Medana Zamfir

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.

Purification and characterization of a bacteriocin produced by Lactobacillus acidophilus IBB 801

M. ZAMFIR, R. CALLEWAERT, P.C. CORNEA, L. SAVU, I. VATAFU and L. DE VUYST.1999.Lactobacillus acidophilus IBB 801 produces a small bacteriocin, designated acidophilin 801, with an estimated molecular mass of less than 6·5 kDa. It displays a narrow inhibitory spectrum (only related lactobacilli but including the Gram‐negative pathogenic bacteria Escherichia coli Row and Salmonella panama 1467) with a bactericidal activity. The antimicrobial activity of cell‐free culture supernatant fluid was insensitive to catalase but sensitive to proteolytic enzymes such as trypsin, proteinase K and pronase, heat‐stable (30 min at 121 °C), and maintained in a wide pH range. The proteinaceous compound was isolated from cell‐free culture supernatant fluid and purified. Crude bacteriocin was isolated as a floating pellicle after ammonium sulphate precipitation (40% saturation) and partially purified by extraction/precipitation with chloroform/methanol (2/1, v/v). Further purification to homogeneity was performed by reversed phase Fast Performance Liquid Chromatography. The amino acid composition was determined. Amino acid sequencing revealed that the N‐terminal end was blocked.

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.

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.

Bacterial community dynamics, lactic acid bacteria species diversity and metabolite kinetics of traditional Romanian vegetable fermentations

BACKGROUND Artisanal vegetable fermentations are very popular in Eastern European countries. Fresh vegetables undergo a spontaneous fermentation in the presence of salt, which is mainly carried out by lactic acid bacteria (LAB). RESULTS Culture-dependent and culture-independent analyses of end-samples of various spontaneous vegetable fermentations carried out in houses of the Chiodju region (central Romania) revealed Lactobacillus plantarum and Lactobacillus brevis as the most frequently isolated LAB species. Leuconostoc mesenteroides and Leuconostoc citreum were also found. Furthermore, the community dynamics of spontaneous cauliflower and mixed-vegetable (green tomatoes, carrots and cauliflower) fermentations revealed three steps: an initial phase characterised by the presence of Enterobacteriaceae and a wide LAB species diversity, encompassing Weissella species; a second phase from day 3 onwards wherein L. citreum and Lb. brevis occurred; and a final phase characterised by the prevalence of Lb. brevis and Lb. plantarum. Metabolite target analysis revealed that glucose and fructose were mostly depleted at the end of fermentation. The main products of carbohydrate metabolism were lactic acid, acetic acid, ethanol and small amounts of mannitol, indicating heterolactate fermentation. CONCLUSION Given their prevalence at the end of vegetable fermentations, Lb. brevis and Lb. plantarum appear to be good candidate starter cultures for controlled vegetable fermentation processes.

Characterization of some bacteriocins produced by lactic acid bacteria isolated from fermented foods

Lactic acid bacteria (LAB) isolated from different sources (dairy products, fruits, fresh and fermented vegetables, fermented cereals) were screened for antimicrobial activity against other bacteria, including potential pathogens and food spoiling bacteria. Six strains have been shown to produce bacteriocins: Lactococcus lactis 19.3, Lactobacillus plantarum 26.1, Enterococcus durans 41.2, isolated from dairy products and Lactobacillus amylolyticus P40 and P50, and Lactobacillus oris P49, isolated from bors. Among the six bacteriocins, there were both heat stable, low molecular mass polypeptides, with a broad inhibitory spectrum, probably belonging to class II bacteriocins, and heat labile, high molecular mass proteins, with a very narrow inhibitory spectrum, most probably belonging to class III bacteriocins. A synergistic effect of some bacteriocins mixtures was observed. We can conclude that fermented foods are still important sources of new functional LAB. Among the six characterized bacteriocins, there might be some novel compounds with interesting features. Moreover, the bacteriocin-producing strains isolated in our study may find applications as protective cultures.

