Sourdough fermentation process

Einkorn flour (1.75 % fat, 64.9 % carbohydrate, 9.7 % fibre and 9.6 % protein; Dogalsan, Ankara, Turkey) was used as material and all the chemicals were purchased from Sigma-Aldrich, Merck (St. Louis, MO, USA), unless otherwise stated. A mixture of 100 g of flour and 100 mL of tap water was prepared and allowed to ferment at 36 °C for 120 h to obtain traditional einkorn sourdough. After fermentation, a fresh mixture of water and flour was inoculated with the matured sourdough from the previous day in a 1:1 ratio, with five daily replenishments (21).

Isolation and identification of microbiota

Serial dilutions were prepared in 0.85 % NaCl solutions up to a dilution of 10^-8 to isolate pure colonies. Subsequently, 0.1 mL of each dilution was spread on de Man, Rogosa and Sharpe (MRS) (Merck Milipore, Darmstadt, Germany) and M17 (Merck Milipore) agar plates for lactic acid bacteria and potato dextrose agar (PDA) for yeasts. The plates were then placed in anaerobic jars (Merck Millipore) containing Anaerocoult® A (Merck Millipore) and incubated at 36 °C for 72 h. Isolates were purified based on colony characteristics and selected colonies were stored at -86 °C in a medium containing 15 % glycerol. The isolates were identified using conventional methods such as Gram staining and catalase tests. Additionally, the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics GmbH, Bremen, Germany) technique was used to determine the genus and species of the isolates at Mustafa Kemal University (Centre for Implementation and Research of Plant Health Clinic, Hatay, Turkey). Protein was extracted from bacterial isolates on MRS and M17 agar plates by formic acid method (22). Protein spectra were obtained by MALDI-TOF Biotyper and then compared with the protein spectra of reference bacterial isolates in the microbial library of the device using MALDI Biotyper Real-Time Classification (RTC) software (Biotyper 3.0; Microflex LT; Bruker Daltonics GmbH).

Cultivation and polysaccharide extraction

MRS and M17 broth were used for cultivation and production of polysaccharides by lactic acid bacteria. The bacterial isolates were cultured in 10 mL of MRS and M17 broth with a 1 % inoculum and then incubated at 36 °C for 72 h. After fermentation, the biomass was separated and then trichloroacetic acid (Isolab Laborgeräte GmbH, Weinheim, Germany) was added to a final concentration of 4 % (m/V) to remove contaminants. Chilled ethanol was added at three times the supernatant volume to precipitate the polysaccharide at 4 °C overnight (23). Centrifugation (Allegra X30; Beckman Coulter, Brea, CA, USA) at 3900×g for 15 min was performed after each step to separate biomass, impurities and polysaccharide. The partially purified polysaccharide sample was then lyophilised for further analysis.

Determination of total sugar and protein content

Different solvents were used to determine the solubility of the polysaccharide and it was soluble only in water. The total sugar content was quantified using the phenol-sulfur method (24) with glucose as standard. The total protein content was determined using the Bradford method with bovine serum albumin as standard (25), and the spectrum was scanned in the range of 200−1000 nm.

Elemental analysis

The elemental composition was analysed with an elemental analyser (Truespec Micro; Leco, St. Joseph, MI, USA) to determine the content of nitrogen, carbon, hydrogen and oxygen in the sample.

Monosaccharide composition

The composition of monosaccharides was analysed using gas chromatography-mass spectrometry (GC-MS model 7890A; Agilent Technologies, Santa Clara, CA, USA) by the breakdown of polysaccharide structures and the subsequent volatilisation of the monosaccharides (26). Glucose, fructose, mannose, xylose, rhamnose and arabinose were used as standards and a similar methodology was applied, except for acid hydrolysis.

Quantification of zeta potential

Zeta potential of the polysaccharide was measured at 25 °C using dynamic light scattering analysis. Malvern Zetasizer Nano Zsp (Malvern, UK) was used for this measurement.

X-ray diffraction analysis

A polysaccharide sample of 100 mg was analysed using the SmartLab XRD instrument (Rigaku, Tokyo, Japan) with a scanning speed of 2°/min in an angular span from 30° to 100°.

Cytotoxicity of polysaccharides

Cytotoxicity was evaluated at the Drug Administration and Research Center at Istanbul Bezmialem University, Turkey. To assess cell viability, the MTT assay was carried out on the CCD-1079Sk fibroblast cell line at a cell count of 10⁴ cell/well. The cells were cultured at 37 °C with 5 % CO₂ for 24 h. A control group was maintained in DMEM-F12 supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin (27).

