γ-aminobutyric acid production by Kluyveromyces marxianus strains.

AIMS: Kluyveromyces marxianus dairy strains were tested for γ-aminobutyric acid (GABA) production. The genes involved in GABA catabolism (UGA1 and UGA2) and anabolism (GAD1) were found in K. marxianus genome. Their relative expression was evaluated with primer designed ad hoc. METHODS AND RESULTS: Strains were grouped on the basis of GAD1 gene sequence. Representative strains for each group were tested for GABA production by high-performance liquid chromatography. All strains produced it at low levels. qRT-PCR showed the absence of a relation between GABA production and GAD1 gene expression. UGA1 and UGA2 genes were not upregulated and low amounts of succinic acid were detected. CONCLUSIONS: All strains released a low amount of GABA suggesting that probably it was stored within cells. The different behaviour of strains in terms of GABA and succinic acid production as well as gene expression highlighted the genetic and phenotypic biodiversity of this species. SIGNIFICANCE AND IMPACT OF THE STUDY: GABA production and genes involved in its catabolism and anabolism were described in a population of dairy K. marxianus for the first time. The variability observed in terms of genetic and phenotypic biodiversity is important especially to exploit this non-conventional yeast as microbial platform.

Characterization of specialized flocculent yeasts to improve sparkling wine fermentation.

AIMS: Flocculent wine yeasts were characterized for the expression of FLO1, FLO5, FLO8, AMN1 and RGA1 genes, growth kinetics and physicochemical properties of the cell surface during a 6-month sparkling wine fermentation period. METHODS AND RESULTS: The expression of FLO1, FLO5, FLO8, AMN1 and RGA1 genes was determined by RT-qPCR. The physicochemical characterization of yeast surface properties was evaluated by the microbial adhesion to solvents method. FLO5 gene was the most expressed one and a linear correlation with the flocculent degree was found. Flocculent strains were more hydrophobic than the commercial wine strain EC1118. CONCLUSIONS: Gene expressions and the ability to face secondary wine fermentation conditions were strain dependent. The importance of FLO5 gene in developing the high flocculent characteristic of wine yeasts was highlighted. Cell surface properties depended on the time of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: Better knowledge about the expression of some genes encoding the flocculent phenotype which could be useful to select suitable starter cultures to improve sparkling wine technology was achieved. A step forward in understanding the complexity and strain-specific nature of flocculation phenotype was done.

Identification of critical genes for growth in olive brine by transposon mutagenesis of Lactobacillus pentosus C11.

Olive brine represents a stressful environment due to the high NaCl concentration, presence of phenolic compounds known as antimicrobials, and low availability of nutrients. Thus, only a few strains of lactic acid bacteria (LAB) are adapted to grow in and ferment table olives. To identify the mechanisms by which these few strains are able to grow in olive brine, Lactobacillus pentosus C11, a particularly resistant strain isolated from naturally fermented table olives, was mutagenized by random transposition using the P(junc)-TpaseIS1223 system (H. Licandro-Seraut, S. Brinster, M. van de Guchte, H. Scornec, E. Maguin, P. Sansonetti, J. F. Cavin, and P. Serror, Appl. Environ. Microbiol. 78:5417-5423, 2012). A library of 6,000 mutants was generated and screened for adaptation and subsequent growth in a medium, named BSM (brine screening medium), which presents the stressful conditions encountered in olive brine. Five transposition mutants impaired in growth on BSM were identified. Transposition occurred in two open reading frames and in three transcription terminators affecting stability of transcripts. Thus, several essential genes for adaptation and growth of L. pentosus C11 in olive brine were identified.

Relationships between microbial population dynamics and putrescine and cadaverine accumulation during dry fermented sausage ripening.

AIMS: To evaluate the concomitant effects of three technological variables (fermentation temperature, NaCl and glucose added to the meat batter) on diamines (cadaverine, putrescine and histamine) accumulation and microbial changes during ripening of dry fermented sausages. METHODS AND RESULTS: The variables were modulated according to an experimental design and predictive mathematical models were obtained. The models indicated that the sausages were characterized by low histamine amount independently on the applied conditions. In contrast, putrescine and cadaverine accumulation was considerable and significantly affected by the three variables. The microbial population dynamics suggest that lactic acid bacteria (LAB) and microstaphylococci are favoured by increasing glucose concentration until 0.7 g kg(-1), while Enterobacteriaceae are negatively influenced by NaCl concentration and, to a lesser extent, by fermentation temperature. CONCLUSIONS: Data obtained showed a relationship between Enterobacteriaceae growth and cadaverine and putrescine accumulation in sausages during ripening. The conditions more favourable for LAB and microstaphylococci induced a reduced growth of Enterobacteriaceae with a consequent reduced accumulation of putrescine and cadaverine. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of systematic experimental designs allows to individuate the technological conditions suitable to keep the aminogenic microflora under control, thus reducing the risk of diamines production during traditional fermented food manufacture.

