Exopolysaccharide (EPS) - Wiley Online Library

Journal of Applied Microbiology 2001, 91, 470±477

Exopolysaccharide (EPS) biosynthesis by Lactobacillus sakei 0±1: production kinetics, enzyme activities and EPS yields B. Degeest, B. Janssens and L. De Vuyst Research Group of Industrial Microbiology, Fermentation Technology and Downstream Processing (IMDO), Department of Applied Biological Sciences, Vrije Universiteit Brussel (VUB), Brussels, Belgium 751/1/01: received 29 February 2001, revised 5 April 2001 and accepted 5 April 2001

B . D E G E E S T , B . J A N S S E N S A N D L . D E V U Y S T . 2001.

Aims: To determine optimal exopolysaccharide (EPS) production conditions of the mesophilic lactic acid bacterium strain Lactobacillus sakei 0±1 and to detect possible links between EPS yields and the activity of relevant enzymes. Methods and Results: Fermentation experiments at different temperatures using either glucose or lactose were carried out. EPS production took place during the exponential growth phase. Low temperatures, applying glucose as carbohydrate source, resulted in the best bacterial growth, the highest amounts of EPS and the highest speci®c EPS production. Activities of 10 important enzymes involved in the EPS biosynthesis and the energy formation of Lact. sakei 0±1 were measured. The obtained results revealed that there is a clear link for some enzymes with EPS biosynthesis. It was also demonstrated clearly that the presence of rhamnose in the EPS building blocks is due to high activities of the enzymes involved in the rhamnose synthetic branch. Conclusions: EPS production in Lact. sakei 0±1 is growth-associated and displays primary metabolite kinetics. Glucose as carbohydrate source and low temperatures enhance the EPS production. The enzymes involved in the biosynthesis of the activated sugar nucleotides play a major role in determining the monomeric composition of the synthesized EPS. Signi®cance and Impact of the Study: The proposed results contribute to a better understanding of the physiological factors in¯uencing EPS production and the key enzymes involved in EPS biosynthesis by Lact. sakei. INTRODUCTION In recent years, the importance of lactic acid bacteria (LAB), both thermophilic (e.g. Streptococcus thermophilus) and mesophilic (e.g. Lactococcus lactis) strains, as producers of exopolysaccharides (EPS) has been demonstrated clearly (De Vuyst and Degeest 1999). It has been shown that EPS can play a signi®cant role as natural texturizers, for instance in the rheology of stirred yoghurt (Rawson and Marshall 1997). They can further function as thickeners, stabilizers, emulsi®ers, bodying agents, gelling agents or fat replacers in Correspondence to: Luc de Vuyst, Research Group of Industrial Microbiology, Fermentation Technology and Downstream Processing (IMDO), Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussels, Belgium. (e-mail: [email protected]).

several food products (De Vuyst and Degeest 1999). In addition, EPS produced by LAB, which are common in fermented foods, are claimed to have various bene®cial physiological effects on humans (Oda et al. 1983, 1991b, 1996, 1998; Kitazawa et al. 1991a; Nakajima et al. 1992). Although more studies on EPS production of both mesophilic and thermophilic LAB strains have been performed recently, a major drawback of their EPS biosynthesis remains the low and unstable production (Degeest and De Vuyst 1999). Lact. sakei strains have been studied extensively in the last decade. Besides their role as a starter culture in meat fermentations, they have a great potential as a solution for preserving fermented meat products from the outgrowth of pathogenic bacteria (e.g. Listeria monocytogenes) by producing bacteriocins (sakacins) (Leroy and De Vuyst 2000). ã 2001 The Society for Applied Microbiology