Microbial composition of einkorn sourdough

The data in Table 1 show the values obtained in the production of einkorn sourdough. The cell counts of yeast and lactic acid bacteria (LAB) were determined both before and after the fermentation and showed that sourdough production was successful after a 10-day fermentation. After this period, the intrinsic microbial content of the einkorn was found to increase approx. 8-fold for the LAB and 2-fold for the yeasts, resulting in a total lactic acid bacteria and yeast cell counts of 8.32 and 4.08 log (CFU/g), respectively. Previous studies by Lim et al. (28) found LAB cell counts of 8.0–9.3 log (CFU/g) post-sourdough production, while Moroni et al. (29) reported yeast cell counts of 3.0–7.3 log (CFU/g) and lactic acid bacteria cell counts of 9.0–9.9 log (CFU/g).

The composition of the microbial community in einkorn sourdough is shown in Table S1, Table S2 and Table S3. As a result of the MALDI-TOF analysis, log score or match values were obtained for each isolate. These values ranged from 0 to 3 and each value indicated that the isolate was similar to specific bacteria in the library based on data from the NCBI database (30). The successful production of sourdough from einkorn was confirmed by analysing 22 isolates, of which 16 were Saccharomyces cerevisiae (70 %) and 6 were Candida lusitaniae (30 %) (Table S1). In addition, among the 56 isolates obtained after LAB isolation (Table S2 and Table S3), 43 were Lactobacillus plantarum (75 %), 5 were L. paraplantarum (8.7 %), 4 were L. brevis (7 %), 2 were Pediococcus pentosaceus (3.4 %), 1 was Enterococcus faecium (1.8 %) and 1 was E. durans (1.8 %). Saeed et al. (31) emphasised the dominance of L. brevis and L. plantarum as important lactic acid bacteria for sourdough production, while S. cerevisiae was the only species required for sourdough production. Ferraz et al. (32) reported that the quality of sourdough was improved by the co-fermentation of L. plantarum and S. cerevisiae, but the lactic acid bacteria had a direct effect on acidification and the final product. Cakır et al. (20) isolated lactic acid bacteria from einkorn sourdough and reported that the dominant lactic acid bacteria belonged to the species P. pentosaceus, L. brevis, L. paraplantarum and L. plantarum. Wieser et al. (33) reported that L. plantarum and E. faecalis played a joint role in sourdough production and were effective in gluten proteolysis, in contrast to the non-proteolytic L. plantarum and E. faecalis.

Extracellular polysaccharide synthesis by lactic acid bacteria

The study investigated the polysaccharide production of lactic acid bacteria obtained from einkorn sourdough. The results, expressed as dry mass in Table S4, showed that exopolysaccharide concentrations varied between 0.4 and 2.4 g/L. Only one strain (SL-1) did not produce any polysaccharides. In contrast, all other strains, including L. plantarum, L. paraplantarum, L. brevis, P. pentosaceus, E. faecium and E. durans showed the ability to produce exopolysaccharides. Liu et al. (34) identified Lactobacillus spp. and L. plantarum as exopolysaccharide producers isolated from traditional sourdough.

Numerous studies have also shown that different strains of Lactobacillus spp. and other lactic acid bacteria found in different types of sourdough have the ability to produce polysaccharides (35, 36). Abedfar et al. (37) isolated lactic acid bacteria from wheat bran sourdough and identified L. plantarum and P. pentosaceus as the predominant species. The production of polysaccharides by P. pentosaceus was 0.26 g/L and by L. plantarum 0.4 g/L. Ogunsakin et al. (38) identified P. pentosaceus SA8 and P. pentosaceus LD7 as producers of polysaccharides. Manini et al. (39) reported that L. plantarum, L. brevis and P. pentosaceus were able to produce exopolysaccharides in sourdough when cultivated on different carbon sources such as glucose, sucrose, raffinose, maltose, lactose and starch. Ispirli et al. (40) isolated and characterised E. durans as a polysaccharide producer from koumiss and kurut. Additionally, Jung et al. (41) identified E. faecium as a polysaccharide producer in sourdough.

Polysaccharide characterisation

The primary organism identified among the isolates was E. durans SL70, which produced extracellular polysaccharides when isolated from einkorn sourdough. The exopolysaccharide from E. durans SL70 in M17 medium, designated EPS-SL70, had a total carbohydrate concentration of (3.7±0.3) g/L and a protein concentration of 0.12 g/L. The absence of a peak at 260 nm indicates the absence of nucleic acid (Fig. S1). In a study by Vosough et al. (42), Enterococcus spp. were identified in Iranian kishk, and the carbohydrate and protein concentrations of EPS from E. durans K48 ranged from 0.76 to 2.39 g/L and 0.2 to 0.52 mg/L, respectively.

