12,715 research outputs found
Study of Oenococcus oeni to improve wine quality
No full textLactic acid bacteria (LAB) are, besides yeasts, the best adapted microbial family to wine conditions. Many genera have been isolated both from grape must and wine, and they represent an important resource in winemaking since most of them are able to perform malolactic fermentation (MLF), the conversion of L-malic acid into L-lactic acid, which is often required to obtain wines with positive flavor and taste characteristics, but has to be avoided in some cases.
Among LAB, Oenococcus oeni is without any doubt the best adapted species to the wine environment, and is often used as a starter to perform MLF. However, this step in winemaking is often difficult to induce and control. Moreover, this microorganism requires up to 10 days to grow and develop countable colonies on plate using classical microbiological methods to enumerate viable cells, and the control of the inoculation, as well as the evaluation of the presence or absence of O. oeni in a sample, requires usually a considerable amount of time. For these reasons one of the purposes of this research project was the development of a Propidium monoazide - quantitative PCR (PMA-qPCR) technique for the fast enumeration of O. oeni in must and wine, and the results obtained show how the developed technique is able to provide a detection limit (0.33 log CFU/mL in must and 0.69 log CFU/mL in wine) which is lower than all of the other molecular biology techniques developed until now.
Furthermore, to better understand which conditions are the most favorable for a successful MLF, a Reverse Transcription – quantitative PCR (RT-qPCR) technique has been developed to study the gene expression levels of the mleA gene, encoding for the malolactic enzyme, in O. oeni. The results obtained show that co-inoculation with Saccharomyces cerevisiae and high concentrations of ethanol in the medium are the best conditions to ensure high levels of transcription of the mleA gene.
Besides the capacity of performing MLF, LAB are capable to influence the aromatic complexity of wine thanks to the release of volatile compounds due to the activity of the β-glucosidase enzyme, which has been isolated in various strains, including O. oeni. For this reason, the last purpose of this work has been the development of a RT-qPCR technique to find out which winemaking practice (sequential inoculation or co-inoculation) is the best to ensure high levels of transcription of the gene encoding for the β-glucosidase enzyme. Results point out that during co-inoculation higher levels of expression are registered.
Therefore, and although winemakers try often to avoid this practice, co-inoculation can be considered the best winemaking scenario to ensure both rapid completion of MLF and expression of β-glucosidase encoding gene, which can lead to the release of positive aromatic volatile compounds in wine.I batteri lattici (LAB) sono, insieme ai lieviti, la famiglia microbica meglio adattata alle avverse condizioni chimiche e chimico-fisiche presenti nel vino. Molti generi sono stati isolati sia dal mosto che dal vino, ed essi rappresentano un’importante risorsa nelle pratiche enologiche, dal momento che molti sono in grado di effettuare la fermentazione malolattica (FML), la conversione dell’acido L-malico in acido L-lattico, che è spesso necessaria per ottenere vini con caratteristiche desiderate di sapore e gusto, ma che in alcuni casi deve essere evitata.
Tra i LAB, Oenococcus oeni è senza dubbio il meglio adattato alle condizioni del vino, ed è spesso usato come starter per eseguire la FML. Tuttavia questa fase è spesso difficile da indurre e controllare nella produzione del vino. In più questo microorganismo richiede fino a 10 giorni per crescere e dare origine a colonie visibili e contabili in piastra utilizzando metodi di microbiologia classica per contare le cellule vitali, e il controllo dell’inoculo, così come la valutazione della presenza o dell’assenza di O. oeni in un campione, richiede di solito una significativa quantità di tempo. Per queste ragioni uno degli scopi di questo progetto di ricerca è stato lo sviluppo di una tecnica di PCR quantitativa basata sull’uso di Propidio Monoazide (PMA-qPCR) per la rapida enumerazione di O. oeni in mosto e in vino, e i risultati ottenuti mostrano che tale tecnica presenta un limite di rilevabilità del microorganismo (0.33 log UFC/mL in mosto e 0.69 log UFC/mL in vino) più basso rispetto a tutte le altre tecniche di biologia molecolare finora sviluppate.
In seguito, per meglio comprendere quali condizioni siano le più favorevoli affinchè la FML venga eseguita con successo, è stata sviluppata una tecnica di trascrizione inversa seguita da PCR quantitativa (RT-qPCR) per studiare i livelli di espressione genica del gene mleA, codificante per l’enzima malolattico, in O. oeni. I risultati ottenuti indicano che il coinoculo con Saccharomyces cerevisiae e alte concentrazioni di etanolo nel mezzo sono le condizioni migliori per assicurare alti livelli di trascrizione del gene mleA.
