maltose and glucose by sourdough

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1 FEMS Microbiology Letters 109 (1993) Federation of European Microbiological Societies /93/$06.00 Published by Elsevier 237 FEMSLE Utilisation of isolated from maltose and glucose by sourdough Peter Stolz, Georg B6cker, Rudi F. Vogel and Walter P. Hammes Institut fiir Lebensmitteltechnologie, Universitdt Hohenheim, Stuttgart, FRG (Received 18 February 1993; revision received 2 March 1993; accepted 8 March 1993) lactobacilli Abstract." The utilisation of glucose and maltose was investigated with Lactobacillus strains isolated from sourdough starters. These preparations have been in continuous use for a long period to produce sourdough from rye, wheat and sorghum. The major metabolic products formed by resting cells from glucose or maltose were lactate, ethanol and acetate. Upon fermentation of maltose, resting cells of Lactobacillus sanfrancisco, L. reuteri, L. fermentum and Lactobacillus sp. released up to 13.8 mm glucose after 8 h. The ratio of released glucose per mol of utilised maltose was up to 1 : 1. Glucose formation was high when starved cells of L. sanfrancisco and Lactobacillus sp. were used. This is consistent with maltose utilisation via maltose phosphorylase which phosphorylates maltose without the expenditure of ATP and thus allows the cell to waste glucose in the presence of abundant maltose. The glucose formed may be utilised by the lactobacilli or other microorganisms, e.g. yeasts. However, the release of glucose into the medium by sourdough lactobacilli prevents competitors from utilising the abundant maltose by glucose repression. In strains of L. sanfrancisco, maltose utilisation was very effective and not subject to glucose repression. Therefore, they overgrow other microorganisms sharing this habitat. Wild isolates of L. sanfrancisco were initially unable to grow on glucose. Upon growth on maltose such strains required adaptation times of up to 150 h to grow on glucose. After subsequent transfer of glucose-grown cells to fresh medium the strains resumed growth both on glucose or maltose. They readily lost their ability to grow on glucose upon exposure to maltose. L. sanfrancisco exhibited biphasic growth characteristics on media containing glucose, maltose or both carbon sources. Evidence is provided that biphasic growth and metabolite formation are dependent on the redox potential. Key words: Lactobacillus sanfrancisco; Sourdough fermentation; Maltose metabolism; Glucose utilisation; Ecology Introduction Sourdough starter preparations are traditionally prepared under non-aseptic but well-defined conditions. During consecutive cycles of the respective process the fermenting organisms are Correspondence to: R.F. Vogel, Institut for Lebensmitteltechnologie, Universit~it Hohenheim, Garbenstr. 25, W-7000 Stuttgart 70, FRG. continuously propagated and strains are selected which are most adapted to this environment [1]. These strains formerly identified as Lactobacillus brevis ssp. lindneri [2,3] were allotted to L. sanfrancisco [4], which was described as a new species prevailing in wheat sourdough [5]. Previous studies revealed that only two strains of L. sanfrancisco and one strain of L. brevis represented 99.9% of the microflora of rye sourdough at cell counts of approx [6]. These lactobacilli share the habitat with the yeast Candida milleri

2 238 which was present at cell counts of < 10 6 cfu g-1. The combination of L. sanfrancisco and Candida milleri was also found in wheat sourdough [7]. Similar microbial associations were detected in sorghum sourdough for the production of Kisra which is the major staple food in Sudan. The main organisms present in this type of sourdough were few strains of L. amylovorus, L. reuteri and L. fermentum and one strain of Candida krusei [8]. This combination of maltosefermenting lactobacilli and maltose-negative yeasts was found, although the conditions present in rye and wheat, or sorghum dough are different with respect to environmental factors (e.g. temperature, water activity, redox potential and availability of substrates). The metabolic activities of the associated organisms e.g. starch degradation and maltose fermentation allow synergism [8]. The metabolic products formed by the numerically dominating lactobacilli mainly determine the quality of the sourdough and thus the nutritional value, shelf life, texture and flavour of the bread. These metabolites result from the fermentation of low molecular sugars present in the dough of which maltose is prevalent. In this communication, the utilisation was investigated of glucose and maltose by lactobacilli isolated from sourdough. Lactobacilli from other environments were used for comparison. A general principle is Table 1 proposed which is causative for the ability of these organisms to compete in this environment. Materials and Methods