Comparative study on the identification of food-borne yeasts

Morphologically distinct yeast colonies from partially and fully processed fruits and vegetables were isolated over a 3-year period. Identification of 239 strains was achieved by using standard methods, commercial identification kits (API 20C and API YEAST-IDENT), and a simplified system for food-borne yeasts. The identified strains of fruit origin represented 36 species belonging to 19 genera. Among strains of vegetable origin, 34 species representing 17 genera were identified. The simplified identification system and the conventional method provided the same results in 80% of the cases. The commercial identification kits were easy to use but were not appropriate for food-borne yeast species. Computer-assisted identification was helpful.     

Inhibition of Listeria monocytogenes by food-borne yeasts

Many bacteria are known to inhibit food pathogens, such as Listeria monocytogenes, by secreting a variety of bactericidal and bacteriostatic substances. In sharp contrast, it is unknown whether yeast has an inhibitory potential for the growth of pathogenic bacteria in food. A total of 404 yeasts were screened for inhibitory activity against five Listeria monocytogenes strains. Three hundred and four of these yeasts were isolated from smear-ripened cheeses. Most of the yeasts were identified by Fourier transform infrared spectroscopy. Using an agar-membrane screening assay, a fraction of approximately 4% of the 304 red smear cheese isolates clearly inhibited growth of L. monocytogenes. Furthermore, 14 out of these 304 cheese yeasts were cocultivated with L. monocytogenes WSLC 1364 on solid medium to test the antilisterial activity of yeast in direct cell contact with Listeria. All yeasts inhibited L. monocytogenes to a low degree, which is most probably due to competition for nutrients. However, one Candida intermedia strain was able to reduce the listerial cell count by 4 log units. Another four yeasts, assigned to C. intermedia (three strains) and Kluyveromyces marxianus (one strain), repressed growth of L. monocytogenes by 3 log units. Inhibition of L. monocytogenes was clearly pronounced in the cocultivation assay, which simulates the conditions and contamination rates present on smear cheese surfaces. We found no evidence that the unknown inhibitory molecule is able to diffuse through soft agar.     

Comparative study on synteny between yeasts and vertebrates

We studied synteny conservation between 18 yeast species and 13 vertebrate species in order to provide a comparative analysis of the chromosomal plasticity in these 2 phyla. By computing the regions of conserved synteny between all pairwise combinations of species within each group, we show that in vertebrates, the number of conserved synteny blocks exponentially increases along with the divergence between orthologous protein and that concomitantly; the number of genes per block exponentially decreases. The same trends are found in yeasts but only when the mean protein divergence between orthologs remains below 36%. When the average protein divergence exceeds this threshold, the total number of recognizable synteny blocks gradually decreases due to the repeated accumulation of rearrangements. We also show that rearrangement rates are on average 3-fold higher in vertebrates than in yeasts, and are estimated to be of 2 rearrangements/Myr. However, the genome sizes being on average 200 times larger in vertebrates than in yeasts, the normalized rates of chromosome rearrangements (per Mb) are about 50-fold higher in yeast than in vertebrate genomes.     

Transfer of genetic material between pathogenic and food-borne yeasts

Many pathogenic yeast species are asexual and therefore not involved in intra- or interspecies mating. However, high-frequency transfer of plasmid DNA was observed when pathogenic and food-borne yeasts were grown together. This property could play a crucial role in the spread of virulence and drug resistance factors among yeasts.     

Accelerated procedure for the enumeration and identification of food-borne Staphylococcus aureus.

A procedure was developed for accelerating to 29 h the enumeration and identification of both healthy and stressed cells of Staphylococcus aureus in foods. Baird-Parker agar medium was incubated for 24 h; S. aureus was identified within 5 additional h by using a simplified thermonuclease test.     

