307 Annals Agric. Sci., Ain Shams Univ., Cairo, 54(2), , (Received August 23, 2009) (Accepted September 6, 2009)

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1 37 Annals Agric. Sci., Ain Shams Univ., Cairo, 54(2), , 29 DEXTRAN PRODUCTION BY SOME LOCAL Leuconostoc mesenteroides STRAINS [24] Abdel-Azeem, Hoda 1 H.M.; Gehan F. Galal 2 and Enas A. Hassan 2 1- Desert Research Center Unit of Microbiology, Matariya, Cairo, Egypt 2- Department of Agricultural Microbiology, Faculty of Agriculture, Ain Shams University, Shoubra El-Kheima, Cairo, Egypt Keywords: Dextran, Leuconostoc mesenteroides, Viscosity, Nutritional requirement, Environmental factor, Static batch culture ABSTRACT Eighty dextran-producing bacteria were obtained from different food stuffes and tested for dextran production. These isolates were classified into four categories namely, high, moderate, weak and inactive according to their efficiency to produce dextran. The most efficient dextran-producing isolates (first category) were presented in the percentage of 7.5% and isolated from sugar cane juice, tomato juice, milk and infected lettuce. McCleskey s medium was superior for dextran production by these isolates than other tested media. The production of dextran was obtained during the end of logarithmic phase till the start of stationary phase (1-16 hr). Only one isolate (No. IL 1) recorded the highest dextran yield as well as conversion coefficient on the natural medium (WBE 3). This isolate was completely identified as strain of Leuconostoc mesenteroides subsp. mesenteroides. The effect of some nutritional and environmental requirements of the dextran production by L. mesenteroides (IL 1) using surface culture technique were studied. The results indicated that the maximum dextran production was obtained by growing the isolates on wheat bran medium containing 1 ml L -1 wheat bran extract (5%), sucrose 1%, yeast extract.5%, tryptone.25%, K 2HPO 4.5% and ph = 8. then incubated at 25ºC for 16 hrs. INTRODUCTION Dextran is a polysaccharide consisting of glucose monomers linked mainly (95%) by (1-6) bonds. Commercial application for dextran is generally in the pharmaceutical industry, but new applications are being considered in the food and textile industries (Shamala and Prasad, 1995). The effect of nutritional requirements and environmental factors on the production of dextran by Leuconostoc mesenteroides growing in different media as a batch culture was studied by many investigators (Santos et al 25, Kim et al 2 and Son et al 28). The bacterium is grown in a sucrose-rich media releasing an enzyme, dextransucrase, which converts excess sucrose to dextran and fructose, when high sucrose concentration is used to produce dextran and fructose, broth viscosity becomes high and process control becomes more difficult. Acceptor molecules, such as maltose present in the culture media can influence dextran molecular weight by allowing the growing chain to be separated from the enzyme active site and transfer to the acceptor (Dols et al 1998). Santos et al (25) found that dextran molecular weight decreases when complementary sugar (maltose, lactose and galactose) together with sucrose were used. Whereas, Kim and Robyt (1995) get four mutants produced extracellular dextransucrases on glucose with higher activities ( times) than the parent strain on sucrose. Differences in physical appearance, solubility and susceptibility to endo-dextranase hydrolysis of the (Received August 23, 29) (Accepted September 6, 29)

2 38 Abdel-Azeem, Hoda; Gehan Galal and Enas Hassan dextrans prepared by different mutants grown on glucose and sucrose were found. In fermentation by Leuconostoc sp., the factors that affect dextran production include the type and concentration of salts, temperature, ph and sucrose concentration (Kim et al 2). Also, Son et al (28) stated that the type and production of dextran were greatly affected by the sucrose concentration, food ingredients and fermentation time. The culture broth with the highest consistency, was obtained from the defined medium containing 2% sucrose, 1.5% skim milk and.5% potato powder and it showed typical pseudoplastic behavior with a ph of 4.7. Santos et al (2) stated that the optimum conditions for dextran and fructose production were T = 35ºC and ph 6.5. Whereas, Kim et al (23) reported that the temperature had very little effect on