Growth and metabolites production by Penicillium brevicompactum in yoghurt.

Penicillium brevicompactum, commonly encountered in the indoor air, is known to produce a mycotoxin, mycophenolic acid (MPA). This mould has been isolated from a wide range of foods; considering that we had previously isolated this species from contaminated yoghurt, in this study we have evaluated its growth in yoghurt sweetened with sucrose, fructose and fructose added with fruit pieces. Fungal growth was evaluated monitoring CO(2) production in the headspace during yoghurt storage at 4+/-1, 8+/-1 and 10+/-1 degrees C throughout 21 days. P. brevicompactum grew well in the samples sweetened with fructose at 8 and 10 degrees C. The addition of sucrose influenced the growth negatively, particularly at 4 degrees C. Volatile Organic Compounds (VOC) and MPA production was determined at 8 degrees C in inoculated and uninoculated yoghurt, as well as in liquid malt extract. Differences in VOC profiles and in MPA production were correlated with the age of the fungus and with the growth medium. This study points out for the first time the early qualitative changes in volatile production patterns of a common indoor mould, grown in yoghurt, as well as the production of MPA during storage at refrigeration temperatures.

In Lactobacillus pentosus, the olive brine adaptation genes are required for biofilm formation.

Lactobacillus pentosus is one of the few lactic acid bacteria (LAB) species capable of surviving in olive brine, and thus desirable during table olive fermentation. We have recently generated mutants of the efficient strain L. pentosus C11 by transposon mutagenesis and identified five mutants unable to survive and adapt to olive brine conditions. Since biofilm formation represents one of the main bacterial strategy to survive in stressful environments, in this study, the capacity of adhesion and formation of biofilm on olive skin was investigated for this strain and five derivative mutants which are interrupted in metabolic genes (enoA1 and gpi), and in genes of unknown function ("oba" genes). Confocal microscopy together with bacteria count revealed that the sessile state represented the prevailing L. pentosus C11 life-style during table olive fermentation. The characterization of cell surface properties showed that mutants present less hydrophobic and basic properties than the wild type (WT). In fact, their ability to adhere to both abiotic (polystyrene plates) and biotic (olive skin) surfaces was lower than that of the WT. Confocal microscopy revealed that mutants adhered sparsely to the olive skin instead of building a thin, multilayer biofilm. Moreover, RT-qPCR showed that the three genes enoA1, gpi and obaC were upregulated in the olive biofilm compared to the planktonic state. Thus enoA1, gpi and "oba" genes are necessary in L. pentosus to form an organized biofilm on the olive skin.

T wave normalization in infarct-related electrocardiographic leads during exercise testing for detection of residual viability: comparison with positron emission tomography.

OBJECTIVES: We investigated the sensitivity and specificity of exercise-induced T wave normalization (TWN) in infarct-related electrocardiographic leads (IRLs) for detection of residual viability in the infarct area. BACKGROUND: The meaning of exercise-induced TWN on IRLs is not yet well understood. Recent reports suggest that TWN during dobutamine echocardiography could indicate the presence of viable myocardium. METHODS: We evaluated 40 consecutive patients with a recent acute myocardial infarction and negative T waves in at least two IRLs. All patients underwent exercise testing; positron emission tomography (PET) with nitrogen-13 ammonia and fluorine-18 fluorodeoxyglucose; and coronary angiography. RESULTS: Twenty-four patients showed exercise-induced TWN: 18 at a work load < or =50 W (group la) and 6 at a work load > or =75 W (group 1b); 16 patients did not show TWN (group 2). On the PET study, viability in the infarct area was present in 17 patients (94%) from group la, in only 1 (16%) from group 1b and in 4 (25%) from group 2 (p < 0.0001). The sensitivity, specificity and diagnostic accuracy of exercise-induced TWN, in comparison with residual viability, were, respectively, 82%, 67%, 75% for TWN at every work load and 77%, 94%, 85% for TWN at a work load < or =50 W. Moreover, the sensitivity and diagnostic accuracy of TWN at the low work load were higher for anterior infarctions (87% and 88%, respectively). CONCLUSIONS: Exercise-induced TWN on IRLs at low work loads is a sensitive and specific index for the presence of residual viability in the infarct area. Sensitivity and diagnostic accuracy of this sign are higher for anterior infarctions.