Major metabolites formed by resting cells of lactobacilli upon fermentation of maltose Microorganisms and culture conditions The strains of microorganisms used during this study are listed in Table 1. Lactobacillus sp. LTH1735 and L. reuteri LTH3120 were grown in sanfrancisco medium (SM) [6]. All other strains were grown in mmrs containing the following components (g 1-1): tryptone (10), yeast extract (10), maltose (20), K2HPO4.3H20 (2), cysteine- HC1 (0.5), MgSOa.7H20 (0.1), MnSO4.4H20 (0.05), Tween 80 (1). The ph was adjusted to 6.2. All incubations were performed in screw cap bottles at 30 C. The ability of strains of L. sanfrancisco to grow on glucose or maltose as sole carbon source or on a combination of both was investigated in mmrs containing a total of 20 g 1-1 of the respective sugars. The presence of oxygen in the medium was indicated by a colour change of the redox indicator resazurine which was added at a concentration of 1 mg 1-1. Preparation of resting cells Cells were grown to the late exponential phase, harvested by centrifugation and washed in PBS- Strain Maltose Lactate Acetate Ethanol Glucose Molar ratio glc/mal L. sanfrancisco ATCC L. sanfrancisco LTH L. sanfrancisco LTH L. reuteri LTH Lactobacillus sp. LTH Lactobacillus sp. LTH Lactobacillus sp. LTH Lactobacillus sp. LTH L. fermentum ATCC L. casei DSM L. plantarurn DSM L. sake LTH glc, glucose; mal, maltose. The concentrations of the sugars are given in mmol 1-1. The negative values for maltose indicate maltose consumption.

3 239 maltose at ph 6.0 consisting of 700 ml solution I containing (g 1-1): NaCI (8), KCI (0.2), Na2HPO 4 2H20 (2.2), KHzPO 4 (12); 100 ml solution II: CaC12 (0.1); 100 ml solution III: MgClz-6H20 (0.2); 100 ml solution IV: maltose (20). All solutions were mixed after autoclaving separately. Subsequently, the cells were suspended in PBS containing 50 mm maltose to an OD578 of 4.0. For the preparation of starved resting cells, PBS without maltose was used in the washing procedure, i.e. the starvation time was approximately 1 h. Determination of sugars and organic acids Resting cells were incubated anaerobically at 30 C. Samples (1 ml) were taken for HPLC analysis after 0.5, 1, 3, 5, 8 and 22 h. The cells were removed by centrifugation and the proteins present in the supernatant were precipitated during more than 2 h after addition of 50 ~1 of 60% perchloric acid. The proteins were removed by centrifugation and the sugars and organic acids in the supernatant were analysed by HPLC as described previously [8]. Results Release of glucose by sourdough lactobacilli The metabolism of glucose and maltose was investigated of strains of L. sanfrancisco and Lactobacillus sp. isolated from rye and wheat and L. reuteri isolated from sorghum sourdough. Lactobacillus sp. strains were identified as a taxonomically separate cluster of strains which represented up to 80% of the microflora in some sourdoughs. The major metabolites formed from maltose by resting cells of sourdough lactobacilli upon 8 h fermentation of maltose are listed in Table 1. L. casei DSM 20011, L. fermentum ATCC 14931, L. plantarum DSM and L. sake LTH 677 were used for comparison. L. casei, L. plantarurn, L. sake and Lactobacillus sp. LTH 3125 produced mainly lactate, whereas the major products formed by L. sanfrancisco, L. reuteri, Lactobacillus sp. LTH 1731, 1735 and 2585 were lactate and ethanol in addition to varying amounts of acetate. Glycerol, 1,3-pro- 0 =E g o "= 30 g= = =,.. :--: 0 I I I I I time (h) Fig. 1. Time course of maltose utilisation and release of glucose by resting cells of L. sanfrancisco LTH 2581 and L. reuteri LTH Concentration of maltose/glucose in fermentations with L. sanfrancisco (t3, II) L. reuteri (, e). pane-diole, and succinate, were detected as minor metabolites. In addition to these metabolites some strains formed significant amounts of glucose from maltose and released it into the buffer medium. L. reuteri LTH 3120 and L. fermentum ATCC formed nearly equimolar amounts of glucose per maltose utilised. No glucose formation was observed with L. sanfrancisco ATCC 27651, L. plantarurn DSM and L. sake LTH 677. The time course of maltose utilisation and release of glucose by resting cells of L. sanfrancisco LTH 2581 and L. reuteri LTH 3120 is depicted in Fig. 1. All strains of L. sanfrancisco and Lactobacillus sp. LTH 1735 exhibited an increased release of glucose upon maltose fermentation when starved cells were used (Table 2). This response was not detected with starved Table 2 Release of glucose by starved resting cells of lactobacilli upon fermentation of maltose Strain ~ 0 Maltose Glucose Molar ratio glc/mal L, sanfrancisco ATCC L. sanfrancisco LTH L, sanfrancisco LTH Lactobacillus sp. LTH L. reuteri LTH glc, glucose; mal, maltose. The concentrations of the sugars are given in mmol 1-1. The negative values for maltose indicate maltose consumption.

