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.

Trends in social inequality in loneliness among adolescents 1991-2014.

BACKGROUND: Loneliness and social inequality in health are important public health concerns. We examined (i) trends in loneliness among adolescents from 1991 to 2014 in Denmark and (ii) trends in social inequality in loneliness. METHODS: Study population: 11-15-year olds in random samples of schools in 1991, 1994, 1998, 2006 and 2014, n = 19 096. Loneliness was measured by a single item and social background by parents' occupational social class (OSC). We calculated absolute (%) differences in loneliness between high and low OSC and relative differences by odds ratio for loneliness. RESULTS: Across all surveys, 6.3% reported feeling lonely. The prevalence increased from 4.4% in 1991 to 7.2% in 2014. The prevalence of loneliness in high, middle and low OSC was 5.8, 5.9 and 8.0%. The increase in loneliness was more pronounced in higher than lower OSC, resulting in a decreasing absolute social inequality in loneliness. The statistical interaction between OSC and survey year was significant, P = 0.0176, i.e. the relative social inequality in loneliness also decreased from 1991 to 2014. CONCLUSION: The prevalence of loneliness increased from 1991 to 2014. The social inequality in loneliness decreased in both absolute and relative terms because of a rising prevalence of loneliness among children from high OSC.

Transcriptional responses in Lactococcus lactis subsp. cremoris to the changes in oxygen and redox potential during milk acidification.

UNLABELLED: Milk acidification and metabolic activity of the starter cultures are affected by oxygen; however, molecular factors related to the redox changes are poorly defined. The objective of the study was to investigate transcriptional responses in Lactococcus lactis subsp. cremoris CHCCO2 grown in milk to the shifts of oxygen and redox potential (Eh7 ). Transcriptomic studies were performed with the use of Illumina HiSeq 2000 mRNA sequencing and validated by the real-time quantitative PCR. In total 105 differentially expressed genes were assigned functional gene names. Most of the differentially expressed genes were detected during aerobic reduction phase. Upregulated genes were implicated in lactose utilization, glycogen biosynthesis, amino sugar metabolism, oxidation-reduction, pyrimidine biosynthesis and DNA integration processes. Genes of purine nucleotide biosynthesis and genes encoding amino acid, multidrug resistance and ion ABC transporters were mostly downregulated, while oligopeptide transporter genes were reduced during oxygen depletion and induced at minimum Eh7 . SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding of gene responses in starter cultures to the changes of oxidation-reduction state is important for the better control and reproducibility of dairy fermentations. We applied mRNA sequencing by Illumina HiSeq 2000 to investigate gene expression profile in a dairy strain of Lactococcus lactis subsp. cremoris during milk acidification. Novelty of this study lies in linking transcriptional responses to oxygen depletion and the changes of redox potential with the fermentation kinetics and clarification of molecular factors specifically expressed in milk which might be essential for bacterial performance and the final quality of cheeses.

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.

Advanced paternal age and mortality of offspring under 5 years of age: a register-based cohort study.

STUDY QUESTION: Do children born to fathers of advanced age have an increased risk of dying before the age of 5 years? SUMMARY ANSWER: Children born to fathers aged 40 years or more have an increased risk of dying in early childhood due to an excess risk of fatal congenital anomalies, malignancies and external causes. WHAT IS KNOWN ALREADY: Advanced paternal age has previously been associated with adverse reproductive outcomes and some long-term health problems in the offspring. This is possibly due to specific point mutations, a condition known to increase in the sperm with increasing paternal age. STUDY DESIGN, SIZE, DURATION: A Danish population-based register study, designed as a prospective cohort study, of 1 575 521 live born children born from 1978 to 2004. The age of the child (in days) was used as the underlying time and the children entered the cohort the day they were born and were followed until 31 December 2009. The children were censored on date of turning 5 years, date of death or date of emigration, whichever occurred first. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data from population-covering registers from Statistics Denmark including the Integrated Database for Labour Market Research, the Medical Birth Registry and the Registry of Causes of Death was linked using the unique civil registry number. Hazard ratios (HR) with 95% confidence intervals (CI) were used to estimate the risk of under-five mortality. The effect of paternal age was examined using restricted cubic splines and paternal age groups. MAIN RESULTS AND THE ROLE OF CHANCE: Compared with children born to fathers aged 30-34 years, a statistically significant excess risk was found for children born to fathers aged 40-44 years [HR: 1.10 (95% CI: 1.00-1.21)] and children born to fathers aged 45+ years [HR: 1.16 (95% CI: 1.02-1.32)]. When only looking at 1-5 year olds, the relative risk (HR) among children born to fathers aged 40-44 years increased to 1.24 (95% CI: 1.00-1.53) and the risk in the oldest paternal age group (45+ years) rose to 1.65 (95% CI: 1.24-2.18). The results suggest that the elevated risk for children of fathers aged 40 years or more was primarily attributed to an elevated risk of dying from congenital malformations, malignancies and external causes. LIMITATIONS, REASONS FOR CAUTION: Specific causes of death might be misclassified; however, this is not likely to be dependent on paternal age. In some cases, the biological father may differ from the father registered. This misclassification is most likely non-differential. WIDER IMPLICATIONS OF THE FINDINGS: The excess risk of mortality among children born to older fathers is in accordance with the literature. The association needs further attention as it can provide valuable knowledge of the etiology of genetic diseases. Also, the association could become of greater importance in the future if the proportion of fathers aged 40+ years keeps growing. STUDY FUNDING/COMPETING INTEREST (S): None.

