Evaluation of the Non-Conventional Yeast Starmerella Bacillaris as Biocontrol Agent and Selected Starter for Alcoholic Beverages Production

Yeasts are the main protagonists of the alcoholic fermentation and strongly influence the final characteristics of wine and other alcoholic beverages. In winemaking, during spontaneous fermentation, this process is carried out by indigenous yeasts present on the grape berry and there is a progressive growth pattern of different yeast species, with the final stages invariably being dominated by the alcohol-tolerant strains of Saccharomyces cerevisiae. This species is universally known as the ‘wine yeast’ and is widely preferred for initiating wine fermentations. From a technological point of view spontaneous grape juice fermentations sometime become stuck or sluggish. This lack of reproducibility and predictability has favored, in the past, the use of yeast starters, generally composed of single strains of S. cerevisiae. Selected S. cerevisiae strains predominate during must fermentation, ensure rapid and reliable grape juice fermentation and, as consequence, consistent and predictable wine quality. However, there has been increasing recognition that wines made with Saccharomyces starter cultures are less complex, producing standardized wines. In fact, during spontaneous fermentation many other species (the so call non–Saccharomyces yeasts) are present in the grape must and traditionally they were considered deleterious as responsible for some wine off-flavors. Recently, the role of non-Saccharomyces species has been re-evaluated as some of them has been proposed as co-starter to simulate natural must fermentation and to confer greater complexity and specificity to the wine. In this context, the thesis project was developed in order to select new non-Saccharomyces yeast strains from fermenting musts of overripe grape berries. This stage of grape ripening is characterized to be more susceptible to infection of fungal pathogens such as Botrytis cinerea, whom yeasts present on the berry surface have to compete with. Thirty-six isolates were identified as Starmerella bacillaris isolated from destemmed dried grape of Raboso Piave variety, grown on Bagnoli DOC (Appellation of Origin) area (North-East of Italy). This species is recently proven to be of oenological interest mainly for its high glycerol production. Among them, 14 strains were phenotypically characterized and, for the first time, the antifungal activity of S. bacillaris species against B. cinerea and P. expansum were demonstrated. Moreover, fermentation performances of S. bacillaris strains were evaluated in synthetic, grape and apple musts (for the first time in cider production). When tested in sequential fermentations with S. cerevisiae this yeast improved the quality of wine and cider. The obtained results indicate that S. bacillaris can be proposed as biocontrol agent on grape in vineyard and during postharvest on stored apples and its presence on fruit surface positively influences the following juice fermentation. Therefore the released of this yeast in the vineyard can increase the sustainability of the production process and the final wine quality. The obtained results provide a solid basis for a new management of yeast used for winemaking and other alcoholic beverage production and open new prospects for a more integrated strategy to increase wine quality. Then, in this thesis, new S. bacillaris characteristics were evaluated in order to improve alcoholic beverages quality, such as mannoproteins and glutathione production. The quantification of these compounds was determined at the end of fermentation in synthetic must. S. bacillaris was proved to produce different mannoproteins with functional characteristics related to wine stability. Mannoproteins extracted from S. bacillaris cultures presented great potential in increasing the protein stabilization, while if they were produced during sequential fermentation with S. cerevisiae, enhanced the tartaric stabilization. Moreover, in this thesis, a very high glutathione content was demonstrated in S. bacillaris cells after fermentation. Therefore, yeast lees obtained from S. bacillaris fermentations can be proposed as as glutathione source. In the last part of this thesis the aim was to understand the genomic determinants of S. bacillaris technological traits described in the previous parts. Therefore. the whole-genome sequence of two relevant S. bacillaris strains was performed. By means of genome comparisons between the two S. bacillaris strains and between S. bacillaris, S. cerevisiae and other oenological yeasts, specific genes and metabolic pathways involved in technologically-relevant traits were identified and studied

"Lactobacillus paracasei" probiotic properties and survivability under stress-induced by processing and storage of ice cream bar or ice-lolly

