Laboratori de Microbiologia

El document forma part dels materials docents programats mitjançant l'ajut del Servei de Política Lingüística de la Universitat de València - Idioma ValenciàAquest document ofereix una visió específica de les diferents pràctiques de laboratori que es desenvolupen dins de l´àrea de Microbiologia. L´objectiu és proporcionar a l´alumnat un material gràfic i de contingut, en referència als medis de cultiu, eines, equipament, procediments i bacteris més comunament assajats al laboratori, facilitant la transmissió de coneixements dins d´aquest camp d´estudi. Aquest material proporciona també discussió i contextualització dels problemes i experiments plantejats al laboratori, justificant així la importància dels coneixements que es pretenen assolir

Mètodes d´identificació bacteriana al laboratori de microbiologia

El document forma part dels materials docents programats mitjançant l'ajut del Servei de Política Lingüística de la Universitat de ValènciaEl present document fa una revisió completa dels mètodes emprats amb més freqüència al laboratori de microbiologia, en relació a la identificació bacteriana, fent ús, fonamentalment, de: 1-Mètodes fenotípics 2-Mètodes moleculars 3-Mètodes proteòmics Amb aquest document es pretén donar a l´alumne eines de coneixement bàsic, que puga emprar tant en l´activitat acadèmica com professional de futur.Main phenotypic, molecular and proteomic techniques, used in bacterial identification, are reviewed in the present document. By means the scientific information included, basic knowledge and new skills can be acquired by the students with the aim to be applied in the development of their future professional careers

Helicobacter pylori Is Present at Quantifiable Levels in Raw Vegetables in the Mediterranean Area of Spain

[EN] Vegetables are one of the main foodstuffs consumed in the Mediterranean diet. However, raw vegetables have been associated with relevant foodborne outbreaks worldwide. Accurate knowledge of the microbiological quantitative risks associated with these matrices is crucial in order to define effective control measures, avoiding the survival and dissemination of foodborne pathogens through the different food chain stages. The aim of the present study is the assessment of the prevalence of Helicobacter pylori (a unique carcinogenic biological agent recognized to date) on leafy vegetables (spinach, lettuce, and chard) by means of the detection of the specific pathogenicity vacA gene. A real-time quantitative polymerase chain reaction (qPCR) optimized approach was used to detect H. pylori-positive samples and the concentration of this pathogen (with a limit of detection equal to 10 cells). One hundred raw vegetable samples were acquired in markets corresponding to the Spanish Mediterranean area. Sliced vegetable leaves were homogenized and centrifuged, and DNA was extracted from the homogenates. qPCR results confirmed 20 out of 100 H. pylori-positive samples, with melting temperature (T-m) values in the range of 84.8-86.5 degrees C (T-m vacA H. pylori = 85 degrees C). Amplicons were cut, purified, and sequenced to confirm the homology with the H. pylori vacA gene. A total of 17 out of 100 vegetable samples (12/45 (26.6%) lettuce, 2/21 (9.5%) spinach, and 3/34 (8.8%) chard samples) were finally confirmed as H. pylori-positive. Contamination levels were in the range of 1.5 +/- 0.3 to 2.5 +/- 0.1 log(10) cycles (36-335 CFU/g leafy vegetables). Our results show that H. pylori is detected by qPCR at levels close to infectious doses in fresh vegetables, thus posing a food safety hazard.This research was funded by the Ministerio de Ciencia e Innovacion, Spain, grant number PID2019-105691RB-I00. M. Garcia-Ferrus is the recipient of a PEJ2018-003746A grant from Ministerio de Economia, Industria y Competitividad, Spain. Prof. Pina-Perez is also grateful to the GVA202/031 grant project.García-Ferrús, M.; González Pellicer, A.; Pina-Pérez, MC.; Ferrús Pérez, MA. (2022). Helicobacter pylori Is Present at Quantifiable Levels in Raw Vegetables in the Mediterranean Area of Spain. Agriculture. 12(3):1-7. https://doi.org/10.3390/agriculture120303391712

Escherichia coli O157:H7 and Salmonella Typhimurium inactivation by the effect of mandarin, lemon, and orange by-products in reference medium and in oat-fruit juice mixed beverage

