Characterization and antimicrobial spectrum of bacteriocins produced by lactic acid bacteria isolated from traditional Bulgarian dairy products.

AIMS: To isolate bacteriocin-producing lactic acid bacteria (LAB) with high wide spectrum antibacterial activity and to characterize their inhibitory peptides. METHOD AND RESULTS: Seven LAB strains [Lactobacillus casei ssp. rhamnosus (PC5), Lactobacillus delbrueckii ssp. bulgaricus (BB18), Lactococcus lactis ssp. lactis (BCM5, BK15), Enterococcus faecium (MH3), Lactobacillus plantarum (BR12), Lactobacillus casei ssp. casei (BCZ2)], isolated from authentic Bulgarian dairy products were capable of producing bacteriocins, inhibiting the widest range of pathogenic bacteria. The bacteriocins were resistant to heating at 121 degrees C for 15 min, stable at pH 2-10, sensitive to protease, insensitive to alpha-amylase and lipase. Two of bacteriocins produced by Lact. bulgaricus BB18 (bulgaricin BB18) and E. faecium MH3 (enterocin MH3) were purified and the molecular masses were determined. The N-terminal amino acid sequence of bulgaricin BB18 did not show strong homology to other known bacteriocins. CONCLUSIONS: Lactobacillus bulgaricus BB18 and E. faecium MH3 produce two novel bacteriocins highly similar to the pediocin-like nonlantibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: The two bacteriocins are potential antimicrobial agents and, in conjunction with their producers, may have use in applications to contribute a positive effect on the balance of intestinal microflora. Furthermore, bulgaricin BB18 strongly inhibits Helicobacter pylori.

Creation of chiral structures inside fused silica glass.

Circularly polarized (CP) femtosecond laser light focused into fused silica produces a permanent recording of optical helicity. Material modification is in the form of highly ordered submicrometer chiral structures whose handedness follows the handedness of the CP light. The evolution of the ordered structures from chaotic modification is described.

Bacteriocin production by strain Lactobacillus delbrueckii ssp. bulgaricus BB18 during continuous prefermentation of yogurt starter culture and subsequent batch coagulation of milk.

By screening for bacteriocin-producing lactic acid bacteria of 1,428 strains isolated from authentic Bulgarian dairy products, Lb. bulgaricus BB18 strain obtained from kefir grain was selected. Out of 11 yogurt starters containing Lb. bulgaricus BB18 and S. thermophilus strains resistant to bacteriocin secreted by Lb. bulgaricus BB18 a yogurt culture (S. thermophilus 11A+Lb. bulgaricus BB18) with high growth and bacteriocinogenic activity in milk was selected. Continuous (pH-stat 5.7) prefermentation processes were carried out in milk at 37 degrees C in a 2l MBR bioreactor (MBR AG, Zurich, Switzerland) with an IMCS controller for agitation speed, temperature, dissolved oxygen, CO2 and pH. Prefermented milk with pH 5.7 coagulated in a thermostat at 37 degrees C until pH 4.8-4.9. S. thermophilus 11A and Lb. bulgaricus BB18 grew independently in a continuous mode at similar and sufficiently high-dilution rates (D=1.83 h(-1)-S. thermophilus 11A; D=1.80 h(-1)-Lb. bulgaricus BB18). The yogurt cultures developed in a stream at a high-dilution rate (D=2.03-2.28 h(-1)). The progress of both processes (growth and bacteriocin production) depended on the initial ratio between the two microorganisms. The continuous prefermentation process promoted conditions for efficient fermentation and bacteriocinogenesis of the starter culture during the batch process: strong reduction of the times for bacteriocin production and coagulation of milk (to 4.5-5.0 h); high cell productivity (lactobacilli-4x10(12) CFU ml(-1), streptococci-6x10(12) CFU ml(-1)); high productivity of bacteriocins (4,500 BU ml(-1))-1.7 times higher than the bacteriocinogenic activity of the batch starter culture.

Femtosecond laser erasing and rewriting of self-organized planar nanocracks in fused silica glass.

Tightly focused, linearly polarized, femtosecond laser radiation can produce highly birefringent nanograting structures inside fused silica glass. Here we report that when the polarization direction of the femtosecond light is changed, old nanogratings are erased and simultaneously replaced with new ones whose orientation is solely determined by the polarization of the rewrite beam. We also show that these volume nanogratings can be rewritten 1000 times with little degradation in their quality.