the dextran molecular weight but it has a significant effect on the degree of branching, at 4ºC and increased to 14.7% at 45ºC. Both the molecular weight (Mw) and the degree of branching not significantly affected by different ph values between 4.5 and 6.. Shamala and Prasad (1995) noted that temperature of 23-26ºC unaerated fermentation of 1-2 % sucrose by L. dextranicum FRW-1 increased culture viscosity. The aim of the present study is to evaluate the potential of using local bacterial strains for dextran production. The study focused on isolation of dextran producing bacteria from different foodstuffs and identified as wells as, optimization conditions for dextran production using static, batch culture technique. 1. Samples MATERIALS AND METHODS Sample of sugar cane, Apple, Tomato juices as well as cucumber, lettuce, milk and pickles (lemon & carrot) were collected from local markets of Cairo, Egypt. The samples were directly transferred into the laboratory for the isolation of dextran producing bacteria. 2. Media used - McCleskey s medium (McCleskey et al 1947) was used for isolation and dextran production from dextran- producing bacteria. - Kurt s medium (Kurt and Curt, 1983) was used for dextran production. - Wheat bran extract media (Shamala and Prasad, 1995). Three media containing wheat bran extract (WBE1, WBE2 & WBE3 media) were used for dextran production. - WBE1 medium containing sucrose, 1 g; Yeast extract, 5 g and WBE, 1 ml/l. - WBE2 medium containing sucrose, 1 g; Tryptone, 1.5 g; and WBE 1 ml/l. - WBE3 medium containing sucrose, 1g; Y. extract, 5 g; Tryptone, 2.5 g; K 2HPO 4, 5 g; WBE, 1 ml; Distilled water, 9 ml and ph 6.7. Wheat bran extract (WBE) was prepared by soaking 1 g of wheat bran in 8 ml distilled water under stirring for 1 h. The soaked slurries were filtered and the residues were washed and filtered again to collect 8 ml filtrate, the ph of extract was adjusted to 5.5, heated till boiling,cooled and centrifuged at 8 rpm. - Preservation medium (Shamala and Prasad, 1995) was used for propagation and preservation of different dextran producing cultures. 3. Isolation of dextran producing bacteria The representative samples of infected cucumber and infected lettuce (each weighing 1 g) or juices, milk and pickles lemon (1 ml each) were mixed or diluted by 1 ml sterile tap water and thoroughly shaken for 1 minutes. Streak and pour plate methods were used for isolation of dextranproducing bacteria from these samples on McCleskey s medium.the developed colonies on the plates (incubated at 25ºC for 1-3 days) were picked under aseptic conditions, purified and microscopically examined. Isolates were maintained on slants of the preservation medium at 5ºC. 4. Maintenance of cultures Stock culture slants were maintained at 5ºC on preservation medium after incubation at 25ºC for 24 hr. 5. Standard inoculum Standard inoculum was prepared by inoculation of conical flasks (25 ml in volume) containing 5 ml of preservation medium with a loop of tested culture. The inoculated flask was incubated at 25ºC for 24 hrs. The content of this flask was used as a standard inoculum (O.D. ranged from.2 x 1 to.3 x 1) for production experiments. 6. Screening for efficient dextran producing bacteria All isolates were tested for their culture viscosities to select the most efficient dextran-producing

3 Dextran production 39 isolates.conical flasks 25 ml containing 1 ml McCleskey s broth were inoculated with the tested isolate and incubated at 25ºC for 72 hrs as a static batch culture, 1 ml representative sample was withdrawn every day for testing viscosity by viscometer. Efficient isolates were selected for further studies. 7. Selection of suitable medium for dextran production Five media for dextran production being McClesky s medium (McCleskey et al 1947), Kurt s medium (Kurt and Curt, 1983) and WBE, media No. 1,2 & 3 (Shamala and Prasad, 1995) were used in this experiment for dextran production in order to select the most suitable media for screening high dextran production. The fermentation and determination of culture viscosity were done as mentioned before. 