A Response Surface Methodology study on the role of factors affecting growth and volatile phenol production by Brettanomyces bruxellensis ISA 2211 in wine.

The present study was aimed at determining the effect of glucose, ethanol and sulphur dioxide on the growth and volatile phenol production by Brettanomyces bruxellensis in red wines using a response surface methodology approach. Sulphur dioxide proved to have a significant (p < 0.05) negative linear and quadratic effect on growth and 4-ethylphenol production. Concentrations of sulphur dioxide higher than 20 mg L(-1), at pH 3.50, induced immediate loss of cell culturability under growth permissive levels of ethanol. Under high ethanol concentrations (14% v/v), the lag phase increased from 3 to 10 days, growth being fully arrested at 15% (v/v). Glucose up to 10 g L(-1) was found to be a significant factor (quadratic level) in biomass increase under low ethanol (<12.5% v/v) and low sulphite concentrations. However, when cells were inactivated by sulphur dioxide and ethanol, glucose (up to 10 g L(-1)) did not prevent cell death. Production of more than 50 µg L(-1) day(-1) of 4-ethylphenol was only observed in the presence of high numbers (10(6) CFU mL(-1)) of culturable cells, being stimulated by increasing glucose concentrations.

The microbial ecology of wine grape berries.

Grapes have a complex microbial ecology including filamentous fungi, yeasts and bacteria with different physiological characteristics and effects upon wine production. Some species are only found in grapes, such as parasitic fungi and environmental bacteria, while others have the ability to survive and grow in wines, constituting the wine microbial consortium. This consortium covers yeast species, lactic acid bacteria and acetic acid bacteria. The proportion of these microorganisms depends on the grape ripening stage and on the availability of nutrients. Grape berries are susceptible to fungal parasites until véraison after which the microbiota of truly intact berries is similar to that of plant leaves, which is dominated by basidiomycetous yeasts (e.g. Cryptococcus spp., Rhodotorula spp. Sporobolomyces spp.) and the yeast-like fungus Aureobasidium pullulans. The cuticle of visually intact berries may bear microfissures and softens with ripening, increasing nutrient availability and explaining the possible dominance by the oxidative or weakly fermentative ascomycetous populations (e.g. Candida spp., Hanseniaspora spp., Metschnikowia spp., Pichia spp.) approaching harvest time. When grape skin is clearly damaged, the availability of high sugar concentrations on the berry surface favours the increase of ascomycetes with higher fermentative activity like Pichia spp. and Zygoascus hellenicus, including dangerous wine spoilage yeasts (e.g. Zygosaccharomyces spp., Torulaspora spp.), and of acetic acid bacteria (e.g. Gluconobacter spp., Acetobacter spp.). The sugar fermenting species Saccharomyces cerevisiae is rarely found on unblemished berries, being favoured by grape damage. Lactic acid bacteria are minor partners of grape microbiota and while being the typical agent of malolactic fermentation, Oenococcus oeni has been seldom isolated from grapes in the vineyard. Environmental ubiquitous bacteria of the genus Enterobacter spp., Enterococcus spp., Bacillus spp., Burkholderia spp., Serratia spp., Staphylococcus spp., among others, have been isolated from grapes but do not have the ability to grow in wines. Saprophytic moulds, like Botrytis cinerea, causing grey rot, or Aspergillus spp., possibly producing ochratoxin, are only active in the vineyard, although their metabolites may affect wine quality during grape processing. The impact of damaged grapes in yeast ecology has been underestimated mostly because of inaccurate grape sampling. Injured berries hidden in apparently sound bunches explain the recovery of a higher number of species when whole bunches are picked. Grape health status is the main factor affecting the microbial ecology of grapes, increasing both microbial numbers and species diversity. Therefore, the influence of abiotic (e.g. climate, rain, hail), biotic (e.g. insects, birds, phytopathogenic and saprophytic moulds) and viticultural (e.g. fungicides) factors is dependent on their primary damaging effect.

Evaluation of the inhibitory effect of dimethyl dicarbonate (DMDC) against wine microorganisms.

