1. Optimization of Levan Production from Zymomonas mobilis and Effects of Plasticizers on Mechanical Properties of the Edible Levan Films Y. GOKSUNGUR *, T. BAYSAL*, S. DAGBAGLI * *, N. S. GIRAY***, S. ŞILBIR * *Ege University, Department of Food Engineering, Izmir, TURKEY * * Celal Bayar University, Department of Food Engineering, Izmir, TURKEY * * * Ministry of Food, Agriculture and Livestock, Food Control Laboratuvary, Izmir, TURKEY e-mail: selim.silbir@ege.edu.trselim.silbir@ege.edu.tr ABSTRACT Levan is a polymer of fructose linked by a β-(2→6) fructofuranosidic bond and found in many plants and microbial products. Microbial levan is of commercial importance, which offers a variety of industrial applications in the fields of cosmetics, foods and pharmaceuticals; it can be used as industrial gums, blood plasma extenders and sweeteners. Potential applications of levan have also been proposed as an emulsifier, formulation aid, stabilizer and thickener, surface-finishing agent, encapsulating agent, and carrier for flavor and fragrances. The production of levan from synthetic medium by Zymomonas mobilis NRRL B-14023 was investigated in flask cultures. The effect of different carbon concentration, pH and different nitrogen sources on levan synthesis was determined and the highest levan was obtained when 300 g/l saccharose concentration, pH 5.0 and yeast extract were used as nitrogen sources. From produced and optimized microbial levan an edible, active, food protective levan film obtained. Different sugars and polyols were used as plasticizers. Physical (thickness and appearance) and mechanical (tensile strength and percent elongation) properties of edible levan films has been also investigated. INTRODUCTION Levan is one of two main types of fructans, which are naturally occurring homopolymers of fructose. Its main chain is composed of repeating five-member fructofuranosyl rings connected by β(2/6) links (Figure 1). Branching of the main chain results when fructofuranosyl rings connect through β (2/1) linkages. Levans produced by different organisms differ in their molecular weight and degree of branching. Levans from plants generally have molecular weights ranging from about 2000 to 33,000 Da. Bacterial levans are much larger than those produced by plants, with multiple branches and molecular weights ranging from 2 to 100 million Da. Though plant fructans are available, many microorganisms like Zymomonas mobilis, Bacillus subtilis, Bacillus polymyxa, and Acetobacter xylinum also produce extracellular levan of high molecular weight when grown on sucrose. Levan can be used as emulsifier, stabilizer and thickener, an encapsulating agent, osmoregulator, cryoprotector makes levan attractive for application in many fields. MATERIALS AND METHODS Organism, culture media and materials Zymomonas mobilis NRRL B-14023 used throughout this study was kindly supplied by the U.S. Department of Agriculture, National Center for Agricultural Utilization Research. Medium composition for inoculum were (in g/l): Sucrose, 50; Yeast Extract, 7.0; KH 2 PO 4, 2.5; (NH 4 ) 2 SO 4, 1.6; MgSO 4.7H 2 O, 1.0. Medium composition for fermentation were (in g/l): Sucrose, 150-300; Yeast Extract, 2.5; KH 2 PO 4,1.0; (NH 4 ) 2 SO 4, 1.0; MgSO 4.7H 2 O, 0.5. The flasks were incubated at 28 °C for 48 h in incubator. Analytical techniques Levan concentration was measured using the 3,5-dinitrosalicylic acid (DNS) method. Total sugar was determined according to the phenol sulfuric acid method using sucrose as the standard. Biomass was determined by measuring optical density at 590nm. RESULTS AND DISCUSSION Levan production The production of levan from synthetic medium by Zymomonas mobilis NRRL B-14023 was investigated in flask cultures. The effect of different carbon concentration, pH and different nitrogen sources on levan synthesis was determined and the highest levan was obtained at 300 g/l sucrose concentration, pH 5.0 and yeast extract was used as nitrogen sources. Figure 5. Effect of pH on Levan Production (T=28 ˚C, Initial sub. con. = 150 g/l) Figure 4. Effect of initial sucrose concentration on Levan Production (pH=5.0, T=28 ˚C) Figure 6. Effect of Nitrogen Sources on Levan Production (pH=5.0, T=28 ˚C, Initial subs. con.= 150 g/l. The nitrogen sources were used on equal nitrogen bases corresponding to 2.5 g/l yeast extract.) 1.Yeast extract, 2. Corn Step liquor, 3. Pepton, 4. Tripton Levan Film Production Levan extracted from fermentation was dried at 28°C for 24 h. For levan film solution 2.5% levan was dissolved in water for 30 min. Then 12 ml of solution poured into the Petri dishes, dried in drying-oven at 50 °C for 24 h. Figure 2. Levan film with sorbitol Levan Film Production- Mechanical Properties Poured levan films can be seen in Figure 5 and Figure 6. Sorbitol and glucose were used as plasticizers. Physical and mechanical properties shown in Table 1. Tensile measurements for tensile strength, elongation were performed with the Instron testing system (model 4411, Instron Engineering Corp., Canton, MA) following the ASTM standard D882 (ASTM 1998b). Thickness of the films was measured using a hand- held micrometer at least 10 random positions on the film. Film-PlasticizerElongation (%)Tensile Strenght (MPa) AppearanceThickness (mm) 2.5% levan+2.5% sorbitol 32.37±3.4733.36±1.64Bright, clear, homogeneous. Can be used as film and coating 0,24 2.5% levan+2.5% glucose 25.54±3.6040.41±1.67Bright, clear, homogeneous. Can be used as film and coating but more brittle. 0,21 Figure 3. Levan film with glucose Table 1. Mechanical and physical Properties of Levan Films Figure 1. Structures of Levan