The quorum-sensing molecule 2-phenylethanol impaired conidial germination, hyphal membrane integrity and growth of Penicillium expansum and Penicillium nordicum.

AIMS: The aim of the study was to investigate the antifungal effects of a quorum sensing-molecule, 2-phenylethanol, against the food spoilage moulds Penicillium expansum and Penicillium nordicum. METHODS AND RESULTS: Conidial germination of the tested Penicillium spp. (three strains in total) were inhibited by treatments with 2-phenylethanol in a concentration-dependent manner. Germinated conidia was significantly reduced from 4·4-16·7% at 7·5 mmol l-1 and completely inhibited at 15 mmol l-1 2-phenylethanol. Integrity of conidial cell membranes was unaffected by 2-phenylethanol resulting in reversible inhibition pattern of germination. In contrast, membrane permeability of actively growing hyphae was severely compromised, showing 63·5 - 75·7% membrane damage upon treatment with 15 mmol l-1 2-phenylethanol. The overall inhibitory effect of 2-phenylethanol on colony development and growth of P. expansum and P. nordicum was additionally confirmed. CONCLUSIONS: 2-phenylethanol inhibits conidial germination and growth of P. expansum and P. nordicum in a nonlethal, reversible and concentration-dependent manner. SIGNIFICANCE AND IMPACT OF THE STUDY: The study indicates that 2-phenylethanol can find potential application as an antifungal agent for biological control of moulds in the food industry.

Non-Saccharomyces yeasts protect against epithelial cell barrier disruption induced by Salmonella enterica subsp. enterica serovar Typhimurium.

UNLABELLED: The human gastrointestinal epithelium makes up the largest barrier separating the body from the external environment. Whereas invasive pathogens cause epithelial barrier disruption, probiotic micro-organisms modulate tight junction regulation and improve epithelial barrier function. In addition, probiotic strains may be able to reduce epithelial barrier disruption caused by pathogenic species. The aim of this study was to explore non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Benchmarking against established probiotic strains, we evaluated the ability of four nonpathogenic yeast species to modulate transepithelial electrical resistance (TER) across a monolayer of differentiated human colonocytes (Caco-2 cells). Further, we assessed yeast modulation of a Salmonella Typhimurium-induced epithelial cell barrier function insult. Our findings demonstrate distinct patterns of non-Saccharomyces yeast modulation of epithelial cell barrier function. While the established probiotic yeast Saccharomyces boulardii increased TER across a Caco-2 monolayer by 30%, Kluyveromyces marxianus exhibited significantly stronger properties of TER enhancement (50% TER increase). In addition, our data demonstrate significant yeast-mediated modulation of Salmonella-induced epithelial cell barrier disruption and identify K. marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates distinct patterns of non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Further, our data demonstrate significant yeast-mediated modulation of Salmonella Typhimurium-induced epithelial cell barrier disruption and identify Kluyveromyces marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. This study is the first to demonstrate significant non-Saccharomyces yeast-mediated epithelial cell barrier protection from Salmonella invasion, thus encouraging future efforts aimed at confirming the observed effects in vivo and driving further strain development towards novel yeast probiotics.

The role of lager beer yeast in oxidative stability of model beer.

AIMS: In this study, we investigated the relationship between the ability of lager brewing yeast strains to tolerate oxidative stress and their ability to produce oxidative stable model beer. METHODS AND RESULTS: Screening of 21 lager brewing yeast strains against diamide and paraquat showed that the oxidative stress resistance was strain dependent. Fermentation of model wort in European Brewing Convention tubes using three yeast strains with varying oxidative stress resistances resulted in three model beers with different rates of radical formation as measured by electron spin resonance in forced ageing experiments. Interestingly, the strain with the lowest oxidative stress resistance and lowest secretion of thioredoxin, as measured by Western blotting, resulted in the highest uptake of iron, as measured by inductively coupled plasma-mass spectrometry, and the slowest formation of radicals in the model beers. CONCLUSIONS: A more oxidative stable beer is not obtained by a more-oxidative-stress-tolerant lager brewing yeast strain, exhibiting a higher secretion of thioredoxin, but rather by a less-oxidative-stress-tolerant strain, exhibiting a higher iron uptake. SIGNIFICANCE AND IMPACT OF THE STUDY: To obtain lager beers with enhanced oxidative stability, yeast strains should be screened for their low oxidative stress tolerance and/or high ability to take up iron rather than for their high oxidative stress tolerance and/or high ability to secrete thioredoxin.