Use of Yarrowia lipolytica strains for the treatment of olive mill wastewater.

The principal aim of this research was to evaluate the ability of different Yarrowia lipolytica strains, having different origin, to grow in olive mill wastewater (OMW) and reduce its COD level. All the strains were able to grow in undiluted OMW; the comparison between the data obtained in a semi-synthetic medium and in OMW suggests that lipases with different specificity can be produced in relation to the medium composition. Under the adopted conditions, the reduction of the OMW COD values varied from 1.47% and 41.22% of the initial value. Some strains determined a significant reduction of polyphenol content, while other ones caused its apparent increase. Moreover, some Y. lipolytica strains, isolated from chilled foods, produced the highest citric acid concentrations. These results evidenced that some Y. lipolytica strains are good candidates for the reduction of the pollution potential of OMW and for the production of enzymes and metabolites such as lipase and citric acid.

Stress echocardiography and exercise electrocardiography for risk stratification after non-Q-wave uncomplicated myocardial infarction.

The aim of our study was to compare the prognostic value of stress echocardiography and exercise electrocardiography after uncomplicated non-Q-wave acute myocardial infarction in a series of 68 consecutive patients. Our data show that stress echocardiography and exercise electrocardiography offer similar prognostic information after uncomplicated non-Q-wave AMI.

Noninvasive risk stratification in women with uncomplicated acute myocardial infarction.

The aim of our study was to compare the prognostic value of stress echocardiography and exercise electrocardiography after uncomplicated non-Q-wave acute myocardial infarction in a series of 89 female patients. Our data show that stress echocardiography has independent predictive value in a female patient population recovering from uncomplicated acute myocardial infarction.

Acetoin production in Saccharomyces cerevisiae wine yeasts.

One hundred strains of Saccharomyces cerevisiae were examined for the capacity to produce acetoin in synthetic medium and in grape must. The low production of acetoin was found to be the more common pattern in this species. Most strains exhibited a similar distribution in both media, production ranging from non-detectable amounts to 12 mg l-1. Only four strains produced high quantities of acetoin, up to 29.5 mg l-1 in synthetic medium and up to 194.6 mg l-1 in grape must. This biometric study showed the existence of two phenotypes, "low and high acetoin production", that could be selected for conferring a desirable flavour of the final product.

Acetaldehyde production in Saccharomyces cerevisiae wine yeasts.

Eighty-six strains of Saccharomyces cerevisiae were investigated for their ability to produce acetaldehyde in synthetic medium and in grape must. Acetaldehyde production did not differ significantly between the two media, ranging from a few mg/l to about 60 mg/l, and was found to be a strain characteristic. The fermentation temperature of 30 degrees C considerably increased the acetaldehyde produced. This study allowed us to assign the strains to different phenotypes: low, medium and high acetaldehyde producers. The low and high phenotypes differed considerably also in the production of acetic acid, acetoin and higher alcohols and can be useful for studying acetaldehyde production in S. cerevisiae, both from the technological and genetic point of view.

Yeasts from Water Buffalo Mozzarella, a traditional cheese of the Mediterranean area.

Countries of the Mediterranean area are characterized by production of artisanal cheeses, obtained from goat, sheep, cow and buffalo raw milk. The numbers and species of yeasts in the different cheeses are variable, but some species are more frequently detected than others. Kluyveromyces marxianus, K. lactis with their anamorph, Candida kefir, Debaryomyces hansenii and C. famata, C. colliculosa and C. catenulata are dominant species in several cheeses. However, Saccharomyces cerevisiae is often detected in pasta filata cheeses, such as Water Buffalo Mozzarella (WBM) or Cacio Cavallo Podolico. Recently, a comprehensive study of yeasts isolated from Mozzarella cheese produced in Basilicata (Southern Italy) has been carried out. The study has focused on lactose and/or galactose fermenting species (Kluyveromyces and Saccharomyces) to evaluate their role on the functional and sensory properties of the product. End products in milk were evaluated and the biodiversity in terms of production of sulphur dioxide, higher alcohols, ethyl acetate, and acetaldehyde was studied. In particular, S. cerevisiae strains from Water Buffalo Mozzarella cheese, compared to strains isolated from different habitats, such as wine, exhibited considerable difference in the production of some volatile compounds. The diversity observed could be related to the particular microhabitat of S. cerevisiae occurring in whey cheese of water buffalo milk.

