Comparison of real-time PCR and nested PCR based on the HlyA gene for the detection of Listeria monocytogenes. Application on cheese samples.

The aim of the present study was to compare the performance of a nested polymerase chain reaction (nPCR) and a real-time PCR based on the amplification of the HlyA gene from Listeria monocytogenes using a plasmid DNA standard. Nested PCR was developed with an internal amplification control (IAC). Both techniques were validated in soft cheese samples by comparing their results with the results of the microbiological reference method ISO 11290-1:2017. Cheese samples artificially contaminated with 3.5 to 3,500 UFC/25 g were processed by ISO 11290-1:2017 and, at several times of culture, DNA samples were extracted. All cheeses contaminated with L. monocytogenes were positive for the microbiological method 96 h post contamination and for nPCR and real-time PCR 48 h post contamination. At this time, the HlyA gene was amplified in all contaminated samples. Both molecular techniques showed the same sensitivity, 30 copies/reaction or 3.5 UFC/25 g, when plasmid DNA standard or artificially contaminated cheese samples were used. Finally, eighty soft cheese samples obtained from local retail stores and tested by three methods were negative, indicating a 100% concordance in results. The development of an nPCR with IAC reinforces the reliability of the negative results without increasing the costs of the reaction. Besides, nPCR showed less sensitivity to the presence of inhibitory substances in the reaction. The use of one of these molecular techniques could be easily coupled to the microbiological method, serving as a screening method in the food industry for hygiene monitoring and early identification of contaminated foods.

Thermosonication as a pretreatment of raw milk for Minas frescal cheese production.

Minas frescal cheese is extremely popular in Brazil, with high perishability and acceptability. Among emerging technologies, ultrasound stands out for its satisfactory results regarding microbiological safety and technological and sensory aspects. The combined mild temperature application, called thermosonication, can generate even more promising results. In this study, a high-intensity ultrasound system combined with thermal heating (TS, thermosonication) was applied for the treatment of raw milk to produce Minas Frescal cheese. US energy was delivered to raw milk samples using a probe operating at a 20 kHz of frequency and nominal power of 160, 400, and 640 W. The TS system was compared with conventional pasteurization (HTST, high-temperature short-time pasteurization) at 72 to 75 °C and 15 s. Soft cheeses were prepared with different samples: (a) raw milk (control), b)conventionally pasteurized milk (HTST), and c) TS treat milk in different nominal power (TS160, TS400, and TS640). The produced cheeses were evaluated for microbiological behavior, rheology, color parameters, and bioactive compounds. TS treatment in milk resulted in higher microbial inactivation and stability during storage, improved color parameters (higher lightness (L*), and whiteness index (WI). TS treatment also showed a higher generation of bioactive compounds (higher antioxidant, and inhibitory activities of α-amylase, α-glucosidase, and angiotensin-converting enzymes) than HTST. The impact of TS on rheological properties was similar to HTST, resulting in more brittle and less firm products than the cheese produced with raw milk. The positive effects were more prominent using a nominal power of 400 W (TS400). Therefore, TS proved to be a promising process for processing milk for Minas Frescal cheese production.

Detection and genotyping of Listeria monocytogenes in artisanal soft cheeses from Ecuador.

Listeria monocytogenes is one of the most important bacteria associated with foodborne diseases, soft cheese being an important L. monocytogenes vehicle. In Ecuador, soft cheese is consumed in 84.3% of urban households. We determined the prevalence of L. monocytogenes and serogroups in 260 fresh artisanal soft cheese samples collected in 18 of 24 Ecuadorian provinces. Listeria spp. detection was carried out by culture-dependent and independent methods; 14.23% of samples were positive for L. monocytogenes. Serogroup IVb was found in 83.78% of the food isolates. Serogroups IIb and IIa were present in 8.11% of our isolates. To our knowledge, this is the first report of L. monocytogenes serogroups associated with food in Ecuador; we also found serogroup similarities between cheese isolates and clinical isolates.

Effect of flaxseed (Linum usitatissimum) and soybean (Glycine max) oils in Egyptian lactating buffalo and cow diets on the milk and soft cheese quality

Produce and compare soft cheese with potential benefits of human health from Egyptian buffalo's and cow's milk was studied. Eight Egyptian lactating buffalos and cows were fed a total mixed ration supplemented with either 0% oil (CD), 2% flaxseed oil (DFO), 2% soybean oil (DSO), or 2% of their mixture (1:1, DFSO) according to a double 4 x 4 Latin Square design. Milk yield was similar between buffalo's diets but was higher in cows fed a DFO, DSO or DFSO resulting in 11.15, 8.21% or 8.97% increases compared with the control diet, respectively. Milk composition was not significantly affected in both buffalos and cows fed diets. The DFO, DSO or DFSO displayed decreased short-chain fatty acids, especially DSO and DFSO (3.73 and 3.33%, respectively) when compared to CD for buffalo milk (6.32%). The DSO and DFSO were more effective for increasing unsaturated fatty acids followed by the DFSO in buffalo's milk fat (42.31 and 41.90 %), whereas DFO and DFSO were more effective in cow's milk fat (39.67 and 39.84%), respectively. DFO, DSO or DFSO had no significant effect on the yield, composition and sensory properties of resultant soft cheese compared to the CD for both lactating cows and buffalos. During storage, a diet rich in unsaturated fatty acids enhances protein proteolysis and antioxidant activity of soft cheese during storage compared to the CD especially for soft cheese produced from buffalo's milk.(AU)