4 240 10' E 1 c o 0,1 O,Ol, ~,, ~,, ~,, O time (11) Fig. 2. Adaptation of strains of L. sanfrancisco to glucose. The time course is depicted of the increase of OD57 s. OD578 of the culture of L. sanfrancisco strains growing on maltose/glucose as carbon source: ATCC (A, ), LTH 1729 ([3, ), LTH 2581 (, o). cells of L. reuteri LTH 3120 which revealed reduced release of glucose and a balanced metabolite formation from maltose which was in contrast to an unbalanced metabolite formation observed with starved cells of L. sanfrancisco (data not shown). Under these conditions not yet identified peaks were observed during HPLC analysis which were also responsible for the stoichiometrically unbalanced ratio of utilised maltose to produced metabolites with non-starved cells of Lactobacil- /us sp. LTH 1735 and 2585 (Table 1). Release of glucose was also observed with growing cells of L. sanfrancisco, L. fermentum and Lactobacillus sp. on mmrs containing maltose, at values of mmol 1-1. E ,1 0,01 lumnla m nn n - - Growth of L. sanfrancisco on maltose or glucose Strains of L. sanfrancisco were precultured overnight on mmrs containing maltose or glucose and transferred to mmrs containing maltose or glucose, respectively. In Fig. 2, the time course is depicted of the increase of OD578 upon growth of L. sanfrancisco in these media. The strains grew well on maltose, whereas a lag phase of 20, 110 and 150 h was observed on glucose with L. sanfrancisco LTH 2581, LTH 1729 and ATCC 27651, respectively. When the strains had acquired the ability to ferment glucose they were able to resume growth on maltose and glucose when transferred to fresh media containing one of these sugars (Fig. 3). However, they lost their O time (h) Fig. 3. Growth of L. sanfrancisco ATCC on mmrs containing maltose, maltose and glucose or glucose. The time course is depicted of the increase of OD57 s. The arrows indicate the transfer of cells to fresh medium. Growth on medium containing maltose ( ), glucose (~), maltose and glucose ( ).

5 3,00 10,00 2, ""'""--" ~ 2,00 1,00 O c ~ 1,50,oo 0,50 o, oo 0,00 0, time (h) Fig. 4. Biphasic growth of L. sanfrancisco ATCC on mmrs containing maltose, and the concomitant change in the molar ratio of acetate/ethanol. The ratio of acetate to ethanol is depicted as grey bars; growth on mmrs containing maltose (10 g-t) (i). After 30 h the fermentation conditions were strictly anaerobic. ability to grow on glucose upon exposure to maltose. Biphasic growth of L. sanfrancisco on glucose and maltose The growth of L. sanfrancisco ATCC was investigated with maltose, glucose or both carbon sources, and the metabolites formed were determined. The time course of growth and metabolite formation is depicted in Figs. 3 and 4. The cell yield measured by the maximum OD578 obtained with cells growing on glucose as sole carbon source or glucose and maltose was 50% of the value obtained with maltose when the quantity of glucose moieties was equal in both media (Fig. 3). In fermentations containing maltose and glucose, both sugars were utilised simultaneously, and no preference for glucose or maltose was observed. Nevertheless, growth of the culture exhibited biphasic characteristics irrespective of the carbon source. During the fermentation the redox potential decreased and a change was observed in the metabolic products formed (Fig. 4). In the initial phase mainly acetate and lactate were formed. Subsequently, lactate and ethanol were formed. At this stage strictly anaerobic con- 241 ditions prevailed, whereas oxygen was present during the initial phase of fermentation in screw cap bottles. Upon transfer of cells from the stationary phase into fresh medium the same biphasic growth was observed. After inoculation at higher cell densities the metabolic switch was observed after a shorter time (Fig. 3). Discussion The maltose metabolism of Lactobacillus strains isolated from sourdough revealed some peculiar features which can affect the microbial ecology in the dough and the sensorial properties of the bread. By tradition, bakers consider the ratio of lactate to acetate as a characteristic criterion for bread quality [1]. This ratio is controlled by technological measures, e.g. the formula, temperature and batch size. During dough mixing, the entry of oxygen leads to an increase of the redox potential. The present investigation indicates