Contribution of Gal- lactic acid bacteria to Saccharomyces cerevisiae metabolic activity in milk.

The contemporaneous presence of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus affected the growth kinetics of Saccharomyces cerevisiae PZ2 and the metabolic products of their growth were quantitatively and qualitatively different from those produced by single strains inoculated alone. S. cerevisiae can grow in milk without using lactose or galactose. In particular, the presence of peptides seems to be sufficient to ensure its growth. The growth of S. cerevisiae with lactic acid bacteria is characterised by stimulatory effects that involve both yeast and bacteria. However, the release of galactose by lactic acid bacteria does not seem to be the core metabolic event of these stimulatory effects on S. cerevisiae.

Proteolytic, lipolytic and molecular characterisation of Yarrowia lipolytica isolated from cheese.

This work studied the qualitative and quantitative proteolytic and lipolytic activities of Yarrowia lipolytica strains isolated from two cheese types. Randomly amplified polymorphic DNA-PCR (RAPD-PCR) analysis was used to compare the cheese strains of Y. lipolytica with strains isolated from other food products and with the type strain of the species in order to investigate the genetic diversity and occurrence of specific environmental groups. Diversity of proteolytic and especially lipolytic activity within Y. lipolytica strains isolated from dairy products was observed. In particular, the degree of specificity for saturated or unsaturated fatty acids as well as for even- or odd-numbered carbon free fatty acids (FFAs) varied among the strains. The RAPD-PCR profiles showed low genetic relatedness between many of the food isolates and the type strain of the species. Such genetic variability needs to be further evaluated. Most of the Y. lipolytica strains appeared to be specific to the particular environment from which they were isolated. However, phenotypic characteristics having technological importance in dairy products and, particularly, lipolytic activities did not correspond to the genetic differences observed by RAPD-PCR analysis.

Variability of the lipolytic activity in Yarrowia lipolytica and its dependence on environmental conditions.

This work was aimed to the evaluation of the variability of lipolytic activity in Yarrowia lipolytica strains, as well as to asses for a selected strain, the response to the changes of physico-chemical variables (such as pH, NaCl and lipid content), in order to obtain predictive models describing their effects on the lipolysis pattern. The strains tested, having different environmental origin, showed different patterns of the free fatty acids (FFA) released. The clustering of the free fatty acids profiles evidenced that the unweighted average distance within the strains of the same species did not exceeded 30%. However, the lipolytic activity of some strains generated FFA profiles that differentiated from the majority of the strains considered. Also, when a single strain was inoculated in model systems in which pH, NaCl and milk fat were modulated according to a Central Composite Design (CCD), chemico-physical characteristics of the system led to marked variations in the lipolytic activity with consequent changes in individual fatty acids released. In most cases, when the same Y. lipolytica strain was used, under the experimental conditions adopted, the modulation of the lactic acid, NaCl and lipid content did not generate differences in the fatty acid release exceeding 20-21%. However, some combinations of factors remarkably affected lipase expression or activity, and generated differences in the fatty acid released higher than those observed among different strains of the same species.

Effects of pH, temperature and NaCl concentration on the growth kinetics, proteolytic activity and biogenic amine production of Enterococcus faecalis.

In this work, the combined effects of temperature, pH and NaCl concentration on the growth dynamics of Enterococcus faecalis EF37, its proteolytic activity and its production of biogenic amines have been studied. The effects of the selected variables have been analysed using a Central Composite Design. The production of biogenic amines, under the adopted conditions, was found to be mainly dependent on the extent of growth of E. faecalis. Its proteolytic activity was not a limiting factor for the final amine production, because in the system studied (skim milk) an excess of precursors was guaranteed. Quantitatively, the most important biogenic amine produced was 2-phenylethylamine but substantial amounts of tyramine were detected in all the samples. This work confirms that the main biological feature influencing the biogenic amine formation is the extent of growth of microorganisms, like E. faecalis, characterised by decarboxylase activity. In the traditional and artisanal cheeses produced using raw milk, enterococci usually reach levels of 10(7) cells/g. With this perspective, it is important that the presence of biogenic amines due to the activities of these microorganisms is maintained within safe levels, without affecting the positive effects of enterococci on the final organoleptic characteristics of the cheese.

Exercise technetium 99m sestamibi single-photon emission computed tomography late after coronary artery bypass surgery: long-term follow-up.