8. Biological activity of dextran producing bacteria In this investigation, the most efficient dextranproducing isolates were grown in Erlenmeyer flasks (5 ml in volume) containing 1 ml suitable medium and incubated at 25ºC for 24 hrs as a static batch culture. Samples (1 ml) were taken from the growing culture periodically every 2 hrs under aseptic conditions to determine the optical density of growth, culture viscosity and ph. The relation between time and optical density (growth curve) was plotted on semi-log paper. Growth parameters such as specific growth rate, hourly growth rate, generation time, multiplication rate and number of generation were calculated from the exponential phase. At the end of fermentation period (24 hr), 1 ml sample of culture fluid were diluted with distilled water to 5 ml and mixed to reduce the viscosity and then centrifuged at 14 rpm for 3 minutes, the sediment (bacterial cells) was washed twice with distilled water, then dried at 7ºC to a constant weight. The residual sugar was determined in the supernatant. Dextran produced was precipitated by ethanol from supernatant, dried and determined as dry weight. Dextran yield, conversion coefficient, productivity, specific viscosity rate, hourly viscosity rate, sugar utilization efficiency and yield factor were calculated. 9. Ultraviolet irradiation According to the method of Carlton and Brown (1981), vegetative cells (in phosphate buffer suspension) were dispensed into 5 ml aliquots in a sterile glass Petri dishes (8 mm in diameter) then exposed to a prewarmed (15-2 minutes before use) short-wave ultraviolet lamp (254 nm) at 2 cm distance. The bacterial suspensions in Petri dishes were stirred with vigorous magnetic stirring for 1,2,3,4 and 6 minutes. Immediately after irradiation, 1 ml sample of each treatment, suspended into 9 ml nutrient broth and incubated for two hours, under dim light to avoid photoreactivation, at 32ºC to allow segregation of the newly mutated chromosome from the nonmutated one in the same cell. Three replicates for each exposure period were carried out. After 2 hours, each irradiated bacterial culture was serially diluted and plated onto nutrient agar. 1. Factors affecting dextran production The propagation was carried out in Erlenmyer flask (25 ml in volume) containing 1 ml medium. These flasks were inoculated with 5 ml standard inoculum and incubated for 16 hrs at 25ºC. At the end of incubation period, viscosities of cultures, growth density as optical density and final ph values were determined Sucrose concentrations Three trial of sucrose concentrations, i.e 1, 2 & 3% were used to study their effect on dextran production by L. mesenteroides subsp. mesenteroides (IL1) and its mutants Nitrogen source The nitrogen sources applied were yeast extract, tryptone, peptone, casein, beef extract, ammonium phosphate, ammonium sulphate, ammonium nitrate, ammonium chloride and a mixture of yeast extract and tryptone. WBE3 medium without the nitrogen source was used as a basal medium. The amount of nitrogen compound added to the media was calculated to give a final concentration of 1.2 g nitrogen/l Wheat bran concentrations Different wheat bran concentrations ranging from 5 to 2% were used to study their effect on dextran production Wheat bran extract s volume Different wheat bran extract volumes ranged from 2.5 to 2% were added to WBE3 medium in order to select the best treatment for dextran production by L. mesenteroides subsp. mesenteroides.

4 31 Abdel-Azeem, Hoda; Gehan Galal and Enas Hassan 1.5. Initial ph Eleven levels of initial ph (ranging from 4.5 to 9.5) were chosen for studying their effect on dextran production Incubation temperature Six different degree temperature, i.e. 2, 22, 24, 26, 28 and 3ºC were investigated to detect the optimum temperature for dextran production Inoculum size Different volumes of standard inoculum ranging from 1 to 6 ml (O.D ranged from.2 x 1 to.3 x 1) were used to inoculate 1 ml WBE3 medium to detect the suitable inoculum size. Then followed the previous procedures till the end of the incubation period in order to determine viscosity, optical density and ph of cultures. 11. Chemical determination - Dextran was determined either viscometrically or by dry weight culture. Viscosity as an index of dextran production was measured at 25ºC with a (Cole-C Parmer) rotational viscometer at a constant speed of.6 rpm using spindle number 5. - The method of Bailey and Oxford (1958) was followed for precipitation and purification of dextran. - Total sugar was determined using Anthrone method according to Trevelyan and Harrison (1956). 