ABSTRACT: Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host. The aim of this study was to identify genotypically lactobacilli strains isolated from newborn stools and selecting strain based on probiotic properties (gastrointestinal tolerance, antibiotic susceptibility, inhibition of pathogen biofilm formation, absence of alfa or gamma-blood hemolysis, and lysozyme sensibility) and technological properties of surviving either in ice cream bar or ice-lolly. Reduction of 1.2log cfu ml-1 of the Lactobacillus paracasei strain was observed after exposure through in vitro gastrointestinal conditions. It inhibited biofilms of Escherichia coli, Salmonella Typhimurium and Candida albicans by mechanisms of competition, exclusion and displacement, and was resistant up to 3000μg ml-1 of egg white lysozyme. It presented neither alfa nor gamma-hemolysis or was antibiotic resistant to usual antibiotics for human use. Microbial survivability in ice cream bar or ice-lolly was assessed up to 21 days of storage at -18°C. Viability was maintained in ice cream bar, but there was a reduction of almost 2.0logs in ice-lolly

Diretriz sobre Diagnóstico e Tratamento da Cardiomiopatia Hipertrófica – 2024

Hypertrophic cardiomyopathy (HCM) is a form of genetically caused heart muscle disease, characterized by the thickening of the ventricular walls. Diagnosis requires detection through imaging methods (Echocardiogram or Cardiac Magnetic Resonance) showing any segment of the left ventricular wall with a thickness > 15 mm, without any other probable cause. Genetic analysis allows the identification of mutations in genes encoding different structures of the sarcomere responsible for the development of HCM in about 60% of cases, enabling screening of family members and genetic counseling, as an important part of patient and family management. Several concepts about HCM have recently been reviewed, including its prevalence of 1 in 250 individuals, hence not a rare but rather underdiagnosed disease. The vast majority of patients are asymptomatic. In symptomatic cases, obstruction of the left ventricular outflow tract (LVOT) is the primary disorder responsible for symptoms, and its presence should be investigated in all cases. In those where resting echocardiogram or Valsalva maneuver does not detect significant intraventricular gradient (> 30 mmHg), they should undergo stress echocardiography to detect LVOT obstruction. Patients with limiting symptoms and severe LVOT obstruction, refractory to beta-blockers and verapamil, should receive septal reduction therapies or use new drugs inhibiting cardiac myosin. Finally, appropriately identified patients at increased risk of sudden death may receive prophylactic measure with implantable cardioverter-defibrillator (ICD) implantation.La miocardiopatía hipertrófica (MCH) es una forma de enfermedad cardíaca de origen genético, caracterizada por el engrosamiento de las paredes ventriculares. El diagnóstico requiere la detección mediante métodos de imagen (Ecocardiograma o Resonancia Magnética Cardíaca) que muestren algún segmento de la pared ventricular izquierda con un grosor > 15 mm, sin otra causa probable. El análisis genético permite identificar mutaciones en genes que codifican diferentes estructuras del sarcómero responsables del desarrollo de la MCH en aproximadamente el 60% de los casos, lo que permite el tamizaje de familiares y el asesoramiento genético, como parte importante del manejo de pacientes y familiares. Varios conceptos sobre la MCH han sido revisados recientemente, incluida su prevalencia de 1 entre 250 individuos, por lo tanto, no es una enfermedad rara, sino subdiagnosticada. La gran mayoría de los pacientes son asintomáticos. En los casos sintomáticos, la obstrucción del tracto de salida ventricular izquierdo (TSVI) es el trastorno principal responsable de los síntomas, y su presencia debe investigarse en todos los casos. En aquellos en los que el ecocardiograma en reposo o la maniobra de Valsalva no detecta un gradiente intraventricular significativo (> 30 mmHg), deben someterse a ecocardiografía de esfuerzo para detectar la obstrucción del TSVI. Los pacientes con síntomas limitantes y obstrucción grave del TSVI, refractarios al uso de betabloqueantes y verapamilo, deben recibir terapias de reducción septal o usar nuevos medicamentos inhibidores de la miosina cardíaca. Finalmente, los pacientes adecuadamente identificados con un riesgo aumentado de muerte súbita pueden recibir medidas profilácticas con el implante de un cardioversor-desfibrilador implantable (CDI).A cardiomiopatia hipertrófica (CMH) é uma forma de doença do músculo cardíaco de causa genética, caracterizada pela hipertrofia das paredes ventriculares. O diagnóstico requer detecção por métodos de imagem (Ecocardiograma ou Ressonância Magnética Cardíaca) de qualquer segmento da parede do ventrículo esquerdo com espessura > 15 mm, sem outra causa provável. A análise genética permite identificar mutações de genes codificantes de diferentes estruturas do sarcômero responsáveis pelo desenvolvimento da CMH em cerca de 60% dos casos, permitindo o rastreio de familiares e aconselhamento genético, como parte importante do manejo dos pacientes e familiares. Vários conceitos sobre a CMH foram recentemente revistos, incluindo sua prevalência de 1 em 250 indivíduos, não sendo, portanto, uma doença rara, mas subdiagnosticada. A vasta maioria dos pacientes é assintomática. Naqueles sintomáticos, a obstrução do trato de saída do ventrículo esquerdo (OTSVE) é o principal distúrbio responsável pelos sintomas, devendo-se investigar a sua presença em todos os casos. Naqueles em que o ecocardiograma em repouso ou com Manobra de Valsalva não detecta gradiente intraventricular significativo (> 30 mmHg), devem ser submetidos à ecocardiografia com esforço físico para detecção da OTSVE.   Pacientes com sintomas limitantes e grave OTSVE, refratários ao uso de betabloqueadores e verapamil, devem receber terapias de redução septal ou uso de novas drogas inibidoras da miosina cardíaca. Por fim, os pacientes adequadamente identificados com risco aumentado de morta súbita podem receber medida profilática com implante de cardiodesfibrilador implantável (CDI)

