Most transcription factors fulfill their role in protein complexes. As a consequence, information about their interaction capacity sheds light on a protein's function and the molecular mechanism ...underlying this activity. The yeast two-hybrid GAL4 (Y2H) assay is a powerful method to unravel and identify the composition of protein complexes. This in vivo based system makes use of two functional protein domains of the GAL4 transcription factor, each fused to a protein of interest. Upon interaction between the two proteins under study, a transcriptional activator gets reconstituted and reporter genes get activated, allowing the yeast to grow on selective medium. In this chapter protocols are given for Y2H library screening, directed Y2H screening, Y2H matrix screening, and YnH screening involving more than two proteins.
The performance of 3 blood culture bottles (BACTEC Plus Aerobic/F, Plus Anaerobic/F, and Anaerobic Lytic/F) were analyzed with clinical specimens collected from 688 Emergency Department patients. A ...total of 270 strains belonging to 33 species were identified, with E. coli and S. aureus as the most frequently detected. Overall recovery rate (RR) of bacteria and yeast was equivalent in the Plus Aerobic/F vials (208 of 270 isolates; 77.0%) and Anaerobic Lytic/F vials (206 isolates; 76.3%) and significantly better than in the Plus Anaerobic/F vials (189 isolates; 70.0%). Median time to detection (TTD) was earliest with the Anaerobic Lytic/F vials (12.0h) compared with the Plus Aerobic/F (14.6h) and Plus Anaerobic/F vials (15.4h). Positivity rate (PR) was similar for Anaerobic Lytic/F vials (76.9%) and Plus Aerobic/F vials (76.5%), but better if compared with Plus Anaerobic/F vials (69.4%). The PR and TTD for the combination of Plus Aerobic/F with Anaerobic Lytic/F (94.5% and 12.3h, respectively) was significantly better than with Plus Aerobic/F with Plus Anaerobic/F (87.8% and 14.1h).
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
This article reports on the investigation on the bioactivity of eugenol, limonene, and citrus extract against three bacteria (Lactobacillus plantarum, Lactobacillus brevis, and Bacillus coagulans) ...and three yeasts (Saccharomyces bayanus, Pichia membranifaciens, and Rhodotorula bacarum), representing the spoilage microflora of fruit juices. The experiments were performed with laboratory media by using a microdilution method. Data were fitted using the Gompertz equation, and the kinetic parameters were used to evaluate the MIC and the dose-dependent effect (at suboptimal doses for each essential oil). Citrus extract was the most effective essential oil, and the results suggested the following susceptibility hierarchy, from the most sensitive microorganism to the most resistant one (values in parentheses represent MICs): S. bayanus (2 ppm) > R. bacarum (3 ppm) > P. membranifaciens (5 ppm) > B. coagulans (cells, 20 ppm) > L. brevis (40 ppm) > L. plantarum (>40 ppm).
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CEKLJ, GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Levels of protein carbonylation in peach fruits inoculated with four antagonistic yeasts (
Pichia membranaefaciens,
Cryptococcus laurentii,
Candida guilliermondii and
Rhodotorula glutinis) were ...significantly reduced in response to reactive oxygen species (ROS) caused by
Monilinia fructicola. In control fruit without yeast treatments, proteins carbonylation obviously increased after inoculation with
M. fructicola, ranging from molecular mass 20 to 120 kDa. However, in yeast-treated fruits, no proteins carbonylation was detected at 1 d, only a small quantity of carbonylation ranging from 28.5 to 45 kDa was found at 2 d. Antagonistic yeasts significantly stimulated the activities of chitinase, β-1,3-glucanase, catalase (CAT), peroxidase (POD) and the expressions of relevant genes during all storage periods. These results suggest that yeast treatments may be related to alleviating proteins carbonylation and mitigating pathogen-induced oxidative damage, which result in decrease of fruit decay and imply that antioxidant defense response may be involved in the mechanisms of microbial biocontrol agents against fungal pathogen.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The non-conventional yeast Arxula adeninivorans uses 1-butanol as a carbon source and has recently attracted attention as a promising organism for 1-butanol production. Alcohol dehydrogenases (adhp) ...are important catalysts in 1-butanol metabolism, but only Aadh1p from Arxula has been characterized. This enzyme is involved in ethanol synthesis but has a low impact on 1-butanol degradation.
In this study, we identified and characterized a second adhp from A. adeninivorans (Aadh2p). Compared to Saccharomyces cerevisiae ADHs' (ScAdh) protein sequences it originates from the same ancestral node as ScAdh6p, 7p and 4p. It is also localized in the cytoplasm and uses NAD(H) as cofactor. The enzyme has its highest activity with medium chain-length alcohols and maximum activity with 1-butanol with the catalytic efficiency of the purified enzyme being 42 and 43,000 times higher than with ethanol and acetaldehyde, respectively. Arxula adeninivorans strain G1212/YRC102-AADH2, which expresses the AADH2 gene under the control of the strong constitutive TEF1 promoter was constructed. It achieved an ADH activity of up to 8000 U/L and 500 U/g dry cell weight (dcw) which is in contrast to the control strain G1212/YRC102 which had an ADH activity of up to 1400 U/L and 200 U/g dcw. Gene expression analysis showed that AADH2 derepression or induction using non-fermentable carbon-sources such as ethanol, pyruvate, glycerol or 1-butanol did occur. Compared to G1212/YRC102 AADH2 knock-out strain had a slower growth rate and lower 1-butanol consumption if 1-butanol was used as sole carbon source and AADH2-transformants did not grow at all in the same conditions. However, addition of the branched-chain amino acids leucine, isoleucine and valine allowed the transformants to use 1-butanol as carbon source. The addition of these amino acids to the control strain and Δaadh2 mutant cultures had the effect of accelerating 1-butanol consumption.
Our results confirm that Aadh2p plays a major role in A. adeninivorans 1-butanol metabolism. It is upregulated by up to 60-fold when the cells grow on 1-butanol, whereas only minor changes were found in the relative expression level for Aadh1p. Thus the constitutive overexpression of the AADH2 gene could be useful in the production of 1-butanol by A. adeninivorans, although it is likely that other ADHs will have to be knocked-out to prevent 1-butanol oxidation.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
In the present study, natural antimicrobials chitosan and thyme, and their combination, were evaluated for their effect on the shelf life of a ready-to-cook (RTC) chicken-pepper kebab (skewer) stored ...under modified atmosphere packaging (MAP) conditions at 4 +/- 0.5 degrees C for 14 days. The following treatments were examined: control samples stored under aerobic packaging (A), samples stored under MAP (M), samples treated with 1.5% chitosan (vol/wt) and stored under MAP (M-CH), samples treated with 0.2% thyme essential oil (vol/wt) (M-T), and samples treated with 1.5% chitosan (vol/wt) and 0.2% thyme essential oil (vol/wt) and stored under MAP (M-CH-T). Treatment M-CH-T significantly affected aerobic plate counts and counts of lactic acid bacteria, Pseudomonas spp., Brochothrix thermosphacta, Enterobacteriaceae, and yeasts and molds during the entire storage period. Similarly, lipid oxidation of the RTC product was retarded (M-CH-T treatment) during storage, whereas redness was maintained in M-T, M-CH, and M-CH-T samples. Based primarily on sensory data (taste attribute), M-CH and M-T treatments extended RTC product shelf life by 6 days, whereas M-CH-T treatment resulted in a product with a shelf life of 14 days that maintained acceptable sensory characteristics (shelf life of the control was 6 days).
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CEKLJ, GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Diploid yeast, like most eukaryotes, can undergo meiotic differentiation to form haploid gametes. Meiotic differentiation and cell growth (proliferation) are mutually exclusive programs, and in yeast ...the switch between growth and meiosis is controlled by nutritional signals. The signaling pathways that mediate nutritional controls on meiotic initiation fall into three broad classes: those that respond to nutrient starvation, those that respond to non-fermentable carbon sources, and those that respond to glucose. At the onset of meiosis, nutritional signaling pathways converge on transcriptional regulation of two genes: IME1, which encodes a transcription factor; and IME2, which encodes a protein kinase. Transcription of IME1 and IME2 trigger initiation of meiosis, and the expression of these two genes is linked with one other, with expression of later meiotic genes and with early meiotic events such as DNA replication. In addition, the signaling pathways that control IME1 and IME2 expression are themselves integrated through a variety of mechanisms. Thus the signal network that controls the switch from growth to meiotic differentiation provides a signaling code that translates different combinations of extracellular signals into appropriate cellular responses.
Effect of fat replacers on kefir quality Ertekin, Bilge; Guzel-Seydim, Zeynep B
Journal of the science of food and agriculture,
15 March 2010, Volume:
90, Issue:
4
Journal Article
Peer reviewed
Open access
The purpose of the study was to determine the effects of fat replacers on the quality of non-fat kefir. Skim milk fortified with Dairy Lo® (DL) and inulin (INU) was fermented with kefir grains to ...manufacture kefir. The results of compositional, microbiological, rheological and sensorial analyses were compared with whole kefir (WK) and non-fat kefir (NFK) controls. Results for dry matter, pH and lactic acid ranged between 82.4 and 109.1 g kg⁻¹, 4.26 and 4.40, and 7.0 and 9.2 g L⁻¹, respectively. Acetaldehyde and ethanol contents of samples were between 2.89 and 7.28 mg L⁻¹, and 151.46 and 323.89 mg L⁻¹, respectively. In all samples, Lactobacillus spp., Streptococcus spp. and yeast counts were between 9.1 and 9.9, 9.3 and 9.9, and 5.2 and 5.6 log cfu mL⁻¹, respectively. Kefir samples had non-Newtonian behaviour and pseudoplastic fluid with thixotropy. At the first day, DL had the highest apparent viscosity (3.119 Pa s) while NFK had the lowest value (1.830 Pa s). In the sensory evaluation, odour and taste scores of samples were not different. Dairy Lo® and inulin could be used without any adverse effect for the production of non-fat kefir.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Viburnum opulus
is a plant with fruits that are rich in biologically active substances, making it valuable to the food and pharmaceutical industries. Here, we present our study of the total phenolic ...and anthocyanin contents and antimicrobial activity in the fruit juice of six
V. opulus
L. accessions. The cultivar ‘Krasnaya Grozd’ was notable for its exceptionally large amount of total phenolics, 1168 mg/100 g, with anthocyanins comprising 3–5 % of the total phenolic content. The evaluation of the antimicrobial properties confirmed that the juice of
V. opulus
fruits strongly inhibited the growth of a wide range of human pathogenic bacteria, both Gram-negative (
Salmonella typhimurium
and
S. agona
) and Gram-positive (
Staphylococcus aureus
,
Lysteria monocytogenes
, and
Enterococcus faecalis
) organisms. Conversely, the yeasts
Debaryomyces hansenii
and
Torulaspora delbrueckii
showed complete resistance to the fruit juice, whereas a low sensitivity was demonstrated by
Trichosporon cutaneum, Kluyveromyces marxianus
var.
lactis
,
Saccharomyces cerevisiae
,
S. cerevisiae
12R, and
Candida parapsilosis
.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The aims of this work were to identify and characterize for some important technological properties the yeast species present throughout the ripening process of Pecorino Crotonese, a traditional ...cheese produced in a well defined area of Southern Italy. In particular, the strain technological properties considered include fermentation/assimilation of galactose and lactose, assimilation of lactate and citrate in the presence of different NaCl concentrations, hydrolysis of butter fat, skim milk, gelatine and casein, production of brown pigments in cheese agar and ability to produce biogenic amines. High yeast levels were recorded in cheese samples already after 5
h of brining (about 5
log
cfu/g) and these concentration remained constant during ripening. The yeast isolates belonged to restrict number of yeast species. While
Kluyveromyces lactis and
Saccharomyces cerevisiae were isolated prevalently in the first stages of Pecorino Crotonese production,
Yarrowia lipolytica and
Debaryomyces hansenii dominated during the later stages of maturation. Otherwise, the latter two were very NaCl resistant species. In fact,
D. hansenii strains conserved the ability to assimilate lactose and galactose in the presence of 10% NaCl, while almost all the strains of
Y. lipolytica isolated assimilated citrate and lactate up to 7.5% NaCl.
Y. lipolytica isolates evidenced also the highest proteolytic and lipolytic activities and the capability to catabolize tyrosine producing brown pigment. In addition they resulted in the highest aminobiogenic potential decarboxylating ornithine, phenylalanine, tyrosine and lysine. However, they were not able to produce histamine, biogenic amine produced by three strains of
D. hansenii.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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