Antagonistic effect of Saccharomyces cerevisiae KTP and Issatchenkia occidentalis ApC on hyphal development and adhesion of Candida albicans.

The morphological transition from yeast to a hyphal form, as well as the adhesion capability to the gastrointestinal tract, are implicated virulent determinant in Candida albicans and could be potential targets for prevention of the opportunistic pathogen. Based on this rationale, two yeast strains Saccharomyces cerevisiae KTP and Issatchenkia occidentalis ApC along with reference strain Saccharomyces boulardii NCDC 363 were screened for the probiotic potential. Characters like pH, temperature, bile, simulated gastrointestinal juice tolerance tests, and Caco-2 cell line adhesion assay were determined in the present study. Further, the evaluation of its impact on C. albicans morphological transition and adhesion was assessed using microtitre germ tube test. In terms of probiotic characteristics, both the strains were tolerant to pH 2.5 and the presence of bile (0.3 to 0.6%) with an optimum growth temperature of 37°C. The strain KTP was also resistant to simulated gastric and intestinal juices as compared to control (13% and 41%, respectively) and NCDC 363 (55% and 35%, respectively). In contrast, both the yeasts had reduced adhesiveness to Caco-2 monolayer. Candida virulence in in vitro systems indicated that treatment of live probiotic yeast cells (108 ml) effectively reduced the filamentation and adhesion of C. albicans. The S. cerevisiae KTP had a profound effect on the hyphal development and adhesion when compared to the ApC and NCDC 363. The strain significantly reduced (P < .05) the hyphal growth in co-cultivated (93% and 94%, respectively) and pre-existing hyphae (54% and 68%) of strains C. albicans 183 and 1151. Isolates KTP and ApC also reduced the adhesion (≈ 22% and 41%, respectively) and transition of blastoconidia at two hours of incubation in abiotic surface. This study provides knowledge on the effect of potential probiotic yeasts such as Saccharomyces and non- Saccharomyces strains against virulence characteristic of Candida albicans.

Biomarkers to evaluate the effects of temperature and methanol on recombinant Pichia pastoris

Pichia pastoris is methylotrophic yeast used as an efficient expression system for heterologous protein production. In order to evaluate the effects of temperature (10 and 30 °C) and methanol (1 and 3% (v/v)) on genetically-modified Pichia pastoris, different biomarkers were evaluated: Heat stress (HSF-1 and Hsp70), oxidative stress (OGG1 and TBARS) and antioxidant (GLR). Three yeast cultures were performed: 3X = 3% methanol-10 °C, 4X = 3% methanol-30 °C, and 5X = 1% methanol-10°C. The expression level of HIF-1α, HSF-1, HSP-70 and HSP-90 biomarkers were measured by Western blot and in situ detection was performed by immunocytochemistry. Ours results show that at 3% methanol -30 °C there is an increase of mitochondrial OGG1 (mtOGG1), Glutathione Reductase (GLR) and TBARS. In addition, there was a cytosolic expression of HSF-1 and HSP-70, which indicates a deprotection against nucleolar fragmentation (apoptosis). On the other hand, at 3% methanol -10 °C and 1% and at methanol -10 °C conditions there was nuclear expression of OGG1, lower levels of TBARS and lower expression of GLR, cytosolic expression of HSF-1 and nuclear expression HSP-70. In conclusion, our results suggest that 3% methanol-30 °C is a condition that induces a strong oxidative stress and risk factors of apoptosis in modified-genetically P. pastoris.

Combined effect of selected non-thermal technologies on Escherichia coli and Pichia fermentans inactivation in an apple and cranberry juice blend and on product shelf life.

The combination of novel, non-thermal technologies for preservation purposes is a recent trend in food processing research. In the present study, non-thermal hurdles such as ultraviolet light (UV) (5.3 J/cm²), high intensity light pulses (HILP) (3.3 J/cm²), pulsed electric fields (PEF) (34 kV/cm, 18 Hz, 93 µs) or manothermosonication (MTS) (4bar, 43 °C, 750 W, 20 kHz) were examined. The objective was to establish the potential of these technologies, applied individually or in paired sequences, to inactivate Escherichia coli and Pichia fermentans inoculated in a fresh blend of apple and cranberry juice. The shelf-life evaluation of selected non-thermally treated samples was conducted over 35 days and compared to pasteurised samples and untreated juices. All treatments applied individually significantly reduced (1.8-6.0 log cfu/ml) microbial counts compared to the untreated sample (p<0.01). Furthermore, UV treatment produced significantly greater inactivation (p<0.05) for E. coli compared to P. fermentans. Combinations of non-thermal hurdles consisting of UV or HILP followed by either PEF or MTS resulted in comparable reductions for both microorganisms (p ≥ 0.05) to those observed in thermally pasteurised samples (approx. 6 log cfu/ml). Thermally pasteurised samples had a shelf life exceeding 35 days, while that of UV+PEF and HILP+PEF-treated samples was 14 and 21 days, respectively. These results indicate that combinations of these non-thermal technologies could successfully reduce levels of E. coli and P. fermentans in apple and cranberry juice, although optimisation is required in order to further extend shelf life.