EPS BIOSYNTHESIS BY LACT. SAKEI 0±1

Lact. sakei also has the capacity to reduce biogenic amines accumulation during sausage fermentation (Bover-Cid et al. 2000). In addition, Lact. sakei has the ability to produce exopolysaccharides. This EPS production might be disadvantageous, for instance in the spoilage of meat (Korkeala and BjoÈrkroth 1997). On the other hand, naturally isolated Lact. sakei strains may be useful for the large-scale production of interesting heteropolysaccharides (Van den Berg et al. 1995). Lact. sakei strain 0±1 was isolated from a Belgian salami (Van den Berg et al. 1993). This strain is able to produce up to 1á4 g l)1 of EPS (Van den Berg et al. 1995), until now the highest value reported for a LAB strain producing heteropolysaccharides. However, a considerable variation can be observed in EPS quanti®cation with LAB, depending on the strains and methods used as well as the environmental factors applied (De Vuyst and Degeest 1999). It has further been shown that speci®c EPS production in Lact. sakei 0±1 increases with decreasing temperature (Van den Berg et al. 1995). The EPS produced by Lact. sakei 0±1 is a highmolecular-mass polymer (MM 6 ´ 106 Da) consisting of pentasaccharide repeating units containing D-glucose, L-rhamnose and sn-glycerol 3-phosphate in a ratio of 3 : 2 : 1 (Robijn et al. 1995). The gene cluster putatively involved in EPS production has already been isolated and characterized (Van den Berg 1996). Mutational analysis (chemical) of the EPS biosynthesis by Lact. sakei 0±1 showed that enzymes involved in the biosynthesis of dTDPrhamnose are necessary for EPS production, as well as glycosyltransferases and polymerizing/exporting proteins (Breedveld et al. 1998). However, no linkage has been shown between enzyme activities present in cell extracts and the amounts of EPS isolated at different phases of a fermentation experiment. Indeed, it is important for the application of EPS in food products and processes to investigate the mechanism involved in EPS biosynthesis during fermentation processes. A similar analysis might lead to an ef®cient production of EPS with novel, desired properties. In this study the in¯uence of medium, temperature and carbohydrate source on EPS biosynthesis in Lact. sakei 0±1 have been investigated, as well as the enzymes involved in the biosynthetic pathway of the polymer. Knowledge of these factors is needed to enhance EPS production and to obtain a better understanding of the mechanisms involved in EPS biosynthesis to result, ®nally, in the stable production of high amounts of (engineered) EPS. MATERIALS AND METHODS Bacterial strains, growth conditions and media Lact. sakei 0±1 (kindly provided by Dr Aat Ledeboer, Unilever, Vlaardingen, The Netherlands) was used as the

471

EPS-producing strain throughout this study. The strain was stored at ±80°C in de Man Rogosa Sharpe (MRS) broth, containing 25% (v/v) glycerol (de Man et al. 1960). Before experimental use, the bacteria were propagated twice (24 h at 20°C) in the medium identical to the one used for the fermentations later on. A semide®ned medium (SDM) or modi®ed MRS medium with 20 g l)1 and 75 g l)1 of glucose (unless otherwise indicated), respectively, was used as EPS production medium. SDM consisted of (g l)1): lactalbumin hydrolysate (Oxoid, Basingstoke, UK), 20; Na2HPO4á2H2O, 10; KH2PO4, 12; MnSO4áH2O, 0á05; MgSO4á7H2O, 0á1; Tween 80, 1 ml l)1. It was adjusted to pH 6á5. The modi®ed MRS medium consisted of (g l)1): peptone (Oxoid), 30; yeast extract (Merck), 12; Lab-Lemco (Oxoid), 8; K2HPO4, 2; sodium acetate, 5; triammonium citrate, 2; MgSO4á7H2O, 0á2; MnSO4áH2O, 0á038; and Tween 80, 1 ml l)1. The pH of the modi®ed MRS broth was adjusted to 6á2. MRS agar was prepared by adding 15 g l)1 of agar to commercial MRS broth (Oxoid). Fermentations and analyses Fermentations were carried out in SDM or modi®ed MRS medium in a 5-L Biostatã CT fermentor (B. Braun Biotech, Melsungen, Germany). The fermentor was controlled automatically for temperature, ran with agitation at 50 r.p.m. to keep the fermentation broth homogeneous, and no air was added. The fermentations in SDM medium were carried out with a free pH course. For the fermentation carried out in modi®ed MRS medium, the pH was controlled at a constant value of 6á2. Samples were taken at regular time intervals to determine cell counts (colony forming units or cfu ml)1) on MRS agar plates, and optical density at 600 nm (O.D.600). From SDM medium, the EPS were isolated by removing the cells through centrifugation (5500 g, 40 min, 4°C), adding one volume of chilled pure ethanol to the cell-free culture supernatant, collecting the EPS precipitate after 3±4 h, washing it with another volume of chilled pure ethanol afterwards, and drying it at 105°C. For the fermentation experiment carried out in the more complex MRS medium, the EPS was isolated as described previously (De Vuyst et al. 1998). In both cases, the amount of EPS was determined gravimetrically and expressed as polymer dry mass (PDM). Residual sugars were determined by high pressure liquid chromatography (HPLC) as described before (De Vuyst et al. 1998). The con®dentiality interval for the different sugars was determined as 0á25 g l)1. For the EPS quanti®cation, the standard deviation was calculated as 20á0%. Biokinetic parameters such as the maximum speci®c growth rate (lmax, h)1) and the speci®c EPS production (mg PDM cell)1) were calculated. The lmax was determined by linear regression

ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 91, 470±477

472 B . D E G E E S T ET AL.

glucose (g/l), PDM/50 (mg/l) Time (h)

(b) glucose/2 (g/l), 10 N NaOH PDM/50 (mg/l)

The activities of 10 enzymes either involved in the Embden± Meyerhof±Parnas pathway (phosphoglucose isomerase, 6-phosphofructokinase, fructose 1,6-bisphosphatase) or in the biosynthesis of sugar nucleotides as EPS precursor molecules (a- and b-phosphoglucomutase, UDP-glucose pyrophosphorylase, UDP-galactose 4-epimerase, dTDPglucose pyrophosphorylase, dTDP-glucose 4,6-dehydratase and the combined activities of dTDP-6-deoxy-D-xylo-4hexulose-3,5-epimerase and NADPH:dTDP-6-deoxy-Dxylo-4-hexulose-4-reductase) were determined at different stages of the fermentation course of Lact. sakei 0±1. Therefore, samples were taken every 5 h from a fermentation experiment carried out in modi®ed MRS medium with 7á5% glucose as sole carbohydrate source at 20°C, and at a controlled pH of 6á2 (by automatic addition of 10 N NaOH). Cell extracts were prepared and enzyme measurements were performed in triplicate as described previously, so that mean values and standard deviations (SD) could be calculated (Degeest and De Vuyst 2000).

(a) O.D., pH, log cfu

Enzyme activities

one used by Van den Berg et al. (1995), but with a free pH course. A second fermentation was studied in MRS medium at a controlled pH of 6á2. The fermentation pro®les of Lact. sakei 0±1 in SDM medium and modi®ed MRS medium were very similar (Fig. 1). The values of O.D. and cfus showed that bacterial growth in both media was similar. This could also be concluded from the maximum speci®c growth rates of both fermentations. For SDM medium, it was calculated to be 0á29 h)1 (r2 0á980) and for the modi®ed MRS medium it was 0á28 h)1 (r2 0á971). The end of the exponential growth phase of both fermentations was reached after 20 h (Fig. 1). This corresponded with the highest amounts of EPS, namely 1079 mg PDM l)1 for the SDM fermentation and 1125 mg PDM l)1 for the MRS fermentation. EPS production started for both fermentation experiments almost simultaneously with growth, and reached a maximum at the end of the log phase. The