Accanto alla capacità di eseguire la FML, i LAB sono in grado di influenzare la complessità aromatica del vino grazie al rilascio di composti volatili dovuto all’attività dell’enzima β-glucosidasi, che è stato isolato da diversi ceppi, anche appartenenti alla specie O. oeni. Per questa ragione l’ultimo obiettivo di questo lavoro è stato lo sviluppo di una tecnica RT-qPCR al fine di individuare quale tecnica produttiva (inoculo sequenziale o coinoculo) fosse la migliore per assicurare alti livelli di trascrizione del gene codificante per l’enzima β-glucosidasi. I risultati evidenziano che durante il coinoculo vengono registrati livelli di trascrizione più alti.
Perciò, nonostante spesso i produttori tendano ad evitare questa pratica, il coinoculo può essere considerato lo scenario migliore per assicurare una rapida conclusione della FML e alti livelli di espressione del gene codificante per l’enzima β-glucosidasi, che può condurre al rilascio di composti aromatici volatili favorevoli nel vino
Microbial spoilage of traditional dry sausages produced in small-scale facilities in Friuli, a northeastern region of Italy.
No full textThe aim of this paper was to determine the causes of spoilage and odorous compounds in two different lots of traditional Friuli sausages produced by two small-scale facilities in San Daniele del Friuli. In spoiled sausages the water activity (Aw) was higher than in unspoiled sausages (0.94 vs. 0.91). Moreover, the pH of the spoiled tested sausages remained high despite the presence of acidifying bacteria (6.5±0.4 log CFU g -1 ), such as lactic acid bacteria (LAB) and coagulase negative catalase positive cocci (CNCPC). Conversely, in unspoiled sausages the pH value was 5.5±0.02. For this reason, the spoilage (mephitic type) can be attributed to the high pH (6.3±0.2 units) of the pork meat used, which was classified as a dark, firm, dry type (DFD), to the high Aw, and to the activity of LAB, CNCPC cocci, and Enterobacteriaceae. Because of the high pH and the lack of naturalor added sugars, these bacteria degraded the proteins via amino acid metabolism to produce ammonia, hydrogen sulphur, and methanethiol, which were responsible for the mephitic and putrid odour of the sausages
Use of propidium monoazide for the enumeration of viable Oenococcus oeni in must and wine by quantitative PCR.
No full textMalolactic fermentation is an important step in winemaking, but it has to be avoided in some cases. It’s
carried out by lactic acid bacteria belonging mainly to the genus Oenococcus, which is known to be a slow
growing bacterium. Classical microbiological methods to enumerate viable cells of Oenococcus oeni in
must and wine take 7e9 days to give results.
Moreover, RT-qPCR technique gives accurate quantitative results, but it requires time consuming steps
of RNA extraction and reverse transcription.
In the present work we developed a fast and reliable quantitative PCR (qPCR) method to enumerate
cells of Oenococcus oeni, directly, in must and wine. For the first time we used a propidium monoazide
treatment of samples to enumerate only Oenococcus oeni viable cells. The detection limit of the developed
method is 0.33 log CFU/mL (2.14 CFU/mL) in must, and 0.69 log CFU/mL (4.90 CFU/mL) in wine,
lower than that of the previously developed qPCR protocols
Use of propidium monoazide for the enumeration of viable Brettanomyces bruxellensis in wine and beer by quantitative PCR
No full textBrettanomyces bruxellensis is a current problem in winemaking all over the world, and the question if B.bruxellensis has a positive or negative impact on wine is one of the most controversial discussions in the world. The presence of live B.bruxellensis cells represents the risk of growth and an increase in cell numbers, which is related to the potential production of volatile phenols. In this work, the optimisation of a PMA-quantitative PCR (qPCR) method to enumerate only viable cells was carried out using the standard strain B.bruxellensis DSMZ 70726. The obtained detection limits were 0.83logCFU/mL in red wine, 0.63logCFU/mL in white wine and 0.23logCFU/mL in beer.Moreover, the quantification was also performed by Reverse Transcription quantitative PCR (RT-qPCR), and the results showed a higher detection limit for all of the trials
Prevention of Penicillium nordicum growth and Ochratoxin A on Sausages with ozonated air
No full textMolds play an important role in flavor of fermented sausage production. However mycotoxinogenic
species are common sausage contaminants, too. Penicillium nordicum, represents the main
toxinogenic species isolated from artisanal sausages of different area of Lombardy, a region in the
North of Italy, and of Europe. P.nordicum growing on the surface of sausages, produces Ochratoxin
A (OTA), which penetrating the meat, can be a potential health risk for consumers. The presence of
OTA on casing represent an additional risk, because in Italy consumers are used to eat both meat
and casing of artisanal sausages. To avoid the growth of P.nordicum does not appear effective in
reducing OTA, in particular in artisanal facilities where it is impossible to check the parameters of
Production (Umidity Relative, Temperature, Time). Therefore other systems are needed to prevent
its growth to eliminate OTA from sausage. Different methods are proposed, including brushing and
washing sausages or by spreading starter cultures on the casings before drying and ripening
