Investigating the effect of sterilisation methods on the physical properties and cytocompatibility of methyl cellulose used in combination with alginate for 3D-bioplotting of chondrocytes.

For both the incorporation of cells and future therapeutic applications the sterility of a biomaterial must be ensured. However, common sterilisation techniques are intense and often negatively impact on material physicochemical attributes, which can affect its suitability for tissue engineering and 3D printing. In the present study four sterilisation methods, autoclave, supercritical CO2 (scCO2) treatment, UV- and gamma (γ) irradiation were evaluated regarding their impact on material properties and cellular responses. The investigations were performed on methyl cellulose (MC) as a component of an alginate/methyl cellulose (alg/MC) bioink, used for bioprinting embedded bovine primary chondrocytes (BPCs). In contrast to the autoclave, scCO2 and UV-treatments, the γ-irradiated MC resulted in a strong reduction in alg/MC viscosity and stability after extrusion which made this method unsuitable for precise bioprinting. Gel permeation chromatography analysis revealed a significant reduction in MC molecular mass only after γ-irradiation, which influenced MC chain mobility in the Ca2+-crosslinked alginate network as well as gel composition and microstructure. With regard to cell survival and proteoglycan matrix production, the results determined UV-irradiation and autoclaving as the best candidates for sterilisation. The scCO2-treatment of MC resulted in an unfavourable cell response indicating that this method needs careful optimisation prior to application for cell encapsulation. As proven by consistent FT-IR spectra, chemical alterations could be excluded as a cause for the differences seen between MC treatments on alg/MC behaviour. This investigation provides knowledge for the development of a clinically appropriate 3D-printing-based fabrication process to produce bioengineered tissue for cartilage regeneration.

Capsular Exopolysaccharides from Two Streptococcus thermophilus Strains Differ in Their Moisture Sorption Behavior.

Streptococcus thermophilus is a species frequently used in the manufacture of fermented milk. Apart from acid production, some strains additionally synthesize exopolysaccharides (EPS) which contribute to texture improvement and syneresis reduction, both being attributable to the EPS's high water binding capacity. There are two different types of EPS that may be produced, namely free exopolysaccharides (fEPS) which are secreted into the medium, and capsular EPS (cEPS) which remain attached to the bacterial cell wall. This study aims to analyze their individual contribution to techno-functional properties of fermented milk by determining the moisture sorption behavior of isolated fEPS and cell-attached cEPS from two S. thermophilus strains separately: ST-1G, a producer of non-ropy fEPS and cEPS, and ST-2E, a producer of ropy fEPS and cEPS. Differences in moisture load and sorption kinetics, determined for the first time for microbial EPS, were related to structural and macromolecular properties. The observed data are discussed by using previously published data on the physical properties of stirred fermented milk produced with these two strains. ST-1G EPS showed a higher cEPS fraction, a higher moisture load and slower moisture desorption than EPS produced by ST-2E, thus contributing to lower syneresis in fermented milk. For ST-2E, higher gel viscosity was related to a higher intrinsic viscosity and molecular mass of the ropy fEPS. Both strains produced complex EPS or EPS mixtures with clearly different molecular structures.

Dextrans of Weissella cibaria DSM14295: Microbial production, structure and functionality.

Lactic acid bacteria of the genus Weissella contribute to spontaneous fermentation in, e.g., sourdough or sauerkraut, but are not registered as starter cultures because of their pending safety assessment. Some strains are able to produce high amounts of exopolysaccharides. This study aims to demonstrate the techno-functionality of five dextrans from W. cibaria DSM14295, produced under varying cultivation conditions, with respect to structural and macromolecular properties. A maximum of 23.1 g/L dextran was achieved by applying the "cold shift" temperature regime. The dextrans differed in molecular mass (9-22∙108 Da, determined by HPSEC-RI/MALLS), intrinsic viscosity (52-73 mL/g), degree of branching (3.8-5.7 % at position O3, determined by methylation analysis) and their side chain length and architecture, determined by HPAEC-PAD after enzymatic hydrolysis. Stiffness of acid gels from milk spiked with these dextrans increased linearly with dextran concentration. Principal component analysis showed that dextrans produced in a semi-defined medium are primarily described by moisture sorption and branching properties, whereas dextrans produced in whey permeate were similar because of their functional and macromolecular properties. Overall, dextrans from W. cibaria DSM14295 have a high potential because of the high production yield and their functionality which can be tailored by the conditions during fermentation.

Degradation of Exopolysaccharides from Lactic Acid Bacteria by Thermal, Chemical, Enzymatic and Ultrasound Stresses.

During isolation, exopolysaccharides (EPS) from lactic acid bacteria are subject of thermal, chemical, enzymatic or ultrasound stress of different intensity that may affect macromolecular properties, for instance molecular mass or (intrinsic) viscosity. These parameters are, however, crucial, as they are associated with the technofunctional potential of EPS replacing commercial thickeners in nonfermented products. The aim of this study was to systematically examine treatments EPS are usually exposed to during isolation and to investigate the underlying degradation mechanisms. Solutions (1.0 g/L) of EPS from Streptococcus thermophilus, isolated as gently as possible, and commercial dextran were analyzed for molecular mass distributions as representative measure of molecule alterations. Generally, acid, excessive heat and ultrasonication, intensified by simultaneous application, showed EPS degradation effects. Thus, recommendations are given for isolation protocols. Ultrasonic degradation at 114 W/cm² fitted into the random chain scission model and followed third- (S. thermophilus EPS) or second-order kinetics (dextran). The degradation rate constant reflects the sensitivity to external stresses and was DGCC7710 EPS > DGCC7919 EPS > dextran > ST143 EPS. Due to their exceptional structural heterogeneity, the differences could not be linked to individual features. The resulting molecular mass showed good correlation (r² = 0.99) with dynamic viscosity.