Several microbial species associated with wine were challenged against increasing concentrations of dimethyl dicarbonate (DMDC). The concentration inducing complete cell death upon addition to red wine was regarded as the minimum inhibitory concentration (MIC). In dry red wines with 12% (v/v) ethanol and pH 3.50, the inactivation depended on the initial cell concentration. For an initial inoculum of 500 CFU/ml, the MIC of the yeasts species Schizosaccharomyces pombe, Dekkera bruxellensis, Saccharomyces cerevisiae and Pichia guilliermondii was 100mg/l. The most sensitive strains belong to Zygosaccharomyces bailii, Zygoascus hellenicus and Lachancea thermotolerans, with MIC of 25mg/l DMDC. For inoculation rates of about 10(6)CFU/ml, the maximum dose of DMDC legally authorized (200mg/l) was not effective against the most resistant species. The addition of 100mg/l potassium metabisulphite (PMB), equivalent to 1mg/l molecular sulphur dioxide, increased the inactivation effect of 100mg/l DMDC over initial yeast populations of 10(6)CFU/ml but did not fully kill S. pombe and S. cerevisiae. Lactic acid and acetic acid bacteria were not killed by the addition of 300 mg/l of DMDC. Trials performed in wines before bottling showed that in most samples indigenous bacterial populations were not affected by 200mg/l DMDC. Therefore, under winery practice, DMDC at the maximum dose legally permitted may be regarded as an efficient preservative to control low contamination rates of yeasts but ineffective against lactic acid and acetic acid bacteria.

Ascomycetous yeast species recovered from grapes damaged by honeydew and sour rot.

AIMS: To identify ascomycetous yeasts recovered from sound and damaged grapes by the presence of honeydew or sour rot. METHODS AND RESULTS: In sound grapes, the mean yeast counts ranged from 3.20 +/- 1.04 log CFU g(-1) to 5.87 +/- 0.64 log CFU g(-1). In honeydew grapes, the mean counts ranged from 3.88 +/- 0.80 log CFU g(-1) to 6.64 +/- 0.77 log CFU g(-1). In sour rot grapes counts varied between 6.34 +/- 1.03 and 7.68 +/- 0.38 logCFU g(-1). Hanseniaspora uvarum was the most frequent species from sound samples. In both types of damage, the most frequent species were Candida vanderwaltii, H. uvarum and Zygoascus hellenicus. The latter species was recovered in high frequency because of the utilization of the selective medium DBDM (Dekkera/Brettanomyces differential medium). The scarce isolation frequency of the wine spoilage species Zygosaccharomyces bailii (in sour rotten grapes) and Zygosaccharomyces bisporus (in honeydew affected grapes) could only be demonstrated by the use of the selective medium ZDM (Zygosaccharomyces differential medium). CONCLUSIONS: The isolation of several species only from damaged grapes indicates that damage constituted the main factor determining yeast diversity. The utilization of selective media is required for eliciting the recovery of potentially wine spoilage species. SIGNIFICANCE AND IMPACT OF THE STUDY: The impact of damaged grapes in the yeast ecology of grapes has been underestimated.

Survival patterns of Dekkera bruxellensis in wines and inhibitory effect of sulphur dioxide.

The wine spoilage yeast species Dekkera bruxellensis, after inoculation in red wines, displayed three survival patterns characterized by: i) initial lag phase followed by growth and sequential death; ii) initial death phase leading to reduced viable counts followed by growth and sequential death; and iii) death phase leading to complete loss of viability. These survival patterns were observed for the same strain in different dry red wine blends with 12% (v/v) ethanol and pH 3.50, in the absence of free sulphur dioxide. For the same wine blend, these patterns also varied with the tested strain. Under laboratory conditions the addition of 150 mg/l of potassium metabisulphite (PMB) to dry red wine with 12% (v/v) ethanol and pH 3.50 reduced initial cell counts by more than 6 logarithmic cycles, inducing full death within less than 24 h. Winery trials showed that D. bruxellensis blooms were only prevented in the presence of about 40 mg/l of free sulphur dioxide in dry red wine, with 13.8% (v/v) ethanol and pH 3.42, matured in oak barrels. These different amounts of PMB and sulphur dioxide corresponded to about 1 mg/l of molecular sulphur dioxide. Our results therefore demonstrate that the control of populations of D. bruxellensis growing in red wine can only be achieved under the presence of relatively high doses of molecular sulphur dioxide.

In vitro methods for specific IgE detection in cow's milk allergy.

