Wine yeast selection in the Iberian Peninsula: Saccharomyces and non-Saccharomyces as drivers of innovation in Spanish and Portuguese wine industries.

Yeast selection for the wine industry in Spain started in 1950 for the understanding of the microbial ecology, and for the selection of optimal strains to improve the performance of alcoholic fermentation and the overall wine quality. This process has been strongly developed over the last 30 years, firstly on Saccharomyces cerevisiae, and, lately, with intense activity on non-Saccharomyces. Several thousand yeast strains have been isolated, identified and tested to select those with better performance and/or specific technological properties. The present review proposes a global survey of this massive ex-situ preservation of eukaryotic microorganisms, a reservoir of biotechnological solutions for the wine sector, overviewing relevant screenings that led to the selection of strains from 12 genera and 22 species of oenological significance. In the first part, the attention goes to the selection programmes related to relevant wine-producing areas (i.e. Douro, Extremadura, Galicia, La Mancha and Uclés, Ribera del Duero, Rioja, Sherry area, and Valencia). In the second part, the focus shifted on specific non-Saccharomyces genera/species selected from different Spanish and Portuguese regions, exploited to enhance particular attributes of the wines. A fil rouge of the dissertation is the design of tailored biotechnological solutions for wines typical of given geographic areas.

Wine aromatic compound production and fermentative behaviour within different non-Saccharomyces species and clones.

AIMS: Twenty-five enological yeasts belonging to nine different species (Candida zeylanoides, Cryptococcus uzbekistanensis, Debaryomyces hansenii, Lachancea thermotolerans, Metschnikowia pulcherrima, Torulaspora delbrueckii, Williopsis pratensis, Zygosaccharomyces bailii and Saccharomyces cerevisiae) were screened for aroma formation and fermentative behaviour as part of a non-Saccharomyces yeast selection programme. METHODS AND RESULTS: Pure cultures were inoculated in pasteurized grape juice in order to perform alcoholic fermentations. Some non-Saccharomyces species did not ferment, others did not get established and none of them completed alcoholic fermentations. The physico-chemical parameters of the wines and the abundance of aromatic compounds at the end of alcoholic fermentation highlighted the notable differences in the aroma-forming ability and fermentative behaviour of the different non-Saccharomyces species, but not within clones. CONCLUSIONS: Lower diversity was detected within non-Saccharomyces species than that reported in S. cerevisiae with regard to enological behaviour and aromatic profiles. Metschnikowia pulcherrima and L. thermotolerans are the two species with higher possibilities to become an inoculum. SIGNIFICANCE AND IMPACT OF THE STUDY: Few significant differences were found within clones of the same species, but very important parameters in wine quality, such as volatile acidity, ethyl acetate and acetoin, which would justify selection programmes within those species. The results also demonstrated that T. delbrueckii and L. thermotolerans are two close species in their aromatic profiles.

Genetic and phenotypic intraspecific variability of non-Saccharomyces yeasts populations from La Rioja winegrowing region (Spain).

AIMS: To determine the intraspecific genetic diversity within five non-Saccharomyces yeast species and the diversity in phenotypic characteristic related to their technological properties. METHODS AND RESULTS: Seventy-one non-Saccharomyces yeasts isolated from different fermentations and facilities of the DOCa Rioja (Spain) belonging to five different wine species (Torulaspora delbrueckii, Lachancea thermotolerans, Metschnikowia pulcherrima, Zygosaccharomyces bailii and Williopsis pratensis) were subjected to clonal characterization by RAPD-PCR, which evidenced wide diversity between them. They were also submitted to a screening for some oenological traits related to the improvement of the aroma of the wine and yeast development in musts. Strains within the same species showed different enzyme activities, tolerated different levels of SO2 and possessed different killer phenotypes. These characteristics made them adjust better or worse to specific vinification processes or wine quality criteria. CONCLUSIONS: A significant genetic and phenotypic variation within the non-Saccharomyces species studied was found, which makes necessary to carry out a selection process in each one. SIGNIFICANCE AND IMPACT OF THE STUDY: Williopsis pratensis, a species that has not been thoroughly explored, may deserve further consideration for oenological applications. Due to the wide range of variation within species, the strains adaptation to the SO2 levels in musts has to be taken into account in selection processes.

Comparison of culture media for the recovery of airborne yeast in wineries.

