Leuconostoc mensenteroides NRRL-B512F를 이용한 Dextran 생산에서의

pH와 sucrose 농도의 영향

박준성, 박종만, 이재영, 김철경, 고창웅*, 김남기

성균관대학교 공과대학 화학공학과

삼척대학교 공과대학 화학공학과*

Effects of pH and sucrose concentration on the production of Dextran

by Leuconostoc mensenteroides NRRL-B512F

Joon-Seong Park, Jong-Man Park, Jae-Young Lee, Chul-Kyung, Kim,

Chang-Woong Koh*, Nam-Ki Kim

Department of chemical engineering, SungKyunKwan University

Department of chemical engineering, SamChok National University*

Introduction

Extracellullar polysaccharides produced by microorganisms offer a variety of useful and potentially low-cost. Such polysaccharides with widely different properties are available and more will be found. Therefore it is expected that fermentation polysaccharides will eventually be developed to fulfill many industrial needs and the production cost will be decreased as volume of production rises.

Dextran(C6H10O5) is an extracellullar polysaccharides cosisting of glucose monomer linked mainly □-1,6 bond. When the Leuconostoc mensenteroides NRRL-B512F is grown on the sucrose media and it produces an extracellular enzyme(dextransucrase). Sucrose is the only known for substrate able to induce this enzyme production.

dextransucrase

[eqn: n C_12 H_12 O_11 \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ → \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ (C_6 H_10 O_5 )_n \\\\\\\ + \\\\\\\ nC_6 H_12 O_6]                sucrose                         dextran                 fructose

Dextran has many industrial applications due to its non-ionic character and good stability under normal operating conditions. It is widely used as a blood volume expander in pharmaceutical industry and food inderstry and as a chromatographic media.

The effect of sucrose concentration and pH on the cell growth and the production of dextran were studied in a batch bioreactor with volume of 4.5L(total working volume is 3L). The pH variation from 6.0 to 6.4 and sucrose concentrations variation from 30g/L to 60g/L in this study.

Materials and methods

Leuconostoc mensenteroides NRRL-B512F from Korean Collection for Type Cultures(KCTC) was used in this study. The strain was stored at 4℃ in MRS agar slant. The fermentation inocula were prepared so that the final medium going into the fermenter was 8% (v/v) of the initial fermentation medium. The organnism was transfered from the stock culture to a similar medium but without agar. The inoculum was grown for 12 hours at 28℃ in shaking incubator.

Fermentation are carried out in a 4.5L jar fermenter(Korea Fermenter Company, Incheon, Korea) with control units for temperature, pH and agitation speed. The fermentation medium consist sucrose(30 and 60 g/L) with variable concentrations, yaest extract(10g/L), potassium phosphate(8g/L), and trace element solution(the constituents in the trace element are listed in table 1.)

Table 1. Constituents in the trace elements solution

Constituents	Concentration(g/L)
MgSO4·7H2O	40
NaCl	5
MnSO4·H2O	5
FeSO4·7H2O	5

Cell growth was monitored by measuring the absorbance at 660nm using spectrophotometer. Cell concentration was defined as cell dry weight per liter of culture broth. Dextran was obtained from fermentation solution by ethanol precipitation. And dextran was dried at vaccum oven(at 70℃, 15 hr). Dextran concentration was determined by dry weight(g/L).

The concentration of sucrose is determined by HPLC equipment(□ Porasil, 3.9mm×300mm, Waters, USA) using acetonitrile : water(80:20) as mobile phase and Carbohydrate analysis column.

Results and discussion

Fig.1, Fig.2 and Fig.3 show time profiles of cell concentration and dextran concentration.

At the end of the fermentaion A(Fig.1), 2.72g/L of the dry cell weight and 6.52 g/L of dextran concentration(at initial sucrose conc. = 30g/L), which were equivalent to 4.05 g/L and 15.24 g/L(at initial sucrose conc. = 60 g/L).

At the end of the fermentation B(Fig.2), 2.93g/L of the dry cell weight and 6.49 g/L of dextran concentration(at initial sucrose conc. = 30g/L), which were equivalent to 4.53 g/L and 14.82 g/L(at initial sucrose conc. = 60 g/L).

At the end of the fermentation C(Fig.3), 0.939g/L of the dry cell weight and 8.02 g/L of dextran concentration(at initial sucrose conc. = 30g/L), which were equivalent to 3.23 g/L and 19.31 g/L(at initial sucrose conc. = 60 g/L).

These results show higher sucrose concentration is good for cell growth and dextran production. Luconostoc mensenteroides NRRL-B512F is known for micro aerophilic so fermentation B was aerated at 0.1vvm. Fermentation B demonstrates that aeration was good for cell growth but it decreased dextran production. It can be seen aeration does not have an important effect in the fermentation results. To compared with fermentation A and C, cell growth of fermentation A is higher than fermentation C, but the dextran production of fermentation C is higher than fermentation A. It is considered that dextransucrase is more activated at pH 6.0 than pH 6.4.

Reference

1. R.L Whistler "Industrial Gums" Academic Press., pp. 1-19 (1993)

2. Shin, Y.C "Study on the production of exopolysaccharide pullulan from

Aureobasidium pullulans". Ph.D. Tesis in KAIST (1998)

3. T. R. Shamala, M. S. Prasad, "Preliminary studies on the production of

high and low viscosity dextran by Leuconosoc spp", Process Biochem.

30, pp237-244 (1995)

4.. Peppler, H, J. and D. Perlman, "Microbial technology", Academic press,

pp.464-467 (1979)

5. Robyt and Walseth, "Biotechnology Vol 3", p.555 (1983)

Fermentation B.

Fig.2 Time profiles of dry cell and dextran

concentration at pH = 6.4,  180rpm

and 28℃(aeration(0.1 vvm))

Fermentation A.

Fig.1 Time profiles of dry cell and dextran

concentration at pH = 6.4,

180 rpm and 28℃(noaeration)

Fermentaion C.

Fig.3 Time profiles of dry cell and dextran

concentration under at pH = 6.0

180 rpm and 28℃(noaeration)