Influence of Wine Composition on the Heat Resistance of Potential Spoilage Organisms

Pasteurization studies were conducted on 29 yeast and five lactic acid bacteria. In general the yeasts were more heat resistant in wine than were the bacteria. The one exception was a strain of Lactobacillus fructivorans that gave an average D-value of 1.7 min at 60 C. Alcohol was the wine constituent that had the greatest effect on resistance; D-values for all test species were inversely related to the ethanol concentration. The response of organisms to other factors such as pH, sugar, and sulfur dioxide varied with the species.     

Prevention of Yeast Spoilage in Feed and Food by the Yeast Mycocin HMK

The yeast Williopsis mrakii produces a mycocin or yeast killer toxin designated HMK; this toxin exhibits high thermal stability, high pH stability, and a broad spectrum of activity against other yeasts. We describe construction of a synthetic gene for mycocin HMK and heterologous expression of this toxin in Aspergillus niger. Mycocin HMK was fused to a glucoamylase protein carrier, which resulted in secretion of biologically active mycocin into the culture media. A partial purification protocol was developed, and a comparison with native W. mrakii mycocin showed that the heterologously expressed mycocin had similar physiological properties and an almost identical spectrum of biological activity against a number of yeasts isolated from silage and yoghurt. Two food and feed production systems prone to yeast spoilage were used as models to assess the ability of mycocin HMK to act as a biocontrol agent. The onset of aerobic spoilage in mature maize silage was delayed by application of A. niger mycocin HMK on opening because the toxin inhibited growth of the indigenous spoilage yeasts. This helped maintain both higher lactic acid levels and a lower pH. In yoghurt spiked with dairy spoilage yeasts, A. niger mycocin HMK was active at all of the storage temperatures tested at which yeast growth occurred, and there was no resurgence of resistant yeasts. The higher the yeast growth rate, the more effective the killing action of the mycocin. Thus, mycocin HMK has potential applications in controlling both silage spoilage and yoghurt spoilage caused by yeasts.     

Characteristics of an Organism Causing Spoilage in an Orange Juice Beverage

S ummary . An organism causing an off-flavour in an orange juice drink is described. Morphological and biochemical properties of this organism, which produces a characteristic coral pink growth on most media, are also reported. Possible relationships with similar organisms known to occur in fruit juices and on fruits are discussed.     

Pectobacterium carotovorum subsp. brasiliense Causing Soft Rot on Paprika in Korea

During summer 2011 in South Korea, severe fruit rot of paprika was observed, causing severe economic losses in paprika production. Symptoms of fruit and pedicel decay were consistent with symptoms caused by Pectobacterium carotovorum subsp. brasiliense (Pcb) as recently described in Brazil, the United States, Israel and South Africa. Physiological analysis and pathogenicity test of strains isolated from paprika fruit revealed that the pathogen was the bacterium Pcb. Sequencing and phylogenetic analysis of the 16S rDNA and partial 16S–23S rDNA intergenic spacer region confirmed that the isolates were Pcb. This is the first report of Pcb in Korea, which has a significant economic impact on Korean paprika production.     

A Note on the Identities of Organisms Causing Black Spot Spoilage of Meat

Four fungal species, Cladosporium cladosporioides, C. herbarum, Penicillium hirsutum and Aureobasidium pullulans were isolated from meat spoiled by black spot and shown to produce black spot colonies when inoculated on to sterile meat slices which were incubated at — 1°C. Penicillium hirsutum grew only on the meat surface but the other species penetrated into the tissues, apparently in response to arid conditions at the surface. It is clear from these observations that the traditional association of black spot spoilage with C. herbarum alone is incorrect.     

Studies on Yeast Uricase

A method for obtaining a highly purified preparation of yeast uricase was developed. The procedure included extraction of uricase from the uric ase-induced yeast cells, fractionation with ammonium sulfate and chromatography on a DEAE-cellulose column. The purified yeast uricase was shown to be ultracentrifugally homogeneous. The enzyme acted best at pH 8.5 and was stable in a range from pH 7.0 to 11.0 and at temperatures lower than 40°G. The Michaelis constant for urate was calculated to be 5.88 × 10^?6 m at pH 8.5, borate buffer. Activity and stability of the enzyme, however, were found to be significantly affected by the kind of buffer used. The enzyme was sensitive to heavy metal ions such as mercuric and cupric ions, but the sensitivity was influenced by the kind of buffer used.     

