Biodiversity of exopolysaccharides produced by Streptococcus thermophilus strains is reflected in their production and their molecular and functional characteristics

Abstract

Twenty-six lactic acid bacterium strains isolated from European dairy products were identified as Streptococcus thermophilus and characterized by bacterial growth and exopolysaccharide (EPS)-producing capacity in milk and enriched milk medium. In addition, the acidification rates of the different strains were compared with their milk clotting behaviors. The majority of the strains grew better when yeast extract and peptone were added to the milk medium, although the presence of interfering glucomannans was shown, making this medium unsuitable for EPS screening. EPS production was found to be strain dependent, with the majority of the strains producing between 20 and 100 mg of polymer dry mass per liter of fermented milk medium. Furthermore, no straightforward relationship between the apparent viscosity and EPS production could be detected in fermented milk medium. An analysis of the molecular masses of the isolated EPS by gel permeation chromatography revealed a large variety, ranging from 10 to >2,000 kDa. A distinction could be made between high-molecular-mass EPS (>1,000 kDa) and low-molecular-mass EPS (<1,000 kDa). Based on the molecular size of the EPS, three groups of EPS-producing strains were distinguished. Monomer analysis of the EPS by high-performance anion-exchange chromatography with amperometric detection was demonstrated to be a fast and simple method. All of the EPS from the S. thermophilus strains tested were classified into six groups according to their monomer compositions. Apart from galactose and glucose, other monomers, such as (N-acetyl)galactosamine, (N-acetyl)glucosamine, and rhamnose, were also found as repeating unit constituents. Three strains were found to produce EPS containing (N-acetyl)glucosamine, which to our knowledge was never found before in an EPS from S. thermophilus. Furthermore, within each group, differences in monomer ratios were observed, indicating possible novel EPS structures. Finally, large differences between the consistencies of EPS solutions from five different strains were assigned to differences in their molecular masses and structures.

FIG. 1.

Different monomers (standard solution of 10 ppm) were separated by HPAEC in a CarboPac PA10 column (Dionex) after elution at a fixed flow rate of 1 ml · min⁻¹ with 18 mM NaOH. The response of the different monomers was measured by pulsed amperometry.

FIG. 2.

Gel permeation chromatograms from polysaccharides isolated from a baseline, nonfermented enriched milk medium (10.0% [wt/vol] skimmed milk powder, 1.0% [wt/vol] peptone, 0.5% [wt/vol] yeast extract) (A), fermented milk medium (10.0% [wt/vol] skimmed milk powder) (B), and fermented enriched milk medium (C and D). (B and C) Chromatograms of EPS from S. thermophilus ST 111; (D) chromatogram of EPS from S. thermophilus ST 113. Vertical lines represent the molecular mass markers as follows: 1, 1,800 kDa; 2, 670 kDa; 3, 270 kDa; 4, 150 kDa; and 5, 80 kDa.

FIG. 3.

Apparent viscosity (A) and EPS measurements (B) during fermentations of S. thermophilus strains in milk medium (10.0% [wt/vol] skimmed milk powder) at 42°C. Samples were taken at different pH values and after 12 and 24 h of fermentation. Black bars, S. thermophilus LY03; white bars, S. thermophilus CH101; striped bars, S. thermophilus STD; gray bars, S. thermophilus ST 113; dotted bars, S. thermophilus ST 111.

FIG. 4.

Influence of different EPS concentrations on the consistencies (K) of the EPS solutions. Strains were grown in milk medium (10.0% [wt/vol] skimmed milk powder) supplemented with lactalbumine hydrolysate (1.6% [wt/vol]) at 42°C for 12 h and at a constant pH of 6.2. ▪, guar gum; ▴, nonfloating EPS of S. thermophilus LY03; ▵, floating EPS of S. thermophilus LY03; ⧫, nonfloating EPS of S. thermophilus ST 111; ◊, floating EPS of S. thermophilus ST 111; □, nonfloating EPS of S. thermophilus ST 113; •, nonfloating EPS of S. thermophilus STD (second y axis); ○, nonfloating EPS of S. thermophilus CH101.