Introduction

Sourdough is a traditional product that has been used for thousands of years in breadmaking for the improvement of the dough properties (Collar et al. 1998), organoleptic characteristics (Hansen & Hansen 1996; Damiani et al. 1996), nutritional value (Liljeberg et al. 1995; Lopez et al 2001) and shelf life of the bread (Corsetti et al. 1998a; 1998b; 2000; Martinez-Anaya et al. 1990; Lavermicocca et al. 2000). These positive effects have been related to the microbial flora, which is mainly composed of yeasts and lactic acid bacteria. The yeast that is most often isolated from spontaneous cereal fermentations is Saccharomyces cerevisiae, while the respective lactic acid bacteria are Lactobacillus sanfranciscensis, L. brevis and L. plantarum (Gobbetti 1998).

In general, the noncompetitive environment for the main carbon source seems to be one of the prerequisites for the stability of yeast/LAB associations in food fermentations. In the case of sourdough, the stability of the specific microbial consortium has been attributed to the manner of the flour mono- and disaccharide fermentation by these microorganisms. The presence of maltose phosphorylase (Stolz et al. 1993), the ability to utilize fructose as an electron acceptor (Gobbetti et al. 1995; Stolz et al. 1995a) and the absence of glucose repression, provide an ecological advantage to L. sanfranciscensis over the other competitor species. Maltose is hydrolysed by maltose phosphorylase resulting in the excretion of glucose in order to avoid excessive intracellular concentrations. Excretion of glucose takes place in concomitance with maltose availability, and after maltose has been depleted the consumption of the excreted glucose begins. The glucose excreted during sourdough fermentation may be utilized by maltose-negative yeasts or may prevent the other competitors from utilizing abundant maltose by glucose repression, thereby giving an ecological advantage to L. sanfranciscensis (Gobbetti 1998). Utilization of fructose as an electron acceptor provides L. sanfranciscensis with additional energy in the form of ATP and on the other hand deprives the competing strains of the carbon source. Hydrolysis of sucrose and the concomitant release of fructose and glucose by sucrose-fermenting strains as well as glucose repression phenomena ensure the stability of this ecosystem (Gobbetti 1998; Vogel et al. 1999).

In traditional Greek wheat sourdoughs, S. cerevisiae, L. sanfranciscensis, L. brevis seem to form a stable consortium, as they were present in significant numbers in all sourdoughs that have been examined (Paramithiotis et al. 2000, De Vuyst et al. 2002). Other yeast species such as Pichia membranaefaciens and Yarrowia lipolytica and lactic acid bacteria such as L. paralimentarius, W. cibaria, P. pentosaceus and Enterococcus faecium make Greek wheat sourdoughs very interesting from the ecological point of view (Paramithiotis 2001). L. sanfranciscensis and L. brevis have been widely isolated from traditional sourdoughs worldwide and the properties that govern their omnipresence have been more or less studied. As far as W. cibaria, L. paralimentarius and P. pentosaceus are concerned, literature is lacking in data referring to their competitiveness in the respective environment.

In this study, single cultures of the heterofermentative lactic acid bacteria L. sanfranciscensis, L. brevis, W. cibaria, and the homofermentative L. paralimentarius and P. pentosaceus were grown in liquid media containing glucose, fructose, maltose and sucrose, either as a single carbon source or in combination always in the presence of glucose. The mode of carbohydrate metabolism and its effect on the metabolites produced were studied in order to justify the competitiveness of certain species in the sourdough environment.

Materials and methods

Microorganisms

The strains used throughout this study were L. sanfranciscensis ACA-DC 3366, L. brevis ACA-DC 3407, W. cibaria ACA-DC 3385, L. paralimentarius ACA-DC 3414 and P. pentosaceus ACA-DC 3391. All strains were isolated from traditional Greek wheat sourdoughs and stored at −80 °C in de Man-Rogosa-Sharpe (MRS) broth (Biokar, Beauvais, France) containing 25% (v/v) glycerol (Sigma Chemical Co, St Louis, MO, USA). Before experimental use strains were propagated twice in the appropriate medium (see below) at 30 °C for 24 h.