The antimicrobial capability of three water extracts of citrus peels was evaluated against Salmonella enterica serovar Typhimurium and Escherichia coli O157:H7 at various concentrations (0.5, 1, 5, 10%) and temperatures (5, 10, 22 °C) in a reference medium. The best of them was mandarin by-product, achieving a maximum inactivation level against S. Typhimurium (8 log10 cycles) with 5% at 5 °C. Also, this by-product had the highest total polyphenol content. Mandarin by-product showed a bactericidal effect in a food matrix also at 5 °C (≈2 log10 cycles). All results were adjusted to the Weibull model and the b values indicated that the higher concentration of mandarin, the greater the inactivation rate in reference medium, without significant differences between 5 and 10%. Similarly, in the food matrix, the inactivation rate of S. Typhimurium was higher when the mandarin by-product was added. Therefore, the mandarin by-product could be used as a control measure of S. Typhimurium in pasteurized products, which are stored under refrigeration.M. Sanz-Puig is grateful to the CSIC for providing a contract as a researcher working actively on the projects with reference IPT-2011-1724-060000 and AGL 2013–48993-C2-2-R, funded by the Ministry of Economy and Competitiveness and with FEDER funds. M.C. Pina-Pérez is grateful to the CSIC for providing a doctoral contract.Peer reviewe

Combined effect of high hydrostatic pressure (HHP) and antimicrobial from agro-industrial by-products against S. typhimurium

The inactivation potential of HHP treatment (200 MPa-2 min) was evaluated against Salmonella enterica serovar Typhimurium in cauliflower and mandarin by-product infusions at 37 and 10 °C. By-product infusions exerted a strong antimicrobial effect used alone, achieving 5 log cycles of bacterial reduction for cauliflower by-product infusion after 10 h and for mandarin by-product infusion after 80 h, at 37 °C. The HHP treatment caused only one log cycle of cellular damage, but when inoculated cauliflower or mandarin by-product infusions were subjected to HHP treatment the antimicrobial effect against S. Typhimurium was enhanced, achieving 5 log cycles of inactivation in 6 h at 37 °C in both cases. Inactivation curves were adjusted to the Weibull equation and the kinetic parameters (b and n) were obtained. When HHP treatment was combined with by-product infusions, the inactivation rates were greater than when either of the by-product infusions was added separately. In conclusion, a synergistic antimicrobial effect against S. Typhimurium appeared to take place when HHP treatment was combined with cauliflower or mandarin by-product infusion. These infusions could be considered as an additional microbial control measure to guarantee the food safety and food quality of pasteurized food products that are stored under refrigeration.M. Sanz-Puig is grateful to the CSIC for providing a contract as a researcher working actively on project AGL 2013–48993-C2-2-R. The present research work was funded by the Ministry of Economy and Competitiveness and with FEDER funds (AGL 2013-48993-C2-2-R).Peer reviewe

Use of Antimicrobials from Plants in Feed as a Control Measure for Pathogenic Microorganisms

Animal Feed has become an increasing critical component of the integrated food chain, in 2010 about 1000 mt of animal feed was produced globally and 150 mt in the EU27. The animal feed has an important impact in the human health. The farm or feedlot is the origin of microorganisms introduced onto carcasses during slaughter and dressing. It appears that changes in diet and management practices could precipitate increased shedding of pathogens. Additionally, antibiotics are used in animals, not only for treatment or prevent diseases, but also to promote growth. As a result of the use of antibiotics, food can contain antibiotic-resistant bacteria and resistance genes with important public health consequences. Although antibiotics are banned as growth promoters in the European Union and some other countries, this is not the case throughout the WHO European Region. Travel and the globalization of trade further increase the risk of spreading antibiotic-resistant bacteria.Peer reviewe

Effect of pulsed electric fields (PEF) combined with natural antimicrobial by-products against S. typhimurium

