Purity Control of the Production of Baker''s Yeast Employing a Fluorescent Antiserum

A simple staining procedure for the rapid detection of wild yeasts contaminating baker's yeast during the course of industrial production is described. Fluorescein-labeled, specific antiserum against Saccharomyces cerevisiae is applied to smears of baker's yeast which are then examined by ultraviolet microscopy. Optimal results are obtained with the combined phase contrast and fluorescence which makes the S. cerevisiae appear green, whereas cells of wild yeasts are visible in bright red counterstain. With this method, wild yeasts could be identified in baker's yeast at a dilution of 1:10,000.     

Construction from a single parent of baker's yeast strains with high freeze tolerance and fermentative activity in both lean and sweet doughs.

From a freeze-tolerant baker's yeast (Saccharomyces cerevisiae), 2,333 spore clones were obtained. To improve the leavening ability in lean dough of the parent strain, we selected 555 of the high-maltose-fermentative spore clones by using a method in which a soft agar solution containing maltose and bromocresol purple was overlaid on yeast colonies. By measuring the gassing power in the dough, we selected 66 spore clones with a good leavening ability in lean dough and a total of 694 hybrids were constructed by crossing them. Among these hybrids, we obtained 50 novel freeze-tolerant strains with good leavening ability in all lean, regular, and sweet doughs comparable to that of commercial baker's yeast. Hybrids with improved leavening ability or freeze tolerance compared with the parent yeast and commercial baker's yeasts were also obtained. These results suggest that hybridization between spore clones derived from a single parent strain is effective for improving the properties of baker's yeasts.     

Chitin in the cell wall of yeasts

Summary According to literature a glucan is the main cell wall constituent of baker's yeast and probably of all yeasts. Chitin is reported in some filamentousEndomycetaceae, but is said to be absent in all other yeasts tested.A modified chitosan sulphate crystallization test for detecting chitin in yeasts and the relevant characteristics of these crystals were described. It was applied to 29 yeast species in addition to baker's and brewer's yeast. ExceptSchizosaccharomyces octosporus all were found to contain chitin as a cell wall constituent. However, the amount was very small, varied in different species and seemed to increase in older cultures.Chitin has also been demonstrated as glucosamine on paper chromatograms of concentrated hydrolysates of the cell wall residue, obtained after treating baker's yeast with boiling dilute alkali and acid.     

Factors which affect the frequency of sporulation and tetrad formation in Saccharomyces cerevisiae baker's yeasts.

To clarify the role that respiration, the mitochondrial genome, and interactions of mitochondria and nucleus play on sporulation and to improve the sporogenic ability of several baker's yeasts, an investigation of the effects of different media and culture conditions on baker's yeast sporulation was undertaken. When standard protocols were followed, the sporulation frequency varied between 20 and 60% and the frequency of four-spore asci varied between 1 and 6%. Different presporulation and sporulation media, the use of solid versus liquid media, and incubation at 22 versus 30 degrees C were checked, and the cells were collected from presporulation media in either exponential or stationary phase. Best results, yielding sporulation and four-spore ascus formation frequencies up to 97 and 60%, respectively, were obtained by collection of the cells in exponential phase from liquid presporulation medium with 10% glucose and transfer of them to sporulation medium with 0.5% potassium acetate at 22 degrees C. Under these conditions, the most important factor was the growth phase (exponential versus stationary) at which cells from presporulation medium were collected. Changes in sporulation frequencies were also measured after transfer of mitochondria from different sources to baker's yeasts. When mitochondria from laboratory, baker's, and wine yeasts were transferred to baker's and laboratory petite strains, sporulation and four-spore ascus formation frequencies dropped dramatically either to no sporulation at all or to less than 50% in both parameters. This transfer also resulted in an increase in the frequency of petite mutant formation but yielded similar growth and respiration rates in glycerol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enumeration of intestinal enterococci and interfering organisms with Slanetz-Bartley agar, KF streptococcus agar and the MUST method

The recovery of intestinal species of enterococci and streptococci and potentially interfering nonfaecal species was measured on KF streptococcus agar, Slanetz-Bartley agar and in a medium based on 4-methylumbelliferyl-β- d -glucoside (MUST method) using pure cultures. Both of the solid media yielded high recoveries of the target species. Their selectivity was better at elevated incubation temperature but nonfaecal Enterococcus and Staphylococcus species were not eliminated even at the elevated temperature. The MUST method tended to give slightly lower recoveries than the agar cultivation methods with some target species at 44°C but recoveries were better at 41°C.     

