Frederik Vaningelgem

Recent developments in the biosynthesis and applications of heteropolysaccharides from lactic acid bacteria

Abstract Microbial exopolysaccharides (EPS) occur as capsules or as secreted slime. They represent a small fraction of todays biopolymer market with factors limiting their use being mainly associated with economical production. Efficient production and reduction in recovery costs requires knowledge of biosynthesis and adoption of appropriate bioprocess technology. EPS from generally recognized as safe food grade microorganisms, particularly lactic acid bacteria (LAB), have potential as food additives or as functional food ingredients with both health and economic benefits. Many different heteropolysaccharides (HePS) are secreted by LAB regarding sugar composition and molecular size but they show few common structural features, which raises questions about the relationship between structure and texture. HePS are made by polymerizing repeating units formed in the cytoplasm. These are assembled at the membrane by specific glycosyltransferases (GTF) through the sequential addition of sugar nucleotides. The latter are delivered as building blocks and attached to the growing repeating unit that is anchored on a lipid carrier. After completion, the repeating unit is externalized and polymerized. The enzymes and proteins involved in biosynthesis and secretion are not necessarily unique to HePS formation. These processes involve a genetic organization that includes specific eps genes and “housekeeping” genes involved in sugar nucleotide biosynthesis. The intentional and controlled use of HePS from LAB or use of strains producing HePS in situ is important in the food industry. However, instability in production and variability of HePS yields are well documented. Therefore, a well understood optimized carbon flux and supply of sugar nucleotides, knowledge of the GTF, and the functional expression of combinations of genes from different origin into stable, industrial strains open interesting ways to polysaccharide engineering.

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.

Microbial physiology, fermentation kinetics, and process engineering of heteropolysaccharide production by lactic acid bacteria

Many species of lactic acid bacteria (LAB) produce extracellular heterotype polysaccharides, the so-called heteropolysaccharides (HePS). Biosynthesis and secretion of the HePS from the LAB occur during different growth phases, and both the amount and type of the polymer is influenced by growth conditions. The total yield of exopolysaccharides produced by the LAB depends on the composition of the medium (carbon and nitrogen sources, growth factors, etc.) and the conditions in which the strains grow, i.e. temperature, pH, oxygen tension, and incubation time. It is never higher than 1.5 g of polymer dry mass per litre of fermentation medium. Whereas mesophilic strains produce maximal amounts of HePS under conditions not optimal for growth, the HePS production from thermophilic LAB strains is growth-associated, i.e. maximum production during growth and under conditions optimal for growth. The HePS degradation often takes place upon prolonged incubation of the HePS-producing LAB strains due to glycohydrolase activity. Primary, secondary, and tertiary modelling unravel the functionality of the HePS-producing LAB strains in a food environment. Finally, appropriate process engineering can lead to an industrial breakthrough of the HePS production and applications: a high and stable high-molecular-mass HePS production by appropriate feeding strategies through fed-batch cultivation on the one hand, and the application of a two-step fermentation process in yoghurt manufacture on the other hand.

Exopolysaccharide-producing Streptococcus thermophilus strains as functional starter cultures in the production of fermented milks

Relationships between exopolysaccharide (EPS) production (amount, molecular mass and sugar composition of the EPS) by different Streptococcus thermophilus strains as a functional starter culture, and textural characteristics (viscosity) of fermented milk and yoghurt have been studied. Five interesting heteropolysaccharide-producing strains have been tested. Both S. thermophilus LY03 and S. thermophilus CH101 produced the highest amounts of EPS and also displayed the highest apparent viscosities in fermented milk. S. thermophilus ST 111 and S. thermophilus STD differed considerably in EPS yields, but not in apparent viscosities of fermented milk. In addition, S. thermophilus ST 111 displayed a high variability in EPS amounts when cultivated in milk. In milk medium, S. thermophilus LY03 produced two heteropolysaccharides, a high-molecular-mass (HMM) EPS and a low-molecular-mass (LMM) EPS of the same composition (Gal/Glu/GalNAc=3.4:1.4:1.0). S. thermophilus ST 111 produced only a HMM-EPS (Gal/Rha=2.5:1.0), while S. thermophilus CH 101 (Gal/Glu=1.0:1.0), S. thermophilus ST 113 (Gal/Glu/Rha/GalNAc=1.7:3.9:1.5:1.0) and S. thermophilus STD (Gal/Glu/Rha/GalNAc=3.5:6.2:1.2:1.0) produced only LMM-EPS. Both HMM-EPS and LMM-EPS solutions (S. thermophilus LY03) demonstrated a pseudoplastic character; HMM-EPS solutions of 0.2% (m/v) displayed a high consistency as well. Although its production of high EPS amounts, S. thermophilus LY03 resulted in relatively thin yoghurts, so that texture values did not directly correlate with EPS production capacity. Once structure/function relationships are known, one can determine the molecular properties of the isolated and purified EPS (molecular size, structural characteristics) from candidate strains to predict their potential in texture formation. For a final selection of interesting EPS-producing starter strains one should test the EPS production under yoghurt manufacturing conditions.