BACKGROUND AND HYPOTHESIS: The prognostic value of exercise technetium 99m sestamibi single-photon emission computed tomography (SPECT) imaging in patients with previous bypass surgery is unknown. The aim of our study was to assess the prognostic information obtained with exercise scintigraphy performed for routine follow-up or reappearance of symptoms. METHODS: We studied 75 patients referred to our Center at a mean of 38 +/- 53 months from the revascularization procedure and prospectively followed them for 38 +/- 24 months. RESULTS: Fifteen patients (20%) had events at follow-up: there were 4 cardiac deaths, 3 nonfatal acute myocardial infarctions, 8 late revascularization procedures (4 percutaneous transluminal angioplasty and 4 repeat bypass surgery). Univariate analysis identified a history of typical angina (p = 0.001), a clinically positive ergometric test (p = 0.009), peak exercise heart rate (p = 0.0003), percentage of maximal predicted heart rate (p = 0.0001), peak exercise double product (p = 0.048), therapy during exercise (p = 0.003), scintigraphic summed reversibility score (i.e., the summation of the segmental differences between stress and rest) (p = 0.014), as significant predictors of events. Three multivariate models were built, with clinical variables (Model 1, chi square 15.97), ergometric variables (Model 2, chi square 19.66), and with scintigraphic variables added to clinical/ergometric variables (Model 3, chi square 31.13). The scintigraphic variable selected in the model as significant predictor of events was the summed reversibility score (p = 0.008). CONCLUSIONS: Exercise sestamibi SPECT scintigraphy provides optimal prognostic information after clinical and ergometric parameters in patients with previous bypass surgery.

[Pharmacologic effects and safety profile of bethanechol in the differential diagnosis of pseudo-anginal thoracic pain]. / Effetti farmacologici e profilo di sicurezza del betanecolo cloruro nella diagnosi differenziale del dolore toracico pseudo-anginoso.

The use of bethanecol has recently been proposed to improve the diagnostic accuracy of esophageal manometry in identifying the origin of symptoms in patients with non cardiac chest pain. In this study we report our experience in 30 patients who underwent esophageal functional studies. Despite its low diagnostic value, bethanecol test (two subsequential doses of 50 micrograms/kg) demonstrated an excellent safety profile, there were few side effects, and patients tolerance was good. The Holter electrocardiographic study showed an isolated case of transient atrioventricular block.

[Combined use of bicycle ergometry test and echo-dipyridamole in stratification of risk after thrombolysed myocardial infarct]. / Uso combinato di test al cicloergometro ed eco-dipiridamolo per la stratificazione del rischio dopo infarto miocardico trombolisato.

AIM: Verify the value of the combined use of Dipyridamole Stress Echocardiography (DSE) and Exercise Testing (ET) for predicting cardiac events after Thrombolysed Myocardial Infarction (TMI). STUDY DESIGN: Prospective, with a medium follow-up of 221 days (range 30-446). CLINICAL SETTINGS: Coronary Care Unit with Cardiology Service. PATIENTS: 32 consecutive patients, 24 men and 8 women, mean age 61 years (range 39-72) with acute myocardial infarction. INTERVENTIONS: Systemic thrombolysis with PTPA. DSE and ET in therapeutic wash out 12 +/- 3 days after admission to the Coronary Care Unit. RESULTS: 9 patients had positive ET, 18 patients had negative ET, 5 patients did not perform ET. 19 patients had positive DSE, 13 patients had negative DSE. Patients with positive DSE had in 6 cases (group 1) WMA remote from the infarction area (WMAR), in 13 cases (group 2) WMA appeared in the infarct related area. Cardiac events occurred in 15 patients: 1 cardiovascular death, 1 myocardial re-infarction, 13 cases of unstable angina. 2 patients with negative DSE had cardiac events. All patients in group 1 had events, 1 patient with a previous positive ET, 3 patients with previous negative ET. 2 patients in this group didn't perform ET. 7 patients in group 2 had cardiac events, in all cases with a previous positive ET. 1 single patient in this group with negative ET had events. CONCLUSIONS: 1) Patients without WMA at DSE are at low risk for cardiac events after thrombolysed myocardial infarction. 2) Presence of WMAR appears to be associated with a higher risk, whatever the result of ET may be. 3) Patients without WMAR can be further stratified by ET in therapeutic wash out. High risk patients seem to be those with positive DSE and positive ET. 4) Combined use of DSE and ET seem to allow optimal risk stratification after thrombolysed myocardial infarction.