Lysine-overproducing mutants of Saccharomyces cerevisiae baker's yeast isolated in continuous culture.

Saccharomyces cerevisiae baker's yeast mutants which produce 3 to 17 times as much lysine as the wild type, depending on the nitrogen source, have been selected. The baker's yeast strain was growth in a pH-regulated chemostat in minimal medium with proline as the nitrogen source, supplemented with increasing concentrations of the toxic analog of the lysine S-2-aminoethyl-L-cysteine (AEC). The lysine-overproducing mutants, which were isolated as AEC-resistant mutants, were also resistant to high external concentrations of lysine and to alpha-aminoadipate and seemed to be affected in the lysine biosynthetic pathway but not in the biosynthetic pathways of other amino acids. Lysine overproduction by one of the mutants seemed to be due to, at least, the loss of repression of the homocitrate synthase encoded by the LYS20 gene. The mutant grew slower than the wild type, and its dough-raising capacity was reduced in in vitro assays, probably due to the toxic effects of lysine accumulation or of an intermediate produced in the pathway. This mutant can be added as a food supplement to enrich the nutritive qualities of bakery products, and its resistance to alpha-aminoadipate, AEC, and lysine can be used as a dominant marker.     

The enumeration of sublethally injured populations of Staphylococcus aureus in foods

Substantiating earlier investigations, pure cultures of Staphylococcus aureus were found to be equally well recovered on Baird-Parker agar at 37°C as at 42°C, whereas Micrococcus spp. are suppressed at the latter temperature to an extent exceeding 5 log₁₀ cycles. It was also established that egg yolk dissimilation by Staph. aureus is intensified at 42°C. Heat treated (60°C) populations of Staph aureus were quantitatively recovered on Baird-Parker agar at 42°C, though acid-injured populations were not. Acid-injury (2% lactic acid at 37°C) could be completely restored by solid medium repaiar during at least 6 h at 23°C on tryptone soya peptone yeast extract egg yolk pyruvate agar. Pure culture studies were confirmed in surveys on trade samples of foods.     

THE ENUMERATION OF FAECAL STREPTOCOCCI IN FOODS, USING PACKER'S CRYSTAL VIOLET SODIUM AZIDE BLOOD AGAR

SUMMARY: Packer's crystal violet sodium azide blood agar (Packer, 1943) used in poured plates at 36·1°, gave satisfactory recovery of pure cultures of Lancefield group D streptococci and completely inhibited the growth of 11 other species of aerobic and anaerobic food bacteria, including Strep. lactis (5 strains). Later, however, one group N streptococcus was obtained which did grow in Packer's agar at 36·1°. To eliminate this organism the incubation temperature had to be increased to 39·5°, using agar strips (Stirling et al. 1950) incubated in a water bath to secure strict temperature control. Under these conditions the recovery of typical group D streptococci was never consistently below 50% of the count in tryptone dextrose yeast extract agar at 31·1°.     

Candida krusei in Baker's yeast production

Summary Candida krusei is a harmful contaminant in baker's yeast manufacture, because it grows much faster than Saccharomyces cerevisiae under production conditions. This investigation showed that C. krusei utilizes the ethanol produced by baker's yeast as sole carbon source when molasses is used as a substrate. When the alcohol concentration in the effluent air is used as a parameter for controlling the aeration of the culture, conditions become favourable for the dominance of wild yeast because some of the ethanol produced by the baker's yeast is consumed immediately by C. krusei and aeration is then automatically reduced, leading to increased growth of the wild yeast.     