Phytase-active yeasts from grain-based food and beer.

AIMS: To screen yeast strains isolated from grain-based food and beer for phytase activity to identify high phytase-active strains. METHODS AND RESULTS: The screening of phytase-positive strains was carried out at conditions optimal for leavening of bread dough (pH 5·5 and 30°C), in order to identify strains that could be used for the baking industry. Two growth-based tests were used for the initial testing of phytase-active strains. Tested strains belonged to six species: Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces bayanus, Kazachstania exigua (former name Saccharomyces exiguus), Candida krusei (teleomorph Issachenkia orientalis) and Arxula adeninivorans. On the basis of initial testing results, 14 strains were selected for the further determination of extracellular and intracellular (cytoplasmic and/or cell-wall bound) phytase activities. The most prominent strains for extracellular phytase production were found to be S. pastorianus KVL008 (a lager beer strain), followed by S. cerevisiae KVL015 (an ale beer strain) and C. krusei P2 (isolated from sorghum beer). Intracellular phytase activities were relatively low in all tested strains. CONCLUSIONS: Herein, for the first time, beer-related strains of S. pastorianus and S. cerevisiae are reported as phytase-positive strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The high level of extracellular phytase activity by the strains mentioned previously suggests them to be strains for the production of wholemeal bread with high content of bioavailable minerals.

Ammonia production and its possible role as a mediator of communication for Debaryomyces hansenii and other cheese-relevant yeast species.

Ammonia production by yeasts may contribute to an increase in pH during the ripening of surface-ripened cheeses. The increase in pH has a stimulatory effect on the growth of secondary bacterial flora. Ammonia production of single colonies of Debaryomyces hansenii, Saccharomyces cerevisiae, Yarrowia lipolytica, and Geotrichum candidum was determined on glycerol medium (GM) agar and cheese agar. The ammonia production was found to vary, especially among yeast species, but also within strains of D. hansenii. In addition, variations in ammonia production were found between GM agar and cheese agar. Ammonia production was positively correlated to pH measured around colonies, which suggests ammonia production as an additional technological parameter for selection of secondary starter cultures for cheese ripening. Furthermore, ammonia appeared to act as a signaling molecule in D. hansenii as reported for other yeasts. On GM agar and cheese agar, D. hansenii showed ammonia production oriented toward neighboring colonies when colonies were grown close to other colonies of the same species; however, the time to oriented ammonia production differed among strains and media. In addition, an increase of ammonia production was determined for double colonies compared with single colonies of D. hansenii on GM agar. In general, similar levels of ammonia production were determined for both single and double colonies of D. hansenii on cheese agar.

Identification of amino acids involved in the Flo11p-mediated adhesion of Saccharomyces cerevisiae to a polystyrene surface using phage display with competitive elution.

AIMS: To identify the main amino acids involved in the Flo11p-mediated adhesion of Saccharomyces cerevisiae to the polystyrene surface PolySorp. METHODS AND RESULTS: Using a combination of phage display and competitive elution revealed that 12-mer peptides of phages from competitive panning with S. cerevisiae FLO11 wild-type (TBR1) cells had a higher consensus than those from competitive panning with S. cerevisiae flo11Delta mutant (TBR5) cells, suggesting that the wild-type cells interact with the plastic surface in a stronger and more similar way than the mutant cells. Tryptophan and proline were more abundant in the peptides of phages from competitive elution with FLO11 cells than in those from competitive elution with flo11Delta cells. Furthermore, two phages with hydrophobic peptides containing 1 or 2 tryptophan, and 3 or 5 proline, residues inhibited the adhesion of FLO11 cells to PolySorp more than a phage with a hydrophobic peptide containing no tryptophan and only two proline residues. CONCLUSIONS: Our results suggest a key role of tryptophan and proline in the hydrophobic interactions between Flo11p on the S. cerevisiae cell surface and the PolySorp surface. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study may contribute to the development of novel strategies to limit yeast infections in hospitals and other medical environments.