(Iacumin et al., 2009). By applying both systems, OTA concentration was reduced but nevertheless
it is used to find artisanal sausages with OTA on the casing or inside the meat. Moreover the use of
starter cultures is not choose by some producers, because they think that the sausages must be done
without any additives or ingredients excepted salt, pepper and nitrate and nitrite. Considering the
OTA (Italian Law limit < 1 μg/Kg) and A.ochraceus represent a problem for artisanal sausages, the
aim of this study was to prevent the growth of P.nordicum to eliminate the risk of OTA in sausages
with the use of ozonated air during the first stage of ripening. The use of ozonated air has prevented
the growth of P.nordicum and consequently the presence of OTA on sausages. This is the second
report of the use of ozonated air to limit mold growth on meat products (Iacumin et al., 2012). The
experiment demonstrated that ozone is an effective method to prevent potentially toxigenic mold
contamination and growth. At the end of ripening, no OTA was found on casings of treated samples
whereas it was found on not-treated ones ( > 1 μg/Kg). The use of ozone did not influence the
ripening, the physico-chemical parameters, the peroxide value and the sensorial characteristic of the
sausages. Our data suggest that the presence of OTA on sausage surfaces could represent a food
health risk only when the molds are not removed from casings. To reduce the health risk, for
consumers that are used to eat both meat and casing of artisanal sausages, we suggest to limit the
growth by treatment with ozonated air during drying and ripening. However, despite, these good
result, we think that the use of starter molds is the best technology to eliminate the risk of OTA and
OTA producing mold. To eliminate completely the risk, it is sufficient to inoculate starter mold
cultures after the casing and after the end of drying. In this way the mold cultures inhibites the OTA
producing molds during all the period of ripening.
Keywords: fermented Sausages, Ochratoxin A, Penicillum nordicum, ozonated ai
Alterazioni di prosciutti cotti prodotti con tecnologia Cook-in e Ship-in.
No full textThe spoilage of cooked hams obtained
from Cook-in and Ship-in technology
was investigated. The spoilage was due
to the growth of Lactobacillus sakei and
Leuconostoc mesenteroides, which grew
during the production and the 4°-6°C
storage period of cooked hams. The spoilage
consisted in the production of cavities, due
to CO2, greening and various molecules:
hydrocarbons, aldehydes, ketones,
carboxylic acids, alcohols, esters and
sulphur compounds
Evaluation of express level of mleA gene in Oenocuccs oeni under different experimental conditions
No full textMalolactic fermentation usually occurs simultaneously or after alcoholic fermentation and is known to improve wine quality trough deacidification, production of desirable flavors and aromas, and enhancement of microbial stability.
In this study we developed a RT-qPCR (reverse transcription quantitative PCR) method to evaluate the gene expression levels of mleA gene in Oenococcus oeni, in a synthetic medium, under different experimental conditions such as different concentrations of malic acid, ethanol, lactic acid. Samples were collected four times a day until the end of malolactic fermentation, and subjected to plate count evaluation, RT-qPCR and enzymatic assays for malic and lactic acid determination, sugars and ethanol.
All trials were carried out in triplicate. Gene expression levels were calculated using the MNE (mean normalized expression) method, according to the MIQE.
Results show that in absence of malic acid mleA gene expression remains stable at a baseline level, while in presence of malic acid and ethanol the expression profile shows a positive peak at the moment of maximum slope of the curve of decrease in concentration of malic acid. Conversely, in presence of lactic acid the expression profile is stable to the baseline level.
In conclusion, ethanol do not affect the gene expression profile, while lactic acid seems to be involved in gene regulation of mleA
MABS validation through repeated execution and data mining analysis
No full textAgent Based Modelling is the most interesting and advanced approach for simulating a complex system: in a social context, the single parts and the whole are often very hard to describe in detail. Besides, there are agent based formalisms which allow to study the emergency of social behaviour with the creation and study of models, known as artificial societies. Thanks to the ever increasing computational power, it's been possible to use such models to create software, based on intelligent agents, which aggregate behaviour is complex and difficult to predict, and can be used in open and distributed systems. Data mining is born in the last decades in order to help users in finding useful knowledge from the otherwise overwhelming amount of data available nowadays from the web and the data collected every day by companies. Data Mining techniques can therefore be the keystone to reveal non-trivial knowledge expressed by the initial assumption used to build the micro-level of the model and the structure of the society of agents that emerged from the simulation
Use of propidium monoazide (PMA) for the enumeration of viable Brettanomyces bruxellensis in wine and beer by quantitative PCR.
No full text- …