O.D., log cfu

(indicated by the correlation coef®cient r2) from the plots of ln O.D.600 vs time. Both a fermentation with free pH starting at pH 6á5 (SDM) and a fermentation controlled at pH 6á2 (by automatic addition of 10 N NaOH) (MRS) were performed to study growth and EPS production kinetics of Lact. sakei 0±1. The in¯uence of the cultivation temperature on bacterial growth, carbohydrate consumption and EPS production of Lact. sakei 0±1, applying either lactose (2%) or glucose (2%) as sole carbohydrate sources in SDM medium, was investigated on a 250 ml scale as stationary cultures in a temperature range from 15 to 42°C. Samples were taken after 15, 24 and 48 h to determine the O.D.600, pH, amount of EPS, cfus and the residual carbohydrate content. To study the in¯uence of the carbohydrate source, fermentation experiments were carried out in SDM medium. Either 2% glucose or 2% galactose or 2% lactose was used as sole carbohydrate source. All fermentations were carried out at 20°C. Samples were taken after 24 h to determine cfus, O.D.600 and the amount of EPS. The monomeric composition of the isolated EPS was determined as described previously (De Vuyst et al. 1998). The maximum speci®c growth rate was calculated as described above.

Time (h)

RESULTS Fermentation course of Lact. sakei 0±1 in SDM and MRS medium To study the fermentation pro®le of Lact. sakei 0±1, we started with a semide®ned (SDM) medium similar to the

Fig. 1 Batch fermentation pro®le of Lactobacillus sakei 0±1 growth and exopolysaccharide production at 20°C in (a) a semide®ned (SDM) medium with 2á0% glucose at free pH (initial pH of 6á5) and (b) modi®ed MRS medium with 7á5% glucose at a controlled pH of 6á2. (j), O.D. at 600 nm; (h), colony forming units (cfu); (r), pH (a) or added base (b); (s) polymer dry mass; (d) residual glucose concentration



polymer seemed to degrade from that point on. Glucose was only partially consumed in both fermentation experiments. Only 12 g were used in the case of SDM, while more glucose (25 g) was consumed in the MRS fermentation due to the controlled pH. In¯uence of temperature on growth and EPS production of Lact. sakei 0±1, applying either glucose or lactose as carbohydrate source The in¯uence of different cultivation temperatures on both growth and EPS production of Lact. sakei 0±1, grown on lactose or glucose as the sole carbohydrate source, was studied in a range from 15 to 42°C. The results are presented in Table 1. The temperatures of 15°C and 25°C were best for bacterial growth and EPS production. At 15°C a high amount of EPS could be isolated for both carbohydrate sources. At 25°C the amounts of EPS were much higher for glucose compared to lactose. Also the bacterial growth was generally better when using glucose, as could be observed from the O.D.600 values, cfus and pH. At 30°C bacterial growth was not optimal for both carbohydrate sources, and the EPS production was low. At 42°C, no EPS production was observed for either of the carbohydrate sources tested. This is not surprising, since almost no bacterial growth took place at this temperature. Indeed, cfus were always lower than 105 ml)1, and almost no acidi®cation was observed. The speci®c EPS production decreased with increasing temperatures for both carbohydrate sources. In all cases, more glucose was consumed as compared to lactose

473

after 48 h of fermentation. For none of the experiments, the carbohydrate source was exhausted, indicating that the carbohydrate/nitrogen ratio of the medium was not optimal. In¯uence of the carbohydrate source on bacterial growth and EPS production of Lact. sakei 0±1 To test fermentability of the carbohydrate source with respect to growth, EPS production and EPS monomeric composition, three fermentations were carried out in SDM medium, applying either a monosaccharide (glucose or galactose) or a disaccharide (lactose) as the sole carbohydrate source (Table 2). When comparing the maximum speci®c growth rates, glucose was clearly the best carbohydrate for growth. Also the amount of EPS produced and the speci®c EPS production were highest for glucose. When comparing galactose and lactose, no difference in maximum speci®c growth rate was found. The speci®c EPS production, however, was higher for galactose compared to lactose. The carbohydrate source had no in¯uence on the EPS monomeric composition. For all sugars both monomers glucose and rhamnose were found in a 3 : 2 ratio. Enzyme activities involved in the EPS biosynthesis of Lact. sakei 0±1 Table 3 represents the activity of different enzymes measured at the different time points of the fermentation displayed in Fig. 1. The activity of a-phosphoglucomutase seemed to be linked to the amount of EPS at the different