Relationship between growth and pH gradients of individual cells of Debaryomyces hansenii as influenced by NaCl and solid substrate.

AIMS: To examine the relationship between the growth and pH gradients of Debaryomyces hansenii at a single-cell level. METHODS AND RESULTS: Using bioimaging techniques, the cell areas and early pH gradients (Delta pH(10)), i.e. the pH gradients determined 10 min after initiation of experiments, were determined for single cells of two D. hansenii strains in fluid and on solid (agar) substrate with and without 8% (w/v) NaCl. The combination of NaCl and solid substrate prolonged the growth initiation of both D. hansenii strains additively. In all our experiments, primarily two groups of cells existed; a vital group consisting of growing single cells with intact early pH gradients, and a group of dead cells without early pH gradients. CONCLUSIONS: Our results show that growth initiation of the D. hansenii cells is severely affected by NaCl and to a lesser extent by the type of substrate in an additive and strain dependent way. Moreover, the early pH gradient of a vital D. hansenii cell cannot be correlated with the rate of its subsequent growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study reveals new knowledge on the growth and pH gradients of D. hansenii on solid surfaces in the presence of NaCl.

Intracellular pH homeostasis plays a role in the NaCl tolerance of Debaryomyces hansenii strains.

The effects of NaCl stress on cell area and intracellular pH (pHi) of individual cells of two Debaryomyces hansenii strains were investigated. Our results show that one of the strains was more NaCl tolerant than the other, as determined by the rate of growth initiation. Whereas NaCl stress caused similar cell shrinkages (30-35%), it caused different pHi changes of the two D. hansenii strains; i.e., in the more NaCl-tolerant strain, pHi homeostasis was maintained, whereas in the less NaCl-tolerant strain, intracellular acidification occurred. Thus, cell shrinkage could not explain the different intracellular acidifications in the two strains. Instead, we introduce the concept of yeasts having an intracellular pKa (pK(a,i)) value, since permeabilized D. hansenii cells had a very high buffer capacity at a certain pH. Our results demonstrate that the more NaCl-tolerant strain was better able to maintain its pK(a,i) close to its pHi homeostasis level during NaCl stress. In turn, these findings indicate that the closer a D. hansenii strain can keep its pK(a,i) to its pHi homeostasis level, the better it may manage NaCl stress. Furthermore, our results suggest that the NaCl-induced effects on pHi were mainly due to hyperosmotic stress and not ionic stress.

Identification of genes and proteins induced during the lag and early exponential phase of lager brewing yeasts.

AIMS: The aim of the present study is to identify genes and proteins whose expression is induced in lager brewing yeast during the lag phase and early exponential growth. METHODS AND RESULTS: Two-dimensional gel electrophoresis was used to identify proteins induced during the lag and early exponential phase of lager brewing yeast in minimal medium. The identified, early-induced proteins were Ade17p, Eno2p, Ilv5gp, Sam1p, Rps21p and Ssa2p. For most of these proteins, the patterns of induction differed from those of the corresponding genes. However, the genes had similar early expression patterns in minimal medium as observed during lager brewing conditions. The expression of previously identified early-induced genes in Saccharomyces cerevisiae grown in minimal medium, ADO1, ALD6, ASC1, ERG4, GPP1, RPL25, SSB1 and YKL056C, was also early induced in lager yeast under brewing conditions. CONCLUSIONS: The results indicate that the above-mentioned genes in general are induced during the lag phase and early exponential growth in Saccharomyces yeasts. The processes in which these genes take part are likely to play an important role during growth initiation. SIGNIFICANCE AND IMPACT OF THE STUDY: Increased knowledge regarding the early growth phase of lager brewing yeast was obtained. Further, the universality of the identified expression patterns suggests new methodologies for optimization and control of growth initiation during brewing fermentations.