12. Calculation The specific growth rate (µ) and doubling time (td) were cauculated from the exponential phase according to Painter & Marr (1963). Yield factor, carbon utilization efficiency and dextran parameters (conversion coefficient, yield and productivity) were also calculated. RESULTS AND DISCUSSION 1. Isolation and selection of dextran-producing bacteria In this study, eighty dextran producing bacteria were isolated from different foodstuffs and tested on McCleskey s medium for dextran production. These isolates were characterized by forming round, convex and slimy colonies with smooth margins. Cells were gram-positive cocci, nonmotile and occurred in pairs or chains. Table (1) shows that the highest percentage of bacterial isolates was obtained from sugar cane juice followed by pickles lemon being 43.75% and 18.75%, respectively. 1% of the total isolates were isolated from tomato juice or infected cucumber. The lowest percentage of isolates was obtained from apple juice or infected lettuce being 5%. The culture viscosity of these isolates varied from one isolate to another as well as fermentation times (from one to three days). So, these isolates were classified into four categories according to their culture viscosity produced. These groups namely, high, moderate, weak and inactive dextran-producing isolates which gave culture viscosity ranged from 4 to > 8 cp, 1 to > 4 cp, 1 to >1 cp and >1 cp, with the incidence percentage of 7.5, 15, and 31.25%, respectively. Also, it could be noticed that most efficient dextran-producing isolates were obtained from sugar cane juice, tomato juice, milk and infected lettuce which represent 3.75% (No. SCJ7, SCJ25 & SCJ28), 1.25% (No. TJ2), 1.25% (No. M3) and 1.25% (No. IL1) of the total isolates, so, these isolates were selected for further studies. 2. Selection of suitable medium for dextranproduction Data presented in Table (2) clearly show that the culture viscosities obtained by different cultures grown on McClesky s medium during 3 days fermentation period at 25ºC were higher than the other four tested media except isolate IL1 which gave approximately the same values of viscosity on wheat bran extract medium No. 3. The viscosity of McCleskey s medium varied greatly from one isolate to an other. The isolates No. IL1, TJ2, M3 and SCJ28 gave the highest culture viscosity being 779, 7785, 74 and 73 cp after the first day of incubation. Also, SCJ25 recorded the highest viscosity being 7792 cp after 3 days of incubation whereas SCJ7 gave the lowest figure on the same medium. So, it could be stated that McClskey s medium was chosen as preferable medium for dextran production by all isolates and also WBE3 medium was chosen for isolate No. IL1. Therefore these isolates were tested for their biological activity expressed as growth parameters and dextran parameters on their preferable media. 3. Biological activity of dextran-producing isolates Results illustrated by Fig. (1) clearly show that isolates grew exponentially during the first 2-1 hrs

5 Dextran production 311 Table 1. The percentage of active, moderate, weak and inactive dextran producing isolates from different foodstuffs Foodstuffs Total No. Isolates % High Moderate Weak Inactive No. % No. % No. % No. % Sugarcane juice Tomato juice Apple juice Milk Pickles lemon Infected cucumber Infected lettuce Total * High = Isolates producing culture viscosity ranged from 4 - >8. ** Moderate = Isolates producing culture viscosity ranged from 1 = > 4 cp * Weak = Isolates producing culture viscosity ranged from 1 to > 1 cp. ** Inactive = isolates producing culture viscosity ranged from 15 > 1 cp cp = Centipoises incubation period on McCleskey s medium. Thereafter the growth rate decreased gradually (phase of decleraing growth) to be more constant (stationary phase) at the last ten hours of incubation. The highest figures of growth (expressed as optical density) were recorded for IL1, M3 and SCJ28 isolates being 2.6, 2.21 and 2., respectively after 1 hrs of incubation at 25ºC on McClesky s medium. Also, these isolates recorded the highest figures of specific growth rate, hourly growth rate, number of generation and multiplication rate as shown in Table (3). On WBE3 medium, isolate No. IL1 grew exponentially during the first 2-24 hr to reach the maximum growth (as optical density) being 2.35 after 24 hr incubation at 25ºC. The growth parameters obtained by this treatment