Osmotic stress response in the wine yeast Dekkera bruxellensis

Dekkera bruxellensis is mainly associated with lambic beer fermentation and wine production and may contribute in a positive or negative manner to the flavor development. This yeast is able to produce phenolic compounds, such as 4-ethylguaiacol and 4-ethylphenol which could spoil the wine, depending on their concentration. In this work we have investigated how this yeast responds when exposed to conditions causing osmotic stress, as high sorbitol or salt concentrations. We observed that osmotic stress determined the production and accumulation of intracellular glycerol, and the expression of NADH-dependent glycerol-3-phosphate dehydrogenase (GPD) activity was elevated. The involvement of the HOG MAPK pathway in response to this stress condition was also investigated. We show that in D. bruxellensis Hog1 protein is activated by phosphorylation under hyperosmotic conditions, highlighting the conserved role of HOG MAP kinase signaling pathway in the osmotic stress response.     

Molecular identification and osmotolerant profile of wine yeasts that ferment a high sugar grape must

The objective of this study was to examine the Saccharomyces and non-Saccharomyces yeast populations involved in a spontaneous fermentation of a traditional high sugar must (Vino cotto) produced in central Italy. Molecular identification of a total of 78 isolates was achieved by a combination of PCR-RFLP of the 5.8S ITS rRNA region and sequencing of the D1/D2 domain of the 26S rRNA gene. In addition, the isolates were differentiated by RAPD-PCR. Only a restricted number of osmotolerant yeast species, i.e. Candida apicola, Candida zemplinina and Zygosaccharomyces bailii, were found throughout all the fermentation process, while Saccharomyces cerevisiae prevailed after 15 days of fermentation. A physiological characterization of isolates was performed in relation to the resistance to osmotic stress and ethanol concentration. The osmotolerant features of C. apicola, C. zemplinina and Z. bailii were confirmed, while S. cerevisiae strains showed three patterns of growth in response to different glucose concentrations (2%, 20%, 40% and 60% w/v). The ability of some C. apicola and C. zemplinina strains to grow at 14% v/v ethanol is noteworthy. The finding that some yeast biotypes with higher multiple stress tolerance can persist in the entire winemaking process suggests possible future candidates as starter for Vino cotto production.     

Response surface optimization of lipid and protein extractions from <Emphasis Type="Italic">Spirulina platensis</Emphasis> using ultrasound assisted osmotic shock method

In this study, we optimized the process for extracting lipids and proteins from wet biomasses of Spirulina sp. using a 4-kHz ultrasonic osmotic shock method with ultrasound enhancement at a constant frequency of 40 kHz. Optimization was conducted using a response surface methodology (RSM) at an osmotic NaCl concentration of 10–30%, solvent:biomass ratio of 5–15 v/w, and extraction times of 20–50 min. The present osmotic shock method with ultrasound irradiation increased lipid yields to 6.65% in the presence of 11.9% NaCl, a solvent:biomass ratio of 12:1 v/w, and a 22-min extraction time, and protein yields to 43.96% with 15.12% NaCl, a solvent:biomass ratio of 10:1 v/w, and a 30-min extraction time.     

Behaviour of Listeria monocytogenes isolates through gastro-intestinal tract passage simulation, before and after two sub-lethal stresses

The effects of previous exposure to sub-lethal acidic and osmotic stresses on the survival of Listeria monocytogenes during exposure to gastro-intestinal (GI) tract simulation, was investigated. Six L. monocytogenes strains isolated from cheeses were selected and exposed to high salt concentrations or acidic conditions and their viability compared in quick and slow digestions. The results demonstrated that (i) all isolates were more sensitive to the exposure to acidic than to osmotic sub-lethal conditions (ii) significant differences (p < 0.05) between the two types of digestion were observed; in slow digestion, the log reduction was higher for all the tested isolates (iii) all isolates were inhibited in the presence of bile salts for both types of digestion (iv) differences between quick and slow digestion were not observed (p > 0.05) after exposure to either osmotic or acidic stress (v) a higher cellular inactivation (p < 0.001) was observed during the passage through the GI tract simulation after exposure to osmotic than to acidic stresses and (vi) neither osmotic nor acidic sub-lethal stresses conferred resistance to simulated GI tract conditions.     