Table 1 In¯uence of different cultivation temperatures on both growth and EPS production of Lactobacillus sakei 0±1 grown in a semide®ned (SDM) medium with either 2á0% glucose or 2á0% lactose as carbohydrate source. Values observed for both carbohydrates are separated by `/' (glucose/lactose)

T (°C)

Time (h)

O.D.600

15 15 15 15 25 25 25 25 30 30 30 30 42 42 42 42

0 15 24 48 0 15 24 48 0 15 24 48 0 15 24 48

0á03/0á02 1á17/0á10 1á88/1á70 1á98/1á96 0á14/0á14 1á98/1á85 2á02/1á90 2á02/1á99 0á14/0á14 1á88/1á74 1á92/1á87 1á92/1á86 0á03/0á02 0á12/0á15 0á16/0á18 0á09/0á11

pH

Amt EPS (mg PDM l)1)

Cell count (cfu ml)1)

Residual carbohydrate (g l)1)

Speci®c EPS production (mg PDM cell)1)


0/0 0/0 800/0 450/240 0/0 460/40 120/30 0/0 0/0 70/8 200/10 0/0 0/0 0/0 0/0 0/0

2á8 107/4á6 106 1á1 109/2á3 108 1á5 109/1á2 108 8á3 109/1á4 109 2á8 109/1á5/109 4á0 109/2á0 109 2á3 109/2á3 109 1á2 109/3á0 105 2á3 107/6á4 107 3á1 108/8á0 108 3á3 108/1á2 109 2á2 107/1á4 107 < 105/ < 105 < 105/ < 105 < 105/ < 105 < 105/ < 105


0/0 0/0 5á3 10)10/0 5á4 10)11/1á7 10)10 0/0 1á2 10)10/2á0 10)11 5á2 10)11/1á3 10)11 0/0 0/0 2á3 10)10/4á3 10)10 6á1 10)7/8á3 10)9 0/0 0/0 0/0 0/0 0/0



Table 2 In¯uence of different carbohydrates on growth, EPS production and the EPS monomeric composition of a Lactobacillus sakei 0±1 strain grown in a semide®ned (SDM) medium at 20°C. Values for cfus, amounts of EPS, speci®c EPS production and EPS monomeric composition were determined after 24 h of incubation

Carbohydrate

lmax (h)1) (r2)

Cell count (cfu ml)1)

Glucose Lactose Galactose

0á29 (0á980) 0á22 (0á994) 0á24 (0á990)

2á0 1010 2á3 1010 2á1 1010

Amt EPS (mg PDM l)1)

Speci®c EPS production (mg PDM cell)1)

Monomeric composition of the EPS (glucose : rhamnose)

1580 1313 1423

7á90 10)11 5á71 10)11 6á80 10)11

3:2 3:2 3:2

Table 3 Activities of enzymes involved in the Embden±Meyerhof±Parnas pathway and the pathway leading to the biosynthesis of sugar nucleotides (for EPS) in cell extracts of the strain Lactobacillus sakei 0±1 grown in modi®ed MRS at 20°C and a controlled pH of 6á2 Avg activity [nmol (mg of cell protein))1 min)1]

S.D.

at indicated time point

Enzyme

1h

5h

10 h

15 h

20 h

25 h

a-phosphoglucomutase b-phosphoglucomutase UDP-glucose pyrophosphorylase UDP-galactose 4-epimerase dTDP-glucose pyrophosphorylase Dehydratase Epimerase reductase Phosphoglucose isomerase 6-phosphofruktokinase Fructose 1,6-bisphosphatase