were.33 h -1, 1.354, 2.29, 3.49 h and.44 for specific growth rate, hourly growth rate, doubling time, number of generation and multiplication rate, respectively. Slight increase in culture viscosity of all the tested isolates was observed during the first 8 hrs of incubation, while the rate of increase was higher at the end of exponential phase and during stationary phase to reach the maximum value after 16 hrs. The highest range of culture viscosity was observed on McCleskey s medium by IL1 and TJ2 being cp and followed by M3 & SCJ28 being cp, whereas IL.1 gave 71 cp on WNE3 medium. Regarding to the relation between consumed sugar and growth or dextran production after 24 hrs on McCleskey s or WBE3 med., data presented in Table (4) clearly show that all the tested isolates consumed high amount of sucrose ranged between 6. to 74. gl -1 and recorded high percentage of sugar utilization efficiency. The lowest consumed sucrose was observed by IL.1 on WBE3 medium being 6 gl -1. After 24 hrs fermentation period, all the tested isolates gave biomass dry weight ranged from gl -1 whereas the amount of dextran dry weight ranged from gl -1. Although the isolates No. TJ2 and IL.1 gave the highest dextran yield and dextran productivity on McClesky s med., but isolate No. IL.1 gave the highest yield factor and conversion coefficient being 4.15% & 21.6% respectively on WBE3 medium. From the aforementioned results it could be noticed that the dextran production were concurrent with the growth during the end of logarithmic phase till the start of the stationary phase (1-16 hr) for all the tested isolates either on McCleskey s medium or on WBE3 medium. These results also indicate that 16 hr fermentation period was the favourest for dextran production by all the tested isolates. Inspite of isolate No. IL1 gave slight decrease in dextran production on WBR3 medium comparing to McCleskey s medium, but the former medium should be taken in consideration due to the low

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7 Culture viscosity (cp) Optical density (OD) Culture viscosity (cp) Optical density (OD) Culture viscosity (cp) Optical density (OD) Dextran production IL 1 in WBE medium Incubation period (hours) IL 1 in WBE medium Incubation time (hours) In McClesky medium ASJ 25 SCJ Incubation period (hours) In McClesky medium ASJ 25 SCJ Incubation time (hours) In McClesky medium M 3 TJ 2 IL Incubation time (hours) In McClesky medium Incubation time (hours) Fig. 1. Culture viscosity and growth curves of the most efficient dextran producing cultures grown on McClesky medium and wheat bran extract medium (WBE) during 24 hours incubation at 25 C M 3 TJ 2 IL 1 Table 3. Specific growth rate (µ), hourly growth rate (HGR), doubling time (td) number of generation (N) and multiplication rate (MR) of the most efficient dextranproducing isolates grown on McClesky s and WBE3 media at 25ºC Bacterial isolates µ (h -1 ) HGR td (h) N MR McClesky s medium SCJ25 SCJ28 M3 TJ2 IL1 WBE3 med. IL

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9 Dextran production 315 cost price of the constituent medium. Therefore, the isolate No. IL.1 was identified according to Bergey s Manual (1994) which proved to be Leuconostoc mesenteroides subsp. mesenteroides. 4. Ultraviolet irradiation Data presented in Table (5) show the survival percentage of L. mesenteroides (IL.1) after exposing at different times to UV (254 mm) at 2 cm distance. The survival percentages were 86.4, 65.6, 5., 2. and.1 after exposing to UV for 1, 2, 3, 4 and 6 minute respectively. All the mutant strains were tested for their ability to grow on different concentrations of sucrose namely 1, 2 and 3%. Five mutant strains varied in their behaviour. 5. Factors affecting the production of dextran The natural medium (WBE3) was used to select the most suitable ingredients and environmental factors for securing high dextran production by Leuconostoc mesenteroides (IL.1) and its mutants No. 1,2,3 & Sucrose concentration Data presented in Table (6) show that there was a gradual decrease in culture viscosity of all strains except mutant No. 4 (C6) and mutant No. 5 (A5) with the increase of sucrose concentration from 1% to 3% except L. mesenteroides mutants No. 4 (C6) and No. 5 (A5) which recorded the highest culture viscosity at 2 and 3% sucrose, respectively. The highest culture viscosity was obtained by