Effect of flaxseed (Linum usitatissimum) and soybean (Glycine max) oils in Egyptian lactating buffalo and cow diets on the milk and soft cheese quality

Produce and compare soft cheese with potential benefits of human health from Egyptian buffalo's and cow's milk was studied. Eight Egyptian lactating buffalos and cows were fed a total mixed ration supplemented with either 0% oil (CD), 2% flaxseed oil (DFO), 2% soybean oil (DSO), or 2% of their mixture (1:1, DFSO) according to a double 4 x 4 Latin Square design. Milk yield was similar between buffalo's diets but was higher in cows fed a DFO, DSO or DFSO resulting in 11.15, 8.21% or 8.97% increases compared with the control diet, respectively. Milk composition was not significantly affected in both buffalos and cows fed diets. The DFO, DSO or DFSO displayed decreased short-chain fatty acids, especially DSO and DFSO (3.73 and 3.33%, respectively) when compared to CD for buffalo milk (6.32%). The DSO and DFSO were more effective for increasing unsaturated fatty acids followed by the DFSO in buffalo's milk fat (42.31 and 41.90 %), whereas DFO and DFSO were more effective in cow's milk fat (39.67 and 39.84%), respectively. DFO, DSO or DFSO had no significant effect on the yield, composition and sensory properties of resultant soft cheese compared to the CD for both lactating cows and buffalos. During storage, a diet rich in unsaturated fatty acids enhances protein proteolysis and antioxidant activity of soft cheese during storage compared to the CD especially for soft cheese produced from buffalo's milk.

A comparison of dynamic tertiary and competition models for describing the fate of Listeria monocytogenes in Minas fresh cheese during refrigerated storage.

This study compares dynamic tertiary and competition models for L. monocytogenes growth as a function of intrinsic properties of a traditional Brazilian soft cheese and the inhibitory effect of lactic acid bacteria (LAB) during refrigerated storage. Cheeses were prepared from raw or pasteurized milk with or without the addition of selected LAB with known anti-listerial activity. Cheeses were analyzed for LAB and L. monocytogenes counts, pH and water activity (aw) throughout cold storage. Two approaches were used to describe the effect of LAB on L. monocytogenes: a Huang-Cardinal model that considers the effect of pH and aw variation in a dynamic kinetic analysis framework; and microbial competition models, including Lotka-Volterra and Jameson-effect variants, describing the simultaneous growth of L. monocytogenes and LAB. The Jameson-effect with γ and the Lotka-Volterra models produced models with statistically significant coefficients that characterized the inhibitory effect of selected LAB on L. monocytogenes in Minas fresh cheese. The Huang-Cardinal model [pH] outperformed both competition models. Taking aw change into account did not improve the fit quality of the Huang-Cardinal [pH] model. These models for Minas soft cheese should be valuable for future microbial risk assessments for this culturally important traditional cheese.

Exploiting antagonistic activity of fruit-derived Lactobacillus to control pathogenic bacteria in fresh cheese and chicken meat.

This study assessed the antagonistic activity of fruit-derived lactic acid bacteria (LAB) strains against food-related bacteria and the effects of the highest organic acids LAB producers on the survival of Listeria monocytogenes and Salmonella Enteritidis PT4 in cheese and chicken meat, respectively. The production of organic acids by the Lactobacillus strains in the tested food matrices was also monitored. All tested LAB strains showed antagonistic activity in vitro on the growth of pathogenic or spoiling food-related bacteria, particularly on L. monocytogenes and/or S. Enteritidis PT4, through the action of non-proteinaceous substances. The highest amounts of acetic and lactic acid were detected in cell free culture supernatants of L. paracasei 108 and L. plantarum 201. In "Minas Frescal" cheese, L. plantarum 49 and L. paracasei 108 decreased the counts of L. monocytogenes, and L. plantarum 201 showed bacteriostatic effects on this pathogen over time. L. paracasei 108 decreased the counts of S. Enteritidis PT4 in ground chicken breast; L. plantarum 49 and L. plantarum 201 failed to decrease the counts of this pathogen. Decreases in counts of L. monocytogenes or S. Enteritidis in "Minas Frescal" cheese and ground chicken breast, respectively, were related with increases in lactic and acetic acid contents and decreases in pH values. L. plantarum 49 and L. paracasei 108 could be used as biopreservation tools in cheese and chicken breast meat, respectively.