Growth conditions

Actively growing culture (inoculum level 1.0%, v/v) was used to inoculate the eight media used. Initially, modified MRS broth containing either 110 mM glucose (M1), or 110 mM fructose (M2), or 55 mM maltose (M3) or 55 mM sucrose (M4) as sole carbon source was used. Then, modified MRS broth was prepared containing 110 mM glucose and 110 mM fructose (M5), 110 mM glucose and 55 mM maltose (M6), 110 mM glucose and 55 mM sucrose (M7). Finally, in the modified MRS broth all four carbohydrates were present in the above mentioned concentration (M8). The initial pH of the media was adjusted to 6.5. Growth was carried out at 30 °C for 36 h and monitored by measuring the optical density at 600 nm. The maximum specific growth rate μmax was determined by linear regression (indicated by the correlation coefficient r2) from the plots of ln (OD/ODo) vs. time. Experiments were performed in triplicate and the average values are presented.

Analysis of metabolites

Sourdough samples were taken over a period of 36 h. Maltose, sucrose, glucose, fructose, ethanol, lactic and acetic acid, glycerol and mannitol were determined by high-performance liquid chromatography analysis (Varian Associates Inc., Palo Alto, CA, USA). First, cells were removed by centrifugation (5000g for 10 min, 4 °C; Heraeus Sepatech Biofuge 22R, Hanau, Germany). The supernatant (1 ml) was mixed with 50 μl perchloric acid (70% v/v, −30 °C) (Sigma) and kept at 4 °C for 24 h. Protein agglomerates were removed by centrifugation (5000g for 60 min, 4 °C), the supernatant was filtered through a 0.22 μm Millex Syringe Filter (Millipore, Billerica, Mass., USA) and 20 μl of the solution were injected into an Aminex HPX-87H column (300 × 7.8 mm; Bio-Rad, Hercules, CA, USA.) connected to a refractive index detector (model LC 1240; GBC Scientific Equipment Pty. Ltd. Dandenong, Victoria, Australia). Elution was performed at 35 °C with 5 mM H2SO4 at a flow rate of 0.5 ml/min. Discrimination between fructose and mannitol was achieved by injecting 20 μl samples into an Aminex HPX-87C and eluting at 75 °C with bi-distilled water at a flow rate of 0.6 ml/min. Data were collected and analysed by using a 746 data module (Waters Corporation, Milford, MA, USA). Quantitative analysis was carried out by standard curves designed for each compound.

Results and discussion

Results are summarized in Tables 1 and 2, where exact data are given for the 36 h samples. The stoichiometry determined between the initial and the final products was valid throughout the growth in all media tested.

Table 1 Growth characteristics, sugar consumption and metabolite production during growth of L. sanfranciscensis, L. brevis and W. cibaria for 36 h at 30 °C
Full size table
Table 2 Growth characteristics, sugar consumption and metabolite production during growth of L. paralimentarius and P. pentosaceus for 36 h at 30 °C
Full size table

Lactobacillus sanfranciscensis strain could ferment glucose, fructose and maltose but not sucrose, when these were used as sole carbon sources or in combination (Table 1). The lowest maximum specific growth rate was observed with glucose; however, the final cell mass in this case was slightly higher than with fructose. On the other hand, maltose enhanced both maximum specific growth rate and final cell mass. Final cell mass when media containing glucose combined with fructose or maltose were used, reached the levels obtained with only maltose, even with lower maximum specific growth rate in the case of the medium with glucose and maltose. Finally, optimal growth and maximum specific growth rate were achieved in medium where all carbon sources were combined (Table 1).

When glucose and maltose were supplied as sole carbon sources, they were fully catabolized and stoichiometrically converted to lactic acid, ethanol and acetic acid at a ratio approaching 1:1:0.04 (Table 1). The catabolism of maltose took place without concomitant extracellular accumulation of glucose. When both glucose and maltose were present, simultaneous co-metabolism at the same rate was observed, resulting in the same end products at the same ratio as above; however, only maltose was fully catabolized. When fructose was present either as sole carbon source or in combination with glucose and/or maltose, it was fully catabolized, being mainly reduced to mannitol. The presence of another carbon source resulted in an increased amount of fructose utilized as an electron acceptor. Furthermore, mannitol production favored acetate against ethanol production, and in the case of the medium where fructose was the sole carbon source, lactic acid and acetic acid were produced at a ratio of 1:1. In all cases, the presence of sucrose had no effect on maximum specific growth rate, final cell mass or metabolite production.