Random amplified polymorphic DNA and restriction enzyme analysis of PCR amplified rDNA in taxonomy: two identification techniques for food-borne yeasts

The random amplified polymorphic DNA (RAPD) assay and the restriction enzyme analysis of PCR amplified rDNA are compared for the identification of the common spoilage yeasts Zygosaccharomyces bailii, Z. rouxii, Saccharomyces cerevisiae, Candida valida and C. lipolytica. Both techniques proved to be adequate tools for yeast identification. Since the RAPD does provide less stable patterns than restriction enzyme analysis of PCR amplified rDNA, and a large amount of data had to be compared without data reduction, Principal Component Analysis (PCA) was applied successfully for clustering the RAPD patterns. The success of PCA is highly influenced by the primer used in RAPD and the amount of reference samples. A large amount of reference samples improves the performance of clustering in PCA. The primer of choice was shown to be important with respect to the discriminatory power of the RAPD method. Some primers used enabled discrimination on the subspecies level. The results collected with both typing methods justify the conclusion that the present typing system can be applied for taxonomical purposes.     

Comparative studies on mitochondrial development in yeasts

Summary High molecular weight mitochondrial RNA from Saccharomyces cerevisiae can be isolated rapidly and in relatively high yield from mitochondria prepared from cells prefixed with glutaraldehyde and disrupted mechanically. The RNA has lower electrophoretic mobilities than corresponding species from cytoplasmic ribosomes, and can also be distinguished from cytoplasmic RNA on the basis of the sensitivity of the mobility to temperature. RNA from cytoplasmic ribosomes and mitochondria of Candida parapsilosis shows a similar differential response to temperature.Mitochondrial ribosomes in Saccharomyces cerevisiae do not appear to be distinguishable from the cytoplasmic particles on the basis of sedimentation velocity. They can be identified, however, by pulse-labelling cells in the presence of cycloheximide. Cytoplasmic ribosomes under these conditions do not label. The labelling of mitochondrial ribosomes is sensitive to chloramphenicol, and is dispersed over the polysomal or ribosomal aggregate region of density gradients.     

Comparative study of the ultrastructural characteristics of Torulopsis candida yeasts grown on glucose and hexadecane]

Electron-microscopic examination of the ultrastructure of Torulopsis candida cells grown on glucose and hexadecane revealed a well-developed network of canals in the cell wall of yeasts grown on hexadecane. These canals appeared in the non-adapted cells at the initial hours of the cultivation and completed their formation at the end of the logarithmic phase. The investigation by the freeze-etching showed the exocytosis of "secretory granules" to take place at the periplasma of the cells, and the morphological relationship of the granules to the canals in the cell wall.     

产胞外多糖酵母菌株的筛选鉴定及发酵产糖

【目的】微生物胞外多糖大多具有良好的功能和特性,但对酵母胞外多糖的研究甚少。本研究从自然界中筛选出产胞外多糖的酵母菌株,并对其发酵产糖条件进行初步研究。【方法】利用平板涂布法从自然界中分离得到酵母菌株,苯酚硫酸法测定菌株胞外多糖的产量,筛选出胞外多糖高产菌株,并对其进行5.8SrDNA分类鉴定,最后优化其产糖培养基组成。【结果】对从葡萄、蜜枣、土壤样品中分离得到的132株酵母进行筛选,最终得到3株高产胞外多糖的酵母菌株Z14、Z20和L25。经5.8S rDNA序列测定及系统发育分析,从左优红葡萄中分离得到的Z14和Z20与东方伊萨酵母(Issatchenkia orientalis)处于同一分支,相似性达到99%以上;从落叶松近表层土壤分离得到的L25与土生隐球酵母(Cryptococcus humicolus)处于同一分支,相似性为98.8%。经优化,利于Z20胞外多糖合成的最优发酵培养基配方为:葡萄糖8%,(NH4)SO40.2%,KH2PO40.1%,酵母浸粉0.1%,CaCl20.01%。在初始pH6.0,发酵温度28℃,摇床转数160 r/min条件下,在此培养基中发酵4 d后胞外多糖产量可达2.046 g/L,比复筛时的产量1.137 g/L提高了79.9%。【结论】文献已报道某些属的酵母可以生产胞外多糖,经本文研究发现Issatchenkia属的酵母也可以合成胞外多糖,并且改变基础产糖培养基的成分可以显着提高Z20胞外多糖的产量。     

Efficacy of the Ryu nonstaining KOH technique for rapidly determining gram reactions of food-borne and waterborne bacteria and yeasts.