BACKGROUND: A new method for determining serum specific IgE (IMMULITE 2000 3gAllergy) has recently become available. OBJECTIVE: To evaluate the clinical performance of IMMULITE 2000 in the diagnosis of cow's milk allergy compared with that of UniCAP. Additionally, we verified the behavior of both methods at two diagnostic decision points proposed by other authors. METHODS: The study population consisted of 31 children with cow's milk allergy (group A) and a control group of 19 atopic children without food allergy (group B). A blood sample from each child was tested using both methods and the results were compared. RESULTS: In group A, the values for cow's milk IgE ranged from 0.35 kU/L (the lowest common detection limit) to above 100 kU/L. In group B, the values were less than 1.1 kU/L for IMMULITE 2000 and less than 1.6 kU/L for UniCAP. An agreement of 90% in IgE classes was obtained. Both methods demonstrated exactly the same diagnostic performance (sensitivity: 100%; specificity: 78.9%; negative predictive value: 100%; positive predictive value: 84.6%; efficiency: 90.2%). The evaluation of the two methods at the two different decision points proposed in the literature showed a better positive predictive value with UniCAP, but we obtained equivalent performance with IMMULITE 2000 by choosing higher cutoff values. CONCLUSIONS: We conclude that IMMULITE 2000 is as effective as UniCAP in the diagnosis of cow's milk allergy. Both methods can be used to obtain site-specific decision points that are population, age and disease dependent.

Cold urticaria and infectious mononucleosis in children.

Physical urticaria includes a heterogeneous group of disorders characterized by the development of urticarial lesions and/or angioedema after exposure to certain physical stimuli. The authors present the case of a child with severe acquired cold urticaria secondary to infectious mononucleosis. Avoidance of exposure to cold was recommended; prophylactic treatment with ketotifen and cetirizine was begun and a self-administered epinephrine kit was prescribed. The results of ice cube test and symptoms significantly improved. Physical urticaria, which involves complex pathogenesis, clinical course and therapy, may be potentially life threatening. Evaluation and diagnosis are especially important in children. To our knowledge this is the first description of persistent severe cold-induced urticaria associated with infectious mononucleosis in a child.

An inter-laboratory evaluation of selective media for the detection and enumeration of yeasts from blue-veined cheese.

Five countries representative of laboratories 1-5 evaluated 11 different selective media, designed to suppress mould and bacterial growth and support yeasts growth, for the recovery of yeast populations from blue veined cheeses. In addition, qualitative results were also incorporated. The yeast enumeration values were subjected to statistical analysis using analysis of variance (ANOVA) and the Tukey-Kramer multiple comparison test. With the exception of Laboratory 3, none of the other laboratories was successful in recovering yeasts on all the media. Six of the media proved inadequate for the enumeration of yeasts in the mould invested environment and were therefore omitted from statistical analysis. No significant differences in quantitative data obtained on Rose-Bengal Chloramphenicol Agar (RBCA), Dichloran Rose-Bengal Chloramphenicol Agar (DRBC), Dichloran 18% Glycerol Agar (DG18), and Malt extract agar supplemented with NaCl and oxytetracycline (MES) were detected by four of the collaborating laboratories whereas one laboratory found RBCA to be superior for yeast enumeration. DG18 and Malt Extract Agar with Biphenyl (MEB), however, were ranked superior based on qualitative results compared to the other media, attributed to distinctive individual yeast colonies and mould inhibition. RBCA, DRBC, DG18, and MES on the other hand, all proved to be adequate in supporting yeast colony development for quantitative analysis in samples obtained from blue veined cheeses.

Spoilage yeasts in the wine industry.

Yeasts play a central role in the spoilage of foods and beverages, mainly those with high acidity and reduced water activity (a(w)). A few species are capable of spoiling foods produced according to good manufacturing practices (GMPs). These can survive and grow under stress conditions where other microorganisms are not competitive. However, many of the aspects determining yeast spoilage have yet to be clarified. This critical review uses the wine industry as a case study where serious microbiological problems are caused by yeasts. First, the limitations of the available tools to assess the presence of spoilage yeasts in foods are discussed. Next, yeasts and factors promoting their colonisation in grapes and wines are discussed from the ecological perspective, demonstrating that a deeper knowledge of vineyard and winery ecosystems is essential to establish the origin of wine spoilage yeasts, their routes of contamination, critical points of yeast infection, and of course, their control. Further, zymological indicators are discussed as important tools to assess the microbiological quality of wines, although they are rarely used by the wine industry. The concepts of the susceptibility of wine to spoilage yeasts and wine stability are addressed based on scientific knowledge and industrial practices for monitoring yeast contamination. A discussion on acceptable levels of yeasts and microbiological criteria in the wine industry is supported by data obtained from wineries, wholesalers, and the scientific literature.Finally, future directions for applied research are proposed, involving collaboration between scientists and industry to improve the quality of wine and methods for monitoring the presence of yeast.

The wide diversity of structurally similar wine proteins.