Lactobacillus brevis strain could ferment only maltose as sole carbon source. Although maltose was not fully catabolized, it was stoichiometrically converted to lactic acid, ethanol and acetic acid at a ratio 1:1:0.02 (Table 1). As in the case of L. sanfranciscensis no glucose was accumulated extracellularly. Although fructose could not be used as a sole carbon source, in the presence of maltose it was utilized as an electron acceptor, thus improving both final cell mass and maximum specific growth rate; moreover, it favored acetate production against lactate and ethanol, resulting in the alternation of the end product ratio to 1:0.2:0.1. As in the case of L. sanfranciscensis, presence of glucose and/or sucrose, i.e. the non-utilizable carbohydrates by this strain, had no effect on maximum specific growth rate, final cell mass and production of metabolites.

Weissella cibaria strain could ferment glucose, fructose, maltose and sucrose when these were present either as sole carbon source or in combination. The lowest and maximal maximum specific growth rate and final cell mass were observed with fructose and maltose as sole carbon sources, respectively. Values in between were obtained when glucose was combined with any other sugar(s) (Table 1).

Fermentation of sugars by W. cibaria, when these were used as sole carbon sources, was variable, i.e. maltose 82%, glucose 69%, fructose 25% and sucrose 58%. On the other hand, when all sugars were combined a clear-cut preference to glucose was observed. In particular, when glucose was combined with fructose or maltose, W. cibaria initially fermented only glucose; fermentation of fructose or maltose initiated only after glucose concentration reached values lower than 90.7 and 81.1 mM, respectively, and after the strain has reached the middle of its exponential growth phase (data not shown). When glucose was combined with sucrose, simultaneous co-metabolism was observed, with glucose however being metabolized more rapidly. When all sugars were present, the strain entered its exponential growth phase by fermenting only glucose. Then, fermentation of fructose, maltose and sucrose initiated at a very slow rate. In all cases, the fermented sugars were stoichiometrically converted to lactic acid, ethanol and acetic acid, at a molecular ratio approaching 1:0.7:0.06 (Table 1).

Lactobacillus paralimentarius strain could ferment glucose, fructose and maltose but not sucrose, when these were used either as sole carbon sources or in combination. Although maximum specific growth rate was the same in all media, slightly higher final cell mass was observed in media containing glucose and maltose either as sole carbon sources or in combination, and slightly lower final cell mass in media containing fructose either as sole carbon source or in combination (Table 2).

When glucose, fructose or maltose was supplied as sole carbon sources, they were stoichiometrically converted to lactic acid. This was not the case when two or more fermentable carbon sources were present in the medium. In that case only a fraction of the catabolised carbohydrates were retrieved as lactic acid (Table 2). When glucose was combined with fructose, L. paralimentarius strain entered the exponential growth phase by fermenting only glucose; then, fermentation of fructose took place after glucose concentration reached values lower than 97.1 mM (data not shown). From that point, fermentation of both monosaccharides took place at the same rate. On the other hand, when glucose was combined with maltose, simultaneous fermentation was observed; maltose was fermented at a very slow rate. The same evidence was observed when glucose, fructose and maltose were combined. In all cases, the presence of sucrose had no effect on maximum specific growth rate, final cell mass or metabolite production.

Pediococcus pentosaceus strain could ferment glucose, fructose and maltose but not sucrose, when these were used as sole carbon sources. Although final cell mass was the same in all media, maximum specific growth rate was slightly higher when two fermentable carbohydrates were combined, compared to the media with only one carbon source, and even higher when all fermentable carbohydrates were combined (Table 2).

When glucose, fructose or maltose was supplied as sole carbon sources, they were stoichiometrically converted to lactic acid. Presence of two or more fermentable carbohydrates resulted in the same observation as in the case of L. paralimentarius strain: only a part of the fermented carbon sources was retrieved as lactic acid (Table 2). When glucose was combined with fructose and/or maltose, a definite preference to glucose was observed. In particular, when glucose was combined with fructose or maltose, P. pentosaceus initially fermented only glucose; fermentation of fructose or maltose initiated only after glucose concentration reached values lower than 82.8 and 90.0 mM, respectively and after the strain has reached the middle of its exponential growth phase (data not shown). From that point, the fermentation rate of glucose was higher than that of fructose and maltose. As in the case of L. paralimentarius, the same observations were made when glucose, fructose and maltose were combined. In all cases, presence of sucrose had no effect on maximum specific growth rate, final cell mass or metabolite production.

