Characterization of S-layer proteins produced by lactobacilli isolated from Romanian artisan fermented products.

AIMS: To characterize S-layer proteins produced by four lactobacilli isolated from Romanian artisan fermented products. METHODS AND RESULTS: Four lactobacilli strains have been shown to produce S-layer proteins, both under optimal and stressfull conditions. The presence of S-layer proteins was confirmed by transmission electron microscopy. Removal of S-layer proteins caused a loss of the bacterial resistance to stress conditions and of the autoaggregation ability. Liquid chromatography-mass spectrometry analysis identified peptides corresponding to Slp M sequence in case of Levilactobacillus brevis 403, and peptides corresponding to Slp A sequence in case of Lactobacillus helveticus 34.9. The analysis confirmed molecular masses of ∼51 and 48 kDa, respectively, for the two proteins, and gave information about their pI, of about 9.4-9.6. A specific PCR amplification was obtained for the genome of Lact. helveticus 34.9 with slpA primers, and the amplicon sequence was 95.31% identical to slpA gene. CONCLUSIONS: Our findings indicate that certain environmental stress conditions can induce the S-layer production, which helps the producing cells to survive under unfavorable conditions.

Non-Dairy Fermented Beverages Produced with Functional Lactic Acid Bacteria.

At present, there is an increasing interest in beverages of non-dairy origin, as alternatives to those based on milk, but having similar health-promoting properties. Fermentation with specific bacteria or consortia may enhance the functionality of these products. In our study, selected lactic acid bacteria, that have been previously shown to possess functional properties (antimicrobial activity, probiotic potential), were used for the fermentation of wheat bran combined with root vegetables. Strains were investigated for their safety, while the obtained beverages were characterized in terms of microbial content, physical, chemical, nutritional, and functional properties. None of the strains harbors virulence genes, but all of them possess genes for survival at low pH, starch metabolism, and vitamin biosynthesis. Three strains (Lactiplantibacillus plantarum BR9, L. plantarum P35, and Lactobacillus acidophilus IBB801) and two substrates (5% wheat bran with 10% red beetroot/carrots) were selected based on a preliminary assessment of the beverage's sensory acceptability. These strains showed good growth and stability over time in the stored beverages. No enterobacteria were detected at the end of fermentations, while the final pH was, in most cases, below 3.5. Free phenolics, flavonoids, and DPPH scavenging effect increased during fermentation in all drinks, reaching 24h values that were much higher than in the unfermented substrates. Most of the obtained drinks were able to prevent the growth of certain pathogens, including Listeria monocytogenes ATCC 19111, Salmonella enterica ATCC 14028, Staphylococcus aureus ATCC 25923, and Escherichia coli ATCC 25922. The obtained beverages would combine the nutritiveness of the raw ingredients with the beneficial effect of fermentation (increasing shelf life, health-promoting effect, pleasant flavor, etc.). They would also fill a gap in the non-dairy probiotics sector, which is constantly increasing due to the increasing number of vegan people or people that cannot consume dairy products.

Lactobacillus Strains for Vegetable Juice Fermentation-Quality and Health Aspects.

Vegetable juices are new carrier variants for beneficial bacteria, representing an alternative to dairy-fermented products, especially for vegan, strict vegetarian, or allergic consumers. The aim of this study was to characterize several Romanian native lactic acid bacteria (LAB) strains to select valuable nutritional and probiotic strains for vegetable juice fermentation. Nineteen LAB strains were analyzed for antibiotic susceptibility (disc-diffusion method), the presence of antibiotic resistance genes, the presence of functional genes. and the production of organic acids by HPLC. Antibiotic resistant strains were observed only with ampicillin (Amp10) and kanamycin (K30), 79% and 32%, respectively, with results partially confirmed by molecular analysis. Multiplex PCR revealed the presence of LBA1272, dltD, folP, agl, α-amy, malL, and ribA genes, related to stress resistance, starch metabolism, and production of vitamins, except for folK. HPLC analyses were performed on beet roots (SF), tomato (TM), and a mixture of carrots, celery, and beet (MTS) juices. High values of lactic acid were recorded in all cases of LAB fermentation (5034-14,176 µg/mL). The maximum values recorded for acetic acid did not exceed 2.5 mg/mL having a positive influence on the product's taste.