In the present work, single grape variety wines, Moscatel and Arinto, were used. Analysis by denaturing polyacrylamide gel electrophoresis of the wine proteins revealed the presence of only a few polypeptides ranging in molecular mass from 15 to 30 kDa. However, a more detailed examination of the whole protein fraction, by a combination of techniques, showed that these wines contain a very large number (many tens and, possibly, many more) of distinct polypeptides, exhibiting similar molecular masses but different electrical charges. The results obtained using highly specific antibodies and N-terminal sequencing indicate that there is structural similarity among most of the wine polypeptides. These observations can be explained by the existence of a common precursor to most or all of the wine proteins, which could generate all of the detected polypeptides by limited proteolysis. Comparison of the N-terminal sequences of the polypeptides isolated from Moscatel wine with proteins from other sources revealed a high degree of homology to pathogenesis-related proteins.

Brown pigments produced by Yarrowia lipolytica result from extracellular accumulation of homogentisic acid.

Yarrowia lipolytica produces brown extracellular pigments that correlate with tyrosine catabolism. During tyrosine depletion, the yeast accumulated homogentisic acid, p-hydroxyphenylethanol, and p-hydroxyphenylacetic acid in the medium. Homogentisic acid accumulated under all aeration conditions tested, but its concentration decreased as aeration decreased. With moderate aeration, equimolar concentrations of alcohol and p-hydroxyphenylacetic acid (1:1) were detected, but with lower aeration the alcohol concentration was twice that of the acid (2:1). p-Hydroxyphenylethanol and p-hydroxyphenylacetic acid may result from the spontaneous disproportionation of the corresponding aldehyde, p-hydroxyphenylacetaldehyde. The catabolic pathway of tyrosine in Y. lipolytica involves the formation of p-hydroxyphenylacetaldehyde, which is oxidized to p-hydroxyphenylacetic acid and then further oxidized to homogentisic acid. Brown pigments are produced when homogentisic acid accumulates in the medium. This acid can spontaneously oxidize and polymerize, leading to the formation of pyomelanins. Mn(2+) accelerated and intensified the oxidative polymerization of homogentisic acid, and lactic acid enhanced the stimulating role of Mn(2+). Alkaline conditions also accelerated pigment formation. The proposed tyrosine catabolism pathway appears to be unique for yeast, and this is the first report of a yeast producing pigments involving homogentisic acid.

Development and use of a new medium to detect yeasts of the genera Dekkera/Brettanomyces.

AIMS: The objectives of this work were to develop a selective and/or differential medium able to efficiently recover Dekkera/Brettanomyces sp. from wine-related environments and to determine the relationship between these yeasts and the 4-ethylphenol content in a wide range of wines. METHODS AND RESULTS: The selectivity of the developed medium was provided by the addition of ethanol, as single carbon source, and cycloheximide. The inclusion of bromocresol green evidenced acid-producing strains. The inclusion of p-coumaric acid, substrate for the production of 4-ethylphenol, enabled the differentiation by smell of Dekkera/Brettanomyces sp. from all other yeast species growing in the medium. The medium was used either by plating after membrane filtration or by the Most Probable Number (MPN) technique. In 29 white and 88 red randomly collected wines, these yeasts were found only in red wines at levels up to 2500 MPN ml-1, but constituted less than 1% of the total microbial flora. In red wines, 84% showed detectable amounts of 4-ethylphenol up to 4430 microg l-1 while 28% of the white wines showed detectable levels up to 403 microg l-1. CONCLUSION: The use of the medium proposed in this work evidenced the presence of low relative populations of Dekkera/Brettanomyces sp. even in wines contaminated by fast-growing yeasts and moulds. SIGNIFICANCE AND IMPACT OF THE STUDY: Further ecological studies on Dekkera/Brettanomyces sp. should take into account the use of highly specific culture media in order to establish their true occurrence in nature.

Production of brown tyrosine pigments by the yeast Yarrowia lipolytica.

AIMS: To study the mechanism of production of brown pigments from tyrosine in the yeast Yarrowia lipolytica. METHODS AND RESULTS: Pigment formation was followed during growth in tyrosine medium, and the presence of the pigment precursor in the medium was assessed by evaluating pigment formation after removing the cells at different times of incubation. It was observed that the pigment precursor accumulated outside the cells during the exponential phase of growth, but pigment formation only occurred during the stationary phase of growth and resulted from the oxidation of the precursor. Pigment formation was repressed by glucose and L-glutamine, and promoted by lactic acid, L-asparagine and glycine. Spectra of 1H and 13C-NMR revealed that the brown pigment was derived from tyrosine and was a polymer composed of a core of aromatic residues. CONCLUSION: The results indicate that pigments result from the extracellular accumulation and auto-oxidation of an intermediate of tyrosine catabolism. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the mechanism of pigment production from tyrosine in a yeast species.