Femtosecond laser writing of porous capillaries inside fused silica glass.

We demonstrate that within a restricted optical pulse duration-pulse energy parameter space tightly focused femtosecond laser radiation can be used to fabricate porous capillaries in bulk fused silica glass by simply moving the laser focus through the material. We show that the rate of penetration of liquids into the porous capillaries can be controlled by the laser polarization, which determines their morphology. The fluid propagation is measured using the form birefringence of nanocrack/nanovoid structures produced inside the capillaries. We also demonstrate the nanofiltration capabilities of the capillaries by separating the relatively small molecules of Rhodamine 6G dye from their solvent.

Optically produced arrays of planar nanostructures inside fused silica.

Linearly polarized femtosecond light pulses, focused inside fused silica to an intensity that leads to multiphoton ionization, produce arrayed planes of modified material having their normal parallel to the laser polarization. The planes are < or = 10 nm thick and are spaced at approximately lambda/2 in the medium for free space wavelengths of both 800 and 400 nm. By slowly scanning the sample under a fixed laser focus, order is maintained over macroscopic distances for all angles between the polarization and scan direction. With the laser polarization parallel to the scan direction we produce long-range Bragg-like gratings. We discuss how local field enhancement influences dielectric ionization, describe how this leads to nanoplane growth, why the planes are arrayed, and how long-range order is maintained.

Memory in nonlinear ionization of transparent solids.

We demonstrate a shot-to-shot reduction in the threshold laser intensity for ionization of bulk glasses illuminated by intense femtosecond pulses. For SiO2 the threshold change serves as positive feedback reenforcing the process that produced it. This constitutes a memory in nonlinear ionization of the material. The threshold change saturates with the number of pulses incident at a given spot. Irrespective of the pulse energy, the magnitude of the saturated threshold change is constant (approximately 20%). However, the number of shots required to reach saturation does depend on the pulse energy. Recognition of a memory in ionization is vital to understand multishot optical or electrical breakdown phenomena in dielectrics.

Polarization-selective etching in femtosecond laser-assisted microfluidic channel fabrication in fused silica.

We fabricate microchannels in fused silica by femtosecond laser irradiation followed by etching in diluted hydrofluoric acid. We show a dramatic dependence of the etch rate on the laser polarization, spanning 2 orders of magnitude. We establish the existence of an energy-per-pulse threshold at which etching of the laser-modified zones becomes highly polarization selective. The enhanced selective etching is due to long-range, periodic, polarization-dependent nanostructures formed in the laser-modified material.

Exopolysaccharides produced by mixed culture of yeast Rhodotorula rubra GED10 and yogurt bacteria (Streptococcus thermophilus 13a + Lactobacillus bulgaricus 2-11).

AIMS: The studies of the production of exopolysaccharides by lactose-negative yeast and a yogurt starter co-cultivated in a natural substrate containing lactose may be considered of interest because they reveal the possibilities for high-efficiency synthesis of biopolymers by mixed cultivation. METHODS AND RESULTS: The mixed culture Rhodotorula rubra GED10 + (Streptococcus thermophilus 13a + Lactobacillus bulgaricus 2-11) was cultivated in cheese whey ultrafiltrate (WU) (44.0 g lactose l(-1)) at initial pH 6.0, 28 degrees C, under intensive aeration (air-flow rate 1.0 l l(-1) min(-1), agitation 220 rev min(-1)) in a MBR AG fermentor. The mixed culture manifested the highest activity for synthesis of exopolysaccharides (19.3 g l(-1)) and cell mass (21.0 g l(-1)) at the 84th hour. The yogurt starter synthesized neutral exopolysaccharides, while the mixed culture yeast + yogurt starter produced acidic exopolysaccharides containing uronic acid (6%). The neutral sugar composition was identified as mannose, glucose, galactose, xylose and arabinose. Mannose dominated in the polymer composition (83%) that was produced only by the yeast (97%). CONCLUSIONS: Lactose in the WU can be effectively utilized by a co-culture of lactose-negative yeast-yogurt starter for synthesis of exopolysaccharides. SIGNIFICANCE AND IMPACT OF THE STUDY: The present findings propose an alternative use of WU as a cost-effective carbohydrate substrate, and suggest that the lactose-negative yeast Rhodotorula rubra can have industrial application as producers of exopolysaccharides.