Minimal Medium Recovery of Chilled Salmonella Heidelberg

1. Salmonella heidelberg , chilled from 37 to 5°C in glucose-salts broth, grew better on a simple medium (glucose-salts agar) than on a complex medium (Tryptic Soy Agar + 0·5% yeast extract).
2. The organisms recovered the ability to grow on the complex medium after a further 8 h incubation at 5°C.
3. RNA synthesis would appear to be a critical factor in the recovery process.     

A note on the effect of sporulation conditions on the resistance of Bacillus spores to heat and chemicals

Spores of Bacillus subtilis SA22 harvested after 22 d incubation on nutrient agar at 30°C were more resistant to 0–04% peracetic acid at 20°C than spores harvested following 2 d incubation. Similarly, spores of B. subtilis globigii B17, harvested after 7 d incubation on a sporulation agar were up to 10 times less resistant to 0.04% peracetic acid at 20°C than spores harvested after 35 d incubation. An increase in resistance to heating at 100°C and to exposure to 17.7% hydrogen peroxide at 20°C occurred as the age of B. subtilis SA22 spores prior to harvesting increased, whereas differences in resistance were not observed with spores of B. subtilis globigii B17.     

Isolation of oligotrophic yeasts from supraglacial environments of different altitude on the Gulkana Glacier (Alaska)

Psychrophilic yeasts have been isolated from supra- and subglacial ice at many sites worldwide. To understand the ecology of psychrophilic yeasts on glaciers, we focused on their adaptation to wide range of nutrient concentrations and their distribution with altitude on the Gulkana Glacier in Alaska. We found various culturable psychrophilic yeasts on the ice surfaces of the glacier, and 11 species were isolated with incubation at 4?°C in four different dilutions of agar medium. Some of our isolated species (Rhodotorula psychrophenolica, Rhodotorula aff. psychrophenolica, Rhodotorula glacialis, and Basidiomycota sp. 1) can grow on the low dissolved organic matter (DOC) concentrations medium (7.6?mg?L(-1) ) which is close to the typical level of supraglacial melt water, suggesting that these species can inhabit in any supraglacial meltwater. Otherwise, most of other species were isolated only from higher DOC concentration medium (183?mg?L(-1) -18.3?g?L(-1) ), suggesting that these are inhabitant around the cryoconite, because DOC concentrations in melted surface-ice contained cryoconite is much higher than in melted water. Similarity of altitudinal distribution between culturable yeast and algal biomass suggests that the ecological role played by the cold-adapted yeasts is as organic matter decomposers and nutrient cyclers in glacier ecosystem.     

Cytochrome c from Schizosaccharomyces pombe. 1. Purification, spectral properties, and amino-acid composition.

Cytochrome c from the fission yeast Schizosaccharomyces pombe has been purified. Its chromatographic and spectral properties are reported and compared to those of iso-1-cytochrome c from baker's yeast; the amino-acid composition is described. Schiz. pombe cytochrome c has a much lower affinity for Amberlite IRP64 than Sacch. cerevisiae iso-1-cytochrome c. Its alpha absorption band splits into three maxima (calpha1, calpha2, and calpha3) at -190 degrees C; this is unusual in yeasts, as shown by the low-temperature whole-cell absorption spectra which were examined in various yeast genera, species, and strains. A minor component can be separated by Amberlite chromatography. It exhibits the same low-temperature splitting of the alpha absorption band as the main fraction and it has a similar amino-acid composition with a notable exception: it is an unmethylated form of the cytochrome.     

