Metabolism of fructose as an electron acceptor by Leuconostoc mesenteroides

f'rocess Biochemistry Vol. 33, No. 7, pp. 735-739, 199S © 1998 Elsevier Scicrlcc Ltd. All rights reserved Printed in Great Britain 11032-95~2/t,~8 $ see front mallei ELSEVIER

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S0032-9592(98~00041-7

Metabolism of fructose as an electron acceptor by Leuconostoc mesenteroides H. Erten Department of Food Engineering, Faculty of Agriculture, University of Cukurow~, 01330Adana. Turkey (Received 27 February 1998: accepted 8 March 1998)

Abstract

The utilisation of fructose as an electron acceptor was studied in Leuconostoc mesenteroides NCIMB 8023 anti Leuconostoc mesenteroides NCFB 811 under anaerobic conditions. Only small amounts of fructose as an electron acceptor were converted to mannitol. Both bacteria produced lactate, ethanol, acetate along with mannitol during the metabolism of fructose in the absence of oxygen. Fermentation of a mixture of glucose and fructose resulted in the production of the same metabolic end-products and small amounts of mannitol were formed. Small concentrations of acetate were also detected, e~ 1998 Elsevier Science Ltd All rights reserved Keywords: Leuconostoc mesenteroides, electron acceptor, fructose metabolism, mannitol production.

Introduction

Materials and methods

Leuconostoc spp. are heterofermentative lactic acid bacteria and ferment glucose to equimolar amounts of D-lactate, ethanol and COz under anaerobic conditions [1]. However, oxygen serves as an electron acceptor under aerobic conditions [2,3] and acetate is formed instead of ethanol in aerated cultures [4-8]. The heterofermentative lactic acid bacteria are not restricted to oxygen and anaerobically, other compounds such as fructose, pyruvate and glycerol can also be used as an alternative electron acceptor [6,8-10]. Leuconostoc (Leuc.) mesenteroides contains mannitol dehydrogenase to convert fructose to mannitol [11, 12]. It produces lactate, acetate, ethanol, mannitol and CO_~ from fructose [11,13]. Fructose is also metabolised to mannitol by Lactobacillus brevis and Lactobacillus buchneri [12]. Unlike Leuc. mesenteroides, these two bacteria are unable to form ethanol, but produce lactate, acetate, COz and mannitol [10-12]. The objective of present study was to examine utilisation of fructose as an electron acceptor in Leuc. mesenteroides during the metabolism of fructose and a mixture of glucose plus fructose under anaerobic conditions.

Organisms The organisms used were Leuc. mesenteroides NCIMB 8023 obtained from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland, UK and Leuc. mesenteroides NCFB 811 obtained from the National Collection of Food Bacteria, Reading, England, UK. The former was isolated from olives fermentation and the latter was from sauerkraut fermentation. The cultures were maintained in All Purpose Tween-Chalk semi Solid Preservation Medium [14]. Medium Medium Glucose Yeast Extract Amino acids (Medium GYA, pH: 6-4) was modified from Medium G Y A of Westby [15]. Medium G Y A contained (g/litre purified water): K2HPO4, 0.52; KH2PO+ 1.15; CaCI>6HzO, I).1)5; MgSO4.7H20, 0.2; NaCI, 0.1; yeast extract (Difco0127-01), 0.1; vitamin free casamino acids (Difco028801), 0.2; L-histidin, 3-1; glucose, I).9; and tracc metals mixture, 3 ml. Trace metals mixturc was prepared according to Owens & Keddie [16]. Medium 735

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G Y A was autoclaved at 121°C for 2 min and incubated at 30°C for 3 days to check the sterility. Medium FYA was similar to Medium GYA, but fructose, 5 mmol/litre, was substituted for glucose. Medium G F Y A was also similar to Medium G Y A but with the addition of fructose, 5 mmol/litre. Chemicals were obtained from Sigma Chemical Ltd or B D H Chemical Ltd, Poole, UK. They were analytical grade or the highest grade available.