Evaluation of the Non-Conventional Yeast Starmerella Bacillaris as Biocontrol Agent and Selected Starter for Alcoholic Beverages Production

Yeasts are the main protagonists of the alcoholic fermentation and strongly influence the final characteristics of wine and other alcoholic beverages. In winemaking, during spontaneous fermentation, this process is carried out by indigenous yeasts present on the grape berry and there is a progressive growth pattern of different yeast species, with the final stages invariably being dominated by the alcohol-tolerant strains of Saccharomyces cerevisiae. This species is universally known as the ‘wine yeast’ and is widely preferred for initiating wine fermentations. From a technological point of view spontaneous grape juice fermentations sometime become stuck or sluggish. This lack of reproducibility and predictability has favored, in the past, the use of yeast starters, generally composed of single strains of S. cerevisiae. Selected S. cerevisiae strains predominate during must fermentation, ensure rapid and reliable grape juice fermentation and, as consequence, consistent and predictable wine quality. However, there has been increasing recognition that wines made with Saccharomyces starter cultures are less complex, producing standardized wines. In fact, during spontaneous fermentation many other species (the so call non–Saccharomyces yeasts) are present in the grape must and traditionally they were considered deleterious as responsible for some wine off-flavors. Recently, the role of non-Saccharomyces species has been re-evaluated as some of them has been proposed as co-starter to simulate natural must fermentation and to confer greater complexity and specificity to the wine. In this context, the thesis project was developed in order to select new non-Saccharomyces yeast strains from fermenting musts of overripe grape berries. This stage of grape ripening is characterized to be more susceptible to infection of fungal pathogens such as Botrytis cinerea, whom yeasts present on the berry surface have to compete with. Thirty-six isolates were identified as Starmerella bacillaris isolated from destemmed dried grape of Raboso Piave variety, grown on Bagnoli DOC (Appellation of Origin) area (North-East of Italy). This species is recently proven to be of oenological interest mainly for its high glycerol production. Among them, 14 strains were phenotypically characterized and, for the first time, the antifungal activity of S. bacillaris species against B. cinerea and P. expansum were demonstrated. Moreover, fermentation performances of S. bacillaris strains were evaluated in synthetic, grape and apple musts (for the first time in cider production). When tested in sequential fermentations with S. cerevisiae this yeast improved the quality of wine and cider. The obtained results indicate that S. bacillaris can be proposed as biocontrol agent on grape in vineyard and during postharvest on stored apples and its presence on fruit surface positively influences the following juice fermentation. Therefore the released of this yeast in the vineyard can increase the sustainability of the production process and the final wine quality. The obtained results provide a solid basis for a new management of yeast used for winemaking and other alcoholic beverage production and open new prospects for a more integrated strategy to increase wine quality. Then, in this thesis, new S. bacillaris characteristics were evaluated in order to improve alcoholic beverages quality, such as mannoproteins and glutathione production. The quantification of these compounds was determined at the end of fermentation in synthetic must. S. bacillaris was proved to produce different mannoproteins with functional characteristics related to wine stability. Mannoproteins extracted from S. bacillaris cultures presented great potential in increasing the protein stabilization, while if they were produced during sequential fermentation with S. cerevisiae, enhanced the tartaric stabilization. Moreover, in this thesis, a very high glutathione content was demonstrated in S. bacillaris cells after fermentation. Therefore, yeast lees obtained from S. bacillaris fermentations can be proposed as as glutathione source. In the last part of this thesis the aim was to understand the genomic determinants of S. bacillaris technological traits described in the previous parts. Therefore. the whole-genome sequence of two relevant S. bacillaris strains was performed. By means of genome comparisons between the two S. bacillaris strains and between S. bacillaris, S. cerevisiae and other oenological yeasts, specific genes and metabolic pathways involved in technologically-relevant traits were identified and studied.I lieviti sono i principali protagonisti della fermentazione alcolica