Characterisation of yeast flora isolated from an artisanal Portuguese ewes' cheese.

The evolution of the yeast flora was studied for an artisanal semi-hard ewes' cheese made from raw milk. Mean log10 yeast counts per gram of cheese body ranged from 2.7 to 6.4, with the higher counts observed after a ripening period of 30 days. The yeast population decreased thereafter and, at the end of curing process, reached values similar to those of the beginning. A total of 344 yeasts strains were randomly isolated from the curd and cheese body during the 60 days long ripening period. Esterase activity was common to almost all isolates (98%) while proteolysis was observed in 12% of the total yeast population. The proportion of strains with positive glucose fermentation increased from 21% in the curd to 75% at the end of the ripening period. A total of 150 isolates representative of the physiological characteristics tested were examined with the API ID 32C system showing different degrees of quality of identification. Only 15% of the strains (23 isolates) were excellently identified being assigned to the species Candida zeylanoides. The most frequent species appeared to be Debaryomyces hansenii (anamorph Candida famata) and Candida intermedia. These two species amounted to 9% of the yeasts in the curd increasing to 86% at the end of the ripening period.

Toxoplasma gondii infection in Brazilian domestic outpatient cats.

The occurrence of Toxoplasma antibodies in domestic outpatient cats in the city of São Paulo was evaluated using the indirect immunofluorescence assay. A total of 248 blood samples obtained from male and female cats seen at the Veterinary Teaching Hospital at the University of São Paulo between February 1996 and January 1997 were tested. Of these, 17.7% were positive, with a 64 titer being detected in most animals. The frequence of Toxoplasma antibodies was significantly higher in older cats, those fed raw meat and those with free access to the outdoor environment. There was no significant difference in reactivity between males and females. We conclude that diet and free access to the outdoor environment were equally important as predisposing factors to the risk of Toxoplasma infection.

Pigment producing yeasts involved in the brown surface discoloration of ewes' cheese.

The appearance of a brown surface discoloration on Portuguese ewes' cheese has never previously been reported on. The regular occurrence of this defect over the past few years has caused serious financial losses to producers, which has led to growing interest in its study. This paper describes a preliminary approach to the problem, based on the hypothesis that pigment producing yeasts are involved. From a group of 51 yeast strains isolated from a number of brown cheese rinds, it was possible to distinguish four pigment producing groups: group I (12 strains), produced an extracellular brown pigment from tyrosine and alkalised the tested media; group II (21 strains), produced a diffusible, reddish-brown pigment from resorcinol and alkalised the tested media; group III (three strains), alkalised the tested media without producing any pigments; group IV (15 strains), neither produced pigments nor alkalised the media. Yarrowia lipolytica and Candida catenulata type strains were also tested and their behaviour was similar to the strains in groups I and IV, respectively. The Filobasidiella neoformans type strain was distinct from all the other groups. The identification methods used for some strains in groups I, II and III suggest that Yarrowia lipolytica species is common to all strains in group I, and that Debaryomyces hansenii is present in both groups II and III. A study of the effect of several metal ions on the production of the brown pigment from tyrosine indicated Mn2+ to be a strong activator. Evidence is provided suggesting that the browning process may be related to tyrosine Yarrowia lipolytica metabolism.

Soluble CD30, dehydroepiandrosterone sulfate and dehydroepiandrosterone in atopic and non atopic children.

Elevated levels of soluble CD30-sCD30 (possibly related to Th2) are seen in atopy and other diseases. Dehydroepiandrosterone (DHEA) may regulate Th1/Th2 balance. The aim was to study the relationship between the levels of sCD30, DHEAS and DHEA in atopic (A) and non atopic (B) children, to assess immune-endocrine interactions. We studied 36 children (8-15 years), 18 (A) monosensitised to Dermatophagoides Pteronyssinus (Dpt.), and 18 (B) with isolate rhinitis (R) or asthma (BA) and R (33.3%) without differences in age and sex. They were all HIV negative and free from any medication. It was determined sCD30, DHEAS and DHEA. There was no statistically significant differences between A and B for the levels of DHEAS, DHEA suggesting that the role of this regulation in Th2 type is not important in patients with allergic disease in this age group. sCD30: A-mean 121.1U/ml; B-mean 53.9U/ml. This difference is statistically significant-p = 0.02. sCD30 in patients only with R showed also significant differences-p = 0.01. These results could reflect the Th2 profile. There is an overlap of the values (for R < 80 U/ml; for BA + R < 140 U/ml). Due to this only values > 80 U/ml for R and > 140 U/ml for BA + R could be considered discriminating for atopy.