Biochemical Studies on Yeast

The present authors obtained direct proof of the occurrence of glucose dehydrogenase in yeast. Optimum pH of the glucose dehydrogenation system in yeast was about 7.0. After dialysis of the salting out preparation, the dialysate revealed only a trace of activity. The addition of DPN or TPN restored the activity. The majority of the yeast glucose dehydrogenase precipitated below about 0.70 ammonium sulfate saturation, and there was no marked activity in 0.30 ammonium sulfate saturation. The yeast glucose dehydrogenase was observed to be highly specific for β-d-glucose.     

Biochemical Studies on Yeast

The growth of Zygosaccharomyces soja was strongly inhibited by the addition of 10^?2 m-monoiodoacetic acid and 10^?3 m-potassium cyanide. The formation of a considerable amount of riboflavin was recognized after 5 days cultivation of this organism in presence of the aforementioned two inhibitors, and its growth was considerably improved after the formation and accumulation of riboflavin. Furthermore, the effect of the aforementioned two inhibitors upon the growth of this organism in young stage was compensated with the addition of riboflavin.     

Rhizoctonia spp. Causing Root and Hypocotyl Rot in Phaseolus vulgaris in Cuba

Sixty isolates of Rhizoctonia spp. were obtained from Cuban bean fields during the period 2004–2007. Isolates were characterized with different techniques, including nuclei staining, pectic zymogram, PCR–RFLP analysis of the rDNA–ITS region and sequencing of the rDNA–ITS region. The majority of the isolates were identified as multinucleate Rhizoctonia solani isolates, representing two different anastomosis groups (AGs), AG 2‐2 WB and AG 4 HGI; the remaining isolates were binucleate Rhizoctonia isolates and belonged to AG F and AG A. AG 4 HGI isolates were equally distributed in all soil types; AG 2‐2 isolates were more frequently isolated from cambisols, whereas AG F isolates were related to calcisols. Pathogenicity experiments in vitro and in the greenhouse, revealed that binucleate isolates only caused root rot, whereas R. solani isolates were able to cause root rot and hypocotyl rot. Furthermore, differences in virulence level were observed between R. solani and binucleate isolates and among different AGs. Isolates of R. solani AG 4 HGI and R. solani AG 2‐2 WB were the most aggressive, binucleate isolates of AG F were intermediate aggressive, whereas a binucleate isolate of AG A was weakly aggressive. In contrast with other reports about R. solani in bean, web blight symptoms were never observed during this study.     

木霉(Trichoderma spp.)对三种引起大棚蔬菜病害病原菌的影响

通过木霉属(Trichoderma) 3菌株与双鸭山蔬菜大棚中的黄瓜枯萎病菌(FusariumoxysporumSchlecht.f.cucumerinum)、黄瓜果腐病菌(PhytophthoracapsiciLeonian)、菜豆叶枯病菌(Cladosporiumsp .)的对峙培养试验,结果表明:绿色木霉1(TrichodermaviridePers.exGray 1)可作为双鸭山蔬菜大棚中的黄瓜枯萎病、黄瓜果腐病、菜豆叶枯病3种病害的生物防治拮抗菌加以利用,该拮抗菌对菜豆叶枯病菌抑制效果最好;绿色木霉2 (Tricho dermaviride 2 )对黄瓜果腐病菌抑制效果最好;而哈茨木霉(TrichodermaharzianumRifai)对以上3种病原菌都有抑制效果,对菜豆叶枯病菌抑制效果最好。从试验结果还可看出,绿色木霉2对黄瓜枯萎病菌和菜豆叶枯病菌的生长有促进作用。     

Studies of Giardia spp. and Cryptosporidium spp. in two adjacent watersheds.