e influenzano fortemente le caratteristiche finali del vino e di altre bevande alcoliche. Nella vinificazione, durante una fermentazione spontanea,questo processo viene effettuato da lieviti autoctoni presenti sulla bacca di uva. Alla fine della fermentazione la specie dominate tra i ceppi è Saccharomyces cerevisiae. Questa specie è universalmente conosciuta come il 'lievito di vino' ed è ampiamente preferita per iniziare fermentazioni del vino. Da un punto di vista tecnologico, le fermentazioni spontanee di succo d'uva si sono spesso bloccate. In passato, questa mancanza di riproducibilità e prevedibilità non era favorevole per una produzione di vino di qualità. L’uso di ceppo di lievito, generalmente composti da singoli ceppi come ad esempio S. cerevisiae aiuta a controllare questo problema. I ceppi selezionati di S. cerevisiae predominano durante la fermentazione del mosto, assicurando una rapida e affidabile fermentazione e, di conseguenza, una qualità del vino coerente e prevedibile. Tuttavia, si è accresciuto la convinzione che i vini ottenuti con culture di starter Saccharomyces siano meno complesse, producendo vini standardizzati. Infatti, durante la fermentazione spontanea, nel mosto d'uva sono presenti molte altre specie (i cosiddetti lieviti non Saccharomyces) e tradizionalmente sono stati considerati come responsabili di alcuni sapori no aggradevoli all’assaggio del vino. Recentemente, il ruolo delle specie non Saccharomyces è stato rivalutato poiché alcune di esse sono state proposte come co-avviatore per simulare la fermentazione dei mosti naturali e per conferire maggiore complessità e specificità al vino. In questo contesto, il progetto di tesi è stato sviluppato per selezionare nuovi ceppi di lievito non Saccharomyces da mosti fermentativi di uva passito. Questa fase di maturazione dell'uva è caratterizzata per essere più suscettibile all'infezione di patogeni fungini come Botrytis cinerea. I trentasei isolati sono stati identificati come Starmerella bacillaris , isolati da uve secche della varietà Raboso Piave, coltivate in zona Bagnoli DOC (denominazione d'origine) (nord-est dell'Italia). Questa specie è recentemente dimostrata di interesse enologico soprattutto per la sua elevata produzione di glicerolo. Tra questi, 14 ceppi sono stati caratterizzati fenotipicamente e, per la prima volta, sono stati dimostrati l'attività antifungina delle specie S. bacillaris contro B. cinerea e P. expansum. Inoltre, le prestazioni di fermentazione dei ceppi di S. bacillaris sono state valutate nei mosti sintetici, uve e mele (per la prima volta nella produzione di sidro). Durante la sperimentazione, questo lievito ha migliorato la qualità del vino e del sidro in fermentazioni sequenziali con S. cerevisiae. I risultati ottenuti indicano che S. bacillaris può essere proposto come agente biocontrollo sull'uva nel vigneto e durante la post-raccolta sulle mele conservate: la sua presenza sulla superficie di frutta influenza positivamente la seguente fermentazione dei succhi. Pertanto, il rilascio di questo lievito nel vigneto può aumentare la sostenibilità del processo produttivo e la qualità finale del vino. I risultati ottenuti forniscono una solida base per una nuova gestione del lievito utilizzato per la produzione di vino e altre bevande alcoliche e aprire nuove prospettive per una strategia più integrata per aumentare la qualità del vino. Sono state valutate le nuove caratteristiche di S. bacillaris al fine di migliorare la qualità delle bevande alcoliche, come le manoproteine e la produzione di glutatione. La quantificazione di questi composti è stata determinata alla fine della fermentazione in mosto sintetico. S. bacillaris ha dimostrato di poter produrre mannoproteine diverse, con caratteristiche funzionali legate alla stabilità del vino. Le manoproteine estratte dalle colture di S. bacillaris presentavano un grande potenziale per aumentare la stabilizzazione delle proteine, mentre se venivano prodotte durante la fermentazione sequenziale con S. cerevisiae, aumentavano la stabilizzazione tartarica. Inoltre, in questa tesi, è stato rilevato un contenuto di glutatione molto elevato in cellule S. bacillaris dopo la fermentazione. Pertanto, le fecce ottenute dalle fermentazioni di S. bacillaris possono essere proposte come fonte di glutatione. Nell'ultima parte di questa tesi si intendeva comprendere le determinanti genomiche delle caratteristiche tecnologiche di S. bacillaris descritte nelle precedenti parti. Per questo è stata eseguita la sequenza intero-genoma di due importanti ceppi S. bacillaris. Con i confronti genomici tra i due ceppi di S. bacillaris e tra S. bacillaris, S. cerevisiae e altri lieviti enologici, sono stati identificati e studiati geni specifici e i percorsi metabolici coinvolti in tratti tecnologicamente rilevanti

