The microflora of traditionally prepared sourdoughs typically consists of two to five strains in a single dough, rarely more. As ~20% of the strains screened were found to produce EPS from sucrose, it is likely that any given sourdough contains EPS-producing lactobacilli. The frequencies of fructan- or glucan-positive strains were highest in the phylogenetically closely related species L. reuteri
, L. frumenti
, L. panis
, and L. pontis
originating from type II sourdoughs or the intestinal tract. Van Geel-Schutten et al. (42
) identified two strains of L. reuteri
among 182 lactobacilli of various origins as potent producers of EPS. Outside of the genus Lactobacillus
, glucan and fructan formation have been found in Leuconostoc
spp. and oral streptococci. Strains of species predominating in type II sourdough fermentations are frequently found in the intestinal tracts of mammals and birds (21
). In particular, virtually all species of lactobacilli detected in pig intestines are also recognized as organisms predominating in type II sourdough fermentations (1
), although evidence for the occurrence of L. pontis
and L. panis
in pig intestines is based on culture-independent techniques only. Remarkably, the frequency of EPS-forming strains of lactobacilli is highest in intestinal isolates and species typical of intestinal microbiotae. It is tempting to speculate that glucan and fructan formation are physiological properties with relevance for the growth and survival of lactobacilli in intestinal environments.
Glucans and fructans are formed from sucrose by the activity of a single enzyme, i.e., glucan- or fructansucrase (28
). Two fructosyltransferases from L. reuteri
strain 121 were recently characterized (43
). One enzyme was characterized as levansucrase based on homology searches using partial amino acid sequences, as well as the structural characterization of the polymer formed (43
). Generally, bacterial levansucrases exhibit fructosyltransferase activities resulting in the formation of the inulin-type fructooligosaccharides kestose and nystose (5
). Accordingly, kestose and nystose production were observed in L. reuteri
strain 121, in which levansucrase is the only fructosyltransferase which is transcribed and active (43
). The second fructosyltransferase gene in L. reuteri
121, a silent gene, exhibits high homology to bacterial levansucrases. The gene product exhibited fructosyltransferase activity upon heterologous expression and was tentatively called inulosucrase based on the formation of kestose and nystose (44
). An 800-bp sequence could be amplified in this work from fructan-forming lactobacilli by PCR targeting levansucrases of lactic acid bacteria, and database searches revealed high similarity to known levansucrase genes. These levansucrase genes were present in only 6 of the 15 fructan-forming strains and in none of the fructan-forming L. reuteri
strains. The fructan of the Lev PCR-positive strain L. sanfranciscensis
LTH2590 (equivalent to TMW 1.392) was previously characterized by enzymatic digestion and reported to be of the levan type (8
). An internal fragment of the putative levansucrase gene was amplified from cDNA libraries of L. sanfranciscensis
LTH2590 and TMW 1.53, indicating levansucrase expression in these strains (this study). Taken together, these results suggest that a levansucrase is responsible for fructan formation in L. sanfranciscensis
strain LTH2590, and they may indicate that the EPS produced by other Lev PCR-positive strains is of the levan type. Because out of 35 strains only fructan-forming lactobacilli were detected by Lev PCR, this PCR is a useful tool for rapid screening of lactobacilli for EPS formation.
121 contains genes for two fructansucrases and one glucansucrase, exhibits glucansucrase and levansucrase activities, and produces two types of EPS, glucan and levan, depending on environmental conditions (20
). L. sanfranciscensis
LTH1729 was reported to produce levan (8
) but was Lev PCR negative (this study). Furthermore, eight additional strains were described in this study that produce a fructan(s) not characterized on the structural level but that are Lev PCR negative. Therefore, it can be anticipated that a further characterization of genes responsible for glucan and fructan formation in lactic acid bacteria would provide new types of glycosyltransferases.
Korakli et al. (18
) have shown by the use of 13
C-labeled sucrose the formation of up to 3.6 g of high-molecular-weight fructan per kg of flour by L. sanfranciscensis
LTH2590 in wheat and rye sourdoughs. Fructan formation in dough was detected by a higher 13
C content of WS-PS in the dough and a 10-fold-higher content of fructose in WS-PS upon fermentation than in control doughs. By the use of the same methodology, it could be demonstrated that those strains producing EPS in mMRS-sucrose also formed EPS during sourdough fermentation in the presence of sucrose. The amounts of high-molecular-weight fructan and/or glucan can be estimated to range between 0.3 and 2 g pf EPS/kg of flour based on the glucose and fructose levels in WS-PS. These amounts are comparable to the fructan levels previously reported (18
) and are higher than the levels of heteroexpolysaccharides produced by dairy lactic acid bacteria during growth in milk (10 to 200 mg per liter of milk) (22
). Hydrocolloids, such as xanthan or modified cellulose, significantly affect dough rheology and bread texture at levels of 0.1 to 1% of the flour base (6
), and levan formed by L. sanfranciscensis
LTH2590 was shown to affect the rheological properties of wheat doughs at a level of 0.1% of the flour base (4
). Therefore, the amounts of EPS formed during sourdough fermentation can be assumed to be technologically relevant.
Bacterial levansucrases that have been characterized usually have invertase activity in addition to levansucrase activity (15
). Because fructose is used as an electron acceptor by heterofermentative lactobacilli from sourdough, resulting in concomitant production of acetate instead of ethanol (37
), sourdough fermentation by fructan-forming strains in the presence of sucrose results in higher acetate contents in the dough (18
). The acetate formed affects sensorial qualities and improves the shelf life of the bread. During fermentation of wheat doughs with 12% sucrose and fructan-forming L. sanfranciscensis
, >50% of the sucrose was metabolized during fermentation (18
). Because sourdough preferments are generally included in wheat bread formulas at levels below 30%, 12% sucrose addition at the preferment stage results in sucrose levels at or below 2% of the flour base in the bread dough. The addition of 2% sucrose is commonly used in wheat bread formulas.
EPS from lactic acid bacteria may influence the intestinal flora, because oligofructose and fructans of the levan and inulin types are known to selectively stimulate the growth of bifidobacteria (3
). Possible health benefits achieved through stimulation of the growth and metabolism of bifidobacteria by dietary oligofructose or fructans have been proposed (11
). In this study, it was shown that two-thirds of EPS-producing strains form fructan. The levan produced by L. sanfranciscensis
LTH2590 is metabolized by bifidobacteria (19
) and selectively stimulated the growth of bifidobacteria during cultivation of human fecal microflorae in vitro (8
). However, wheat and rye flours contain ~6.6 and 8.5% arabinoxylans, 1.4 and 2% β-glucans, and 1 and 4% fructans, respectively. Therefore, it remains to be established whether fructans produced during sourdough fermentation exert an additional effect on the composition and activity of the intestinal microflora and human health.
In conclusion, it was shown that the production of EPS from sucrose is a metabolic activity that is widespread among sourdough lactic acid bacteria. Lev PCR produced false-negative, but not false-positive, results and thus allows rapid screening of isolates based on fructan formation. EPS-positive strains formed technologically relevant amounts of EPS during sourdough fermentation. These results will allow the deliberate use of EPS-forming lactobacilli in bread production to achieve the replacement of additives currently used in bread production by glucans or fructans formed in situ. The links between type II sourdough and intestinal microflorae on the levels of species composition and EPS production may prove to be helpful for the further development of pre- and probiotic concepts.