Diversity of stress tolerance in Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus paraplantarum: A multivariate screening study

Sixty-three strains of the taxonomically related species Lactobacillus plantarum subsp. plantarum, L. plantarum subsp. argentoratensis, L. paraplantarum and L. pentosus isolated from sourdoughs and other food and non-food sources and 14 strains of other members of the genus Lactobacillus were screened for their tolerance of acid, alkaline, heat, oxidative, osmotic, detergent and starvation stresses in order to evaluate the diversity of stress response. Most strains of the L. plantarum group were highly tolerant of acid, alkaline and osmotic stress and highly sensitive to detergent stress, while a larger diversity was found for other stress. Multivariate analysis allowed grouping the strains in clusters with similar response patterns. Stress response patterns in the L. plantarum group were similar to those of species of the L. casei/L. paracasei group but clearly different from those of other mesophilic Lactobacillus. No relationship was found between grouping obtained on the basis of stress response patterns and by genotypic fingerprinting (rep-PCR), nor with the taxonomic position or isolation source of the strains. Further experiments with selected strains showed that exponential phase cells were generally but not always more sensitive than stationary phase cells. The ability to grow under stressful conditions showed a slightly better correlation with the ecological conditions prevailing in the isolation niches of the strains.This study will be the basis for further investigations to identify and exploit the basis of diversity in the stress response of lactic acid bacteria.     

Emulsifying and foaming properties of Phaseolus vulgaris and coccineus proteins

Protein isolates from two Phaseolus cultivars, common bean (Phaseolus vulgaris L.) and scarlet runner bean (Phaseolus coccineus L.), were prepared by wet extraction methods (isoelectric precipitation – 4000 rpm, ultrafiltration, extraction with NaCl 2%, and isoelectric precipitation – 9900 rpm). The protein isolates were characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then evaluated for their solubility. The emulsion stability of emulsions produced at pH 7.0 and 5.5 with 1% or 2% or 3% w/v protein isolate was evaluated by average droplet size diameter, viscosity and creaming measurements. Emulsions with 1% protein content were unstable through storage. Emulsions with 3% w/v protein isolate concentration, extracted by ultrafiltration at pH 5.5 from both cultivars, were flocculated; this was more pronounced for coccineus isolates. The foaming properties, for the respective foams, were investigated. Foams with 1% w/v protein showed little foaming ability Ultrafiltration isolates produced more foam, which was especially stable at pH 5.5.     

Biochemical characterization of dextranase from Arthrobacter oxydans and its cloning and expression in Escherichia coli

Appreciably elevated levels of dextranase from Arthrobacter oxydans (AODex) isolated from sugar-cane farm soil was resulted from the culture on the Luria-Bertani (LB) medium containing 1%(w/v) soluble starch, glycerol, or dextran. The responsible gene (aodex) was cloned, its nucleotide sequence was determined, and expression of the encoded protein was achieved in Escherichia coli. An open reading frame was composed of 1,863 bp putatively encoding a 68.3 kDa protein. Recombinant A. oxydans dextranase (rAODex) was purified about 16 fold by nickel-nitrilotriacetic acid affinity column chromatography; K _m value for dextran T2000 was 0.85 mg/mL (w/v). AODex treatment of stale sugar cane juice resulted in a yield of square and light-colored sugar crystals.     

Positioning of cell growth and division after osmotic stress requires a map kinase pathway

The yeast Saccharomyces cerevisiae has a genetic program for selecting and assembling a bud site on the cell cortex. Yeast cells confine their growth to the emerging bud, a process directed by cortical patches of actin filaments within the bud. We have investigated how cells regulate budding in response to osmotic stress, focusing on the role of the high osmolarity glycerol response (HOG) pathway in mediating this regulation. An increase in external osmolarity induces a growth arrest in which actin filaments are lost from the bud. This is followed by a recovery phase in which actin filaments return to their original locations and growth of the original bud resumes. After recovery from osmotic stress, haploid cells retain an axial pattern of bud site selection while diploids change their bipolar budding pattern to an increased bias for forming a bud on the opposite side of the cell from the previous bud site. Mutants lacking the mitogen-activated protein (MAP) kinase encoded by HOG1 or the MAP kinase kinase encoded by PBS2 (previously HOG4) show a similar growth arrest after osmotic stress. However, in the recovery phase, the mutant cells (a) do not restart growth of the original bud but rather start a new bud, (b) fail to restore actin filaments to the original bud but move them to the new one, and (c) show a more random budding pattern. These defects are elicited by an increase in osmolarity and not by other environmental stresses (e.g., heat shock or change in carbon source) that also cause a temporary growth arrest and shift in actin distribution. Thus, the HOG pathway is required for repositioning of the actin cytoskeleton and the normal spatial patterns of cell growth after recovery from osmotic stress.     

