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.
The aim of this study was to investigate whether the biofilm-forming ability and/or the disinfectant susceptibility accounted for the persistence of Listeria monocytogenes in Gorgonzola cheese processing plants. For this purpose, a set of 16 L. monocytogenes isolates collected in the 2004-2007 period was analyzed, including 11 persistent isolates collected in different years, within the collection period, and displaying identical or highly correlated pulsotypes. The evaluation of biofilm-forming ability was assessed using crystal violet (CV) staining and the enumeration of viable cells on stainless steel coupons (SCC). Absorbance values obtained with CV staining for persistent and non-persistent isolates were not significantly different (rm-ANOVA p>0.05) and the cell counts from non-persistent isolates showed to be higher than of persistent isolates (rm-ANOVA p<0.05). A simulation of disinfectant treatments was performed on spot inoculated coupons in clean and dirty conditions, according to EN 13697, and on biofilms on SSC, grown in nutrient-rich (dirty) and limiting (clean) conditions using acid acetic-hydrogen peroxide (P3) and acid citric-hydrogen peroxide (MS) commercial disinfectants. The treatment was considered effective when a 4 Log reduction in viable cell count was observed. The Log reductions of persistent and non-persistent isolates, obtained with both the assays in clean and dirty conditions, were compared and no significant differences were detected (rm-ANOVA p>0.05). A greater influence of organic matter on MS could explain why P3 was efficient in reducing to effective levels the majority of the isolates at the lowest concentration suggested by the manufacturer (0.2% [v/v]), while the same purpose required an higher concentration (1% [v/v]) of MS.In conclusion, our results demonstrate that the persistence of these isolates in Gorgonzola cheese processing plants was linked neither to the biofilm forming ability nor to their susceptibility to hydrogen peroxide-based disinfectants, therefore other factors should contribute to the persistent colonization of the dairies.
Five L. innocua and five L. monocytogenes, including persistent and non-persistent isolates collected from Gorgonzola processing plants, were compared regarding their biofilm-forming ability and their biofilm susceptibility to two hydrogen peroxide (HP) based disinfectants in use at the plants. No significant difference in biofilm-forming ability by both species was observed (P>0.05) in crystal violet staining and viable count assays. The susceptibility to HP disinfectants of the L. monocytogenes and L. innocua biofilms was determined. In order to mimic clean and soiled biofilm forming conditions, biofilms were grown, respectively, in 1/10 diluted TSB-YE and in TSB-YE. The results showed no significant differences between species or conditions (P>0.05) regardless of whether the isolates were classified as persistent or non-persistenttb. A hierarchical clustering based on Principal Component Analysis performed on the tested variables, indicated the presence of two major clusters. Persistent and non-persistent isolates from both species were allocated in both clusters, suggesting that they behaved in a similar way in response to the tested conditions. This study showed that biofilms of in-house L. innocua could monitor the effectiveness of HP-based disinfectants. Moreover, biofilms of L. innocua could be used as surrogates of L. monocytogenes in sanitizer-based biofilm eradication trials simulating dairy processing environments, whenever the use of the pathogen is not an option.
Previously pulsed field gel electrophoresistyped Listeria monocytogenes isolates (N095) were tested by repetitive element sequence-based PCRs (repPRCs). A combined rep-PCR typing approach showed 95 % repeatability, 0.98 discriminatory power, 95.5 % sensitivity, 75 % specificity, 91.2 % predictive positive value, and 85.7 % predictive negative value. Hence, rep-PCR represents an efficient and rapid subtyping method for L. monocytogenes.
The ability of Listeria monocytogenes to survive in different environments and establish persistent contaminations is an important issue for food producers. This study aimed to assess the environmental contamination level in an Italian salami producing plant and to identify possible sources of contamination using pulsed field gel electrophoresis (PFGE) on L. monocytogenes isolates obtained from environmental (n=54) and meat samples (n=9) collected over 9 months. Detection of L. monocytogenes was performed using the UNI EN ISO 11290-1 procedure and every isolate was characterised with PFGE, using AscI and ApaI restriction enzymes. The environmental detection frequencies were constant both in the first (22%) and the second (27%) visit, thus suggesting the presence of strains adapted to the processing plant. Equipments can represent a reservoir of L. monocytogenes from which it can spread into the whole producing plant. The reservoir was documented by PFGE results which showed several persistent strains. Moreover, PFGE proved the cross-contamination between surfaces and semiprocessed products like pastes, which furthermore have been contaminated by L. monocytogenes in 100% of samples in the first two visits and in 33% in the last visit. This study gave evidence that detection methods and PFGE characterisation can be effective tools to detect possible sources and routes of contamination
RESUMO -O presente trabalho teve como objetivo estudar a produção de lipase pela linhagem mutante Aspergillus niger 11T53A14 por fermentação em estado sólido utilizando diferentes subprodutos agroindustriais (borra de café, polpa de macaúba e amêndoa da manga). A fermentação foi realizada em Erlenmeyer de 250 mL contendo 15 g de resíduo inoculados com uma suspensão 10 7 conídios/mL e conduzidos em estufa a 32 o C por 48 horas. Os experimentos foram conduzidos segundo um planejamento fatorial fracionado 2 5-1 , onde as variáveis estudadas foram: volume da solução de HCl 0,1 mol/L, concentração de nitrogênio (sulfato de amônio) e concentração dos subprodutos. As variáveis: concentração de nitrogênio e umidade (volume da solução de HCl) foram estatisticamente significativas. A maior atividade lipásica (81,16 U/g) foi obtido em meio de cultivo composto por 80 mL/100g de meio da solução de HCl, 0,4% de nitrogênio do sulfato de amônio e 11% de borra de café. INTRODUÇÃOAs lipases são enzimas utilizadas em vários setores industriais com ampla aplicação. São classificadas como hidrolases (triacilglicerol acil hidrolases, EC 3.1.1.3) e caracterizam-se por catalisar uma variedade de reações (Jensen, 1983). As lipases de fungos filamentosos são preferencialmente utilizadas para aplicações industriais devido à viabilidade de obtenção, entre os fungos, Aspergillus niger é conhecido como um dos melhores produtores de enzimas extracelulares (Mahadik et al., 2004). A fermentação em estado sólido envolve o crescimento e o metabolismo do substrato sólido úmido na ausência ou quase ausência de água livre e possui algumas vantagens frente a fermentação submersa, tais como: produtividade elevada, custos operacionais reduzidos, meios de fermentação facilitados, equipamentos mais simples e uso de subprodutos agroindustriais como substrato principal (Contesini, 2009). Os subprodutos agroindustriais são geralmente considerados os melhores substratos para a produção de lipase por fermentação em estado sólido, uma vez que são abundantes, baratos e contêm fontes de carbono, nitrogênio e minerais. A borra de café é derivada do preparo da bebida e possui cerca de 18% de óleo (Azevedo, 2007). A macaúba é obtida da extração do óleo e é rica em proteínas, lipídios e carboidratos (Amaro et al., 2015). Os subprodutos do processamento agraindustrial da manga (casca, amêndoa e semente) são gerados em grandes quantidades , eles apresentam concentrações
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