10 2 00 71 00 11 3 00 10 2 820 42 703 31 00

210 12 00 57 8 00 19 3 57 12 90 24 936 11 1244 12 00

502 7 00 193 21 15 5 47 6 120 11 170 16 1205 36 1803 25 21

798 9 00 197 8 33 3 50 4 180 6 169 31 1280 201 1750 120 00

832 1 00 204 4 29 5 52 12 236 2 186 20 1406 176 1720 215 11

833 24 00 199 15 20 6 55 9 240 21 180 12 1420 104 1746 98 00

time points of the fermentation experiment. Indeed, higher activities were observed towards the end of the exponential growth phase, going hand-in-hand with an increase of the amount of EPS. For b-phosphoglucomutase no activity was observed in any stage of the fermentation. The enzyme UDP-glucose pyrophosphorylase and the enzymes leading to the biosynthesis of rhamnose (dTDP-glucose pyrophosphorylase, dTDP-glucose 4,6-dehydratase and the epimerase reductase system) displayed activity that increased concomitant with increasing amounts of EPS. The activity of UDP-galactose 4-epimerase was very low compared to the one of UDP-glucose pyrophosphorylase. Finally, both enzymes involved in energy formation (phosphoglucose isomerase and 6-phosphofructokinase) displayed very high activities, as one would expect from housekeeping enzymes necessary for the survival of the bacterial cell. The enzyme fructose 1,6-bisphosphatase displayed no activity. DISCUSSION Lact. sakei 0±1 produces a heteropolysaccharide consisting of the monosaccharides D-glucose and L-rhamnose in a ratio of 3 : 2. In this study, the potential of Lact. sakei 0±1 to produce EPS was investigated in two different media. EPS production

by Lact. sakei 0±1 is growth-associated, indicating primary metabolite kinetics (De Vuyst et al. 1998). Whereas the amount of sugar converted to EPS is the same in MRS and SDM medium, under pH-controlled and -uncontrolled conditions, respectively, it appears that more glucose is converted to lactic acid at a controlled pH of 6á2. During the stationary phase, the amount of EPS decreased for both media, indicating possible enzymatic degradation. An unstable EPS production is also seen for other lactic acid bacterium strains (Cerning et al. 1988, 1990; Gancel and Novel 1994; Macura and Townsley 1984). Furthermore, it has been shown that the EPS degradation kinetics are only dependent on temperature and pH, indicating an enzymatic breakdown (De Vuyst et al. 1998). Recently, some glycohydrolases responsible for EPS degradation have been isolated from an EPS-producing Lact. rhamnosus strain (Pham et al. 2000). Further, it was shown that the growth temperature of Lact. sakei 0±1 is an important factor that can dramatically in¯uence the amount of EPS produced. The optimal temperature for bacterial growth and for EPS production of Lact. sakei 0±1 seemed to be in the lower range from 15 to 25°C. Several reports on the in¯uence of temperature on EPS production of LAB exist already (De Vuyst and Degeest 1999). For Lact. sakei 0±1, Van den Berg et al. (1995) also observed higher EPS yields at lower tempera-



tures. They concluded that 20°C was the optimum temperature for EPS production. In general, one can state that for thermophilic LAB strains higher temperatures result in an optimal bacterial growth, going hand-in-hand with high EPS yields (De Vuyst et al. 1998; De Vuyst and Degeest 1999; Degeest and De Vuyst 1999, 2000). For mesophilic LAB strains, EPS production seems to take place at conditions less favourable for bacterial growth (Cerning et al. 1992; Kojic et al. 1992; Gancel and Novel 1994; Marshall et al. 1995; Mozzi et al. 1995, 1996; Van den Berg et al. 1995; Gamar et al. 1997; Gassem et al. 1997), which may enable the uncoupling of growth and EPS production in mesophiles (Looijesteijn and Hugenholtz 1999). However, both growth-associated and non-growth-associated production kinetics were observed by Manca de Nadra et al. (1985) and Kojic et al. (1992). Therefore, as stated by Pham et al. (2000), LAB exopolysaccharides should be considered as minor products diverted away from glycolysis rather than as secondary metabolites. Our results further demonstrated that the nature of the carbohydrate source had no effect on the EPS monomeric composition, but in¯uenced the amounts of EPS considerably. Van den Berg et al. (1995) also postulated that growth of Lact. sakei 0±1 on different carbohydrate sources did not change the sugar composition of the EPS produced. Grobben et al. (1996) observed different EPS yields when using either glucose or fructose as the sole carbohydrate source. Finally, this study reports the ®rst demonstration that a linkage exists between enzyme activities present in cell extracts of a mesophilic LAB strain, in casu Lact. sakei 0±1, and the EPS yield. The activities of a-phosphoglucomutase, UDP-glucose pyrophosphorylase, UDP-galactose 4-epimerase, dTDP-glucose pyrophosphorylase, dTDP-glucose 4,6dehydratase and the epimerase reductase system parallel the EPS production course. A correlation between EPS production and the activity of a-phosphoglucomutase was also observed for a Strep. thermophilus strain, while very low activity was measured for this enzyme in a non-EPSproducer (Degeest and De Vuyst, 2000). Therefore, a-phosphoglucomutase is considered the enzyme linking sugar catabolism and sugar anabolism in EPS-producing LAB strains (Degeest and De Vuyst 1999). Until now, b-phosphoglucomutase activity (related to EPS production) has only been demonstrated for Lact. lactis, using maltose as the sole carbohydrate source (SjoÈberg and Hahn-HaÈgerdal 1989). The activity of the rhamnose synthesizing enzymes in cell extracts of Lact. sakei is correlated with the presence of rhamnose in the structure of the EPS produced by this strain. As reported previously, no activities of these enzymes were found in cell extracts of a Strep. thermophilus or a L. delbrueckii subsp. bulgaricus strain that produce EPS lacking rhamnose constituents (Grobben et al. 1996; Degeest & De Vuyst, 2000). Breedveld et al. (1998) investigated