Pilot scale isolation of exopolysaccharides from Streptococcus thermophilus DGCC7710: Impact of methodical details on macromolecular properties and technofunctionality.

Exopolysaccharides (EPS) from Streptococcus thermophilus provide similar technofunctionality such as water binding, viscosity enhancing and emulsifying effects as commercial thickeners at a significant lower concentration. Despite their high technofunctional potential, hetero polysaccharides from lactic acid bacteria are still not commercially used in unfermented foods, as the small amount of synthesised EPS calls for a high isolation effort. This study aims to analyse the macromolecular properties of EPS and cell containing isolates from S. thermophilus DGCC7710 obtained by different isolation protocols, and to link these data to the technofunctionality in model food systems. The EPS content of the isolates was affected by the microfiltration/ultrafiltration membranes used for cell removal/dialysis, respectively, and was 89% at maximum. There was no link between purity of the isolates, molecular mass (3 × 106 Da) and intrinsic viscosity (0.53 - 0.59 mL/mg) of the EPS. After adding EPS containing isolates to milk, gel stiffness after acidification increased by 25% at maximum, depending on the type and concentration of the specific isolate. Partly purified, cell containing isolates were effective at low absolute EPS concentration (approx. 0.1 g/kg) and therefore represent, together with their simple isolation protocol, an interesting approach to introduce microbial EPS into non-fermented products.

How Capsular Exopolysaccharides Affect Cell Surface Properties of Lactic Acid Bacteria.

Some lactic acid bacteria are able to produce exopolysaccharides that, based on localization, can be distinguished in free and capsular or cell-bound exopolysaccharides (CPS). Up to now, the former were the focus of current research, mainly because of the technofunctional benefits they exhibit on fermented dairy products. On the other hand, CPS affect the surface properties of bacteria cells and thus also the textural properties of fermented foods, but data are very scarce. As the cell surface properties are strongly strain dependent, we present a new approach to investigate the impact of CPS on cell surface hydrophobicity and moisture load. CPS positive and negative Streptococcus thermophilus and Weissella cibaria were subjected to ultrasonication suitable to detach CPS without cell damage. The success of the method was verified by scanning electron and light microscopy as well as by cultivation experiments. Before applying ultrasonication cells with CPS exhibiting an increased hydrophilic character, enhanced moisture load, and faster water adsorption compared to the cells after CPS removal, emphasizing the importance of CPS on the textural properties of fermented products. The ultrasonic treatment did not alter the cell surface properties of the CPS negative strains.

Production and molecular structure of heteropolysaccharides from two lactic acid bacteria.

In the dairy industry, exopolysaccharides (EPS) produced in situ from lactic acid bacteria are of great interest because of their contribution to product texture. Some EPS cause ropiness which might be linked to specific physical and chemical EPS properties. EPS show a broad variety of chemical structures and, because analysis is rather complex, it is still a major challenge to establish structure-function relationships. The aim of this study was to produce EPS with different degree of ropiness, perform in-depth structural elucidations and relate this information to their behaviour in aqueous solutions. After cultivation of Streptococcus thermophilus DGCC7919 and Lactococcus lactis LL-2A and subsequent EPS isolation, both EPS showed similar macromolecular properties, but pronounced differences in monosaccharide composition and glycosidic linkages. Our data suggests that mainly the side chains in the EPS from LL-2A might be responsible for a higher ropiness than that observed for EPS from DGCC7919.

Test parameters and cell chain length of Streptococcus thermophilus affect the microbial adhesion to hydrocarbons assay: a methodical approach.

The microbial adhesion to hydrocarbons (MATH) test is one of the most common method to determine the hydrophobicity of cell surfaces. Despite its prevalence, no standard test parameters are used in literature, making a direct comparison of data almost impossible. Criticism also focuses on test parameters that may mask hydrophobic interactions and hence lead to erroneous test results. We methodically investigated the impact of different MATH test parameters on the calculation of the cell surface hydrophobicity of Streptococcus thermophilus, a widespread exopolysaccharide-producing lactic acid bacterium used in the production of fermented milk products. Besides composition and ionic strength of the buffer used for cell re-suspension, we observed a pronounced time dependency of the turbidity of the cell suspension during phase separation due to sedimentation and/or cell lysis. A new modification of the MATH assay was applied to enable the determination of cell surface hydrophobicity of long chain-forming bacteria. As the cell surface hydrophobicity was not altered during exponential growth phase, we assume that the cell surface and its capsular exopolysaccharide layer are not changed during cultivation.

Shear treatment of starter culture medium improves separation behavior of Streptococcus thermophilus cells.

A central step in the production of starter cultures is the separation of the cells from the fermentation medium, which is usually achieved by disk centrifuges. In case of microorganisms which produce exopolysaccharides (e.g., various strains of lactic acid bacteria), the properties of the respective exopolysaccharides may interfere with this separation step. By using six strains of Streptococcus thermophilus the hypothesis was tested that a shear treatment of the fermented culture medium improves subsequent cell separation markedly. Depending on the type of exopolysaccharides (freely present in the medium, or as capsules around the cells) an energy input of up to 2.5 kJ/mL generated with an Ultra-Turrax affected cell chain length of the strains and viscosity of fermentation medium differently. For bacteria producing capsular exopolysaccharides, space- and time-resolved centrifugation experiments revealed an increase of sedimentation velocity after shear treatment. In general, viability of the microorganisms, detected by flow cytometry measurements and fermentation experiments, was not affected by the shearing procedure. The results therefore indicate that strain-targeted shearing is helpful to improve the separability of cells from the fermented media.