A simple and rapid (< 60 s) nonstaining technique with 3% potassium hydroxide to determine Gram reactions was tested with 495 food-borne and waterborne bacteria and yeasts. In KOH, suspensions of gram-negative bacteria become viscous and string out. Gram-positive bacteria are not affected. There was 100% correlation between the KOH string test results and gram-positive and gram-negative strains.     

Comparative study of Candida lipolytica yeasts with various abilities to produce citrate

The growth and the activity of some enzymes were studied in a Candida lipolytica strain 12a which did not synthesize acids in a medium with glucose under the conditions of nitrogen deficiency. The substrate was not assimilated and cyanide-resistant respiration did not develop in the strain under the conditions of profound nitrogen deficiency. The inability of cells to assimilate glucose at the stationary phase of growth resulted, apparently, from an abrupt decrease of phosphofructokinase and pyruvate dehydrogenase activities in the cells. The activities of pyruvate carboxylase and citrate synthase fell down abruptly at the same time.     

Locally isolated yeasts from Malaysia: identification, phylogenetic study and characterization

Yeasts are a convenient platform for many applications. They have been widely used as the expression hosts. There is a need to have a new yeast expression system to contribute the molecular cloning demands. Eight yeast isolates were screened from various environment sources and identified through ribosomal DNA (rDNA) Internal Transcribed Spacer (ITS). Full sequence of the rDNA ITS region for each isolate was BLASTed and phylogenetic study was constructed by using MEGA4. Among the isolates, isolate WB from 'ragi' (used to ferment carbohydrates) could be identified as a new species in order Saccharomycetales according to rDNA ITS region, morphology and biochemical tests. Isolate SO (from spoiled orange), RT (rotten tomato) and RG (different type of 'ragi') were identified as Pichia sp. Isolates R1 and R2, S4 and S5 (from the surrounding of a guava tree) were identified as Issatchenkia sp. and Hanseniaspora sp., respectively. Geneticin, 50 μg/mL, was determined to be the antibiotic marker for all isolates excepted for isolates RT and SO which used 500 μg/mL and 100 μg/mL Zeocin, respectively. Intra-extracellular proteins were screened for lipolytic activity at 30°C and 70°C. Thermostable lipase activity was detected in isolates RT and R1 with 0.6 U/mg and 0.1 U/mg, respectively. In conclusion, a new yeast-vector system for isolate WB can be developed by using phleomycin or geneticin as the drugs resistance marker. Moreover, strains RT and R1 can be investigated as a novel source of a thermostable lipase.     

Comparative genomics of transcriptional regulation in yeasts and its application to identification of a candidate alpha-isopropylmalate transporter

Conservation rates in non-protein-coding regions of five yeast genomes of the genus Saccharomyces were analyzed using multiple whole-genome alignments. This analysis confirmed previously shown decrease in conservation rates observed immediately upstream of the translation start point and downstream of the stop-codon. Further, there was a sharp conservation peak in the upstream regions likely related to the core promoter (-35 bp to +35 bp around TSS) and a conservation peak downstream of the stop-codon whose function is not yet clear. Regulation of leucine and methionine biosynthesis controlled by the global regulator Gcn4p and pathway-specific regulators was analyzed in detail. A candidate alpha-isopropylmalate carrier, YOR271cp, was identified based on conservation of Leu3p binding sites, analysis of ChIP-chip data, protein localization and sequence similarity.