Isolation, characterization, and mode of action of a class III bacteriocin produced by Lactobacillus helveticus 34.9.

Traditionally fermented foods and beverages are still produced and consumed at a large scale in Romania. They are rich sources for novel lactic acid bacteria with functional properties and with potential application in food industry or health. Lactobacillus helveticus 34.9, isolated from a home-made fermented milk is able to inhibit the growth of other bacteria, such as other lactic acid bacteria, but also strains of Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, and Halobacillus hunanensis, a halobacterium isolated from the degraded wall of a Romanian monastery. L. helveticus 34.9 produces a large bacteriocin (35 KDa), active in a wide pH range, but inactivated by heat and proteinase K treatment. It shares about 20% sequence coverage with helveticin J, as determined by LC-MS analysis. Bacteriocin production was enhanced under stress conditions, especially when combined stresses were applied. Its mode of action and degree of inhibition depended on the concentration and on the indicator strain that was used; L. delbrueckii subsp. bulgaricus LMG 6901T cells from a suspension were killed, but the viability of H. hunanensis 5Hum cells was only reduced to 60%, within 8 h. However, the bacteriocin was able to prevent the bacterial growth of both indicator strains when added to the cultivation medium prior inoculation. Scanning electron microscopy images revealed morphological changes induced by the bacteriocin treatment in both sensitive strains, but more severe in the case of L. delbrueckii subsp. bulgaricus. Due to the broad antibacterial spectrum and its production under various stress conditions, the bacteriocin or the producing strain may find application in health, food and non-food related fields, including in the restoration of historical buildings.

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.

S-layer production by Lactobacillus acidophilus IBB 801 under environmental stress conditions.

The ability of microorganisms to synthesize S-layer, the outermost structure of the microbial cell envelope composed of non-covalently bound proteins, has been ascribed to help microorganisms to exert their probiotic properties in the host. In this work, formation of S-layer by the potentially probiotic strain Lactobacillus acidophilus IBB 801 under different stress culture conditions (high incubation temperatures, presence of bile salts or NaCl, and acidic pH) was assayed. A marked S-layer synthesis by L. acidophilus IBB 801 was detected when the strain was grown at 42 °C and in the presence of 0.05 % bile salts or 2.0 % NaCl. The presence of S-layer proteins was further confirmed by transmission electron microscopy and protein identification by MS/MS. The differential expression of the proteome of this strain at 42 °C, when a marked formation of S-layer was detected, revealed the overexpression of six proteins mainly related to general stress and protein biosynthesis and translation, while four proteins detected in lower amounts were involved in DNA repair and energy metabolism. As L. acidophilus IBB 801 produces both a bacteriocin and S-layer proteins, the strain could be of interest to be used in the formulation of functional food products with specific properties.

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.

Stress response of some lactic acid bacteria isolated from Romanian artisan dairy products.

Understanding the mechanisms of stress response and adaptation to stress in the case of lactic acid bacteria (LAB), especially in the case of strains with functional properties, is very important when such strains are potential candidates for starter cultures or probiotics. In this context, our study shows the response of some LAB [four exopolysaccharide (EPS)-producing strains and one strain with potential probiotic effect] to the stresses induced by low and high incubation temperatures, acidity, NaCl, and bile salts, often encountered during the technological processes in food or during the passage through the human gastro-intestinal tract. The strains were able to grow at temperatures up to 40 °C (the mesophilic strains) and 47 °C (the thermophilic strain), in medium with an initial pH of at least 4.0 (Lactobacillus acidophilus IBB801), or in the presence of NaCl up to 10% (Weissella confusa/cibaria 38.2), or bile salts up to 0.2% (L. acidophilus IBB801). The protein and isoenzyme patterns of the strains subjected to various stress conditions presented several differences compared with the control patterns, among which the overexpression of some proteins of about 50-60 kDa, differences in the bands intensity in the case of the intracellular enzymes, or the complete loss of some of these bands. The best survival to low pH values and high temperatures was observed for strain L. acidophilus IBB801, the candidate probiotic strain. The EPS production of the four tested strains was, in general, directly related to the growth, the highest yields being obtained when strains were incubated at 24 °C.

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.

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.