H. Erten

Na2SO4 in high purity water at a flow rate of 0.5 ml/min. 24 pl of sample was injected into H P L C connected with a refractive index (Shodex, RI-71). Peaks were analysed using a model 3390A HP integrator (Hewlett Packard). Results and discussion

Dissimilation of glucose by Leuc. mesenteroides Growth conditions Cultures were grown in screw-capped 300ml bottles containing 250 ml medium under anaerobic conditions at 25°C. The head space was filled with sterile paraffin. The medium was deoxygenated by bubbling oxygen free mixture of 97% N2 and 3% H2 (British Oxygen Co. Ltd, London, UK) passed through an oxy-purge for 20 rain. lnocula were made as follows: two drops from a Pasteur pipette of each microorganism from stock culture, grown in the APT-chalk semi-solid preservation medium at 30°C for 2 days, were transferred to screw-capped 5 0 m l bottles including deoxygenated medium GYA, FYA, GFYA. The head spaces were filled with sterile paraffin. They were incubated for 24 h at 25°C. A 12.5 ml of inoculum from 24 h culture was added to the same medium and incubated at 25°C for 48 h. All experiments were carried out in duplicate.

Determination of concentrations of substrates and metabolic end-products Glucose, fructose, mannitol, lactate, ethanol and acetate were analysed by High Performance Liquid Chromatography (HPLC) according to Nuraida et al. [8] and Nuraida [13] using an Aminex HPX-87H column (Bio-Rad, Richmont, CA) with a cation H + guard column (Bio-Rad 125-0129). The column was held at 45°C. The eluent used was 0.01 N H 2 8 0 4 in high purity water (conductivity _<0.1 lls/cm) at a flow rate of 0"6 ml/min. 50 ~1 of a sample was injected. The peaks were detected using a model 100 Ultra Violet (UV) absorbance detector and an 8430 refractive index (RI)detector with an SP4400 dual channel integrator. The concentrations of substrates and metabolic end-products were obtained from the equations of standard curves. Amounts of glucose, fructose, mannitol and ethanol were calculated from their RI peaks, whereas concentrations of lactate and acetate were determined from their U V peaks. Fructose and mannitol eluted together on Aminex HPX-87H column. Therefore, fructose was determined using a Bio-Rad Aminex HPX-87N column. Mannitol was calculated from subtraction of the fructose concentration determined using the Bio-Rad Aminex HPX-87N column. Aminex HPX-87N column was operated at 85°C. Samples were eluted with 0.015 M

The metabolism of glucose in G Y A Medium under anaerobic conditions by Leuc. mesenteroides at 25°C is given in Table 1. Glucose was fermented to lactate, ethanol and acetate by test bacteria. Leuc. mesenteroides N C I M B 8023 consumed less glucose than did strain NCFB 811. De Moss et al. [17] and Gunsalus & Gibbs [18] stated that glucose was metabolised to 1 tool each of lactate and ethanol under anaerobic conditions by Leuc. mesenteroides, strain 39. Glucose was fermented via the phosphoketolase pathway by heterofermentative lactic acid bacteria [19] and little or no acetate was formed in the absence of air [7]. Leuc. mesenteroides N C D O 518, Leuconostoc spp. Pz 45 and Pz 10 mainly produced lactate and ethanol and only small amount of acetate under anaerobic conditions [8]. The observed results are not quite the same as the results of De Moss et al. [17] and Gunsalus & Gibbs [18] but are in good agreement with the results of Nuraida et al. [8].