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.

Transcriptional analysis of genes associated with stress and adhesion in Lactobacillus acidophilus NCFM during the passage through an in vitro gastrointestinal tract model.

The aim of the present study was to investigate the transcription of genes associated with stress and adhesion in Lactobacillus acidophilus NCFM during the passage through an in vitro gastrointestinal tract model. As acidified milk exerted a protective effect on the bacteria leading to increased survival, the gene expression studies were carried out with pre-inoculation of L. acidophilus NCFM in acidified milk. The induction of the genes encoding the stress-related proteins GroEL, DnaK and ClpP, and adhesion-related genes encoding mucin-binding proteins, fibronectin-binding protein and S-layer was analyzed by real-time PCR. The genes encoding GroEL, DnaK and ClpP were significantly up-regulated (9- to 16-fold) during gastric digestion and declined upon subsequent duodenal digestion. The genes encoding mucin-binding proteins and fibronectin-binding protein were not influenced by saliva and gastric juice, but they were significantly upregulated during incubation in duodenal juice and bile (6- to 7-fold). A significant induction of the gene encoding the S-layer protein was not detected. Our results give a better understanding of the functionality of L. acidophilus NCFM and other probiotics during passage through the gastrointestinal tract; hence, they provide an implementable basis for the selection of prospective probiotic candidates.

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.

Lactic acid bacteria and yeasts associated with gowé production from sorghum in Bénin.

AIMS: To identify the dominant micro-organisms involved in the production of gowé, a fermented beverage, and to select the most appropriate species for starter culture development. METHODS AND RESULTS: Samples of sorghum gowé produced twice at three different production sites were taken at different fermentation times. DNA amplification by internal transcribed spacer-polymerase chain reaction of 288 lactic acid bacteria (LAB) isolates and 16S rRNA gene sequencing of selected strains revealed that the dominant LAB responsible for gowé fermentation were Lactobacillus fermentum, Weissella confusa, Lactobacillus mucosae, Pediococcus acidilactici, Pediococcus pentosaceus and Weissella kimchii. DNA from 200 strains of yeasts was amplified and the D1/D2 domain of the 26S rRNA gene was sequenced for selected isolates, revealing that the yeasts species were Kluyveromyces marxianus, Pichia anomala, Candida krusei and Candida tropicalis. CONCLUSIONS: Gowé processing is characterized by a mixed fermentation dominated by Lact. fermentum, W. confusa and Ped. acidilactici for the LAB and by K. marxianus, P. anomala and C. krusei for the yeasts. SIGNIFICANCE AND IMPACT OF THE STUDY: The diversity of the LAB and yeasts identified offers new opportunities for technology upgrading and products development in gowé production. The identified species can be used as possible starter for a controlled fermentation of gowé.

Antimicrobial activity of Bacillus subtilis and Bacillus pumilus during the fermentation of African locust bean (Parkia biglobosa) for Soumbala production.