Impact of stress conditions on the growth of Lactobacillus acidophilus IBB 801 and production of acidophilin 801.

The influence of different stress conditions, such as low and high temperatures, the presence of salt or ethanol in the medium, on the growth of Lactobacillus acidophilus IBB801 and the production of acidophilin 801 was investigated in this study. The strain was able to grow at up at to 47 degrees C, while higher temperatures were lethal. A slow growth was detected at 24 degrees C, starting after 24 h of incubation, and the strain was able to survive at 10 degrees C for more than 48 h of incubation. The protein profiles revealed by one dimensional SDS-PAGE showed at least four overexpressed and two repressed bands at low temperatures (10 degrees C and 24 degrees C) compared with the profiles at 37 degrees C and 42 degrees C. The bacteriocin activity was the highest when the producing strain was grown at optimum temperature. However, at 10 degrees C, the inhibitory activity showed a slight increase compared with the one reached at 24 degrees C and 47 degrees C. The same increase in activity was observed in the presence of low amounts of salt (5 to 10 g/L). Higher concentrations of salt or ethanol addition to the growth medium had a negative effect on acidophilin biosynthesis.

Screening of lactic acid bacteria isolates from dairy and cereal products for exopolysaccharide production and genes involved.

A total of 174 lactic acid bacteria (LAB) strains isolated from dairy and cereal products were screened for the production of exopolysaccharides (EPS). Therefore, a rapid screening method was developed based on ultrafiltration and gel permeation chromatography. Furthermore, a screening through the polymerase chain reaction (PCR) was performed with primer pairs targeting different genes involved in EPS production. Nine isolates produced a homopolysaccharide of the glucan type, whereas only one strain produced a heteropolysaccharide. The production of a glucan by a strain of Lactococcus lactis and the production of a heteropolysaccharide by a strain of Lactobacillus curvatus are reported for the first time. The PCR screening revealed many positive strains. For three of the ten EPS-producing strains, no corresponding genes could be detected. Furthermore, a lot of strains possessed one or more eps genes but did not produce an EPS. Therefore, a screening on the molecular level should always be accompanied by another screening method that is able to distinguish true EPS producer strains from non-producing ones. Statistical analysis did not reveal any relationship between the type and origin of the strains, the presence or absence of a capsular polysaccharide or EPS, and the presence or absence of eps genes.

Biodiversity of lactic acid bacteria in Romanian dairy products.

Traditionally fermented dairy products are still a very important part of the daily food in Romania, especially for people living in the countryside. To study the biodiversity of lactic acid bacterium strains of these products, 110 samples (raw and fermented milk, sour cream, and cheese) were collected from farm houses, monasteries, and local markets throughout Romania. Lactic acid bacteria (LAB) were isolated using six different cultivation conditions. All 599 isolates were tested for their Gram reaction, catalase activity, and morphology. A rep-PCR fingerprinting technique with the (GTG)5 primer and, in some cases SDS-PAGE of total cell proteins and 16S rRNA gene sequencing were used to cluster and/or identify the LAB. The biodiversity of the isolated strains was correlated with the type of product and/or technology applied. The most frequent LAB found in Romanian raw milk and fermented dairy products were Lactococcus lactis, Leuconostoc spp., and Enterococcus spp. Among the latter, a new species E. saccharominimus was found.

Reclassification of Leuconostoc argentinum as a later synonym of Leuconostoc lactis.

Leuconostoc argentinum, Leuconostoc lactis and ten related strains from Romanian dairy products formed a single cluster, clearly separated from other Leuconostoc species, after numerical analysis of repetitive extragenic palindromic-PCR patterns, whole-cell protein profiles (SDS-PAGE) and fluorescent amplified fragment length polymorphism (FAFLP) band patterns. 16S rRNA gene sequence analysis confirmed a very high similarity between both type strains and representative dairy isolates (>99.6 %). DNA-DNA hybridization experiments revealed high relatedness values between the type strains of L. argentinum and L. lactis and between these strains and representative Romanian strains. These data and the lack of phenotypic distinctive characteristics demonstrate that L. argentinum and L. lactis are synonymous.

Biodiversity of exopolysaccharides produced by Streptococcus thermophilus strains is reflected in their production and their molecular and functional characteristics.

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.