The effect against Salmonella enterica serovar Typhimurium of PEF treatment combined with cauliflower and mandarin by-product infusions at several concentrations (0, 1, 5, and 10% (w/v)) was evaluated at various incubation temperatures (10, 22, and 37 °C). The possible synergistic antimicrobial action of the combined process of Pulsed Electric Field (PEF) technology followed by exposure to the by-product infusions and the occurrence of sublethal cellular damage were also studied. Antimicrobial kinetics of by-product infusions alone or following PEF treatment were fitted to a Weibull model. Both mandarin and cauliflower by-product infusions showed a maximum antimicrobial effect against S. Typhimurium after 10 h at 37 °C when the microorganism was exposed to 10% of by-product infusion, achieving reductions of initial bacterial load up to undetectable levels. The effect of the PEF treatment (20 kV–900 μs) caused a reduction of 4 log cycles of the initial cell population (108 cfu/mL) of S. Typhimurium and 1 log cycle (90%) of cellular damage. Moreover, when the PEF pre-treated S. Typhimurium population was subjected to subsequent incubation in the presence of both by-product [10%] infusions, the microbial inactivation was faster, achieving a reduction of the initial bacterial load (4 log10 cycles) up to undetectable levels in 2 h. The kinetic values of the Weibull model were obtained. The higher the concentration of by-product infusion, temperature, and PEF treatment applied, the greater the kinetic parameter “b” values, which are related to the microbial inactivation rate. Therefore, the addition of cauliflower and mandarin by-product infusions could be a good additional control measure contributing to ensure bacterial counts below recommended limits in pasteurized PEF products during their storage at refrigeration temperatures.M. Sanz-Puig is grateful to the CSIC for providing a contract as a researcher working actively on project AGL 2013-48993-C2-2-R. The present research work was funded by the Ministry of Economy and Competitiveness through project AGL 2013-48993-C2-2-R and with FEDER funds. We are also grateful to INDULLEIDA, S.A. and TRASA, S.L. for providing the by-products that we worked with. Authors acknowledge L. Santos-Carvalho Erasmus Placement scholarship, and L.M. Cunha acknowledges support from the European Union (FEDER funds) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministerio da Educação e Ciência) under the Partnership Agreements PT2020 UID/QUI/50006/2013–POCI/01/0145/FEDER/007265 (LAQV) and UID/AGR/04033/2013–POCI/01/0145/FEDER/006958 (CITAB).Peer reviewe

Improvement of the probiotic growth-stimulating capacity of microalgae extracts by pulsed electric fields treatment

This study aimed to investigate the nutritional character (carbohydrates, proteins, pigments, and phycocyanin), total antioxidant capacity (TAC) and the capability of simulating the growth of Lactobacillus rhamnosus of different extracts from C. vulgaris and A. platensis by means of the application of conventional aqueous extraction procedure and pulsed electric field (PEF) extraction technology. It was confirmed a significantly improved nutritional profile of Chlorella and Spirulina extracts obtained by PEF technology pre-treatment (3 kV/cm, 100 kJ/kg), with specifically higher values in total carbohydrate, Chlorophyll a, Chlorophyll b, and carotenoids content, and TAC. Additionally, Spirulina PEF extract showed a probiotic's growth-stimulating capability of 1 log10 cycle when fermented by Lactobacillus rhamnosus, with a metabolomic profile specifically rich in bioactive short chain fatty acids (SCFAs) and organic acids (3-phenyl lactic acid). The present study points out the applicability of PEF extraction technology under optimized conditions to improve the nutritional and functional character of microalgae and cyanobacterial-derived ingredients

Listeria Monocytogenes inactivation kinetics under microwave and conventional thermal processing in a kiwifruit puree

The inactivation of Listeria monocytogenes in a kiwifruit puree by conventional and microwave heating was studied. Survival curves at three microwave power levels (600 1000 W) and three temperatures (50 60 °C) were obtained. Data were properly fitted by a first-order kinetic model. Processing times under both technologies were corrected to isothermal treatment for the kinetic study. Microwave heating was shown to effectively inactivate L. monocytogenes. In the range of microwave and conventional processing conditions assayed, the 5-log10 reductions of L. monocytogenes recommended by the FDA for pasteurized products were achieved. The level of microwave power applied had a considerable influence on the Listeria monocytogenes inactivation rate. The higher the power level, the faster the inactivation. The inactivation of Listeria monocytogenes under microwave heating at 900 W (D60°C=17.35 s) and 1000 W (D60°C=17.04 s) happened faster than in a conventional thermal process (D60°C=37.45 s). Consequently, microwave heating showed greater effectiveness for Listeria monocytogenes inactivation than conventional heating.The authors thank the Ministerio de Educacion y Ciencia for the financial support given through Projects AGL 2010-22176 and AGL 2010-22206-C02-01 and the Generalitat Valenciana for the financial support given through Project ACOMP/2012/161 and the Grant awarded to the author Maria Benlloch.Benlloch Tinoco, M.; Pina Pérez, MC.; Martínez Navarrete, N.; Rodrigo Aliaga, MD. (2014). Listeria Monocytogenes inactivation kinetics under microwave and conventional thermal processing in a kiwifruit puree. Innovative Food Science and Emerging Technologies. 22:131-136. https://doi.org/10.1016/j.ifset.2014.01.005S1311362