Surface tension of Phaseolus vulgaris and coccineus proteins and effect of polysaccharides on their foaming properties

The surface tension of protein isolates from common bean (Phaseolus vulgaris L.) and scarlet runner bean (Phaseolus coccineus L.), prepared by isoelectric precipitation and ultrafiltration was evaluated, with respect to protein concentration (0.001–0.1% w/v) and pH (pH 4.5, 5.5, 7.0 and 8.0). Surface tension was most reduced, and with a higher rate of reduction at higher protein concentration and at pH 8.0. Foams (1, 2% w/v protein), at the same pH values, with and without the addition of polysaccharides, were studied. The proteins’ foaming behaviour was related to their adsorption behaviour. Arabic gum, locust bean gum (0.1% and 0.25% w/v), xanthan gum and a xanthan/locust bean gum mixture (0.1% w/v) had a positive effect on foam creation. All polysaccharides increased foam stability, probably due to the viscosity increase and to the creation of a network, which prevents the air droplets from coalescence. Isolates from P. coccineus and isolates obtained by ultrafiltration seemed to exhibit better foaming properties.     

Effect of Lepidium perfoliatum seed gum addition on whey protein concentrate stabilized emulsions stored at cold and ambient temperature

In this study the effect of Lepidium perfoliatum seed gum on the properties of whey protein concentrate (WPC) stabilized corn oil-in-water emulsions at pH 7 was investigated. Various concentrations (0–0.6% w/v) of L. perfoliatum seed gum were used together with 2% (w/v) WPC to emulsify corn oil in water at a ratio of 1:5. Quality attributed such as particle size distribution, creaming profile and coalescence rate during storage at 4 and 25 °C; surface and interfacial tension; zeta potential and viscosity of the emulsions were determined. The results indicated that the addition of L. perfoliatum seed gum had no significant effect on zeta potential but the surface and interfacial tension increased with the rise of gum concentration. It was also found that the addition of L. perfoliatum seed gum to WPC emulsions at a critical concentration of 0.2% (w/v) caused flocculation of oil droplets, which resulted in marked increase in particle size and the creaming rate. However at higher gum concentrations beyond this value, the particle size remained constant, apparently because of the high viscosity of the aqueous phase. At all concentrations tested, emulsions stored at 4 °C were more stable except for those containing 0.2% L. perfoliatum seed gum.     

Antagonistic effect of Pseudomonas graminis CPA-7 against foodborne pathogens in fresh-cut apples under simulated commercial conditions

Recently, we reported that the application of the strain CPA-7 of Pseudomonas graminis, previously isolated from apple, could reduce the population of foodborne pathogens on minimally processed (MP) apples and peaches under laboratory conditions. Therefore, the objective of the present work was to find an antioxidant treatment and a packaging atmosphere condition to improve CPA-7 efficacy in reducing a cocktail of four Salmonella and five Listeria monocytogenes strains on MP apples under simulated commercial processing. The effect of CPA-7 application on apple quality and its survival to simulated gastric stress were also evaluated. Ascorbic acid (2%, w/v) and N-acetyl-l-cysteine (1%, w/v) as antioxidant treatments reduced Salmonella, L. monocytogenes and CPA-7 recovery, meanwhile no reduction was observed with NatureSeal^® AS1 (NS, 6%, w/v). The antagonistic strain was effective on NS-treated apple wedges stored at 10 °C with or without modified atmosphere packaging (MAP). Then, in a semi-commercial assay, efficacy of CPA-7 inoculated at 10⁵ and 10⁷ cfu mL⁻¹ against Salmonella and L. monocytogenes strains on MP apples with NS and MAP and stored at 5 and 10 °C was evaluated. Although high CPA-7 concentrations/populations avoided Salmonella growth at 10 °C and lowered L. monocytogenes population increases were observed at both temperatures, the effect was not instantaneous. No effect on apple quality was detected and CPA-7 did not survived to simulated gastric stress throughout storage. Therefore, CPA-7 could avoid pathogens growth on MP apples during storage when use as part of a hurdle technology in combination with disinfection techniques, low storage temperature and MAP.     