Stress in femtosecond-laser-written waveguides in fused silica.

We identify two states of stress induced in waveguides fabricated by femtosecond lasers in fused silica and show how they can be relieved by annealing. In-plane stress and stress concentration are revealed through birefringence and loss measurements. Another kind of laser-induced stress appears in the form of swelling of the glass surface when waveguides are written near the surface and is a manifestation of confined rapid material quenching. By annealing the sample we reduce the losses by approximately 30% (at 633 nm) and decrease the birefringence by a factor of 4 in fused silica.

Synthesis of carotenoids by Rhodotorula rubra GED8 co-cultured with yogurt starter cultures in whey ultrafiltrate.

Two cultures, a yeast ( Rhodorula rubra GED8) and a yogurt starter ( Lactobacillus bulgaricus 2-11+ Streptococcus thermophilus 15HA), were selected for associated growth in whey ultrafiltrate (WU) and active synthesis of carotenoids. In associated cultivation with the yogurt culture L bulgaricus 2-11+S. thermophilus 15HA under intensive aeration (1.3 l(-1)min(-1) air-flow rate) in WU (45 g lactose l(-1)), initial pH 5.5, 30 degrees C, the lactose-negative strain R. rubra GED8 synthesized large amounts of carotenoids (13.09 mg l(-1 )culture fluid). The carotenoid yield was approximately two-fold higher in association with a mixed yogurt culture than in association with pure yogurt bacteria. The major carotenoid pigments comprising the total carotenoids were beta-carotene (50%), torulene (12.3%) and torularhodin (35.2%). Carotenoids with a high beta-carotene content were produced by the microbial association 36 h earlier than by Rhodotorula yeast species. No significant differences were notd in the ratio between the pigments synthesized by R. rubra GED8+ L. bulgaricus 2-11, R. rubra GED8+ S. thermophilus 15HA, and R.rubra GED8+yogurt culture, despite the fact that the total carotenoid concentrations were lower in the mixed cultures with pure yogurt bacteria.

Femtosecond laser fabrication of nanostructures in silica glass.

A femtosecond laser beam focused inside fused silica and other glasses can modify the refractive index of the glass. Chemical etching and atomic-force microscope studies show that the modified region can have a sharp-tipped cone-shaped structure with a tip diameter as small as 100 nm. Placing the structure near the bottom surface of a silica glass sample and applying a selective chemical etch to the bottom surface produces clean, circular, submicrometer-diameter holes. Holes spaced as close to one another as 1.4 microm are demonstrated.

Ultra-high resolution index of refraction profiles of femtosecond laser modified silica structures.

The combination of selective chemical etching and atomic force microscopy has been used for the first time to make ultra-high spatial resolution (20 nm) index of refraction profiles of femtosecond laser modified structures in silica glass.

Lactic acid bacteria and yeasts in kefir grains and kefir made from them.

In an investigation of the changes in the microflora along the pathway: kefir grains (A)-->kefir made from kefir grains (B)-->kefir made from kefir as inoculum (C), the following species of lactic acid bacteria (83-90%) of the microbial count in the grains) were identified: Lactococcus lactis subsp. lactis, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus, Lactobacillus casei subsp. pseudoplantarum and Lactobacillus brevis. Yeasts (10-17%) identified were Kluyveromyces marxianus var. lactis, Saccharomyces cerevisiae, Candida inconspicua and Candida maris. In the microbial population of kefir grains and kefir made from them the homofermentative lactic streptococci (52-65% and 79-86%, respectively) predominated. Within the group of lactobacilli, the homofermentative thermophilic species L. delbrueckii subsp. bulgaricus and L. helveticus (70-87% of the isolated bacilli) predominated. Along the pathway A-->B-->C, the streptococcal proportion in the total kefir microflora increased by 26-30% whereas the lactobacilli decreased by 13-23%. K. marxianus var. lactis was permanently present in kefir grains and kefirs, whereas the dominant lactose-negative yeast in the total yeast flora of the kefir grains dramatically decreased in kefir C.

pi-shifted Sagnac interferometer for characterization of femtosecond first- and second-order polarization mode dispersion.