an influence of the redox potential on growth and metabolite formation of sourdough lactobacilli. The biphasic growth characteristics observed with L. sanfrancisco was concomitant with a change in the metabolites formed. No diauxic growth was observed in media containing both glucose and maltose. At the start of the fermentation, residual oxygen is present in the medium which is reduced, and anaerobic conditions are established. The change from aerobic to anaerobic conditions is visualised by the addition of resazurine to the medium. As long as sufficient oxygen is present acetate is formed in addition to lactate. The ethanol formed in the later fermentation phase (strictly anaerobic) may be necessary to regenerate reduction equivalents, i.e. NAD. When cells were used at a higher inoculation density the metabolic switch occurred earlier because the oxygen was consumed faster. In strains of L. sanfrancisco, maltose fermentation was not repressed by glucose. In contrast, twice the cell yield was obtained with 20 g 1-1 maltose compared with 20 g 1-1 glucose, i.e. with the same number of glucose residues. Thus, utilisation of maltose is more effective than utilisation of glucose. The higher cell yield on maltose and

6 242 the release of glucose at ratios of up to 1:1 glucose per consumed maltose is consistent with maltose utilisation via maltose phosphorylase. The presence of this enzyme was demonstrated in strains of L. brevis isolated from spoiled beer [9,10]. It phosphorylates and cleaves maltose without the expenditure of ATP to form glucose and glucose-l-phosphate which is further metabolised. Thus, the cell can afford 'wasting' of glucose in the presence of abundant maltose, which is the major low-molecular carbon source in beer and in sourdough. Part of the glucose was released into the medium, whereas it was simultaneously metabolised or accumulated intracellulary resulting in a ratio of released glucose to utilised maltose lower than 1. Simultaneous utilisation of glucose and maltose was also observed with growing ceils of L. sanfrancisco. The reason for the lag phase in the growth of L. sanfrancisco ATCC and LTH 1729 during adaptation to glucose in the absence of maltose remains to be elucidated. It may be caused by the presence of different transport systems for maltose and glucose which are regulated by the sugars or by the redox potential. Increasing levels of glucose were detected during the fermentation of sorghum [8] and rye sourdough (data not shown). The apparently futile release of glucose may become an ecological advantage as it prevents maltose positive competitors from utilising the abundant maltose. The release of glucose by lactobaciui is consistent with 'control' of the growth of bacteria and yeasts competing for maltose by feeding or glucose repression. This is supported by the observation that a Saccharomyces cerevisiae strain disappeared from the microbial population of a sourdough during consecutive fermentations even after initial inoculation at high cell densities [ll]. The disappearance may be caused by repression of genes involved in maltose fermentation by the glucose released by sourdough lactobacilli. The release of glucose by L. sanfrancisco prevents competitors from utilising maltose and thus affects the microbial ecology in sourdough. Furthermore, the ability of L. sanfrancisco to compete is supported by effective utilisation of the maltose, which is only available for strains exhibiting this mechanism. Acknowledgement This work was supported by the Commission of the European Communities Contract No. BIOT-CT (SSMA). References 1 Spicher, G. and H. Stephan (1987) Handbuch Sauerteig. BBV, Hamburg. 2 Spicher, G. and R. Schr6der (1978) Z. Lebensm. Unters. Forsch. 167, Spicher, G. (1984) Z. Lebensm. Unters. Forsch. 178, Weiss, N. and Schillinger, U. (1984) Syst. Appl. Microbiol. 4, Kline, L. and Sugihara, T.F. (1971) Appl. Microbiol. 21, B6cker, G., Vogel, R.F. and Hammes, W.P. (1990) Getreide Mehl und Brot 44, Sugihara, T.F., Kline, L. and Miller, M.W. (1971) Appl. Microbiol. 21, Hamad, S., B6cker, G., Vogel, R.F. and Hammes, W.P. (1992) Appl. Microbiol. Biotechnol. 37, Wood, B.J.B. and Rainbow, C. (1960) Biochem. J. 78, Kamogawa, A., Yokobayashi, K. and Fukui, T. (1973) Agr. Biol. Chem. 37, Nout, M.J.R. and Creemers-Molenar, T. (1987) Chem. Microbiol. Technol, Lebensm. 10,