The direct air sampling impaction method on agar was evaluated using aerobiocollectors for the recovery of yeasts present in the winery air. Three culture media with different composition and specificity were studied. In addition, a resuscitation phase was included before the culture in the specificity medium [in the case of the Dekkera-Brettanomyces Differential Medium (DBDM) medium]. Sampling was conducted at different times of the year and in different parts of the wineries, which were different in age and design. Both the Chloramphenicol Glucose Agar (CGA) and Agar Lysine AL media recovered yeasts from the air without any prior resuscitation phase. CGA was able to recover a higher number of colony-forming units of yeasts than the other media. Consequently, to estimate the number of yeasts present in winery air, the best choice of medium would be CGA. The AL medium permitted the growth of the greatest range of genera and species. If the aim is to study the diversity of yeasts present in the air, the most suitable medium is AL. Neither CGA nor AL proved suitable for recovering yeasts of the Brettanomyces genus. The DBDM medium was the only one which provided sufficient specificity for their recovery and identification from the air, although their special characteristics made a prior protocol of resuscitation necessary.

Factors of influence in the distribution of mold in the air in a wine cellar.

UNLABELLED: This paper studies the presence of mold in the air of a vinification and ageing wine cellar. The influence of other factors such as the time of year, the sampling point, and the activity being carried out in the cellar has been analyzed. Neither the type of activity being carried out in the cellar nor the temperature or relative humidity fluctuations throughout the year are determining factors in the presence of mold in the air. For this group of microorganisms, the design of the cellar studied is the fundamental factor. Areas with little ventilation favor high levels of relative humidity and, hence, a higher presence of mold in the air. The mold population in these areas is not very diverse, which indicates that colonization by certain types of mold that have adapted to the conditions established therein is permanent. Areas with greater air flow, constant activity, and frequent cleaning show lower mold populations in the air and of a more varied composition. PRACTICAL APPLICATION: This work shows that given the growing importance of the presence of mold in wine cellars, the design thereof should take into account suitable ventilation of all the areas and control of the relative humidity. Hence, the presence of traditional underground areas for ageing wine, which is justifiable in seasons where temperature and humidity control lead to major technical problems, should be reconsidered in the design of new wine cellars.

Presence of non-Saccharomyces yeasts in cellar equipment and grape juice during harvest time.

The purpose of this study was to analyze the presence of different yeasts in the facilities of four wineries from the D.O.Ca. Rioja region in Spain. The study was conducted through the identification of the yeasts via the PCR-RFLP technique of the ITS region of rDNA. The diversity of non-Saccharomyces yeasts found in wineries has previously only been studied to a limited extent, despite the fact that these yeasts take part both in the start of spontaneous fermentation and in the changes which occur in the wines during their subsequent conservation. Most earlier studies carried out on cellar ecosystems have focussed on the clonal diversity of Saccharomyces cerevisiae. The results obtained in this study indicated that the presence of non-Saccharomyces yeasts in facilities is higher than that of the S. cerevisiae, with percentages of over 60% in all the wineries analyzed. Yeasts belonging to 10 genera and 18 species were isolated, but the only genera present in all four wineries were Cryptococcus, Pichia, and Saccharomyces. The Zygosaccharomyces bailii yeast responsible for taint was detected in one cleaned winery, in both the winemaking equipment and the fermenting must. It was also noted that the quantity and type of yeasts present in the facilities are related to the product used for cleaning them. It is also necessary to point out that the cleaning of the cellars prior to the reception of the grapes does not completely eliminate the yeasts present, so that these can subsequently become part of the vinification process.

Presence of lactic bacteria in the air of a winery during the vinification period.

In this paper we have studied the presence and evolution in the winery air of the lactic bacteria responsible for malolactic fermentation. Sampling took place during the winemaking process (between September 2007 and July 2008) in a winery from the Rioja appellation in Spain. The results obtained indicated that the presence of these microorganisms in the atmosphere was detected when grapes were entering the winery, while malolactic fermentation was taking place, and when liquid containing bacteria was manipulated. The species and clones of the lactic bacteria identified were also related to those present in the vinification tanks at any given stage of the process.

Effect of the addition of inert cellulose substrates to grape must on Saccharomyces cerevisiae diversity and the evolution of alcoholic fermentation.

AIMS: To study the addition of cellulose-based adjuvant as a resource to offset the negative effects produced by grape juice clarification during alcoholic fermentations. METHODS AND RESULTS: The effect of the addition of two kinds of inert cellulose substrates in white wine vinification was investigated in two different musts. In one of these musts, stuck fermentations were detected. One of the types of cellulose examined had a fining effect, which caused a decrease in the number of viable yeasts in the medium and altered the distribution and frequency of the clones, which performed the fermentation. The other cellulose substrate made the medium cloudier but did not alter the distribution of yeasts in comparison with the control. CONCLUSIONS: The behaviour of the inert cellulose substrates on vinification depends on its physical characteristics and its capacity for making the must cloudy. SIGNIFICANCE AND IMPACT OF THE STUDY: The addition of inert cellulose substrates in white wine vinification improves the fermentation process and the quality of wines obtained. This effect is more noticeable in difficult fermentations. One variety of cellulose showed an inhibitory effect on Torulaspora delbrueckii yeasts.

