IN VITRO STUDY OF SOME PREBIOTIC PROPERTIES OF LEVAN-TYPE EXOPOLYSACCHARIDE Ayla ŞENER*, Ayhan TEMİZ Hacettepe University, Department of Food Engineering, Beytepe 06800, Ankara, TURKEY INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSIONS Table 1. Effects of LEV and prebiotic substances on the growth and acidifying activity of La-5 Prebiotic substances Initial cell number (log cfu/ml) Viable cell number (log cfu/ml) after 24 h Increasing in viable cell number (%) pH NC* PC* INU % % % FOS % % % LEV % % % The results showed that the growth performance of La-5 varied with the type of prebiotic (Table 1). All tested prebiotics enhanced the growth of La-5 when compared with the basal medium (NC). At a concentration of 2%, FOS supported the highest growth. Supplementation with INU resulted in the next highest amount of growth. LEV also supported the growth of La-5 in comparison to the basal medium but was less effective than FOS and INU. As seen in Table 2, La-5 did not tolerate pH 1; no viable count was not obtained in any sample at this pH. But at pH 2, La-5 showed more acid tolerance when LEV used in the medium in comparison to FOS, INU and GLU. After 3 h incubation at pH 2, the viable count was 2.43 log cfu/ml for INU and 4.12 log cfu/ml for LEV. No viable count was obtained with FOS or GLU after 3 h incubation at Ph 2. The lag times (LT) in growth of La-5 caused by bile were 1.3, 1.9, 1.0 and 0.4 hours for GLU, FOS, INU and LEV, respectively. The shortest LT was for LEV. The bile resistance of La-5 was the highest when LEV was added to the medium. In conclusion, although LEV did not increase the number of bacteria as much as FOS and INU, it did increase the acid tolerance and decrease the lag time for Lactobacillus acidophilus La-5 growth. These results suggest the potential of LEV as a prebiotic under some conditions. * NC = negative control-the basal growth medium; PC = positive control-the basal growth medium supplemented with 2% glucose In recent years, the effects of prebiotics and probiotics have attracted the interest of both consumers and food manufacturers. Probiotics are benaficial bacteria that confer a health benefit on the host. Prebiotics are non-digestible food ingredients that selectively stimulate the growth and/or activity of probiotic bacteria (Gibson and Roberfroid, 1995). To provide health benefits, probiotics must reach sufficient numbers in the large intestine. Maintaining a proper equilibrium of the microflora in the gastrointestinal tract may be ensured by systematic supplementation of the diet with probiotics, prebiotics or symbiotics that is combination of a pro- and prebiotics. Impediments such as the acidic pH in the stomach, degradative enzymes and bile in the small intestine, and some adverse factors related to food products cause decreasing viability of probiotics. There are two general forms of fructans, levans and inulins. They are distinguished by the type of linkage between the fructose molecules. Inulin is formed by  (2→1) fructosyl-fructose links. Levan, found primarily in microbial products, although recently also found in various grasses and woody-stemmed plants, is formed by  (2→6) fructosyl- fructose links and have the side chains joined to backbone via  (2→1) linkages (Han, 1990). Levan has a low viscosity and high solubility in water. Levan has been shown to be an effective hypocholesterolemic drug, an immune modulator, an antitumour agent, an anti-in­flammatory material and a blood plasma extender. In the food industry, it has potential as a fructose source and for the production of fructooligosaccharides, thickeners, stabilizers, encapsulating agents, flavors and aroma carriers (Han, 1990; Yamamoto et al., 1999; Borsari et al., 2006). Because of the considerable effects of prebiotics on the viability and growth of probiotics, it is important to select suitable prebiotic substances to produce foods containing a combination of prebiotics and probiotics. The aim of this study was in vitro investigation of the effects of levan on the growth performance and on the acid and bile resistance of an important intestinal bacterium, Lactobacillus acidophilus La-5. The bacterial strain used in this study was Lactobacillus acidophilus La-5 obtained from Chr. Hansen. Carbohydrate-free MRS broth was used as a basal growth medium in all analysis. Levan (LEV) was obtained from Montana Polysaccharides Corp. Commercial preparations of prebiotics and carbohydrate used to compare with LEV were fructooligosaccharide (FOS; Dora/Orafti, Turkey), inulin (INU; Dora/Orafti, Turkey) and glucose (GLU; Merck).  