Effect of microencapsulation on survival of Lactobacillus plantarum in simulated gastrointestinal conditions, refrigeration, and yogurt

In the present research the survival of free and microencapsulated cells of a new strain of Lactobacillus plantarum BL011 under stress conditions was tested in sodium alginate or pectin, coated with sodium alginate or chitosan. Results for the simulated gastrointestinal medium (SGT) showed no change in viability of cells in relation to the control. However, the simulated gastric medium (GM) drastically reduced the viability under the tested conditions, with no significant differences between free and immobilized cells. Under refrigerated storage viability of immobilized cells were greatly enhanced compared to the free microorganisms, and the treatments showing the lowest loss of viability were those of 4% (w/v) pectin, 3% (w/v) sodium alginate coated with chitosan and a mixture of 2% (w/v) sodium alginate and 2% (w/v) pectin, respectively. Loss of viability of immobilized L. plantarum in 3% alginate coated with chitosan in yogurt was of 0.55 log cycles during 38 days of storage. The results of this study suggest the efficiency of immobilization techniques to increase the survival of lactobacilli in yogurt under refrigerated storage.     

Investigation of the selective bactericidal effect of several decontaminating solutions on bacterial biofilms including useful,spoilage and/or pathogenic bacteria

The aim of this work was to investigate the selective bactericidal effect of several decontaminating solutions on some spoilage, pathogenic and useful bacteria isolated from a traditional meat workshop.Fourteen decontaminating solutions, i.e., acid, alkaline, osmotic, biocide solutions or their combinations were tested on five bacterial species grown as monospecies biofilms. The solution made of monolaurin (0.075% w/v) and acetic acid at pH 5.4 was the most selective decontaminating solution. It reduced by only 0.2 and 0.4 log Lactobacillus spp. and Staphylococcus carnosus, whereas Pseudomonas fluorescens, Pseudomonas putida and Listeria monocytogenes exhibited reductions of 3.7, 3.2 and 4.2 log, respectively. The acetic solution (pH 5.4) and the solution containing monolaurin (0.075% w/v) and sodium sulfate (12% w/v) were also selective. But their bactericidal effects on Pseudomonas species were relatively small.Four selected solutions were then applied to seven bacterial species grown as multispecies biofilms. The mixture solution of monolaurin (0.075% w/v) and acetic acid at pH 5.4 showed again the best selectivity. Finally, lowering the pH of the acetic solution from 5.4 to 5.2 increased the selective decontamination.     

Isolation,structural features and rheological properties of water‐extractable β‐glucans from different Greek barley cultivars

β‐Glucans were isolated from six Greek barley cultivars (Persefoni, Kos, Thessaloniki, Athinaida, Dimitra and Triptolemos) by water extraction at 47 °C, enzymatic removal of starch and protein and subsequent precipitation of the water‐soluble β‐glucans with 37% (w/v) ammonium sulfate saturation. The purity of barley β‐glucans was high (>93% dry basis) with some small contamination by protein (<3.84%). The molecular size of the β‐glucan isolates was determined by high‐performance size‐exclusion chromatography (HPSEC); the weight‐average molecular weights and the intrinsic viscosities ranged between 0.45 × 10⁶ and 1.32 × 10⁶ and 2.77 and 4.11 dl g^?1, respectively. Structural features of barley β‐glucans were revealed by ¹³C NMR spectroscopy and high‐performance anion‐exchange chromatography (HPAEC) of the oligomers released by the hydrolytic action of lichenase. Lichenase degradation showed that β‐glucans from all barley cultivars consisted of blocks of cellotriosyl and cellotetraosyl units, accounting for 90.6–92.3% of the total oligomers released, with a molar proportion of these units between 2.31 and 2.77. Rheological measurements of aqueous solutions/dispersions of β‐glucans showed the behaviour of non‐interacting polysaccharides and a transition from the typical viscoelastic response to gel‐like properties after a time period that depended on the molecular size of the polysaccharide. The lowest molecular size β‐glucans from the Triptolemos cultivar showed shorter gelation times than their higher molecular weight counterparts. The effect of sugar incorporation (glucose, fructose, sucrose, xylose and ribose), at a concentration of 30% (w/v), to the β‐glucans gels (6% w/v) on compression parameters seemed to be related to the type of sugar used; the pentose sugars substantially reduced gel firming. Copyright © 2004 Society of Chemical Industry     