L. mesenteroides (IL.1) and mutant No. 3 (C4) at 1% sucrose being 7213 and 6715 cp followed by mutant No. 4 (C6) and mutant No. 2 (B3) at 2% and 1% sucrose being 6698 and 631 cp, respectively. The lowest culture viscosity was observed by mutant No. 5 (A5) at all sucrose concentrations. The growth of all strains gave the same trend at different sucrose concentrations. The highest growth which expressed as optical density (O.D) was observed by L. mesenteroides (IL.1) followed by mutant No. 3 (C4) and No. 4 (C6) being 5. and 4.6 or 4.5, at 1% and 2% sucrose respectively. The corresponding figures of final ph values were 3.63, 3.79 and 4.8, respectively. Generally, it could be concluded that 1% sucrose was the best concentration for the growth and dextran production by L. mesenteroides (IL.1) and mutant No. 3 (C4), whereas 2% sucrose was suitable for the growth and dextran production by mutant No. 4 (C6). These results are in agreement with those of Son et al (28) who reported that the highest dextran yield was obtained at 2% sucrose Nitrogen source Generally, it could be stated that all the tested organic sources has a drastic effect on dextran production (viscosity), by all the tested strains comparing to the control treatment. The highest value of culture viscosity, ph & optical density were obtained by L. mesenteroides (IL.1) being 72 cp, 3.91 and 4.5, respectively (Table 7). Table 5. Survival percentage of Leuconostoc mesenteroides (IL.1) after exposing at different time to UV (254 nm) at 2 cm distance Time/min Dilution Not calculated Replicate No. I II III Average Survival rate (%)

10 316 Abdel-Azeem, Hoda; Gehan Galal and Enas Hassan

11 Dextran production 317

12 Optical density and final ph Culture viscosity (cp) Optical density and final ph Culture viscosity (cp) 318 Abdel-Azeem, Hoda; Gehan Galal and Enas Hassan Final ph Optical density (OD) Culture viscosity (cp) B Volume of wheat bran extract (5 %) ml Final ph Optical density (OD) Culture viscosity (cp) C Initial ph Fig. 2. Culture viscosity, final ph and optical density of Leuconostoc mesenteroides (IL1) as influenced by some nutritional and environmental factors i.e. wheat bran extract concentrations (A), volumes ofwheat bran extract (B), initial ph (C), temperature (D) and inoculum size (E) after 16 hours incubation period at25 C

13 Dextran production Wheat bran extract concentration Data illustrated by Fig. (2,A) show that no remarkable differences could be detected between culture viscosity and growth density values in WBE3 medium supplemented with wheat bran extract 5% or 1% (control on WBE3 medium). Increasing wheat bran extract more than 1% resulted in decrease in the yield of dextran and growth density values. So, 5% wheat bran will be added to WBE3 medium instead of 1% in the further studies. The aforementioned results are nearly similar to those obtained by Shamala and Prasad (1995) who stated that the maximum viscosity achieved by L. dextranicum FPW-1 was 69 Cp in 5% wheat bran extract (WBE), then decreased to 28 cp at 2% WBE Volume of wheat bran extract The effect of volume of 5% wheat bran extract on the growth and dextran production by L. mesenteroides (IL.1) was studied by increasing WBE volume from 25 to 2 ml/l. Data in Fig. (2,B) show that the culture viscosity of L. mesenteroides (IL.1) increased with increasing WBE volume till reaching the maximum value being 7647 cp in WBE3 medium containing 1 ml/l 5% wheat bran extract. Increasing the volume of WBE from 1 to 175 ml/l didn't increase the culture viscosity but 2 ml/l decreased the culture viscosity about 11.63%. Data also revealed that the highest growth density and ph values being 5.8 and 4.43 were recorded at higher volume of WBE being 2 and 15 ml/l, respectively Initial ph Results in Fig. (2,C) indicate that ph 8. was the most favourable one for dextran production by L. mesenteroides (IL.1). Moreover, bacterial growth was higher when initial ph ranged from 6.5 to 9. It seems therefore that the initial ph ranged between 7 to 8.5 was suitable for growth and dextran production. At initial ph ranged between 4.5 to 6. or 9.5 no viscosity was produced and lower bacterial growth was recorded. Veljkovic et al (1992) recorded that optimum ph for cell growth and dextran production ranged between 6. to Incubation temperature Data illustrated by Fig. (2,D) clearly show that the culture viscosity and growth