Bacterial growth: LEV and prebiotic substances were tested at concentrations of 0.5%, 1% and 2% (w/v). La-5 was activated in MRS broth (Merck) and transferred (1%) into the basal growth medium supplemented with LEV, INU or FOS. Basal growth media with no carbohydrate or with 2% glucose were used for the negative control (NC) and the positive control (PC) respectively. Initial cell numbers were determined by pour plate method using MRS agar. After incubation at 37°C for 24 h, cell numbers in the culture media were again determined by pour plate method using MRS agar. Culture acidifying activities were determined using a pH meter. Three replicates were made for each test point. The effects of prebiotics on the growth performance of La-5 were evaluated according to the increasing in the number of viable bacteria using the following equation: Increasing in viable = Viable cell number after 24 h - Initial cell number x 100 cell number (%) Initial cell number  Acid resistance: La-5 was activated in MRS broth and subcultered in basal medium supplemented with 2% LEV, FOS, INU or GLU. After 24 h incubation, tubes were centrifuged at 3000 rpm for 15 min. The pellets were washed with sterile distilled water and resuspended in sterile distilled water. Then each culture suspension was transferred into flasks containing 0.05% sodium chloride solutions at pH 1 and 2. Hydrochloric acid (5 M) was used to adjust pH values and 0.05% sodium chloride solution (pH 5.35) was used as a control. The flasks were incubated at 37°C for 0, 0.5, 1, 2 and 3 h. After incubation periods, viable cell numbers in the flasks were determined by pour plate method using MRS agar.  Bile resistance: The bacterial culture was activated in MRS broth and subcultered in basal medium supplemented with 2% LEV, FOS, INU or GLU. After 24 h incubation, each culture was transferred (1%) into MRS broth supplemented with and without 0.5% bile (Oxbile, Merck). Inoculated media were incubated at 37°C and bacterial growth was monitored hourly by measuring absorbance at 620 nm using a UV-Visible spectrophotometer (Agilent 8453). Growth curves were plotted and the times required to attain an increase in the absorbance by 0.3 units were determined for both the control culture and the cultures with bile. The difference in time (hours) between the test culture media and the control was considered the lag time (LT). The delay in growth as a result of inhibition by bile was calculated. References Acknowledgements The authors wish to thank Joan Combie, Ph.D., Montana Polysaccharides Corp. for providing levan to use in this study. * address: SubstancepHViable count (log cfu/ml) 0 h½ h1 h2 h3 h GLU < < < <1 FOS < < < <1 INU < < < < LEV < < < < Table 2. Effects of LEV and prebiotic substances on acid rsistance of La-5  Borsari, R. R. J., Celligoi, M. A. P. C., Buzato, J. B., Silva, R. S. S. F. (2006). Influence of carbon source and the fermentation process on levan production by Zymomonas mobilis analyzed by the surface response method. Ciênc. Tecnol. Aliment., 26(3),  Han, Y. W. (1990). Microbial levan. Advances in Applied Micrbiology, 35,  Gibson, G.R. and Roberfroid, M.B. (1995). Dietary modulation of the human colonic microbiota: Introducing the concept of prebiotics. Journal of Nutrition, 125, 1401–1412.  Yamamoto, M., Y., Takahashi, Y., Kawano, M., Iizuka, M., Matsumoto, T., Saeki, S., Yamaguchi, H. (1999). In vitro digestibility and fermentability of levan and its hypocholesterolemic effects in rats. J. Nutr. Biochem. 10,