Leuconostoc mesenteroides NRRL B-742에 의한

Fructose 로부터Mannitol 생산

김병훈, 유선균, 정상귀, 조용익, 양수연, 김도만, 조동련, 조성용,

최창남, 김승재, 김창용*, 김민경*

전남대학교 공업기술연구소,  물질생물화학공학과*

Mannitol Production from Fructose

by Leuconostoc mesenteroides NRRL B-742

B. H. Kim, S. K. Yoo, S. G. Chung, Y. I. Cho, S. Y. Yang, D. M. Kim,

D. L. Cho, S. Y. Cho, C. N. Choi, S. J. Kim, C. Y. Kim*, M. K. Kim*

Engineering Research Institute, Chonnam National University

*Dept. material and Biochemical Engineering, Chonnam National University

Introduction

Consumer demand for functional foods that promote health is rapidly increasing.  Mannitol as GRAS (generally recognized as safe) is used as a food ingredient, anticaking, stabilizing, thickening, and texturizing and, as a medical treatment for reducing plasma hydrogen peroxide in patients undergoing coronary artery bypass graft surgery (Zager 1994, Yang at al. 1992).  Ability of heterofermentative lactic acid bacteria to reduce fructose to mannitol has been well known and in our laboratory we have found that Leuconostoc mesenteroides NRRL B-742 converts quantitatively fructose to mannitol (Martinez et al. 1963,,Vandamme et al. 1987, Yun et al. 1996).

The object of this study was to examine optimum condition for mannitol production by L. mesenteroides NRRL B-742.

Materials and Methods

The organism used was L. mesenteroides NRRL B-742 and culture medium used for mannitol production consisted of yeast extract  0.5(w/t)%; KH2PO4 3 g/l; FeSO4·H2O 0.01g/l; MnSO4·4H2O 0.01 g/l; MgSO4·7H2O 0.2 g/l; NaCl 0.01 g/l; CaCl2 0.05 g/l and fructose 5 %(w/v).  Fructose was sterilized separately. Batch fermentation was performed in a 8 l bioreactor(Hannil R&D. Korea) with 6 l working volume at a temperature 28℃ and 100rpm. For determination of the effect of aeration on conversion of fructose to mannitol, two fermentaions were performed at aeration (about 0.5 vvm) and non-aeration, respectively.  For determination of the effect of culture pH, culture pH was adjusted to 5.0, 6.0, and 6.5. The culture was inoculated 2 %(v/v) of initial fermentation medium.  Cell growth was measured by optical density at 660nm using a UV spectrophotometry. Quantative analysis of Fructose and mannitol were performed by thin layer chromatography(TLC) method. The TLC plate was irrigated two times with MeCN-EtOAc-1-propanol-water(85:20:20:15, v/v/v/v) at 20℃. The solvent was allowed to ascend to the top of the plate and the plate was removed and thoroughly dried between the two ascents. This method has been developed to separation aldoses and their corresponding alditols. A dipping detection system using alkaline silver nitrate-sodium thiosulfate was used to detect the small amounts of mannitol. The concentrations of fructose and mannitol were analyzed and calculated from area of spot using densitometry method (Total Lab., Phoretix).

Result and discussion

The production kinetics of mannitol by L. meseterides B-742 is illustrated  Fig. 1, (A) aerobic condition at pH 6.0, (B) anaerobic condition at pH 6.0, (C) anaerobic condition at pH 6.5, and (D) anaerobic condition at 5.0. Bioconversion of fructose of mannitol varied dependent on oxygen intensity.  No difference of growth on fructose was found between arobic (□max = 0.43) and anaerobic (□max = 0.44) status of fermentation, this trend was coupled rapid utilization of fructose. At both cases, fructose was consumed completely within 14 to 16 h fermentaion. The accumulation of mannitol was peaked at 14 h at aerobic and 20 h anaerobic, respectively. However, the amount of mannitol at anaerobic (29.2 g/l) was 1.3 fold higher that at aerobic (22.7 g/l). When L. meseterides B-742 remained at resting state (stationery phase), there found notable different fate of mannitol accumulation.  When bacteria rested, continuous aeration accelerated the decrease of mannitol much higher rate than non-aeration.  It indicates that fructose dehydrogenase catalyzes the reversible reaction between fructose and manitol (Sakai et Yamanaka, 1968).   Sequential study was performed for determine the effect of culture pH on conversion of fructose to mannitol.  Culture pH in two batch anaerobic fermentation was maintained at pH 5.0 and 6.5, respectively. The specific growth rate was not affected by varying culture pH, but the

(A)

(B)

(C)

(D)

Figure1. Batch fermentations of L. mesenteroides NRRL B-742. (A) culture pH 6.0 and aeration, (B) culture pH 6.0 and no-aeration, (C) culture pH 6.5 and no-aeration, and (D) culture pH 5.0 and no-aeration.

maximum biomass was the highest at culture pH 6.5. Interestingly, when bacteria cells were rested, no mannitol decrease was found at pH 5.0 and 6.5, it compared to culture pH 6.0.  The reason why the amount of mannitol kept constant at pH 5.0 could be explained that the activity of mannitol dehydrogenase for reduction of mannitol to fructose is the highest around culture pH 8.0 (Martinez, 1962). In culture pH 6.5, fructose was not consumed completely even when bacterial growth reached stationery phase, indicating that reduction of mannitol to fructose was suppressed due to unknown reason. The highest accumulation of mannitol during batch fermentation (20 h) without any loss was found culture pH 5.0 with no aeration.  At this time, conversion yield of fructose to mannitol was about Y = 0.78 (g/g) and productivity was P = 1.57 (g/h/l).

Table 1. Batch fermentations of L. mesenteroides NRRL B-742. (A) culture pH 6.0 and aeration, (B) culture pH 6.0 and no-aeration, (C) culture pH 6.5 and no-aeration, and (D) culture pH 5.0 and no-aeration. * : sepecific growth rate.

Run	culture pH	mannitol	productivity *growth rate
A	6.0	22.7	1.6 0.43
B	6.0	29.1	2.2 0.44
C	6.5	30.5	1.8 0.40
D	5.0	37.8	2.5 0.47

Reference

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