Lactic acid tolerance determined by measurement of intracellular pH of single cells of Candida krusei and Saccharomyces cerevisiae isolated from fermented maize dough.

Strains of Candida krusei and Saccharomyces cerevisiae were grown together at 30 degrees C in MYGP broth, pH 2.5, in the presence of 106.4 mM undissociated lactic acid. The two C. krusei strains investigated grew within 48 h from initial counts of 2 x 10(4) to approximately 10(7) cells/ml whereas the two S. cerevisiae strains investigated survived but did not grow in the presence of 106.4 mM undissociated lactic acid at pH 2.5. To explain the differences in lactic acid tolerance of the two yeast species, we used fluorescence-ratio-imaging microscopy and a perfusion system to determine the short-term intracellular pH (pH(i)) changes in single cells of C. krusei and S. cerevisiae. The changes were investigated both in the presence of low (20.7 mM) and high (106.4 mM) concentrations of undissociated lactic acid. For both the investigated species 20.7 mM undissociated lactic acid did not seem to influence the initial pH(i) which for C. krusei was found to be approximately 8.0 and for S. cerevisiae 6.9-7.5. For both C. krusei strains, perfusion with 106.4 mM undissociated lactic acid induced only weak short-term pH(i) responses with a decrease in pH(i) of less than one pH unit. Contrary, for both strains of S. cerevisiae perfusion with 106.4 mM undissociated lactic acid resulted in a significant decrease in pH(i) from initially 6.9-7.5 to 6.2-6.4 after 1 min and further to a pH(i) of < or = 5.5 after 3 min after which it remained constant. The results obtained show that C. krusei is more resistant to short-term pH(i) changes caused by lactic acid than S. cerevisiae, and this, in turn, may be part of the explanation why C. krusei is more tolerant to lactic acid than S. cerevisiae.

The relative glucose uptake abilities of non-Saccharomyces yeasts play a role in their coexistence with Saccharomyces cerevisiae in mixed cultures.

The growth and glucose uptake of single cultures of the wine-related yeasts Kluyveromyces thermotolerans, Torulaspora delbrueckii, and Saccharomyces cerevisiae were investigated. The yeasts had different specific glucose uptake rates (qs) that depended on the residual glucose concentration and the oxygen availability. In mixed cultures, the qs values of the yeasts were not subject to any interaction effects over a wide range of glucose concentrations. Our results strongly indicate that the relative glucose uptake abilities of both non-Saccharomyces yeasts, i.e. the qs(non-Saccharomyces)/qs(S. cerevisiae) ratios, regulated their abilities to compete for space in mixed cultures with S. cerevisiae, which, in turn, regulated their early deaths. This hypothesis enabled us to explain why K. thermotolerans was less able than T. delbrueckii to coexist with S. cerevisiae in mixed cultures. Furthermore, it enabled us to explain why oxygen increased the abilities of K. thermotolerans and T. delbrueckii to coexist with S. cerevisiae in the mixed cultures.

The effect of oxygen on the survival of non-Saccharomyces yeasts during mixed culture fermentations of grape juice with Saccharomyces cerevisiae.

AIMS: The effect of oxygen on the survival of Torulaspora delbrueckii and Kluyveromyces thermotolerans during mixed culture fermentations in grape juice with Saccharomyces cerevisiae was investigated. METHODS AND RESULTS: Fermentations were carried out in two simple fermentation systems differing in the availability of oxygen. At low available oxygen conditions, T. delbrueckii and K. thermotolerans began to die off after two days of mixed culture fermentation. In filtrates from 2-day-old mixed cultures, single cultures of T. delbrueckii and K. thermotolerans survived and actively produced ethanol to concentrations of approx. 65 and 70 g l-1, respectively, at low available oxygen conditions. Oxygen clearly increased the survival time and decreased the death rate of T. delbrueckii and K. thermotolerans in mixed cultures, whereas it did not affect the growth and survival of S. cerevisiae. CONCLUSION: Our results show that the deaths of T. delbrueckii and K. thermotolerans in mixed cultures at low available oxygen conditions are not due to toxic metabolites produced by the yeasts but rather to the lack of oxygen. Furthermore, they indicate that T. delbrueckii and K. thermotolerans are less tolerant to low available oxygen conditions than S. cerevisiae. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study reveals new knowledge on the mechanisms underlying the succession of yeasts during wine fermentations. This knowledge may be of importance when creating defined, mixed starter cultures for the controlled production of wines with a wide range of flavour compositions.

