Bart Degeest

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.

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.

Correlation of Activities of the Enzymes α-Phosphoglucomutase, UDP-Galactose 4-Epimerase, and UDP-Glucose Pyrophosphorylase with Exopolysaccharide Biosynthesis by Streptococcus thermophilus LY03

ABSTRACT The effects of different carbohydrates or mixtures of carbohydrates as substrates on bacterial growth and exopolysaccharide (EPS) production were studied for the yoghurt starter cultureStreptococcus thermophilus LY03. This strain produces two heteropolysaccharides with the same monomeric composition (galactose and glucose in the ratio 4:1) but with different molecular masses. Lactose and glucose were fermented by S. thermophilusLY03 only when they were used as sole energy and carbohydrate sources. Fructose was also fermented when it was applied in combination with lactose or glucose. Both the amount of EPS produced and the carbohydrate source consumption rates were clearly influenced by the type of energy and carbohydrate source used, while the EPS monomeric composition remained constant (galactose-glucose, 4:1) under all circumstances. A combination of lactose and glucose resulted in the largest amounts of EPS. Measurements of the activities of enzymes involved in EPS biosynthesis, and of those involved in sugar nucleotide biosynthesis and the Embden-Meyerhof-Parnas pathway, demonstrated that the levels of activity of α-phosphoglucomutase, UDP-galactose 4-epimerase, and UDP-glucose pyrophosphorylase are highly correlated with the amount of EPS produced. Furthermore, a weaker relationship or no relationship between the amounts of EPS and the enzymes involved in either the rhamnose nucleotide synthetic branch of the EPS biosynthesis or the pathway leading to glycolysis was observed for S. thermophilus LY03.

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.

Exopolysaccharide (EPS) biosynthesis by Lactobacillus sakei 0-1: production kinetics, enzyme activities and EPS yields.

Aims: To determine optimal exopolysaccharide (EPS) production conditions of the mesophilic lactic acid bacterium strain Lactobacillus sakei 0–1 and to detect possible links between EPS yields and the activity of relevant enzymes.

A novel area of predictive modelling: describing the functionality of beneficial microorganisms in foods

Predictive microbiology generally focuses on the potential outgrowth of spoilage bacteria and foodborne pathogens in foods. Little attention has been paid to the biokinetics of beneficial foodgrade microorganisms, such as lactic acid bacteria. The latter is commonly used in the food fermentation industry, mainly for the in situ production of the antimicrobial lactic acid to extend the shelf life of the food. Furthermore, many strains show additional industrial potential as novel starter cultures since they produce functional metabolites, such as bacteriocins and exopolysaccharides. The production of these functional metabolites has been demonstrated during in vitro experiments, but in many cases these novel starter cultures seem to be less efficient when applied in a food system. A modelling approach may contribute to a better understanding of the tight relation between the food environment and bacterial functionality. Primary modelling can be applied to fit the experimental data concerning cell growth, sugar metabolism, and the production of functional metabolites for a given set of environmental conditions. This led to conclusions concerning the growth-associated production of bacteriocin and exopolysaccharides, the inactivation of these molecules when cell growth levels off, and a minimum cell concentration to trigger on bacteriocin production. Examples deal with the production of the bacteriocin sakacin K by the natural fermented sausage isolate Lactobacillus sakei CTC 494, and the production of heteropolysaccharides by the yoghurt starter culture Streptococcus thermophilus LY03. Secondary modelling of biokinetic parameters quantifies the production of bacteriocin and exopolysaccharides in function of environmental factors. As an example, the specific bacteriocin production by Lb. sakei CTC 494 decreases with increasing sodium chloride concentrations. Furthermore, since the assessment of functionality is frequently hampered by the nature of the food system, mathematical modelling techniques may help to predict the functional behaviour of novel lactic acid bacteria starter cultures in a food matrix, and hence quantify in situ production. For example, a model may simulate cell growth and exopolysaccharide production of S. thermophilus LY03 in a milk environment, where direct measurements are difficult to perform.

Effect of medium composition and temperature and pH changes on exopolysaccharide yields and stability during Streptococcus thermophilus LY03 fermentations

To increase the exopolysaccharide (EPS) yields from Streptococcus thermophilus LY03 and to unravel the nature of the EPS degradation process, fermentation experiments were carried out with this strain in a customized MRS medium, using different additional carbohydrates or amino acids possibly related to growth and EPS production. No significant increase of the EPS yields or activities of the enzymes alpha-phosphoglucomutase, UDP-glucose pyrophosphorylase and UDP-galactose 4-epimerase that are correlated with EPS production, or of the activity of dTDP-glucose pyrophosphorylase involved in the rhamnose synthetic branch of EPS biosynthesis, was observed. The EPS monomer composition remained unchanged for all experiments. Fermentations with a sudden temperature increase or lowered pH were carried out as well to try to avoid EPS degradation upon prolonged fermentation. It was demonstrated that EPS degradation took place enzymatically. Incubations of purified high-molecular-mass EPS with cell-free culture supernatant or cell extracts showed its degradation by enzymes with an endo-activity. This glycohydrolytic activity probably encompasses several enzymes having a molecular mass lower than 50,000 and 10,000 Da, and seems to be rather stable at high temperature and low pH. These results contribute to a better understanding of the physiological and chemical factors influencing EPS production and degradation.

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).

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.