Elemental analysis showed that EPS-SL70 contained carbon (30.2 %), hydrogen (5.2 %) and nitrogen (3.6 %). Zanzan et al. (43) identified a polysaccharide from E. faecium F58 with a carbon content of 44.27 %. Gu et al. (44) reported that the polysaccharide derived from E. durans consisted of carbon (41.08 %) and hydrogen (7.23 %), without nitrogen, suggesting the presence of protein.

FTIR analysis revealed ten distinct bands at specific frequencies. Fig. 1a shows a band profile reminiscent of common polysaccharide structures (42, 45, 46). The frequency at 3280 cm^-1 (79.3 %) was attributed to intracellular hydrogen bonds or hydroxyl groups, while the frequency at 1657 cm^-1 (52.6 %) indicated C=O stretches of amide bonds, confirming the presence of a protein structure within the carbohydrate backbone of the extracellular polysaccharide. The vibration at 1535 cm^-1 (78.3 %) was associated with the carboxyl or carboxylate group. Additionally, the frequencies at 1329 cm^-1 (67.2 %) and 1223 cm^-1 (75.7 %) were associated with C-H stretching deformation. The presence of C–C vibration in the pyranose form of sugars was identified at 1123 cm^-1 (80.8 %) and 1045 cm^-1 (73.8 %). Furthermore, frequencies at 830 cm^-1 (59.2 %), 736 cm^-1 (76.4 %) and 675 cm^-1 (58.1 %) indicated configurations (α and β) in the pyranose form, α-glucans and the carbohydrate skeletal vibrations, respectively.

Fig. 1

The results of: a) FTIR spectrum of E. durans SL70 polysaccharide and b) GC-MS spectra of monosaccharides: 1=rhamnose (10.3 min), 2=arabinose (10.6 min), 3=xylose (11 min), 4=mannose (17.9 min), 5=glucose (18.1 min) and 6=galactose (18.4 min)

The analysis of monosaccharide composition of the polysaccharide revealed that EPS-SL70 was a heteropolysaccharide composed mainly of mannose (60 %), glucose (15 %), rhamnose (13 %), arabinose (5 %), xylose (3 %) and galactose (3 %), as shown in Fig. 1b. E. durans K48 also produced a polysaccharide with a similar structure, but with a higher amount of galactose (42). Lactobacillus spp. showed the ability to produce polysaccharides containing rhamnose, glucose, galactose and mannose units (47). Similarly, Leuconostoc pseudomesenteroides RJ-5 produced extracellular polysaccharides containing mannose, glucose, arabinose, xylose and galactose (48).

The zeta potential, mobility and conductivity of EPS-SL70 were (-15.2±0.6) mV, (-1.2±0.05) cm²/(V∙s) and (1.42±0.08) mS/cm, respectively. The low value of zeta potential was indicative of the negative charge of the polysaccharide backbone. It was concluded that EPS-SL70 is an anionic heteropolysaccharide. S. thermophilus CRL1190 had a cytoprotective polysaccharide with a zeta potential of (-5.4±0.9) mV (49). The polysaccharide kefiran, produced from kefir grains using UHT skimmed milk, was negatively charged in different aqueous solutions and at different pH values (50).

X-ray diffraction, the most widely used technique for the determination of the crystalline or non-crystalline (amorphous) nature of a polymer, was performed to estimate the phase identification of EPS-SL70 and the narrow peaks showed crystalline form (51). EPS-SL70 was found to have a uniform structure with a large extended peak that represents strong crystallinity without amorphous regions (Fig. 2a). The polysaccharides obtained from E. faecium MC-5 (51) and Lactobacillus sp. had the same structure (52)[REMOVED HYPERLINK FIELD].

Fig. 2

XRD pattern (a) and cytotoxicity effect (b) of EPS-SL70 on human fibroblast cell line (CCD-1079Sk) (*p<0.05 and **p<0.01). C=control

Fibroblasts are the central cells of connective tissue and fibroblast dysfunction causes many defects in this tissue. They are affected by various substances and their activity can decrease, leading to connective tissue defects. In this study, a normal human fibroblast cell line (CCD-1079Sk) was used to evaluate the toxicity of EPS-SL70 (Fig. 2b), and no significant effect was observed at all concentrations. According to Hala et al. (53), lactic acid bacteria were determined to be safe when the survival rate was more than 80 %. Therefore, the cytotoxicity of the polysaccharide from E. durans SL70 can be neglected (p<0.05 and p<0.01).