density of L. mesenteroides (IL.1) were increased by increasing incubation temperature till reached the maximum being 7739 cp at 25ºC. It is interesting to notice that increasing the temperature from 25ºC to 26ºC decrease the yield of dextran about 14.2%. Generally, it could be noticed that the high efficiency of L. mesenteroides to produce dextran were attained at the low tested temperature which ranged between 2 to 25ºC. This means that high level of dextransucrase which converted sucrose to dextran was produced at lower temperature than was needed for maximal bacterial growth. These results are not in accordance with those of Kim et al (23) who stated that biosynthetic efficiency of dextran was attained at the same temperature of bacterial growth of Leuconostoc strains being 28ºC. Final ph values showed a sharp drop than initial ph (8.5) ranging between Finally, it could be recommended to incubate the culture of L. mesenteroides (IL.1) for 16 hours at 25ºC in static culture for optimum dextran production Inoculum size Results illustrated by Fig. (2,E) show that inoculation with 3-5% of standard inoculum gave the highest culture viscosity being 7767 cp by L. mesenteroides (IL.1) after 16 hours incubation period at 25ºC. The corresponding figures for growth density and final ph were and respectively. The variation in size of inoculum had a negative effect on ph values where the ph decreased by increasing the size of inoculum. REFERENCES Bailey, R.W. and A.E. Oxford (1958). A quantitative study of the production of dextran from sucrose by rumen strains of Streptococcus bovis. J. Gen. Microbiol., 19: Bergey s Manual (1994). Determinative Bacteriology, 7 th Ed. p John G. Holt; Noel R. Brieg; Peter, H.A. Sneath; James T. Staley; Stanley T. Williams ;Williams and Wilkins (eds.) Baltimore. Carlton, B.C. and B.J. Brown (1981). Gene mutation. In : Manual of Methods for General Microbiology, pp Gerhardt, P. ; R.G.E. Marray; R.N. Costilow; E.W. Nester; WA. Wod; N.R. Krieg and G.H. Phillips (eds.). American Society for Microbiology, Washington, DC Dols, M.; M. Remand-Simeon; R. Willemot; M. Vignon and P. Monsan (1998). Structural characterization of the maltose accepto-products synthesised by Leuconostoc mesenteroides NNRRK B1299 dextransucrase. Carbohydrate Res., 35:

14 32 Abdel-Azeem, Hoda; Gehan Galal and Enas Hassan Kim, D. and J.F. Robyt (1995). Production, selection and characteristics of mutants of Leuconostoc mesenteroides B-742 constitutive for dextransucrases. Enzyme. Microb. Technol. 17(8): Kim, D.; J.F. Robyt; S.Y. Lee; J.H. Lee and Y.M. Kim (23). Dextran molecular size and degree of branching as a function of sucrose concentration and temperature of reaction of L. mesenteroides B-512 FMCM dextransucrase. Carbohydrate Res. 338: Kim, P.; S. Thomas and H. Fogler (2). Effects of ph and trace minerals on long-term starvation of Leuconostoc mesenteroides. Appl. Environ. Microbiol. 66: Kurt, G. and E. Curt (1983). Dextran production by microorganisms or enzymes. European Pat. No AZ. McCleskey, C.S.; L.W. Faville, and R.O. Barnett (1947). Characteristics of Leuconostoc mesenteroides from cane juice. J. Bact. 54: Painter, P.R. and A.G. Marr (1963). Mathematics of microbial populations. Annual Rev. Microbiol. 22: Santos, M.; J. Teixeira and A. Rodrigues (2). Production of dextransucrase, dextran and fructose from sucrose using Leuconostoc mesenteroides NRRL B512 (F). Biochemical Engineering. J. 4: Santos, M.; A. Rodrigues; J.A. Teixeira (25). Production of dextran and fructose from carob pod extract and cheese whey by Leuconostoc mesenteroides NRRL B512 (F). Biochemical Engineering Journal, 25: 1-6. Shamala, T.R. and M.S. Prasad (1995). Preliminary studies on the production of high and low viscosity dextran by Leuconostoc spp. Process. Biochem. 3(3): Son, M.J.; E.K. Jang; O.S. Kwon; J.H. Seo; I.J. Kim; I.S. Lee; S.C. Park and S.P. Lee (28). Characterization of dextran produced from Leuconostoc citreum SS strain isolated from Korean fermented vegetable. Eur. Food Res. Technol. 226: Trevelyan, W.E. and J.S. Harrison (1956). Studies on yeast metabolism. I. Fractionation and micro-determination of cells carbohydrate. Biochem. J., 63: Veljkovic, V.B.; M.L. Lazic; D.J. Rutic; S.M. Jovanovic and D.V. Skala (1992). Effects of aeration on extracellular dextransucrase production by Leuconostoc mesenteroides. Enzyme Microb. Technol. 14: