Nitrogen availability of grape juice limits killer yeast growth and fermentation activity during mixed-culture fermentation with sensitive commercial yeast strains

The competition between selected or commercial killer strains of type K2 and sensitive commercial strains of Saccharomyces cerevisiae was studied under various conditions in sterile grape juice fermentations. The focus of this study was the effect of yeast inoculation levels and the role of assimilable nitrogen nutrition on killer activity. A study of the consumption of free amino nitrogen (FAN) by pure and mixed cultures of killer and sensitive cells showed no differences between the profiles of nitrogen assimilation in all cases, and FAN was practically depleted in the first 2 days of fermentation. The effect of the addition of assimilable nitrogen and the size of inoculum was examined in mixed killer and sensitive strain competitions. Stuck and sluggish wine fermentations were observed to depend on nitrogen availability when the ratio of killer to sensitive cells was low (1:10 to 1:100). A relationship between the initial assimilable nitrogen content of must and the proportion of killer cells during fermentation was shown. An indirect relationship was found between inoculum size and the percentage of killer cells: a smaller inoculum resulted in a higher proportion of killer cells in grape juice fermentations. In all cases, wines obtained with pure-culture fermentations were preferred to mixed-culture fermentations by sensory analysis. The reasons why killer cells do not finish fermentation under competitive conditions with sensitive cells are discussed.     

Detection of maltose fermentation genes in the baking yeast strains of Saccharomyces cerevisiae

The presence of any one of the five unlinked MAL loci (MAL1, MAL2, MAL3, MAL4 and MAL6) confers the ability to ferment maltose on the yeast Saccharomyces cerevisiae. Each locus is composed of three genes encoding maltose permease, alpha-glucosidase and MAL activator. Chromosomal DNA of seven representative baking strains has been separated by pulse-field gel electrophoresis and probed with three genes in MAL6 locus. The DNA bands to which all of the three MAL-derived probes simultaneously hybridized were chromosome VII carrying MAL1 in all of the strains tested, chromosome XI carrying MAL4 in six strains, chromosome III carrying MAL2 in three strains and chromosomes II and VIII carrying MAL3 and MAL6, respectively, in the one strain. The number of MAL loci in baking strains was comparable to those of brewing strains.     

Stability of ramipril in water, apple juice, and applesauce

The stability of ramipril in water, in apple juice, and in applesauce was studied. The contents of a single capsule each of ramipril 1.25, 2.5, and 5 mg were mixed in glass beakers with 120 mL of deionized and filtered water, apple juice, or applesauce. Each mixture was apportioned into 10 120-mL amber polyethylene terephthalate (PET) containers. Five of the containers in each set were stored at 23 degrees C, and samples were taken at 0, 1, 2, 6, 12, and 24 hours. The other five containers were stored at 3 degrees C, and samples were taken at 4, 8, 12, 24, and 48 hours. The samples were analyzed for ramipril concentration by stability-indicating high-performance liquid chromatography (HPLC). The quantity of drug remaining in the PET container after "administration" was determined by mixing the contents of single 5-mg ramipril capsules with 60 mL of apple juice, pouring the mixture into a waste receptacle, rinsing the PET container three separate times with 10 mL of water, and analyzing the pooled fluid from these rinses for ramipril concentration by HPLC. Under no condition did the percentage of ramipril remaining drop below 90%. No peaks for degradation products appeared in the chromatograms. The mean +/- S.D. quantity of ramipril remaining in the PET containers after draining was 0.3 +/- 0.3% for the apple juice. Ramipril from 1.25-, 2.5-, and 5-mg capsules mixed in water, in apple juice, and in applesauce was stable for 24 hours at 23 degrees C and for 48 hours at 3 degrees C.     

Inactivation of Escherichia coli O157:H7 in apple juice by irradiation

Three strains (932, Ent-C9490, and SEA13B88) of Escherichia coli O157:H7 were used to determine the effectiveness of low-dose gamma irradiation for eliminating E. coli O157:H7 from apple juice or cider and to characterize the effect of inducing pH-dependent, stationary-phase acid resistance on radiation resistance. The strains were grown in tryptic soy broth with or without 1% dextrose for 18 h to produce cells that were or were not induced to pH-dependent stationary-phase acid resistance. The bacteria were then transferred to clarified apple juice and irradiated at 2 degrees C with a cesium-137 irradiator. Non-acid-adapted cells had radiation D values (radiation doses needed to decrease a microbial population by 90%) ranging from 0.12 to 0.21 kGy. D values increased to 0.22 to 0.31 kGy for acid-adapted cells. When acid-adapted SEA13B88 cells were tested in five apple juice brands having different levels of suspended solids (absorbances ranging from 0.04 to 2.01 at 550 nm), radiation resistance increased with increasing levels of suspended solids, with D values ranging from 0.26 to 0.35 kGy. Based on these results, a dose of 1.8 kGy should be sufficient to achieve the 5D inactivation of E. coli recommended by the National Advisory Committee for Microbiological Criteria for Foods.     

Development of a yeast trihybrid screen using stable yeast strains and regulated protein expression

We describe a yeast trihybrid system that facilitates rapid screening of cDNA libraries. Novel yeast vectors were developed that direct integration of cDNA encoding the bait and third protein component into the yeast chromosome. A recombinant yeast strain is thus generated (screening strain) and is available for library transformation. Transformation with the library DNA is a single, efficient transformation event, allowing the cDNA library to be represented in one step. Recovery of the library plasmid from the yeast is also simplified, since it is the only episomal plasmid. Assay of trihybrid interaction and identification of positive clones is facilitated by regulating expression of the third protein component using the yeast MET3 promoter, which is repressed in the presence of exogenous methionine. Trihybrid interactions are detected only on media lacking methionine. This trihybrid system uses the standard E. coli LacZ and yeast HIS3 reporter genes and is compatible with most available Gal4 activation domain cDNA libraries. We describe the successful application of this yeast trihybrid system to the study of phosphoprotein interactions involved in T-cell signaling.     

Characterization of absorption and scattering properties for various yeast strains by time-resolved spectroscopy

An understanding of the optical properties of biological media and cells is essential to the development of noninvasive optical studies of tissues. Unicellular organisms offer a unique opportunity to investigate the factors affecting light propagation, since they can be manipulated in ways impossible for more complex biological samples. In this study, we examined optical absorption and scattering properties of strongly multiple scattering yeast suspensions by means of near-infrared (NIR) time-resolved spectroscopy (TRS) and a sample substitution method. We determined the critical parameters for photon migration by varying the cell organelle content, the cell ploidy, the cell size, and the concentration of suspended cells. The results indicate that the photon absorption is insensitive to cell differentiation and that the cell volume is the primary factor determining light-scattering property.     

Transient behaviour of baker's yeast during enforced periodical variation of dissolved oxygen concentration

The aim of the investigation was to find out the influence of the variation of the dissolved oxygen concentration in the microenvironment of yeast cells on their physiological behaviour in small laboratory reactors and estimate their behaviour in large industrial reactors. Since the morphology of the laboratory and industrial yeasts differed considerably, their transient behaviour was investigated and compared. For this purpose, the strain Saccharomyces cerevisiae H620 and an industrial strain were cultivated on synthetic as well as on complex medium in batch operation during periodical variation of the dissolved oxygen concentration, monitoring the most important key parameters. Also the yeast was cultivated in batch as well as in continuous operation, the cell-containing culture medium was recirculated through a nonaerated loop at different recirculation rates (residence times of the cells in the loop), and the key operation variables were monitored. It was found that the transient behaviour of laboratory and industrial yeasts differed slightly. Since cells growing in batch culture are more sensitive to dissolved oxygen concentration variation than cells growing in continuous culture, the transient behavior of cells cultivated in batch operation varied from those in continuous operation. If the anaerobic phase was longer than 1 min, ethanol was produced. However, it was consumed during the aerobiosis again, provided that phase was considerably longer than the anaerobic phase. This means that the yeast cultivation was not influenced by the periodic operation of the dissolved oxygen. Judging from the measurements in large stirred tank and airlift tower loop reactors, in general, the cells would spend more time in the aerobic than in the anaerobic flow region, and they would spend less than 1 min in the anaerobic flow region. Therefore, no considerable effect of the periodically varied dissolved oxygen concentration on the cell cultivation can be expected in large-scale reactors. In the stirred tank-loop-system at high pumping rates/high frequencies of periodically varied dissolved oxygen concentration, unexpectedly, the formation of ethanol was observed, which might be caused by stress imposed on the cells.     

Comparative classification of Acinetobacter baumannii strains using seven different typing methods

A group of 49 Acinetobacter baumannii strains obtained from several hospital outbreaks and some sporadic cases were typed by biotyping, antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), plasmid typing, multilocus enzyme electrophoresis, whole-cell protein profile, and Fourier-transform infrared (FT-IR) spectroscopy. All these methods have shown a high degree of reproducibility and are capable of recognising strains from the same epidemiological event. However, their power to discriminate between epidemiologically unrelated strains varies, with PFGE being superior to the other methods investigated. FT-IR spectroscopy, which has not yet been used for typing of Acinetobacter strains, proved to be a very rapid and highly reproducible method, but was somewhat limited in its discriminating power.     

Differentiation of brewing yeast strains by pyrolysis mass spectrometry and Fourier transform infrared spectroscopy

Two rapid spectroscopic approaches for whole-organism fingerprinting--pyrolysis mass spectrometry (PyMS) and Fourier transform infrared spectroscopy (FT-IR)--were used to analyse 22 production brewery Saccharomyces cerevisiae strains. Multivariate discriminant analysis of the spectral data was then performed to observe relationships between the 22 isolates. Upon visual inspection of the cluster analyses, similar differentiation of the strains was observed for both approaches. Moreover, these phenetic classifications were found to be very similar to those previously obtained using genotypic studies of the same brewing yeasts. Both spectroscopic techniques are rapid (typically 2 min for PyMS and 10 s for FT-IR) and were shown to be capable of the successful discrimination of both ale and lager yeasts. We believe that these whole-organism fingerprinting methods could find application in brewery quality control laboratories.     

Effect of yeast culture and phenolic acids on the physiology of rumen fermentation determined in vitro

Annexin V has a calcium-dependent binding affinity for anionic phospholipids and activated platelets, and prevents prothrombinase activity. We investigated the clinical significance of IgG anti-annexin V antibodies in patients with SLE. The study population consisted of 140 patients with SLE. Sera were examined for IgG anti-annexin V antibodies by ELISA. IgG anti-annexin V antibodies were detected in 27 of 140 patients (19%). Significantly higher incidences of arterial or venous thrombosis, intrauterine fetal loss, and prolonged activated partial thromboplastin time were found in patients with anti-annexin V antibodies than in those without anti-annexin V antibodies. Three patients with thrombosis were found not to have anticardiolipin antibodies, but to show sustained serological reactions for anti-annexin V antibodies, irrespective of prednisolone administration. These results indicated the clinical characteristics of SLE patients with anti-annexin V antibodies, and that these antibodies may be associated with the pathogenesis of thrombotic events.     

Comparative evaluation of macrodilution and alamar colorimetric microdilution broth methods for antifungal susceptibility testing of yeast isolates

A comparative evaluation of the macrodilution method and the Alamar colorimetric method for the susceptibility testing of amphotericin B, fluconazole, and flucytosine was conducted with 134 pathogenic yeasts. The clinical isolates included 28 Candida albicans, 17 Candida tropicalis, 15 Candida parapsilosis, 12 Candida krusei, 10 Candida lusitaniae, 9 Candida guilliermondii, 18 Torulopsis glabrata, and 25 Cryptococcus neoformans isolates. The macrodilution method was performed and interpreted according to the recommendations of the National Committee for Clinical Laboratory Standards (document M27-P), and the Alamar colorimetric method was performed according to the manufacturer's instructions. For the Alamar colorimetric method, MICs were determined at 24 and 48 h of incubation for Candida species and T. glabrata and at 48 and 72 h of incubation for C. neoformans. The overall agreement within +/- 1 dilution for Candida species and T. glabrata against the three antifungal agents was generally good, with the values for amphotericin B, fluconazole, and flucytosine being 85.3, 77.9, and 86.2%, respectively, at the 24-h readings and 69.3, 65.2, and 97.2%, respectively, at the 48-h readings. Most disagreement was noted with fluconazole against C. tropicalis and T. glabrata. Our studies indicate that determination of MICs at 24 h by the Alamar colorimetric method is a valid alternate method for testing amphotericin B, fluconazole, and flucytosine against Candida species but not for testing fluconazole against C. tropicalis and T. glabrata. For flucytosine, much better agreement can be demonstrated against Candida species and T. glabrata at the 48-h readings by the Alamar method. Excellent agreement within +/- dilution can also be observed for amphotericin B, fluconazole, and flucytosine (80, 96, and 96%, respectively) against c. neoformans when the MICs were determined at 72 h by the Alamar method.     

Killer toxins of certain yeast strains have potential growth inhibitory activity on gram-positive pathogenic bacteria

The killer yeast strains which are encountered most frequently among species in the genera Saccharomyces, Candida, Hansenula, Pichia and Kluyveromyces (ten killer strains in toto) were tested for the activity of their toxins on the growth of some pathogenic bacteria (four Gram-positive and six Gram-negative strains) in a test in which purified toxins were not required. Neither toxins of the killer Saccharomyces cerevisiae and Pichia membranefaciens strains were active against the bacterial strains. In contrast the killer toxins of Hansenula anamola, Hansenula mrakii, Candida tropicalis, Kluyveromyces drosphilarum and Kluyveromyces lactis showed potential growth inhibitory effects on Gram-positive pathogenic bacteria. Thus, yeast killer toxins were found to be active only on Gram-positive bacterial cell types.     

New methods for evaluation and analysis of organoleptic qualities of foods and for calculation of changes. 9. Dependence of irradiation flavor of apple juice and concentrate on the gamma radiation dose

Various authors have defined organoleptically the concept "cooking flavour" for heat-treated apple juice and concentrate. In a similar way, the "irradiation flavour" caused by gamma radiation is defined in the present paper. It has been found that the irradiation flavour is attributable to low-boiling substances which appear already in the first fraction if the modified MICKO distillation is performed. Tetrahydrofuran (THF) has been identified as the key substance in irradiation flavour. As further evidence may be quoted the similarity of the intensity of off-flavour which was observed also by gas chromatography at the same times of retention, and the removal of the irradiation flavour by means of a THF-specific agent (mercury acetate). In connection with THF solutions added to non-irradiated apple juice, the authors employed the technique of "subjective olfactometry", using the "absolute value scale", to establish mathematical correlations between the intensity of irradiation flavour and the concentration of off-flavour products in apple juice or concentrate. The dependence of the intensity of irradiation flavour on the THF concentration and the gamma radiation dose, respectively, obeyed an exponential function. Thus, it has been possible to calculate the amounts of THF formed by radiation doses varying from 0.64 to 2.55 Mrad. Furthermore, the authors determined the minimum radiation dose for 12% apple juice and for 67% apple concentrate at which the irradiation flavour is first perceived (treshold of perception).     

Comparative analyses of the secondary structures of synthetic and intracellular yeast MFA2 mRNAs

The overall folded (global) structure of mRNA may be critical to translation and turnover control mechanisms, but it has received little experimental attention. Presented here is a comparative analysis of the basic features of the global secondary structure of a synthetic mRNA and the same intracellular eukaryotic mRNA by dimethyl sulfate (DMS) structure probing. Synthetic MFA2 mRNA of Saccharomyces cerevisiae first was examined by using both enzymes and chemical reagents to determine single-stranded and hybridized regions; RNAs with and without a poly(A) tail were compared. A folding pattern was obtained with the aid of the MFOLD program package that identified the model that best satisfied the probing data. A long-range structural interaction involving the 5' and 3' untranslated regions and causing a juxtaposition of the ends of the RNA, was examined further by a useful technique involving oligo(dT)-cellulose chromatography and antisense oligonucleotides. DMS chemical probing of A and C nucleotides of intracellular MFA2 mRNA was then done. The modification data support a very similar intracellular structure. When low reactivity of A and C residues is found in the synthetic RNA, approximately 70% of the same sites are relatively more resistant to DMS modification in vivo. A slightly higher sensitivity to DMS is found in vivo for some of the A and C nucleotides predicted to be hybridized from the synthetic structural model. With this small mRNA, the translation process and mRNA-binding proteins do not block DMS modifications, and all A and C nucleotides are modified the same or more strongly than with the synthetic RNA.     

Multicellular and aggregative behaviour of Salmonella typhimurium strains is controlled by mutations in the agfD promoter

To investigate the biochemical requirements for in vivo L-DOPA production by cells genetically modified ex vivo in a rat model of Parkinson's disease (PD), rat syngeneic 9L gliosarcoma and primary Fischer dermal fibroblasts (FDFs) were transduced with retroviral vectors encoding the human tyrosine hydroxylase 2 (hTH2) and human GTP cyclohydrolase I (hGTPCHI) cDNAs. As GTPCHI is a rate-limiting enzyme in the pathway for synthesis of the essential TH cofactor, tetrahydrobiopterin (BH4), only hTH2 and GTPCHI cotransduced cultured cells produced L-DOPA in the absence of added BH4. As striatal BH4 levels in 6-hydroxydopamine (6-OHDA)-lesioned rats are minimal, the effects of cotransduction with hTH2 and hGTPCHI on L-DOPA synthesis by striatal grafts of either 9L cells or FDFs in unilateral 6-OHDA-lesioned rats were tested. Microdialysis experiments showed that those subjects that received cells cotransduced with hTH2 and hGTPCHI produced significantly higher levels of L-DOPA than animals that received either hTH2 or untransduced cells. However, animals that received transduced FDF grafts showed a progressive loss of transgene expression until expression was undetectable 5 weeks after engraftment. In FDF-engrafted animals, no differential effect of hTH2 vs hTH2 + hGTPCHI transgene expression on apomorphine-induced rotation was observed. The differences in L-DOPA production found with cells transduced with hTH2 alone and those cotransduced with hTH2 and hGTPCHI show that BH4 is critical to the restoration of the capacity for L-DOPA production and that GTPCHI expression is an effective means of supplying BH4 in this rat model of PD.     

Assessment of the environmental behaviour of antioxidants in folios. Comparative risk analysis for the use of folios in agriculture

The objective of the reported study has been to assess and evaluate as comprehensively as possible the environmental impact of Octadecyl 3(3,5-di-tert-butyl-4-hydroxyphenyl) propionate (CAS no: 2082-79-3, Irganox 1076, Ciba Specialty Chemicals Inc., Additives), which is used as an antioxidant. The potential impact on the compartments soil, groundwater and surface water is to be considered. For comparative purposes, additionally, other chemical compounds being currently under environmental discussion are also taken into account. These comprise pesticides and phthalates which are ubiquitously distributed plasticizers as well as a complexing agent. Since the data basis for each of the compounds under consideration is different, a tiered approach comprising various methodologies of impact assessment has been chosen to achieve the best possible comprehensiveness. The tiers are: 1. Tier: hazard assessment using a scoring system 2. Tier: comparative risk assessment. When interpreting the results of each method, system boundaries as well as underlying assumptions were taken into consideration. Both methodologies showed, that-as compared to the reference substances-there is no relevant environmental and toxicological concern due to low environmental and human hazard from Octadecyl 3(3,5-di-tert-butyl-4-hydroxyphenyl) propionate.     

Modulation of glycerol and ethanol yields during alcoholic fermentation in Saccharomyces cerevisiae strains overexpressed or disrupted for GPD1 encoding glycerol 3-phosphate dehydrogenase

The possibility of the diversion of carbon flux from ethanol towards glycerol in Saccharomyces cerevisiae during alcoholic fermentation was investigated. Variations in the glycerol 3-phosphate dehydrogenase (GPDH) level and similar trends for alcohol dehydrogenase (ADH), pyruvate decarboxylase and glycerol-3-phosphatase were found when low and high glycerol-forming wine yeast strains were compared. GPDH is thus a limiting enzyme for glycerol production. Wine yeast strains with modulated GPD1 (encoding one of the two GPDH isoenzymes) expression were constructed and characterized during fermentation on glucose-rich medium. Engineered strains fermented glucose with a strongly modified [glycerol] : [ethanol] ratio. gpd1delta mutants exhibited a 50% decrease in glycerol production and increased ethanol yield. Overexpression of GPD1 on synthetic must (200 g/l glucose) resulted in a substantial increase in glycerol production ( x 4) at the expense of ethanol. Acetaldehyde accumulated through the competitive regeneration of NADH via GPDH. Accumulation of by-products such as pyruvate, acetate, acetoin, 2,3 butane-diol and succinate was observed, with a marked increase in acetoin production.