Modeling the Thermal Inactivation of <i>Monascus ruber</i> Ascospores Isolated from Green Olive (<i>Arauco</i> Cultivar) Storage Brine: An Alternative Strategy to Reduce Antifungal Chemical Agents

Monascus ruber is an important fungus that causes spoilage in table olives, resulting in the darkening of the brine, the softening of the fruit, increased pH, and apparent mycelial growth. This study aimed to evaluate this resistance, providing a model to determine the optimal processing conditions for mitigating fungal contamination and prolonging shelf life without antifungal agents while optimizing pasteurization to reduce energy consumption. The resistance in brine (3.5% NaCl; pH 3.5) from Arauco cultivar green olives imported from Argentina was assessed. Four predictive models (log linear, log linear + shoulder, log linear + tail, log linear + shoulder + tail) estimated kinetic parameters for each survival curve. Log linear + shoulder + tail provided the best fit for 70 °C and 75 °C, with low RMSE (0.171 and 0.112) and high R2 values (0.98 and 0.99), respectively, while the log linear model was used for 80 °C. Decimal reduction times at 70, 75, and 80 °C were 24.8, 5.4, and 1.6 min, respectively, with a z-value of 8.2 °C. The current regulatory processes are insufficient to eliminate M. ruber at requisite levels, considering reduced antifungal agents

Identification and Selection of Prospective Probiotics for Enhancing Gastrointestinal Digestion: Application in Pharmaceutical Preparations and Dietary Supplements