In vitro attachment of phylloplane yeasts to Botrytis cinerea, Rhizoctonia solani, and Sclerotinia homoeocarpa

The ability of yeasts to attach to hyphae or conidia of phytopathogenic fungi has been speculated to contribute to biocontrol activity on plant surfaces. Attachment of phylloplane yeasts to Botrytis cinerea, Rhizoctonia solani, and Sclerotinia homoeocarpa was determined using in vitro attachment assays. Yeasts were incubated for 2 d on potato dextrose agar (PDA) prior to experimentation. A total of 292 yeasts cultured on PDA were screened for their ability to attach to conidia of B. cinerea; 260 isolates (89.1%) attached to conidia forming large aggregates of cells, and 22 isolates (7.5%) weakly attached to conidia with 1 or 2 yeast cells attached to a few conidia. Ten yeasts (3.4%), including 8 isolates of Cryptococcus laurentii, 1 isolate of Cryptococcus flavescens, and an unidentified species of Cryptococcus, failed to attach to conidia. All non-attaching yeasts produced copious extracellular polysaccharide (EPS) on PDA. Seventeen yeast isolates did not attach to hyphal fragments of B. cinerea, R. solani, and S. homoeocarpa after a 1 h incubation, but attachment was observed after 24 h. Culture medium, but not culture age, significantly affected the attachment of yeast cells to conidia of B. cinerea. The 10 yeast isolates that did not attach to conidia when grown on agar did attach to conidia (20%-57% of conidia with attached yeast cells) when cultured in liquid medium. Attachment of the biocontrol yeast Rhodotorula glutinis PM4 to conidia of B. cinerea was significantly greater at 1 x 10(7) yeast cells x mL(-1) than at lower concentrations of yeast cells. The ability of yeast cells to attach to fungal conidia or hyphae appears to be a common phenotype among phylloplane yeasts.     

The enumeration of thermotrophic types amongst the Enterobacteriaceae colonizing perishable foods

A total of 41 pure cultures of Enterobacteriaceae, comprising 32 thermotrophic and nine psychrotrophic strains, pathogens or marker organisms, were examined for numbers of colony forming units obtained at 37° and 42°5°C (thermotrophs) and 30°C (psychrotrophs), when surface-plated on a rich infusion agar and violet red bile agar. In addition 42 food and water samples, collected in a rural area of the Philippines, were examined by surface inoculating violet red bile AIPC (agar immersion plating and contact; 'dip') slides and incubating at 37° and 42°5°C. At 42°5°C there was almost total recovery of the thermotrophic Enterobacteriaceae, whereas the psychrotrophic strains were completely suppressed. At 37°C the psychrotrophs were only slightly inhibited. The Philippine foods, predominantly cooked meals, milk and drinking water, appeared to be significantly colonized by thermotrophic Enterobacteriaceae. It is concluded that incubation at 42°5°C satisfactorily selects enteropathogenic and other enteric Enterobacteriaceae while suppressing the psychrotrophic types which are mainly of vegetable origin. It is emphasized that, regardless of the temperature used, a resuscitation procedure for Enterobacteriaceae populations that have incurred sublethal injury in food has to precede counts on or in the usual selective media.     

Leavening ability of baker's yeast exposed to hyperosmotic media

To develop a simple and rapid method for enhancing the leavening ability of baker's yeast, we examined the fermentation ability of baker's yeast exposed to hyperosmotic media. When baker's yeast cells were incubated at 25 degrees C for 1 h in a hyperosmotic medium containing 0.5% yeast extract, 0.5% peptone and 20% sucrose, the cells showed a higher fermentation ability in the subsequent fermentation test than those untreated. The increased ratios were from 40 to 60% depending on the strains used. Glucose and fructose showed a similar effect to that of sucrose, but sorbitol was less effective. A high correlation between the intracellular glycerol content and fermentation ability after the osmotic treatment suggested that glycerol accumulated during the hyperosmotic treatment was used in the subsequent fermentation as a substrate, lessened the lag time, and consequently enhanced the fermentation ability. Various baker's yeasts also showed a high leavening ability in dough after the hyperosmotic treatment.     

