Fernanda Mozzi

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.

Biotechnology of lactic acid bacteria: novel applications.

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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.

Functional fermented whey-based beverage using lactic acid bacteria

Whey protein concentrate (WPC) is employed as functional food ingredient because of its nutritional value and emulsifying properties. However, the major whey protein beta-lactoglobulin (BLG) is the main cause of milk allergy. The aim of this study was to formulate a fermented whey beverage using selected lactic acid bacteria and WPC35 (WPC containing 35% of proteins) to obtain a fermented product with low lactose and BLG contents and high essential amino acid concentration. Cell viability, lactose consumption, lactic acid production, proteolytic activity, amino acid release and BLG degradation by the selected strains Lactobacillus acidophilus CRL 636, Lactobacillus delbrueckii subsp. bulgaricus CRL 656 and Streptococcus thermophilus CRL 804, as single or mixed (SLaB) cultures were evaluated in WPC35 (10%, w/v) incubated at 37 degrees C for 24h. Then, the fermented WPC35 was mixed with peach juice and calcium lactate (2%, w/v) and stored at 10 degrees C for 28 days. During fermentation, single cultures grew 1.7-3.1 log CFU/ml and produced 25.1-95.0 mmol/l of lactic acid as consequence of lactose consumption (14.0-41.8 mmol/l) after 12h fermentation. L. delbrueckii subsp. bulgaricus CRL 656 was the most proteolytic strain (626 microg/ml Leu) and released the branched-chain essential amino acids Leu (16 microg/ml), Ile (27 microg/ml) and Val (43 microg/ml). All strains were able to degrade BLG in a range of 41-85% after 12h incubation. The starter culture SLaB grew 3.0 log CFU/ml, showed marked pH reduction, produced 122.0 mmol/l of lactic acid, displayed high proteolytic activity (484 microg/ml Leu) releasing Leu (13 microg/ml), Ile (18 microg/ml) and Val (35 microg/ml), and hydrolyzed 92% of BLG. The addition of calcium lactate to WPC35 maintained the drink pH stable during shelf life; no contamination was detected during this period. After 28 days, a decrease in cell viability of all strains was observed being more pronounced for L. delbrueckii subsp. bulgaricus CRL 656 and L. acidophilus CRL 636 (2.3 and 1.9 log CFU/ml, respectively). The results showed that WPC fermentation by rationally selected lactic acid bacteria might be used for developing functional beverages with improved characteristics such as reduced BLG content and increased branched-chain essential amino acids.

Whey fermentation by thermophilic lactic acid bacteria: evolution of carbohydrates and protein content.

Whey, a by-product of the cheese industry usually disposed as waste, is a source of biological and functional valuable proteins. The aim of this work was to evaluate the potentiality of three lactic acid bacteria strains to design a starter culture for developing functional whey-based drinks. Fermentations were performed at 37 and 42 degrees C for 24h in reconstituted whey powder (RW). Carbohydrates, organic acids and amino acids concentrations during fermentation were evaluated by RP-HPLC. Proteolytic activity was measured by the o-phthaldialdehyde test and hydrolysis of whey proteins was analyzed by Tricine SDS-PAGE. The studied strains grew well (2-3log cfu/ml) independently of the temperature used. Streptococcus thermophilus CRL 804 consumed 12% of the initial lactose concentration and produced the highest amount of lactic acid (45 mmol/l) at 24h. Lactobacillus delbrueckii subsp. bulgaricus CRL 454 was the most proteolytic (91 microg Leu/ml) strain and released the branched chain amino acids Leu and Val. In contrast, Lactobacillus acidophilus CRL 636 and S. thermophilus CRL 804 consumed most of the amino acids present in whey. The studied strains were able to degrade the major whey proteins, alpha-lactalbumin being degraded in a greater extent (2.2-3.4-fold) than beta-lactoglobulin. Two starter cultures were evaluated for their metabolic and proteolytic activities in RW. Both cultures acidified and reduced the lactose content in whey in a greater extent than the strains alone. The amino acid release was higher (86 microg/ml) for the starter SLb (strains CRL 804+CRL 454) than for SLa (strains CRL 804+CRL 636, 37 microg/ml). Regarding alpha-lactalbumin and beta-lactoglobulin degradation, no differences were observed as compared to the values obtained with the single cultures. The starter culture SLb showed high potential to be used for developing fermented whey-based beverages.

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.

Effect of galactose and glucose on the exopolysaccharide production and the activities of biosynthetic enzymes in Lactobacillus casei CRL 87.

Aims: The objective of this work was to study the influence of the sugar source on exopolysaccharide (EPS) production and the activities of the enzymes involved in the synthesis of sugar nucleotides in Lactobacillus casei CRL 87. The relationship between these enzymes and EPS formation was determined.

Functionality of exopolysaccharides produced by lactic acid bacteria in an in vitro gastric system

Aims:  To evaluate whether slime‐exopolysaccharides (EPS) or capsular‐polysaccharide (CPS) production could protect the polymer‐producing strains Streptococcus thermophilus CRL 1190 and Lactobacillus casei CRL 87 against the harsh conditions of an in vitro gastric system (GS). EPS stability on the GS was studied.

Exopolysaccharide production by Lactobacillus casei under controlled pH

SummaryThe exopolysaccharide (EPS) production and growth characteristics of Lactobacillus casei CRL 87 under pH control were studied. Maximum polymer synthesis (488 mg/l) and cell viability (2.4×1010 cfu/ml) occurred when L. casei was cultured at a constant pH of 6.0 and 30°C for 24 h. However, the optimum specific EPS production (3.9×10-5 g EPS/g cell dry weigt) and EPS yield (4.3%) were found at a pH of 4.0.