Fructose metabolism by Leuc. mesenteroides Data presented in Table 2 show that Leuc. mesenteroides N C I M B 8023 and Leuc. mesenteroides NCFB 811 utilised fructose to lactate, ethanol, acetate and mannitol. The formation of metabolic end-products per mol fructose by Leuc. mesenteroides N C I M B 8023 and

Table 1. Metabolic end-products from glucose in cultures of Leuc. mesenteroides in GYA medium under anaerobic conditions at 25°C Strain

NC1MB 8023

NCFB 811

Compounds

Glucose Lactate Ethanol Acetate Glucose Lactate Ethanol Acetate

Concentration* (mmol/litre) Initial1-

Final:[:

4"3 0-25 < 0-2 < 0'02 4.7 0.45 < 0.2 < 0-02

0"95 3.4 2.9 1' 1 < 0"04 4.75 4.5 0.55

*Results are means of determinations of duplicate experiments; tfor initial values, samples were taken in 1-2 rain after inoculation; $final values are for samples taken 48 h later.

Metabolism of fructose by Leuconostoc mesenteroides

737

Table 2. Metabolic end-products from fructose in cultures of

Table4. Metabolic end-products from fructose in Leuco-

Leuc. mesenteroides in FYA medium under anaerobic condi-

nostoc spp. [13]

tions at 25°C Compound Strain

NCIMB 8023

NCFB 811

Compounds

Fructose Lactate Ethanol Acetate Mannitol Fructose Lactate Ethanol Acetate Mannitol

Concentration* (mmol/litre) Initial+

Final~

4.4 0.25 < 0.2 < 0-02 < 0.04 4-6 0.25 < 0.2 < 0.02 < 0.04

< 0"04 3'2 2'5 1'15 1'15 < 0'04 3,25 1.7 1.25 1.2

*Results are means of determinations of duplicate experiments; ?for initial values, samples were taken in 1-2 rain after inoculation: $final values are for samples taken 48 h later.

Metabolic end-products (mol/mol fructose) Strain NCDO 518

Strain Pz 45

Strain Pz 10

Lactate Ethanol

0.6 0-3

0,9 0-7

0.6 0.3

Acetate Mannitol

(1-3

(}-I 0" I

0-2

0-4

Table 5. Metabolic end-products from glucose plus fructose in cultures of Leuc. mesenteroides in GFYA Medium under unaembic conditions at 2.5;°C Strain

Compounds

Leuc. mesenteroides N C F B 811 in this study is given in Table 3. Both strains used small a m o u n t s of fructose as an electron acceptor for the p r o d u c t i o n of mannitol. T h e reduction of 1 mol of fructose led to the f o r m a t i o n of 0 - 2 6 m o l of m a n n i t o l i.e. 26% of fructose was metabolised to mannitol. Small c o n c e n t r a t i o n s of fructose were also converted to acetate. Fructose m e t a b o l i s m differs from glucose m e t a b o lism in lactic acid bacteria. It can be used as an electron acceptor by h c t e r o f e r m e n t a t i v e Lactobacillus spp. and Leuconostoc spp. [9-12] N u r a i d a [13] studied fructose f e r m e n t a t i o n in Leuc. mesenteroides N C D O 518, Leuconostoc sp. Pz 45, and Leuconostoc sp. Pz 10 with u n a e r a t e d cultures (Table 4). Fructose was f e r m e n t e d to lactate, ethanol, acetate, and mannitol. T h e a m o u n t s of e n d - p r o d u c t s differed b e t w e e n the strains and only small a m o u n t s of m a n n i t o l were p r o d u c e d due to the small a m o u n t s of fructose utilised as an electron acceptor. Leuc. mesenteroides, P R L L33 f e r m e n t e d fructose to

Table 3. Metabolic end-products from fructose in cultures of

Leuc. mesenteroides in FYA medium under anaerobic conditions at 25°C Compound

Lactate Ethanol Acetate Mannitol

Metabolic end-products* (mol/mol fructose) Strain NCIMB 8023

Strain NCFB 811

0"67 0"57 0"26 0"26

0-65 0-37 0-27 0-26

*Calculations are based on the consumption of fructose given in Table 2.