A novel configuration for measuring femtosecond polarization mode dispersion (PMD) with a pi-shifted Sagnac interferometer is studied. The advantages of this configuration include a response that is independent of the orientation of the device under test. This independence allows characterization of the first-and second-order PMD by measurement of the wavelength dependence of the differential group delay and of the orientation of the device's principal states of polarization. The method provides an extension of the dynamic range of the Sagnac interferometer technique to few-femtosecond PMD measurement.

Vinorelbine, epirubicin, and methotrexate (VEM) as primary treatment in locally advanced breast cancer.

PURPOSE: This phase II trial of VEM (vinorelbine + epirubicine + methotrexate) in the treatment of locally advanced breast cancer was conducted to obtain downstaging to allow surgery and breast conservation. PATIENTS AND METHODS: This multicenter study recruited 58 patients with locally advanced breast cancer (two patients ineligible); 56 were evaluable for response and tolerance. RESULTS: Downstaging was obtained in 77% of the patients with a pathological complete response (pCR) rate of 9%. At 33 months of follow-up, median survival has not been reached. Neutropenia grade 3-4 was reported in 31% of cycles with 3% of cycles with infection grade 3. Alopecia grade 3 was noticed for 71% of patients. CONCLUSION: VEM represents an effective regimen for patients with locally advanced breast cancer, allowing an important pCR. Moreover, this regimen appears to be particularly well tolerated.

Production and monomer composition of exopolysaccharides by yogurt starter cultures.

As components of starter cultures for Bulgarian yogurt, Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus revealed extensive exopolysaccharide (EPS) production activity when cultivated in whole cow's milk. The polymer-forming activity of thermophilic streptococci was lower (230-270 mg EPS/L) than that of the lactobacilli (400-540 mg EPS/L). Mixed cultures stimulated EPS production in yogurt manufacture, and a maximum concentration of 720-860 mg EPS/L was recorded after full coagulation of milk. The monomer structure of the exopolysaccharides formed by the yogurt starter cultures principally consists of galactose and glucose (1:1), with small amounts of xylose, arabinose, and/or mannose.

Measurement of femtosecond Polarization Mode Dispersion (PMD) using biased p-shifted low-coherence interferometry.

Conventional low-coherence interferometry (LCI) can be employed in the measurement of polarization mode dispersion (PMD) of fiber-optic components and fibers. However, the smallest PMD, which can be measured using this technique, is limited by the coherence length of the source. We propose a biased p-shifted Michelson interferometer where a birefringent crystal is inserted in front of the interferometer to introduce a bias differential group delay (DGD) larger than the coherence time of the source. In this way, the limitation imposed by the source coherence time has been overcome and PMDs much smaller than the source coherence time, in the order of several femtoseconds, can be measured. Experimental results for the PMD have been shown and compared with Jones matrix eigen-analysis. The theoretical model confirms the experimental observations.

Production of amino acids by yogurt bacteria.

The dynamics of free amino acid production by the selected strains Streptococcus thermophilus 13a and Lactobacillus bulgaricus 2-11 were studied in pure and mixed cultivations during yogurt starter culture manufacture. L. bulgaricus 2-11 showed the highest activity for producing free amino acids with high individual concentrations over the first hour of growth (50% of the total amount). By the end of milk's full coagulation (4.5 h), 70% of the total amount of amino acids was released. S. thermophilus 13a showed poor proteolytic properties and consumed up to 70% of the free amino acids produced by L. bulgaricus 2-11 in the process of coagulation of milk with the mixed culture.

Caroteno-protein and exopolysaccharide production by co-cultures of Rhodotorula glutinis and Lactobacillus helveticus.

The lactose-negative yeast Rhodotorula glutinis 22P and the homofermentative lactic acid bacterium Lactobacillus helveticus 12A were cultured together in a cheese whey ultrafiltrate containing 42 g L-1 lactose. The chemical composition of the caroteno-protein has been determined. The carotenoid and protein contents are 248 micrograms g-1 dry cells and 48.2% dry weight. Carotenoids produced by Rhodotorula glutinis 22P have been identified as beta-carotene 15%, torulene 10%, and torularhodin 69%. After separating the cell mass from the microbial association, the exopolysaccharides synthesized by Rhodotorula glutinis 22P were isolated from the supernatant medium in a yield of 9.2 g L-1. The monosaccharide composition of the synthesized biopolymer was predominantly D-mannose (57.5%).