Two adjacent British Columbia, Canada, watersheds with similar topographical features were studied. Both the Black Mountain Irrigation District (BMID) and the Vernon Irrigation District (VID) serve rural agricultural communities which are active in cattle ranching. The present study was carried out in five phases, during which a total of 249 surface water samples were tested in the study watersheds. The aims of these phases were to determine levels of parasite contamination in raw water samples collected from the intakes as well as from other sites in each watershed and to investigate cattle in the watersheds as potential sources of parasite contamination of surface drinking water supplies. Giardia cysts were not detected in the raw water samples collected from lake sources at the headwaters of both watersheds but were found in 100% (70 or 70) of water samples collected at the BMID intake and 97% (68 of 70) of water samples collected at the VID intake. Significantly higher levels (P < 0.05) of Giardia cysts were found at the BMID intake (phase 1, 7 to 2,215 cysts per 100 liters; phase 3, 4.6 to 1,880 cysts per 100 liters) when compared with that of the VID intake (2 to 114 cysts per 100 liters). The BMID watershed has a more complex system of surface water sources than the VID watershed. Cattle have access to creeks in the BMID watershed, whereas access is restricted in the VID watershed. Collection of raw water samples from a creek upstream and downstream of a cattle ranch in the BMID watershed showed that the downstream location had significantly higher (P < 0.05) levels (0.6 to 42.9 cysts per 100 liters and 1.4 to 300.0 oocysts per 100 liters) of both Giardia cysts and Cryptosporidium oocysts than those of the upstream location (0.5 to 34.4 cysts per 100 liters and 0.5 to 34.4 oocysts per 100 liters). Peak concentrations of both parasites coincided with calving activity. Fecal samples, collected from cattle in both watersheds, showed 10% (3 of 30) in the BMID and 50% (5 of 10) in the VID watersheds to be Giardia positive. No Cryptosporidium-positive fecal samples were found. Giardia cysts isolated from the BMID watershed were repeatedly infective to gerbils in contrast to those from the VID watershed. The 10 BMID drinking water Giardia isolates retrieved into culture and biotyped showed zymodeme and karyotype heterogeneity. The differences in patterns of parasite contamination and cattle management practices contribute to the unique watershed characteristics observed between two areas which are topographically similar and geographically adjacent.     

Effect of Yeast Extract Concentration on Viability and Cell Distortion in Rhizobium spp.

A low concentration of yeast extract (0·1%) in liquid media favoured rapid growth and high percentage of viable cells in cultures of Rhizobium japonicum (CB 1809), R. lupini (WU 425), R. meliloti (SU 47), R. trifolii (TA1) and a cowpea strain (CB 756). Concentrations of yeast extract > 0·35% depressed viability and produced distorted cells in all strains except SU 47: TA1 was especially sensitive. When used at 0·5–1% (w/v), each yeast extract (Difco, Oxoid, Vegemite) or casein hydrolysate produced greatly enlarged abnormal cells of TA1, each containing several granules of poly-β-hydroxybutyrate and whorls of intracytoplasmic membranes, and showing greater internal disorganisation than that seen in root nodule bacteroids. Lysogenic and non-lysogenic cultures of R. trifolii were all sensitive to yeast extract, and such sensitivity, for strains of several species, was unrelated to effectiveness in nodulating host plants. Glycine inhibited growth of all strains tested. Several other amino acids occurring in casein hydrolysate inhibited TA1 strongly and induced formation of distorted cells and spheroplasts; this distortion was partly counteracted by adding salts of calcium or magnesium. In media with 0·1% yeast extract the use of mannitol, sucrose, lactose or galactose as alternative carbon sources, each at a concentration of 0·02–1%, did not affect numbers of viable rhizobia or cell shape in all strains tested.     

Glucosamine Resistance in Yeast. I. a Preliminary Genetic Analysis

Mutants of the yeast Saccaromyces cerevisiae which can grow on glycerol medium in the presence of 0.05% D (+) glucosamine have been isolated. Genetic analysis of 13 of these glucosamine resistant (GR) mutants demonstrated two modes of inheritance. One group of mutants (GR 5, 6, 7, 8, 9 and 10) gave results characteristic of non-Mendelian inheritance and it is suggested that these mutants represent one or more new mitochondial loci. Four of the remaining mutants showed clear-cut Mendelian inheritance. These mutants fell into two complementation groups and subsequent mapping experiments demonstrated that two independent loci, gay 1 and gay 2, unlinked to each other or to the centromeres of chromosomes I, II, IV, VIII or IX, were responsible for conferring glucosamine resistance in these mutants.     