0"3

NCIMB 8023

NCFB 81 l

Glucose Fructose Lactate Ethanol Acetate Mannitol Glucose Fructose Lactate Ethanol Acetate Mannitol

Concentration* (mmol/litre) Initial+

Finals

4.6 4-6 04 < 02 < (t.(12

< 0.04 < 0.04 6.75 5.45 1-55 2"4 < 0"04 < 0'04 7 6 13 1-1

< (1"04

4"45 4-45 0.4 < 0.2 < 0.02 < 0.04

*Results are means of determinations of duplicate experiments; +for initial values, samples were taken in 1-2 min after inoculation; $final values are for samples taken 48 h later.

Table 6. Metabolic end-products from glucose plus fructose in cultures of Leuc. mesenteroMes in GFYA Medium under anaerobic conditions at 2_5;°C Compound

Lactate Ethanol Acetate Mannitol

Metabolic end-products* (tool/tool glucose plus fructose) Strain NCIMB 8023

Strain NCFB 811

(}.69 {}.57 {1.17 0.26

0.74 (/.67 (). 15 0-12

*Calculations are based on the consumption of glucose plus fructose given in Table 5.

H. Erten

738 Table 7. Metabolic end-products from glucose plus fructose in cultures of Leuconostoc spp. [13] Compound

Lactate Ethanol Acetate Mannitol

Metabolic end-products* (mol/mol glucose plus fructose) Strain NCDO518

Strain Pz 45

Strain Pz l0

0.7 0.4 0.3 0.3

0.9 0'8 0.1 0.1

1'0 0'7 0.1 0.8

give the same metabolic end-products as those obtained from glucose, except that 30% of fructose was also converted to mannitol. A tool of fructose was metabolised to 0.6 mol lactate, 0"5 mol ethanol, 0.2 tool acetate and 0.3 mol mannitol by Leuc. mesenteroides, PRL L33 [20]. The conversion of l mol fructose by Leuc. mesenteroides, strain 39-ATCC 12291 gave rise to 0.61 mol lactate, 0"48mol ethanol, 0.2tool acetate, 0.66 tool CO,_ and 0.33 mol mannitol. The difference between glucose and fructose dissimilation was that fructose was used as an electron acceptor to yield mannitol. 30-35% of original fructose was converted to mannitol [11]. Martinez et al. [10] and Stanier et al. [12] stated that 1 tool of fructose was fermented to 0.67 mol of mannitol, 0"33 mol of lactate and acetate by Lactobacillus brevis. Distribution of labelled 1,2,6-C j4 in fermentation products from fructose was investigated in Leuc. mesenteroides [11]. Carbon dioxide was mainly produced from fructose 1-C 14, ethanol from 2-C H, and lactate from 6-C 14 and 2-C 14 and acetate from 2-C 14. The fermentation of fructose to metabolic end-products was a similar pathway to that in the glucose metabolism. Fructose and mannitol had the same specific radioactivity and therefore, mannitol produced was not degraded during fructose fermentation. The observed results in this study are in good agreement with the results of Blackwood & Blackley [20] and Busse et al. [11] and are similar to (depending on strain) those of Nuraida [13]. The results differ from those of Martinez et al. [10] and Stanier et al. [12].

Dissimilation of a mixture of glucose and fructose by Leuc. mesenteroides In this present study, the same metabolic end-products of fructose metabolism were produced from a mixture of glucose and fructose i.e. lactate, ethanol, acetate and mannitol were formed (Table 5). The comparison of metabolic end-products on mol of glucose and fructose is given in Table 6. Small amounts of mannitol and acetate were formed. The production of mannitol indi-

cates that some fructose was metabolised as an electron acceptor. The results in this study supports the observations of Nuraida [13] given in Table 7.

Acknowledgements The author is very grateful to the University of Cukurova (Adana, Turkey) for financial support. He also thanks Dr J. D. Owens, Dept. of Food Science and Technology, University of Reading, Reading, UK and Prof. Dr. A. Canba~, Dept. of Food Engineering, University of Cukurova, Adana, Turkey for their cooperation.

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