The increased interest in the sourdough ecosystem results from its positive effect on the quality of the final product and most of it being directly related to the sourdough microflora. The adaptation of the microorganisms that form this microflora to that particular environment is an essential feature for the successful utilization of the sourdough. Yeasts like S. cerevisiae and lactic acid bacteria such as L. sanfranciscensis, L. brevis and L. plantarum are more often isolated from sourdoughs in significant numbers, which suggests that these species form its dominant microflora. In most cases, these microorganisms are encountered together with other yeasts and lactic acid bacteria that form a secondary microflora.

Lactobacillus sanfranciscensis is the most studied sourdough lactic acid bacterium and, with very few exceptions (Martinez-Anaya et al. 1990), is considered to be a key sourdough lactic acid bacterium in the biotechnology of baked sourdough products (Gobbetti & Corsetti 1997). Absence of preference for glucose or maltose, maltose utilization via maltose phosphorylase and utilization of fructose as an electron acceptor are the special features that characterize this lactic acid bacterium and can partly justify its competitiveness and persistence in the sourdough environment. It seems that these features are essential for efficient competitiveness, as they govern the interactions with the sourdough yeasts. In this study, L. sanfranciscensis strain exhibited no preference for glucose when it was combined with maltose. Both sugars were fermented simultaneously and at the same rate. The same observation was also made by previous studies (Hammes et al. 1996; Stolz et al. 1993).

Lactobacillus brevis strain could only ferment maltose, while concerning W. cibaria, L. paralimentarius and P. pentosaceus strains, the preference for glucose fermentation was evident when it was combined with the other carbon sources. In all cases, that preference was stated by the higher fermentation rate of glucose compared to the one of the other sugars, while in most cases strains were fermenting only glucose, and when its concentration lowered below a critical value, depending on the carbohydrate and the species, they commenced fermentation of the other carbohydrates present. Since these species are not isolated very often or in numbers suggesting a significant contribution in the development of the sourdough ecosystem, no such study has been conducted so far, and thus no data concerning their preference for the fermentation of a carbon source are available.

In regard to maltose metabolism, experiments performed with resting cells of L. sanfranciscensis, L. reuteri and L. pontis (Neubauer et al. 1994; Stolz et al. 1995a; b) resulted in extracellular glucose accumulation as an evidence of maltose phosphorylase activity, whereas only a transient release of glucose was observed with growing cells. This was also the case in the present study; no extracellular glucose accumulation was observed, when strains were growing in medium containing maltose as a sole carbon source. Since no experiment took place in order to determine this enzyme in cell extracts, no conclusion can be drawn regarding its existence. It can be hypothesized however, that the nonphosphorylated glucose formed upon the cleavage of maltose by maltose phosphorylase, instead of excretion by the cell, could be immediately converted by hexokinase activity, which in turn is induced by the presence of either glucose or fructose (Stolz et al. 1995b). Regardless of the mechanism by which maltose uptake takes place, absence of extracellular glucose accumulation could play an important role, as far as the interactions with the sourdough yeasts are concerned. Since glucose in the growth medium is not replenished due to excretion by the lactic acid bacteria, it will soon be depleted and that will lead sourdough yeasts to maltose fermentation, creating an antagonism for the carbon source.

Oxygen is the preferred electron acceptor for the L. sanfranciscensis strains (Roecken & Voysey 1995) and when this is depleted, fructose is used as an electron acceptor and reduced to mannitol in the presence of maltose (Stolz et al. 1995a; Gobbetti et al. 1995). Only L. sanfranciscensis and L. brevis strains used in this study could utilize fructose as an electron acceptor. Due to the utilization of fructose as an electron acceptor, an increase in acetate production was observed causing a decrease of the fermentation quotient. This is an important feature, since a fermentation quotient in the range of 2.0–2.7 is essential for the perception of the pleasant odor in rye breads and the enhancement of the inhibitory effects of sourdoughs against microorganisms surviving the baking process in wheat bread production (Roecken 1996). The incomplete carbon recoveries that took place, especially when carbon sources were combined, were another interesting observation. This could be either due to the formation of additional end products, undetectable by the analytical step of this study, or to the utilisation of the intermediate metabolic products via other biosynthetic pathways.

Conclusions

The property that definitely differentiates L. sanfranciscensis and L. brevis strains from W. cibaria, L. paralimentarius and P. pentosaceus strains and account for their competitiveness in the sourdough environment is the ability to utilize fructose as an electron acceptor. This property seems to have a decisive role for effective competitiveness in the given environment, while properties such as glucose replenishment through maltose phosphorylase activity and absence of glucose repression, although important, seems not to be prerequisite for the same purpose.