Applicability of Lactobacillus plantarum IMDO 788 as a starter culture to control vegetable fermentations

BACKGROUND Fermentation of vegetables and fruits is a traditional preservation technique, e.g. in Eastern Europe. Although usually spontaneous fermentation processes are applied, the addition of lactic acid bacteria (LAB) starter cultures could accelerate processing and improve the consistency and quality of the end-products. RESULTS The application of Lactobacillus plantarum IMDO 788 as a starter culture strain for cauliflower and mixed vegetable fermentations resulted in accelerated acidification as compared with the spontaneous fermentations. The strain dominated the background microbiota throughout the process, whereas the spontaneous fermentations were characterised by widely variable species diversity. During the spontaneous fermentations, almost all carbohydrates were converted into lactic acid, ethanol, mannitol and acetic acid, indicating the participation of both heterofermentative and homofermentative LAB species. During the starter culture-added fermentations, residual carbohydrates were found and lactic acid and ethanol were the main end-metabolites. Vegetable-associated aromas, ethyl acetate and isoamyl acetate were produced during all fermentations. The high concentration of ethanol and the production of ethyl acetate and isoamyl acetate suggested the involvement of yeasts during all fermentations. CONCLUSION Lactobacillus plantarum IMDO 788 was an adequate starter culture strain for vegetable fermentations, prevailing over endogenous LAB communities. Further optimisation of the starter culture formulation is necessary to avoid yeast growth.

Functional Properties of Lactic Acid Bacteria Isolated from Romanian Fermented Vegetables

A total of 139 lactic acid bacterium (LAB) strains isolated from Romanian traditionally fermented vegetables were screened for the ability to produce exopolysaccharides and for their antagonistic activity against a set of nine LAB strains, three Bacillus strains, and four Gram-negative bacteria. Eighty-five of the tested strains showed a variable antimicrobial activity against Listeria monocytogenes ATCC 1911, 35 of the strains showed a limited inhibition zone against Escherichia coli ATCC25922, and 26 strains against Salmonella enterica ATCC 14024, while 19 strains showed inhibition against one or all three Bacillus strains used as indicators. None of the tested strains showed an antimicrobial activity against Staphylococcus aureus ATCC 25923. Several strains showed antibacterial activity against more than one indicator strain. For instance, Lactobacillus plantarum 307, Lactobacillus brevis 308, and Lactobacillus plantarum/pentosus 358 were active against five of the indicator strains used, while other 23 LAB were active against three indicator strains. In the case of two strains, namely Leuconostoc citreum 344 and Lactobacillus brevis 183, the activity was maintained after neutralizing the pH of the cell-free supernatant likely due to the production of bacteriocins. The gel permeation chromatography-based screening revealed seven EPS-producing LAB strains. Two of the positive strains, namely Leuconostoc citreum 177 and Leuconostoc citreum 52, have been shown to produce large amounts of EPS, of about 20 g/L. All isolated EPS have a high molecular mass, of above 1400 KDa, and a monomer composition dominated by the presence of glucose.

Versatile SPR aptasensor for detection of lysozyme dimer in oligomeric and aggregated mixtures

A Surface Plasmon Resonance (SPR) sensor for the quantitation of lysozyme dimer in monomer-dimer mixtures, reaching a detection limit of 1.4nM dimer, has been developed. The sensor is based on an aptamer which, although developed for the monomeric form, binds also the dimeric form but with a strikingly different kinetics. The aptasensor was calibrated using a dimer obtained by cross-linking. Sensorgrams acquired with the aptasensor in monomer-dimer mixtures were analysed using Principal Components Analysis and Multiple Regression to establish correlations with the dimer content in the mixtures. The method allows the detection of 0.1-1% dimer in monomer solutions without any separation. As an application, the aptasensor was used to qualitatively observe the initial stages of aggregation of lysozyme solutions at 60°C and pH 2, through the variations in lysozyme dimer amounts. Several other methods were used to characterize the lysozyme dimer obtained by cross-linking and confirm the SPR results. This work highlights the versatility of the aptasensor, which can be used, by simply tuning the experimental conditions, for the sensitive detection of either the monomer or the dimer and for the observation of the aggregation process of lysozyme.