475

mutants of Lact. sakei 0±1 and observed that no EPS was synthesized when the strain lacked dTDP-glucose pyrophosphorylase and/or dTDP-glucose 4,6-dehydratase activity, the ®rst and second enzyme of the rhamnose synthetic branch, respectively. The low enzyme activity of UDP-galactose 4-epimerase, in particular as compared to the one observed in an EPS-producing Strep. thermophilus strain could be explained by the fact that no galactose is present in the repeating unit of the EPS of Lact. sakei 0±1 (Degeest and De Vuyst 2000). The enzymes involved in the Embden±Meyerhof±Parnas pathway displayed high activity, indicating that most of the carbohydrate consumed by the cells is used for the production of energy. The fact that the enzyme fructose 1,6-bisphosphatase displayed no activity suggests that all fructose 6-phosphate was converted into lactic acid, and that there was no `backward' ¯ow towards EPS production via this route. To conclude, EPS production in Lact. sakei 0±1 takes place during the exponential growth phase. Low temperatures enhance the speci®c EPS production of Lact. sakei 0±1. Glucose seems to be the best carbohydrate source for EPS production. The enzymes involved in the biosynthesis of the activated sugar nucleotides play a major role in determining the monomeric composition of the synthesized EPS. A link exists between enzyme activities of a-phosphoglucomutase, UDP-glucose pyrophosphorylase, UDP-galactose 4-epimerase, dTDP-glucose pyrophosphorylase, dTDP-glucose 4,6-dehydratase and the epimerase reductase system present in cell extracts of Lact. sakei 0±1 and EPS production. ACKNOWLEDGEMENTS The authors acknowledge ®nancing from the European Commission (grants FAIR-CT-98±4267 and INCO Copernicus IC15-CT98±0905), the Flemish Institute for the Encouragement of Scienti®c and Technological Research in the Industry (IWT), the Fund for Scienti®c Research (FWO ± Flanders) and the Research Council of the Vrije Universiteit Brussel (VUB). Bart Degeest is a recipient of a fellowship from the Flemish Institute for the Encouragement of Scienti®c and Technological Research in the Industry (IWT). REFERENCES Bover-Cid, S., Hugas, M., Izquierdo-Pulido, M. and Vidal-Carou, M.C. (2000) Reduction of biogenic amine formation using a negative amino acid-decarboxylase starter culture for fermentation of Fuet sausages. Journal of Food Protection 63, 237±243. Breedveld, M., Bonting, K. and Dijkhuizen, L. (1998) Mutational analysis of exopolysaccharide biosynthesis by Lactobacillus sakei 0±1. FEMS Microbiology Letters 169, 241±249.



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