Production of ribonucleotides by autolysis of Pichia anomala mutant and some physiological activities.

Various mutants of Pichia anomala were isolated by ethyl methanesulfonate (EMS) treatment and UV irradiation through cycloheximide resistance and KCl sensitivity. The selected mutant HA-2 accumulated a higher content of RNA and grew faster than the wild-type strain in yeast extract-malt (YM) broth. Autolysis of the HA-2 mutant at 60 degrees C and pH 7.0 for 6 h was the best condition to obtain maximum yields of 5'-ribonucleotides, inosinic monophosphate (IMP) (6.2 mg/g biomass) and guanylic monophosphate (GMP) (35.5 mg/g biomass). The yield of adenylic monophosphate (AMP) (7.8 mg/g biomass) was optimal at 60 degrees C at pH 6.5 for 6 h. The inhibitory activity of the angiotensin-converting enzyme and the nitrite-scavenging activity for autolysates of the HA-2 mutant were about 13.0% and 47.0% higher than those of native strain, respectively.

Optimization of the production of Chondrus crispus hexose oxidase in Pichia pastoris.

Hexose oxidase (D-hexose:O(2)-oxidoreductase, EC 1.1.3.5, HOX) normally found in the red alga Chondrus crispus was produced heterologously in different host systems. Full-length HOX polypeptide was produced in Escherichia coli, but no HOX activity could be detected. In contrast, active HOX could be produced in the methylotrophic yeast Pichia pastoris. Several growth physiological and genetic approaches for optimization of hexose oxidase production in P. pastoris were investigated. Our results indicate that specific growth conditions are essential in order to produce active HOX with the correct conformation. Furthermore, HOX seems to be activated by proteolytic cleavage of the full-length polypeptide chain into two fragments, which remain physically associated. Attempts to direct HOX to the extracellular compartment using the widely used secretion signals from Saccharomyces cerevisiae invertase or alpha-mating factor failed. However, we show in this study that HOX is transported out of P. pastoris via a hitherto unknown mechanism and that it is possible to enhance this secretion by mutagenesis from below the detection limit to at least 250 mg extracellular enzyme per liter.

[The role of glutathione in natural and modified radioresistance of yeast cells]. / Rol' gliutationa v prirodnoi i modifitsirovannoi radiorezistentnostidrozhzhevykh kletok.

A study was made of the dependence of different natural and modified radioresistance upon glutathione content of yeast cells (Saccharomyces cerevisiae and Pichia guilliermondii). It was shown that glutathione was only involved in the formation of natural radioresistance in Saccharomyces cerevisiae cells. It was also shown that the increase in the radioresistance of yeast cells under the effect of 2-amino-2-thiazoline was accompanied by the increase in the level of total glutathione in them.

[Action of hypoxic sensitizers on the survival of haplont yeasts irradiated with ionizing radiation]. / Deistve gipoksicheskikh sensibilizatorov na vyzhivaemost' drozhzhei-gaplontov, obluchennykh ioniziruiushchei radiatsiei.

A study was made of the oxygen effect and the radiosensitizing action of metronidazole and misonidazole on hypoxic cells of irradiated yeast haplonts. It was shown that metronidazole did not increase the radiosensitivity of all the strains under study while the sensitizing effectiveness of oxygen and misonidazole approximated the values characteristic of different repair-deficient rad-mutants. Possible causes of the radiosensitizing effects observed are discussed.

[Selection of mutant branched-chain amino acid producers in the methylotrophic yeasts Hansenula polymorpha]. / Selektsiia mutantov-produtsentov aminokislot s razvetvelennoi tsep'iu u metilotrofnykh drozhzhei Hansenula polymorpha.

The object of the work was to test several methods for selection of the methylotrophic yeast Hansenula polymorpha mutants accumulating amino acids with a branched chain (valine, leucine, isoleucine). Selection of strains resistant to norvaline, an analogue of valine and leucine, was found to be the most effective method. The mutants were grown in a medium with methanol and accumulated mainly valine and, in lesser quantities, leucine, isoleucine, aspartate, asparagine and a cysteine derivative. The mutants accumulated up to 110 mg of amino acids per 1 L of the cultural broth, while the wild type strain accumulated only 5 mg/L.