Lactobacillus, and Lactococcus, are used in situ to improve the texture of fermented dairy products such as yogurt and cheese. This group of food-grade bacteria produces a wide variety of structurally different polymers including levan with potential use for new applications. A number of Japanese companies use microbial levans as additives in their milk products containing Lactobacillus species. In addition, the replacement of thickeners or stabilizers which are produced by non-food-grade bacteria, with levan has emerged (Van Kranenburg et al., 1999). The cholesterol- and triacylglycerol-lowering effects of levan have been reported by Yamamoto et al. (1999) and could be applied to develop levans as health foods or nutraceuticals.
4. Outlook and Perspectives
Levan has a great potential as a functional biomaterial in food, feed, cosmetic, pharmaceutical and other industries. However, use of this biopolymer has yet not been practical due to the scarce information about its polymeric properties required for industrial applications and lack of feasible processes for large scale production. For technical applications, fructans with high molecular mass and low degree of branching would be desirable. However, microbial levans and their oligomers have been less well characterized in the area of carbohydrate structure analysis. In order to utilize versatile water-soluble levans, a broader understanding of the behavior of levan is required. For example, the fundamental rheological properties of levan in solutions such as viscosity, thixotropy, dilatancy, elasticity, pseudoelasticity and viscoelasticity will become more important for new applications of levan.
Biggs, D.R. and Hancock, K.R., "In vitro digestion of bacterial and plant fructans and effects on ammonia accumulation in cow and sheep rumen fluids", J. Gen. Appl. Microbiol., 44, 167 (1998).
Calazans, G.M.T., Lima, R.C., de França, F.P. and Lopes, C.E., "Molecular weight and antitumour activity of Zymomonas mobilis levans", Int. J. Biol. Macromol., 27, 245 (2000).
Clarke, M.A., Roberts, E.J. and Garegg, P.J., "New compounds from microbiological products of sucrose", Carbohydr. Polym., 34, 425 (1997).
Guo, J.-H, Skinner, G.W., Harcum, W.W. and Barnum, P.E., "Pharmaceutical applications of naturally occuring water-soluble polymers", PSTT., 1, 254 (1998).
Gamal M. Imam and Nadia M. Abd-Allah, "Fructosan, a new soil conditioning polysaccharide isolated from the metabolites of Bacillus polymyxa AS-1 and its clinical applications", Egypt J. Bot., 17, 19 (1974).
Han, Y.W., "Microbial levan", Adv. Appl. Microbiol., 35, 171(1990).
Hendry, G.A.F. and Wallace, R.K., "The origin, distribution, and evolutionary significance of fructans", Science and Technology of Fructans, Suzuki, M. and Chatterton, N.J., eds., CRC Press, Boca Raton (1993).
Marx, S.P., Winkler, S. and Hartmeier, W., "Metabolization of -(2,6)-linked fructose-oligosaccharides by different bifidobacteria", FEMS Microbiol. Lett., 182, 163 (2000).
Park, J.M., K, S.Y., Song, K.B, K, J.W., Lee, S.B., Nam, Y. W., Shin, J.S., Park, Y.I., Rhee, S.K. and Paek, K.H. "Transgenic tobacco plants expressing the bacterial levansucrase gene show enhanced tolerance to osmotic stress", J. Microbiol. Biotechnol., 9, 213 (1999).
Pilon-Smits, E.A.H., Ebskamp, M.J.M., Paul, M.J., Jeuken, M.J.W., Weisbeek, P.J. and Smeekens, J.C.M., "Improved performance of transgenic fructan-accumulating tobacco under drought stress", Plant Physiol. 107, 125 (1995).
Rhee, S.K., Song, K.B., Yoon, B.D. and Kim, C.H., PCT-KR00-01556, 2000.
Suzuki, M. and Chatterton, N.J., "Science and Technology of Fructans", CRC Press, Boca Raton (1993).
Van Kranenburg, R., Boels, I.C., Kleerebezem, M. and de Vos, W.M., "Genetics and engineering of microbial exopolysaccharides for food: approaches for the production of existing and novel polysaccharides", Curr. Opin. Biotechnol., 10, 498 (1999).
Vijn, I. and Smeekens, S., "Fructan: More than reserve carbohydrate? ", Plant Physiol., 120, 351 (1999).
Vina, I., Karsakevich, A., Gonta, S., Linde, R. and Bekers, M., "Influence of some physicochemical factors on the viscosity of aqueous levan solutions of Zymomonas mobilis", Acta. Biotechnol., 18, 167(1998).
Yamamoto, Y., Takahashi, Y., Kawano, M., Iizuka, M., Matsumoto, T., Saeki, S. and Yamaguchi, H., "In vitro digestibility and fermentability of levan and its hypocholesterolemic effects in rats", J. Nutr. Biochem., 10, 13 (1999).