Relationship between growth of food‐spoilage yeast in high‐sugar environments and sensitivity to high‐intensity pulsed UV light irradiation

The relationship between prior growth of food‐spoilage yeast in high‐sugar environments and their subsequent survival postpulsed UV (PUV) irradiation was investigated. Test yeast were separately grown to early stationary phase in YPD broth containing increasing concentrations of glucose (1–50% w/v) and were flashed with ≤40 pulses of broad‐spectrum light at lamp discharge energy settings of 3.2, 7.2 and 12.8 J (equivalent to UV doses of 0.53, 1.09 and 3.36 μJ cm^?2, respectively) and their inactivation measured. Findings showed that prior growth in high‐sugar conditions (≥30% glucose w/v) enhanced the sensitivity of all nine representative strains of Zygosaccharomyces bailii, Z. rouxii and Saccharomyces cerevisiae yeast to PUV irradiation. Significant differences in inactivation amongst different yeast types also occurred depending on amount of UV dose applied, where the order of increasing sensitivity of osmotically stressed yeast to PUV irradiation was shown to be Z. rouxii, Z. bailii and >S. cerevisiae. For example, a 1.2‐log order difference in CFU mL^?1 reduction occurred between Z. bailii 11 486 and S. cerevisiae 834 when grown in 50% w/v sugar samples and treated with the uppermost test UV dosage of 3.36 μJ cm^?2, where these two yeast strains were reduced by 3.8 and 5.0 log orders, respectively, after this PUV treatment regime compared to untreated controls. The higher the UV dose applied the greater the reduction in yeast numbers. For example, a 1.0‐, 1.4‐ and 4.0‐log order differences in CFU mL^?1 numbers occurred for S. cerevisiae 834 grown in 15% w/v sugar samples and then treated with PUV dose of 0.53, 1.09 and 3.36 μJ cm^?2, respectively. These findings support the development of PUV for the treatment of high‐sugar foods that are prone to spoilage by osmotolerant yeast.     

Isolation and characterization of galactomannan from Dimorphandra gardneriana Tul. seeds as a potential guar gum substitute

A galactomannan was obtained from mature seeds of Dimorphandra gardneriana Tul., the plant from which rutin is extracted. The galactomannan extraction was based on manual separation of the endosperm, water dissolution, centrifugation and precipitation with ethanol. The galactomannan yield obtained (31%) was similar to values reported for other Brazilian seeds and to that of guar gum. The polysaccharide from D. gardneriana seeds (GalDG) was characterized by gas–liquid chromatography (GLC), gel permeation chromatography (GPC), rheology and also by ¹³C and ¹H nuclear magnetic resonance (NMR). The monosaccharide composition in weight % was mannose 64.2, galactose 34.7 and glucose 1.1. Small amounts of protein and uronic acid were found, values being 1.75 and 2.8% (w/w), respectively. The mannose/galactose ratio of GalDG (1.84) is similar to values reported for galactomannans extracted from other Brazilian seeds, and is the M/G value closest to that of guar gum (1.6–1.8). The intrinsic viscosity of galactomannan from D. gardneriana (8.7 dL/g), in water at 25 °C, is lower than the [η] value of guar gum, but the absolute viscosity of the GalDG in aqueous solution at concentrations of 0.1 and 1% (w/v) is higher. The aqueous solution at 1% (w/v) behaves as a pseudoplastic fluid, but a Newtonian behavior was noted for the solution at 0.1%. The high average molar masses, M_w of 3.9 × 10⁷ g/mol and M_n of 1.9 × 10⁷ g/mol, determined by GPC are probably due to molecular aggregation. ¹³C and ¹H NMR spectra (DEPT 135 and HSQC) of GalDG solutions in D₂O were recorded. The patterns of mannose substitution in GalDG and guar gum are similar.