Complex Nature of the Genome in a Wine Spoilage Yeast,Dekkera bruxellensis

When the genome organizations of 30 native isolates belonging to a wine spoilage yeast, Dekkera (Brettanomyces) bruxellensis, a distant relative of Saccharomyces cerevisiae, were examined, the numbers of chromosomes varied drastically, from 4 to at least 9. When single gene probes were used in Southern analysis, the corresponding genes usually mapped to at least two chromosomal bands, excluding a simple haploid organization of the genome. When different loci were sequenced, in most cases, several different haplotypes were obtained for each single isolate, and they belonged to two subtypes. Phylogenetic reconstruction using haplotypes revealed that the sequences from different isolates belonging to one subtype were more similar to each other than to the sequences belonging to the other subtype within the isolate. Reanalysis of the genome sequence also confirmed that partially sequenced strain Y879 is not a simple haploid and that its genome contains approximately 1% polymorphic sites. The present situation could be explained by (i) a hybridization event where two similar but different genomes have recently fused together or (ii) an event where the diploid progenitor of all analyzed strains lost a regular sexual cycle, and the genome started to accumulate mutations.Recent achievements in genome sequencing have revealed that gene contents vary among distantly related organisms but are relatively constant among closely related species. For example, among hemiascomycete yeasts, which originated more than 250 million years ago and include well-studied yeasts such as Saccharomyces cerevisiae and Candida albicans (3, 4), an average genome contains approximately 5,000 genes. Approximately one-half of the protein-coding gene families are preserved in all of the yeasts sequenced to date. However, there is a large variation in the gene order and configuration of chromosomes among different species.Chromosome configuration is usually well preserved among populations belonging to the same species. Only rarely do geographically separated populations, for example, Mus musculus (8, 32), differ in the number and form of chromosomes. The mutability of the genome enhances the adaptability of the species, but it also decreases the viability of the new variant. In addition, these changes can preclude successful reproduction and can be a decisive factor in the emergence of new species (2; for a review, see references 6 and 7).Among closely related yeasts belonging to the Saccharomyces sensu stricto clade (including S. cerevisiae), which originated approximately 20 million years ago, the gene contents are relatively similar (13). Their genomes are almost colinear and consist of 16 chromosomes. Some inter- and intraspecific variations are observed predominantly at the chromosome ends (18, 19). Sensu stricto species are semifertile, meaning that they can successfully mate and produce F1 offspring but that the hybrids are largely sterile. It appears that this clade has still not completed the speciation process (7). The relatively low chromosome variability among Saccharomyces sensu stricto yeasts is probably promoted by regular sexual cycles. These yeasts are diploid, but heterozygosity is almost absent because of the homothallic life-style, which enables haploid spores from the same yeast cell to mate. Only for “sterile” hybrids, such as the lager brewing yeast Saccharomyces pastorianus (Saccharomyces carlsbergensis), originating upon the mating of two different species, has a pronounced heterozygosity been observed (14). The parental genomes came from S. cerevisiae and a close relative, Saccharomyces bayanus. A study of allotetraploid hybrids between a diploid S. cerevisiae strain and a diploid S. bayanus strain demonstrated that these hybrids behave essentially as diploids regarding meiosis and sporulation and had 77% spore viability (1, 22). The extent of intra- and interspecific genome variability is not well known for other yeasts, especially among distant relatives of S. cerevisiae. The only well-studied exception is a pathogen, Candida albicans, that is believed to be predominantly asexual. This yeast diverged from the S. cerevisiae lineage prior to the origin of the efficient homothallic life-style (reviewed in reference 25). The genome is diploid and shows a low level of heterozygosity (12), and large variations in the configurations of the chromosomes among different isolates have been reported (reviewed in reference 29).Dekkera bruxellensis is often isolated in wineries and is well known as a major microbial cause of wine spoilage. The lineages of D. bruxellensis and S. cerevisiae separated at approximately the same time as the lineages of S. cerevisiae and C. albicans separated, approximately 200 million years ago (40). However, D. bruxellensis and S. cerevisiae share several characteristics, such as the production of ethanol, the ability to propagate in the absence of oxygen (anaerobic growth), and petite positivity (the ability to produce offspring without mitochondrial DNA [mtDNA]), that are rarely found among other yeasts (16, 20). So far, a sexual cycle in D. bruxellensis has not been found.In this paper, we analyzed the genome structures of 30 isolates of D. bruxellensis originating from different geographical localities around the world. We show that these isolates have different numbers and sizes of chromosomes and also that the numbers of copies of several analyzed genes and their sequences vary. In addition, we could detect heterozygosity in the partial genome sequence of strain Y879.