Interactions between Zygosaccharomyces mellis and Wallemia sebi in diluted molasses.

The yeast Zygosaccharomyces mellis and the mould Wallemia sebi were isolated from the same sample of crystalline sugar. Interactions between these fungi were investigated using a diluted molasses medium (water activity 0.89, pH 6.0) as a model system for the syrup film covering the surface of moist crystalline sugar. Single and mixed cultures of Z. mellis and W. sebi were incubated at 25 degrees C for 400 h. Our results show that the growth of Z. mellis in single culture was limited by available glucose and fructose, and that W sebi was able to invert sucrose to glucose and fructose in both single and mixed culture. Furthermore, the presence of W. sebi in the mixed culture increased the maximum specific growth rate of Z. mellis from 0.074 to 0.19 h(-1) and the growth yield of Z. mellis from 7.3 x 10(6) to 5.4 x 10(7) cfu/ml. These results indicate that the ability of W. sebi to invert sucrose may stimulate the growth of Z. mellis. Finally, the presence of Z. mellis inhibited the ability of W. sebi to invert sucrose: W. sebi was able to invert 1.0 g sucrose/l per h in single culture but only 0.6 g sucrose/l per h in mixed culture. As predicted by Raoults law, this corresponded to a reduction in the water activity of the growth medium from 0.890 to 0.850 in single culture, and to 0.865 in mixed culture.

Individual cells of Saccharomyces cerevisiae and Zygosaccharomyces bailii exhibit different short-term intracellular pH responses to acetic acid.

The effects of perfusion with 2.7 and 26 mM undissociated acetic acid in the absence or presence of glucose on short-term intracellular pH (pH(i)) changes in individual Saccharormyces cerevisiae and Zygosaccharomyces bailii cells were studied using fluorescence-ratio-imaging microscopy and a perfusion system. In the S. cerevisiae cells, perfusion with acetic acid induced strong short-term pH(i) responses, which were dependent on the undissociated acetic acid concentration and the presence of glucose in the perfusion solutions. In the Z. bailii cells, perfusion with acetic acid induced only very weak short-term pH(i) responses, which were neither dependent on the undissociated acetic acid concentration nor on the presence of glucose in the perfusion solutions. These results clearly show that Z. bailii is more resistant than S. cerevisiae to short-term pH(i) changes caused by acetic acid.

Endocytosis and vacuolar morphology in Saccharomyces cerevisiae are altered in response to ethanol stress or heat shock.

The vital lipophilic dye N-(3-triethylammoniumpropyl)-4-[6-(4-(diethylamino)phenyl]hexatrie nyl ) pyridinium dibromide (FM 4-64) was used to study the effect of ethanol stress and heat shock on endocytosis in the yeast Saccharomyces cerevisiae. Yeast cells stained with FM 4-64 were placed in a culture chamber and the internalization of the dye was monitored by fluorescence microscopy during perfusion of the cells with fresh growth medium. In the absence of ethanol in the perfusion medium, the internalization of FM 4-64 from the plasma membrane to the vacuolar membrane by yeast cells harvested from the exponential phase of growth was completed in 30 min. The presence of 6% (v/v) ethanol in the perfusion medium had no obvious effect on the internalization of FM 4-64 from the plasma membrane, but did lead to an accumulation of the dye in endocytic intermediates. Consequently, vacuolar membrane staining was delayed. Cells stained with FM 4-64 and subjected to heat shock displayed a similar effect, with endocytic intermediates becoming more prominent with the severity of the heat shock. For both ethanol stress and heat shock, vacuolar morphology altered from segregated structures to a single, large organelle. The findings of this study reinforce previous observations that ethanol stress and heat shock induce similar responses in yeast.