Peripheral melt of flap after laser in situ keratomileusis.

BACKGROUND: Laser in situ keratomileusis (LASIK) is an effective procedure to correct myopia. It may have complications related to the flap, such as epithelial ingrowth and stromal melt. METHODS: We report on a patient who developed extensive epithelial ingrowth and partial keratolysis of the flap following LASIK. This complication was treated by lifting the flap and removing the epithelium from within the interface. RESULTS: Progressive keratolysis (stromal melt) can result in irregular astigmatism and loss of vision as well as photophobia and ciliary injection. The pathogenesis is not completely understood although the epithelial ingrowth in the interface is always present, and epithelial-stromal interaction with production of proteases may be involved. CONCLUSION: Epithelial ingrowth may develop in the lamellar interface after LASIK and be associated with melting of the edge of the flap. This undesirable complication can be successfully managed with early surgical removal of the epithelium and proper attachment of the flap.

Rolandic epilepsy and cortical dysplasia: MRI correlation of epileptiform discharges.

An 8 year-old girl presented with simple facial motor seizures. Although the electroencephalogram (EEG) demonstrated left hemisphere centrotemporal spikes with features consistent with benign rolandic epilepsy, magnetic resonance imaging (MRI) indicated a left hemisphere focal cortical dysplasia. MRI-assisted EEG dipole analysis of the spikes suggested that the rolandic fissure rather than the focal cortical dysplasia was the origin of the epileptic spike discharge. This noninvasive method may be a useful adjunct in evaluation of some patients with epilepsy and focal superficial cerebral lesions.

Ecology of inoculated and spontaneous fermentations in Rioja (Spain) musts, examined by mitochondrial DNA restriction analysis.

Analysis of mitochondrial DNA restriction patterns was used to study the introduction of a selected strain of Saccharomyces cerevisiae for fermentation of non-sterile musts of La Rioja (Spain). All of the isolates from the inoculated musts showed the restriction pattern of the selected strain. The same technique was used to study the spontaneous fermentation of musts, showing that a few strains were responsible for the fermentations. One of the strains identified from the spontaneous fermentations had been identified in a previous vintage.

Atypical scabies in HIV-positive patients.

Five cases of Norwegian or keratotic scabies in HIV-positive patients are described. One of these patients was the source of an outbreak in a hospital, ultimately involving 72 persons. Three of our patients had a markedly pruritic eruption. This is unusual in crusted scabies in which pruritus is usually slight or absent. Two of the five patients had unusual CD4 counts of more than 200 cells per cubic millimeter. All our patients responded to lindane and keratolytic agents. When generalized papular, crusted, or eczematoid lesions are observed in HIV-positive patients, particularly if the CD4 count is less than 200/mm3, scabies should be included in the differential diagnosis.

Midazolam in the treatment of refractory neonatal seizures.

Midazolam, a potent short-acting benzodiazepine, is a safe and highly effective agent for the control of status epilepticus. Its efficacy in the control of neonatal seizures, however, has not been determined. Six neonates (aged 1-9 days; gestation, 30-41 weeks) developed seizures from a variety of causes. In each case, seizures persisted for > 12 h despite high-dose phenobarbital therapy with or without the addition of phenytoin. Midazolam was then administered by continuous intravenous infusion (0.1-0.4 mg/kg/h) for 1 to 3 days. Within 1 h of initiation of midazolam, seizures were controlled in all six neonates. Electroencephalographic seizures were abolished in four of six neonates; however, two neonates continued to have electrographic seizures (without clinical accompaniment) for a further 12 h. Blood pressure and pulse rate were not changed after the initiation of midazolam, and adverse reactions were not observed in any neonate. Because more than one third of all neonatal seizures are refractory to high-dose phenobarbital and phenytoin, midazolam administered by continuous intravenous infusion may be a valuable adjunctive therapy.

Parietal occipital edema in hypertensive encephalopathy: a pathogenic mechanism.

Eight patients with hypertensive encephalopathy from diverse etiologies developed cerebral edema in the vertebrobasilar distribution which resolved after blood pressure was lowered. Parietal occipital edema is a recognized feature of hypertensive encephalopathy. The explanation for this regional pathological variation in hypertensive encephalopathy remains undefined. Some evidence suggests that sympathetic innervation of the anterior cerebral vasculature may be protective, and conversely, the relative lack of sympathetic innervation in the vertebrobasilar vasculature may predispose the parietal occipital region to the development of cerebral edema in hypertensive encephalopathy.