Our study investigated the effectiveness of 446 strains of lactic acid bacteria (LAB) belonging to different species and isolated from diverse sources (food, human, and animal) as potential probiotic candidates, with the perspective of producing dietary supplements or pharmacological formulations suitable for enhancing gastrointestinal digestion. The survival capability of all the isolates under harsh gastrointestinal tract conditions was evaluated, in which only 44 strains, named high-resistant, were selected for further food digestibility investigations. All 44 strains hydrolyzed raffinose and exhibited amino and iminopeptidase activities but at various extents, confirming species- and strain-specificity. After partial in vitro digestion mimicking oral and gastric digestive phases, food matrices were incubated with single strains for 24 h. Fermented partially digested matrices provided additional functional properties for some investigated strains by releasing peptides and increasing the release of highly bio-accessible free phenolic compounds. A scoring procedure was proposed as an effective tool to reduce data complexity and quantitively characterize the probiotic potential of each LAB strain, which could be more useful in the selection procedure of powerful probiotics

Correlation between natural microbial load and formation of ropy slime affecting the superficial color of vacuum-packaged cooked sausage

: The present study outlines a comprehensive correlation between the natural microbial load, which is predominantly composed of heat-resistant sporous-forming Bacillus, and the changes in the original properties related to the superficial color of vacuum-packaged cooked sausages. For this purpose, microbial growth curves were plotted by stimulating the growth of the natural microbiota in sausage packages at different temperatures. The correlations were investigated during sample incubation by the instrumental evaluation of color and the ropy slime detection on the sausage surface. The entrance of the natural microbiota into the stationary phase (ca. 9.3 log cfu/g) resulted in changes in the superficial color, which was demonstrated by the discoloration of vacuum-packaged cooked sausages. Therefore, it seems to be a suitable borderline for predictive models applied in durability studies that aim to estimate the period in which vacuum-packaged cooked sausages keep their typical superficial color, anticipating product refusal in markets

Probiotic potential and biofilm inhibitory activity of Lactobacillus casei group strains isolated from infant feces

Probiotic bacteria are receiving growing interest, particularly for the preparation of functional foods. In the present study, eight Lactobacillus strains, newly isolated from infant feces, were investigated for the presence of probiotic properties such as antimicrobial susceptibility, hemolytic activity, resistance to simulated gastro-intestinal conditions, bile salts hydrolytic activity, inhibitory ability against biofilm formation by other bacteria, attachment to HT-29 human cancer cells and anti-cancer activity. All the strains tested highlighted interesting properties, but L. paracasei DTA93 and L. paracasei DTA81 appeared of particular interest. Some properties of these two strains resulted similar, and in some cases superior, to the reference widespread probiotic commercial strain L. rhamnosus GG. Strain L. paracasei DTA81 possesses amazingly high adherence ability to HT-29 cells, about ten times higher than that of L. rhatnnosus GG. Moreover, L. paracasei DTA93 and L. paracasei DTA81 were able to effectively inhibit biofilm formation of Escherichia coli and Listeria innocua

Potentially Postbiotic-Containing Preservative to Extend the Use-By Date of Raw Chicken Sausages and Semifinished Chicken Products

This study aimed to evaluate the use of potentially postbiotic-containing preservative (PPCP), produced in a semiculture fermentation system with Lacticaseibacillus paracasei DTA 83 and Saccharomyces cerevisiae var. boulardii 17, to extend the use-by date of raw chicken sausages and semifinished chicken products. Microorganisms associated with the spoilage of chicken products were stimulated to grow by pair incubation of the products at two different temperatures and with collection at different times. The turbidity method was performed to evaluate the microbial susceptibility to PPCP. PPCP was added in chicken products to obtain an in situ partial inhibitory effect on spoilage microorganisms to extend the use-by date. The in vitro trial showed total inhibition of the microbial growth by adding above 3.0% of PPCP. Although this concentration showed a remarkable inhibitory potential, its addition can severely impact the formulation cost. Thus, the application of doses with partial microbial inhibition may be a suitable strategy for the use of PPCP in chicken products. The results revealed that cold chain management and couse of PPCP in chicken products extended the proposed use-by date, suggesting an alternative food preservation technology for the use of naturally derived compounds