Incubation temperatures, sex ratios and sex determination in a population of Nile crocodiles (Crocodylus niloticus)

A demographic study of the Nile crocodile Crocodylus niloticus at Lake Ngezi, Zimbabwe, revealed that females predominated in all size classes and among embryos. The sex of C. niloticus was shown to be determined by the temperature of egg incubation in constant temperature laboratory experiments. At 31 °C and below only females were produced. The threshold temperature for maleness was between 31 ° and 34 °C, but appeared to vary between clutches. The duration of the incubation period varied with temperature and was 110 days at 28 °C, falling to 85 days at 34 °C. Incubation temperature affected hatchling length, but not mass. Hatchlings from incubation at 34 °C were shorter on average than those from incubation at 28 °C and 31 °C, but by three months had outgrown them. There was no sex-related difference in length in a random sample of 200 two-year-old C. niloticus on a crocodile farm. Mean temperatures in wild nests were consistently lower than 31 °C and therefore the male threshold as determined in the laboratory. Embryonic development was slow and hatching success poor. The shallowest eggs in a nest had higher mean temperatures and more advanced embryos than the deepest eggs. They also experienced daily temperature fluctuations of up to 10 °C during which the maximum occasionally rose to 35 °C. Constant temperature incubation was not a good model of field conditions, but the correlation between nest temperatures and embryonic sex is consistent with temperature-dependent sex determination in the wild.     

Evaluation of selective media for enumeration of yeasts during wine fermentation

The growth of individual species of yeasts during wine fermentations was measured by plating wine samples on malt extract, ethanol sulphite and lysine agars. Colonies of Saccharomyces cerevisiae dominated on plates of malt extract agar and sometimes masked the presence of other non- Saccharomyces species. Lysine agar suppressed the growth of S. cerevisiae and enabled the enumeration of non- Saccharomyces species such as Kloeckera apiculata, Candida stellata and Saccharomycodes ludwigii. The growth of non- Saccharomyces yeasts on ethanol sulphite agar was variable.     

Comparison of the Effects of Liquid Medium Repair and the Incorporation of Catalase in MacConkey Type Media on the Recovery of Enterobacteriaceae Sublethally Stressed by Freezing

Nine pure cultures of species of Enterobacteriaceae were stressed by rapid freezing in tryptone soya broth (TSB) to — 22°C and subsequent storage at that temperature for 7 d. About one to two log cycles kill and at least one additional log cycle sublethal impairment was achieved. Numbers of colonies of these cultures in poured plates of violet red bile glucose (VRBG) agar, with 67 u/ml of catalase added at 47°C, were only slightly higher than those in plain VRBG, both incubated overnight at 30°C. Two hours incubation of TSB suspensions at 17–25° C resulted in almost complete restoration of the ability of cells to develop colonies in VRBG, without, however, leading to any significant multiplication.
Similar experiments with 32 samples of frozen minced meat, 27 samples of frozen surface water, 18 of frozen chicken liver and 14 of fresh sausage substantiated the results obtained in the studies on pure cultures.
In the experiments with the nine pure cultures the influence of the nutrient composition of the solid enumeration media: 'minimal' agar, TSB agar (TSBA) and Mueller-Hinton agar with Polyvitex nutrient supplement (MHA), on the recovery of Enterobacteriaceae stressed by freezing was also studied. Colony numbers in TSBA and MHA were virtually identical. The glucose mineral salts medium led to lower recovery, indicating that so-called 'minimal medium recovery' of stressed bacterial populations is not a common phenomenon.     

Expression of human antithrombin III in Saccharomyces cerevisiae and Schizosaccharomyces pombe

Recombinant plasmids were constructed that direct the synthesis of human antithrombin III in baker's yeast, Saccharomyces cerevisiae, and the fission yeast, Schizosaccharomyces pombe. The signal sequence of antithrombin III was recognized by both yeast species, and antithrombin III was secreted into the medium. When the signal sequence was replaced by a sequence of ten arbitrary amino acids, the product expressed from such a construct stayed inside the cell. Antithrombin III was glycosylated by the baker's and fission yeast and was immunologically identical to antithrombin III isolated from human plasma. Antithrombin III isolated from the culture media of recombinant yeasts was biologically active, as could be shown by progressive inhibitor activity and heparin cofactor activity.