AIMS: To examine predominant isolates of Bacillus subtilis and B. pumilus isolated from Soumbala for their antimicrobial activity against indicator microorganisms as Micrococcus luteus, Staphyloccocus aureus, Bacillus cereus, Enterococus facium, Listeria monocytogenes, Escherichia coli, Salmonella typhimurium, Shigella dysenteriae, Yersinia enterocolitica, Aspergillus ochraceus and Penicillium roqueforti. METHODS AND RESULTS: Growth inhibition of indicator microorganisms by cells and supernatants of three B. subtilis and two B. pumilus strains was investigated using agar diffusion tests. Inactivation of indicator microorganisms was investigated in laboratory broth and during the fermentation of African locust bean for Soumbala production. The Bacillus isolates showed variable ability of inhibition and inactivation according to the indicator microorganism. The supernatants of pure cultures of B. subtilis inhibited one strain of B. cereus, one of Staph. aureus and E. coli and caused abnormal germination of Aspergillus ochraceus. The supernatant of mixed cultures of B. subtilis and indicators inhibited all the indicators. A treatment with protease eliminated the inhibitions. Isolates of B. subtilis inactivated all the indicators organisms during the fermentation of African locust bean as well as in laboratory broth with about five to eight decimal reduction. CONCLUSION: Bacillus isolates from Soumbala inhibit and inactivate Gram-positive and Gram-negative bacteria as well as ochratoxin A producing fungi during both laboratory cultivation and natural fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: Selection of starter cultures of Bacillus spp. for controlled production of Soumbala.

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.

Yeast populations associated with Ghanaian cocoa fermentations analysed using denaturing gradient gel electrophoresis (DGGE).

The yeast populations associated with the fermentation of Ghanaian cocoa were investigated using denaturing gradient gel electrophoresis (DGGE). Samples were collected at 12-24 h intervals from heap and tray fermentations, at three different fermentation sites and different periods during the season. Eukaryotic universal primers were used to amplify a fragment of the 26S rRNA gene. The DGGE profiles were relatively complex, underlining that the fermentation of cocoa is a complex microbial process. The identities of selected fragments in the denaturing gels were revealed by sequencing. Hanseniaspora guilliermondii, Candida krusei and Pichia membranifaciens were detected from most fermentations, indicating their possible important role in the fermentation of Ghanaian cocoa. Saccharomyces cerevisiae and Candida zemplinina were almost exclusively detected during tray fermentations. The developed DGGE protocol was compared with traditional culture-based isolations. The results were comparable but slightly different, as one yeast species (C. zemplinina) was only detected using DGGE. On the other hand, Trichosporon asahii yielded only faint bands in the denaturing gels, despite the fact that it was detected using culture-based methods. Analysis of pure cultures showed that the targeted region of the 26S rRNA gene was poorly amplified in T. asahii, whereas all other investigated isolates were amplified efficiently using the chosen PCR approach. Cluster analysis revealed that the DGGE profiles clustered according to fermentation method and fermentation site. Furthermore, clustering according to progress in the fermentation was observed. The DGGE technique therefore seems to offer a relatively fast and reliable method for studying yeast population dynamics during cocoa fermentations.

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.

A flow cytometric technique for quantification and differentiation of bacteria in bulk tank milk.

AIMS: The present study describes a flow cytometric technique for quantification and differentiation of bacteria in bulk tank milk according to the main cause of elevated counts. METHODS AND RESULTS: A total of 75 Danish bulk tank milk samples exceeding the grading level of 3.0 x 10(4) CFU ml(-1) were examined by both flow cytometry and traditional microbiological analyses. The correlation coefficient (r) between the two methods was 0.71. For the differential analyses of the dominant bacterial populations four different parameters were used to give a species-characteristic pattern. The four parameters were as follows: staining with Oregon Green conjugated wheat germ agglutinin that binds to the cell wall of bacteria, staining with hexidium iodide that binds to all bacterial DNA, the flow cytometric forward scatter and the flow cytometric side scatter. Three regions in the flow cytometric plot were defined: region 1 includes bacteria mainly associated with poor hygiene, region 2 includes psychrotrophic hygiene bacteria and region 3 includes bacteria mainly related to mastitis. The ability of the flow cytometric technique to predict the main cause of elevated bacterial counts on routine samples was examined. Comparing these results with results obtained by traditional microbiological analyses for identification showed that for 81% of the samples the two techniques agreed on the main cause of an elevated bacterial count. CONCLUSIONS: The ability of the presented flow cytometric technique to enumerate and differentiate bacteria in bulk tank milk according to the main cause of elevated counts was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: This study described the first step in development of a technique suitable for routine analyses of bulk tank milk samples. A technique indicating the main cause of an elevated count will enable the farmer to eliminate the contamination source within a short time limit.

Predominant microflora of downgraded Danish bulk tank milk.

The microflora of downgraded Danish bulk tank milk was examined to identify the main causes of increased microbial counts. Seventy-five representative samples with a microbial count exceeding 3.0 x 10(4) cfu/mL were selected for a more detailed microbial examination. A total of 1237 isolates from these samples were identified. Gram-negative, oxidase-positive bacteria were found in 72% of the samples. Coliforms were found in 20% of the samples, and non-coliforms were found in 49% of the samples. Coryneforms, other gram-positive rods, Lactococcus spp., Micrococcus spp., and coagulase-negative Staphylococcus spp. were found in 28 to 53% of the samples. Bacillus spp., Enterococcus spp., Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus uberis, and yeasts were found in <25% of the samples. Additionally, the isolates were divided into 3 groups, based on the main cause of an elevated microbial count. Microorganisms primarily associated with poor hygiene dominated the microflora in 64% of the samples; bacteria also related to poor hygiene, but in addition associated with growth at low temperatures (psychrotrophic bacteria) dominated the microflora in 28% of the samples; and bacteria mainly associated with mastitis dominated the microflora in 8% of the samples. A bulk tank milk storage period of 48 h instead of 24 h did not affect the proportion of downgraded milk samples and could not be associated with a specific group of microorganisms. Further, no relationship was found between somatic cell counts and the presence of mastitis bacteria.

Genetic diversity of the species Debaryomyces hansenii and the use of chromosome polymorphism for typing of strains isolated from surface-ripened cheeses.

AIMS: To investigate the genetic diversity among strains of Debaryomyces hansenii and further to evaluate chromosome polymorphism determined by pulse-field gel electrophoresis (PFGE) as a tool for strain typing. METHODS AND RESULTS: In total 56 isolates of D. hansenii were analysed by PFGE. The isolates included type strains and other strains obtained from culture collections as well as strains collected during production of Danish surface-ripened cheeses. By use of the PFGE technique the number and size of the chromosomal bands were calculated and the total genome size estimated. The number of chromosomal bands observed was found to vary from five to 10. The most common chromosome number was found to be six and for strains with six chromosomes the total genome size was found to vary from 9.4 to 12.6 Mb. The chromosome numbers for the type strain of each variety of D. hansenii (D. hansenii var. hansenii and D. hansenii var. fabryi) appeared to be six and seven respectively. By use of the PFGE technique it was possible to differentiate between all the investigated CBS strains and the vast majority of the dairy isolates. The dairy isolates that were found to have identical profiles (three of 56 isolates) were all isolated during production of one batch of surface-ripened cheeses and are likely to be the same strain isolated several times during cheese production. Further it was shown that the PFGE analysis did not result in a division of the two D. hansenii varieties, i.e. D. hansenii var. fabryi and D. hansenii var. hansenii into separate groups. CONCLUSION: The present study shows that the chromosomal arrangement of D. hansenii strains is heterogenic and does have a distinct chromosome polymorphism. Further the PFGE technique was proved to have a high discriminative power for strain typing of D. hansenii. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained add to the first knowledge on the genetic diversity of the species D. hansenii. Further the distinct chromosome polymorphism of D. hansenii strains as shown in this study makes the PFGE technique a useful tool for strain typing of D. hansenii, e.g. during cheese production.

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.

Detection of resistance of lactic acid bacteria to a mixture of the hop analogue compounds tetrahydroiso-alpha-acids by noninvasive measurement of intracellular pH.

AIMS: To examine the resistance of beer isolates of lactic acid bacteria (LAB) towards a mixture of tetrahydroiso-alpha-acids (Tetra) by growth experiments as well as by measurement of intracellular pH. METHODS AND RESULTS: Beer LAB isolates were identified to species level by SDS-PAGE of whole-cell proteins. Beer isolates of Lactobacillus brevis showed better ability for growth in the presence of Tetra than nonbeer isolates of the L. brevis or other species of LAB including beer and nonbeer isolates. The antimicrobial effect of Tetra was also examined by noninvasive measurement of intracellular pH by fluorescence ratio imaging microscopy for selected beer isolates of L. brevis and Pediococcus inopinatus. Strains of L. brevis showing limited decrease of intracellular pH during exposure to Tetra also showed better ability for growth in the presence of these compounds as well as in commercial beer products. CONCLUSIONS: It was possible to apply a method for noninvasive measurement of intracellular pH to predict the resistance of beer spoilage LAB towards the Tetra hop analogue compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the usability of a new rapid method for detecting hop-resistant variants of known beer spoilage LAB species.