Electrodiagnosis of infantile botulism.

Analysis of the literature on the electrophysiologic features of infantile botulism was undertaken. Small compound muscle action potential amplitude is a very sensitive feature but lacks specificity. The decremental response to 2- to 3-Hz repetitive nerve stimulation is inconsistent and not a reliable sign. Tetanic and posttetanic facilitation are highly sensitive and highly specific. Absence of posttetanic exhaustion is also highly specific for infant botulism and shared only by hypermagnesemia. We conclude that the findings of low compound muscle action potential amplitude in combination with tetanic facilitation or posttetanic facilitation and absence of posttetanic exhaustion constitute the triad on which the electrodiagnosis of infantile botulism can be supported.

Hemorrhagic cardioembolic stroke: is anticoagulation absolutely contraindicated?

Hemorrhagic cerebral embolism should not be considered an absolute contraindication to immediate low-dose systemic anticoagulation. Low levels of anticoagulation may give some protection from recurrent embolism while minimizing the risks of intracranial bleeding. Until further studies are available, these decisions must be made on a case-by-case basis, supported by limited scientific information.

Transient appearance of periodic EEG discharges in senile dementia.

We describe an elderly patient with probable Alzheimer's disease whose EEG and clinical status improved rapidly coincident with normalization of temperature. This finding suggests that her transient illness, of fever, pneumonia and confusional state, triggered the production of this periodic EEG pattern. With the increased prevalence of dementia in our aging population, we predict that the appearance of a multifocal periodic triphasic sharp wave pattern in the EEG of chronically demented patients (with superimposed infection) will become more common. Only when this pattern persists over time and the patient is not systemically ill, is this pattern a reliable discriminator for CJD. Serial EEGs in this setting (after treatment of the systemic illness) might reveal the transient nature of the periodic EEG change, thus avoiding the erroneous conclusion that the patient's dementia is a manifestation of CJD.

Dipole reversal: an ictal feature of benign partial epilepsy with centrotemporal spikes.

Benign epilepsy with centrotemporal spikes (BECTS) is characterized by brief stereotypical partial seizures with motor and/or sensory symptoms with frequent secondarily generalized seizures. The interictal EEG shows slow, disphasic, high-voltage spikes in the centrotemporal areas. The few published examples of ictal tracings depict focal rhythmic sharp waves and spikes without significant postictal slowing. We report an ictal event in BECTS that is unusual in the evolution and polarity of the ictal discharges. In this subclinical seizure, ictal multiple spike and wave discharges appear as a dipole: they are electropositive in T3-C3 and negative in F3. These surface positive epileptic discharges are unique and require explanation. We postulate that the seizure discharge arises in the depths of the sylvian fissure involving folded cortical areas. This occurs in such a way that the negative component of the discharges is concealed from the scalp electrodes. Thus, a relative positivity is recorded on the surface. This represents a dipole reversal relative to the interictal discharges (characteristic of BECTS). This geometrical explanation allows us to avoid postulating an unusual mechanism of generation of this seizure.

EEG and pathologic findings in patients undergoing brain biopsy for suspected encephalitis.

Previous studies have suggested that EEG may be helpful in the diagnosis of herpes encephalitis (HE). To further define the value of EEG in an acutely ill, febrile, encephalopathic patient suspected as having encephalitis, we reviewed initial preoperative EEGs with the results of cerebral biopsies in 24 consecutive patients suspected of having encephalitis. Statistical analysis demonstrated that EEG patterns have only limited association with biopsy results. Since diseases which mimic encephalitis may also generate identical EEG changes, this is not an unexpected finding. EEG is useful in the evaluation and management of patients with encephalitis. However, the absence of specific wave forms or focal EEG abnormalities in the proper clinical setting should not deter consideration of HE or delay treatment.

Cerebral vasoconstriction as a complication of carotid endarterectomy. Case report.

The case is presented of an elderly man with an acute confusional state occurring soon after he had undergone a right carotid endarterectomy. Angiography demonstrated segmental areas of cerebral vasoconstriction and an electroencephalogram revealed periodic lateralized epileptiform discharges, both involving the right hemisphere. Cerebral hyperperfusion has been implicated in the genesis of several transient neurological syndromes following carotid endarterectomy. This case suggests that cerebral vasoconstriction may also be associated with impairment of cerebrovascular autoregulation observed after this procedure.