Relationship between lipid composition, frequency of ethanol-induced respiratory deficient mutants, and ethanol tolerance in Saccharomyces cerevisiae.

The frequency of ethanol-induced respiratory deficient mutants and lipid composition in two Saccharomyces cerevisiae strains showing different degrees of ethanol tolerance were investigated. The more ethanol-tolerant strain exhibited a lower frequency of ethanol-induced respiratory deficient mutants than the less ethanol-tolerant strain. In addition, the more ethanol-tolerant strain contained a higher ergosterol/phospholipid ratio, a higher proportion of phosphatidylcholine, a lower proportion of phosphatidylethanolamine, a higher incorporation of long-chain fatty acids in total phospholipids, and a slightly higher proportion of unsaturated fatty acids in total phospholipids than the less ethanol-tolerant strain. These results show a clear relationship between the lipid composition, the frequency of ethanol-induced respiratory deficient mutants, and the ethanol tolerance of S. cerevisiae. A possible explanation of this relationship is discussed.

Measurement of the effects of acetic acid and extracellular pH on intracellular pH of nonfermenting, individual Saccharomyces cerevisiae cells by fluorescence microscopy.

The effects of acetic acid and extracellular pH (pHex) on the intracellular pH (pHi) of nonfermenting, individual Saccharomyces cerevisiae cells were studied by using a new experimental setup comprising a fluorescence microscope and a perfusion system. S. cerevisiae cells grown in brewer's wort to the stationary phase were stained with fluorescein diacetate and transferred to a perfusion chamber. The extracellular concentration of undissociated acetic acid at various pHex values was controlled by perfusion with 2 g of total acetic acid per liter at pHex 3.5, 4.5, 5.6, and 6.5 through the chamber by using a high-precision pump. The pHi of individual S. cerevisiae cells during perfusion was measured by fluorescence microscopy and ratio imaging. Potential artifacts, such as fading and efflux of fluorescein, could be neglected within the experimental time used. At pHex 6.5, the pHi of individual S. cerevisiae cells decreased as the extracellular concentration of undissociated acetic acid increased from 0 to 0.035 g/liter, whereas at pHex 3.5, 4.5, and 5.6, the pHi of individual S. cerevisiae cells decreased as the extracellular concentration of undissociated acetic acid increased from 0 to 0.10 g/liter. At concentrations of undissociated acetic acid of more than 0.10 g/liter, the pHi remained constant. The decreases in pHi were dependent on the pHex; i.e., the decreases in pHi at pHex 5.6 and 6.5 were significantly smaller than the decreases in pHi at pHex 3.5 and 4.5.

The effect of ethanol and specific growth rate on the lipid content and composition of Saccharomyces cerevisiae grown anaerobically in a chemostat.

The effects of produced ethanol and specific growth rate on the lipid content and composition of Saccharomyces cerevisiae CBS 2806 were studied using anaerobic chemostat cultures. The cells adapted to increased concentrations of produced ethanol by increasing the proportion of ergosterol at the expense of lanosterol, by increasing the proportion of phosphatidylinositol at the expense of phosphatidylcholine, and by increasing the amount of C18:0 fatty acids in total phospholipids at the expense of C16:0 fatty acids. The produced ethanol had no effect on the phospholipid content nor on the proportion of unsaturated fatty acids in the phospholipids. The specific growth rate had no effect on the phospholipid content, the sterol composition, the phospholipid composition, the fatty acid composition of total phospholipids, or on the proportion of unsaturated fatty acids in the phospholipids of S. cerevisiae. It was not possible to separate the effects of produced ethanol and growth rate on the ergosterol content of the chemostat-grown S. cerevisiae cells.