Diversity of Heteropolysaccharide-Producing Lactic Acid Bacterium Strains and Their Biopolymers

ABSTRACT Thirty-one lactic acid bacterial strains from different species were evaluated for exopolysaccharide (EPS) production in milk. Thermophilic strains produced more EPS than mesophilic ones, but EPS yields were generally low. Ropiness or capsular polysaccharide formation was strain dependent. Six strains produced high-molecular-mass EPS. Polymers were classified into nine groups on the basis of their monomer composition. EPS from Enterococcus strains were isolated and characterized.

Exopolysaccharide-producing strains of thermophilic lactic acid bacteria cluster into groups according to their EPS structure.

Aims: To compare galactose‐negative strains of Streptococcus thermophilus and Lactobacillus delbrueckii subspecies bulgaricus isolated from fermented milk products and known to produce exopolysaccharides (EPSs).

Fermentation conditions affecting the bacterial growth and exopolysaccharide production by Streptococcus thermophilus ST 111 in milk‐based medium

Aims:  To study the effect of different fermentation conditions and to model the effect of temperature and pH on different biokinetic parameters of bacterial growth and exopolysaccharides (EPS) production of Streptococcus thermophilus ST 111 in milk‐based medium.

UDP-N-Acetylglucosamine 4-Epimerase Activity Indicates the Presence of N-Acetylgalactosamine in Exopolysaccharides of Streptococcus thermophilus Strains

ABSTRACT The monomer composition of the exopolysaccharides (EPS) produced byStreptococcus thermophilus LY03 and S. thermophilus Sfi20 were evaluated by high-pressure liquid chromatography with amperometric detection and nuclear magnetic resonance spectroscopy. Both strains produced the same EPS composed of galactose, glucose, and N-acetylgalactosamine. Further, it was demonstrated that the activity of the precursor-producing enzyme UDP-N-acetylglucosamine 4-epimerase, converting UDP-N-acetylglucosamine into UDP-N-acetylgalactosamine, is responsible for the presence of N-acetylgalactosamine in the EPS repeating units of both strains. The activity of UDP-N-acetylglucosamine 4-epimerase was higher in bothS. thermophilus strains than in a non-EPS-producing control strain. However, the level of this activity was not correlated with EPS yields, a result independent of the carbohydrate source applied in the fermentation process. On the other hand, both the amounts of EPS and the carbohydrate consumption rates were influenced by the type of carbohydrate source used during S. thermophilus Sfi20 fermentations. A correlation between activities of the enzymes α-phosphoglucomutase, UDP-glucose pyrophosphorylase, and UDP-galactose 4-epimerase and EPS yields was seen. These experiments confirm earlier observed results for S. thermophilus LY03, although S. thermophilusSfi20 preferentially consumed glucose for EPS production instead of lactose in contrast to the former strain.

Growth and energy generation by Enterococcus faecium FAIR-E 198 during citrate metabolism

Citrate metabolism by Enterococcus faecium FAIR-E 198, isolated from Greek Feta cheese, was studied in various growth media containing citrate either in the presence of glucose, or as the sole carbon source, both under aerobic and anaerobic conditions. In de Man-Rogosa-Sharpe (MRS) broth with increasing citrate concentrations, cometabolism of citrate and glucose took place. Glucose was stoichiometrically converted into lactate, while citrate into acetate. Glucose consumption and biomass yield were enhanced with increasing initial citrate concentrations, even though maximum specific growth rate was not. When citrate was used as the sole carbon source in increasing initial concentrations, the main end product was acetate. Small amounts of lactate, formate, ethanol, and acetoin were also produced. In all cases, no significant differences were observed between aerobic and anaerobic conditions. However, when citrate was used as sole carbon source, formate production was favored in the absence of oxygen. The present work shows that E. faecium is able to utilize citrate in synthetic media, either in the presence of glucose or as the sole carbon source, resulting in energy production and the formation of aroma compounds.

Sensitivity of capsular-producing Streptococcus thermophilus strains to bacteriophage adsorption

Aims:  To determine whether the presence and type of exopolysaccharides (EPS), slime‐EPS or capsular, and the structural characteristics of the polymers produced by Streptococcus thermophilus strains could interfere with or be involved in phage adsorption.