Antimicrobial activity of essential oils against Vancomycin-Resistant enterococci (VRE) and Escherichia coli O157: H7 in feta soft cheese and minced beef meat

Eleven essential oils (EOs) were evaluated for their antibacterial properties, against Vancomycin-Resistant Enterococci (VRE) and E. coli O157:H7. EOs were introduced into Brain Heart Infusion agar (BHI) (15ml) at a concentration of 0.25 to 2 percent (vol/vol) to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for each pathogen evaluated. Results showed that the most active essential oils against bacteria tested were thyme oil, with MIC90 and MBC90 for the VRA strains of 0.25 percent and 0.5 percent, respectively. Eucalyptus, juniper and clove oils were the least potent agent, with MIC90 and MBC90 of 2 percent. Furthermore, the inhibitory effect of these EO were evaluated against VRE and E. coli O157:H7, experimentally inoculated (10³ cfu/g) in Feta soft cheese and minced beef meat, which was mixed with different concentrations (0.1 percent, 0.5 percent and 1 percent) of the EO and stored at 7 ºC for 14 days. Out of eucalyptus, juniper, mint, rosemary, sage, clove and thyme oils tested against target bacteria sage and thyme showed the best results. Clove and mint did not show any effect on VRE and E. coli O157:H7 in both kinds of studied foods. The addition of thyme oil at concentrations of 0.5 and 1 percent caused best significant reduction in the growth rate of VRE and E. coli O157:H7 in cheese and meat at 7 ºC. It is concluded that selected plant EOs can act as potent inhibitors of both microorganisms in a food product. The results revealed the potential of thyme oil as a natural preservative in feta soft cheese and minced beef meat against VRE and E. coli O157:H7 contamination.

Antimicrobial activity of essential oils against vancomycin-resistant enterococci (vre) and Escherichia coli o157:h7 in feta soft cheese and minced beef meat.

Eleven essential oils (EOs) were evaluated for their antibacterial properties, against Vancomycin-Resistant Enterococci (VRE) and E. coli O157:H7. EOs were introduced into Brain Heart Infusion agar (BHI) (15ml) at a concentration of 0.25 to 2% (vol/vol) to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for each pathogen evaluated. Results showed that the most active essential oils against bacteria tested were thyme oil, with MIC90 and MBC90 for the VRA strains of 0.25% and 0.5%, respectively. Eucalyptus, juniper and clove oils were the least potent agent, with MIC90 and MBC90 of 2%. Furthermore, the inhibitory effect of these EO were evaluated against VRE and E. coli O157:H7, experimentally inoculated (10(3) cfu/g) in Feta soft cheese and minced beef meat, which was mixed with different concentrations (0.1%, 0.5% and 1%) of the EO and stored at 7 °C for 14 days. Out of eucalyptus, juniper, mint, rosemary, sage, clove and thyme oils tested against target bacteria sage and thyme showed the best results. Clove and mint did not show any effect on VRE and E. coli O157:H7 in both kinds of studied foods. The addition of thyme oil at concentrations of 0.5 and 1% caused best significant reduction in the growth rate of VRE and E. coli O157:H7 in cheese and meat at 7 (o)C. It is concluded that selected plant EOs can act as potent inhibitors of both microorganisms in a food product. The results revealed the potential of thyme oil as a natural preservative in feta soft cheese and minced beef meat against VRE and E. coli O157:H7 contamination.

Antimicrobial activity of essential oils against Vancomycin-Resistant enterococci (VRE) and Escherichia coli O157:H7 in feta soft cheese and minced beef meat

Eleven essential oils (EOs) were evaluated for their antibacterial properties, against Vancomycin-Resistant Enterococci (VRE) and E. coli O157:H7. EOs were introduced into Brain Heart Infusion agar (BHI) (15ml) at a concentration of 0.25 to 2% (vol/vol) to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for each pathogen evaluated. Results showed that the most active essential oils against bacteria tested were thyme oil, with MIC90 and MBC90 for the VRA strains of 0.25% and 0.5%, respectively. Eucalyptus, juniper and clove oils were the least potent agent, with MIC90 and MBC90 of 2%. Furthermore, the inhibitory effect of these EO were evaluated against VRE and E. coli O157:H7, experimentally inoculated (10³ cfu/g) in Feta soft cheese and minced beef meat, which was mixed with different concentrations (0.1%, 0.5% and 1%) of the EO and stored at 7 ºC for 14 days. Out of eucalyptus, juniper, mint, rosemary, sage, clove and thyme oils tested against target bacteria sage and thyme showed the best results. Clove and mint did not show any effect on VRE and E. coli O157:H7 in both kinds of studied foods. The addition of thyme oil at concentrations of 0.5 and 1% caused best significant reduction in the growth rate of VRE and E. coli O157:H7 in cheese and meat at 7 ºC. It is concluded that selected plant EOs can act as potent inhibitors of both microorganisms in a food product. The results revealed the potential of thyme oil as a natural preservative in feta soft cheese and minced beef meat against VRE and E. coli O157:H7 contamination.

Antimicrobial activity of essential oils against Vancomycin-Resistant enterococci (VRE) and Escherichia coli O157:H7 in feta soft cheese and minced beef meat

Eleven essential oils (EOs) were evaluated for their antibacterial properties, against Vancomycin-Resistant Enterococci (VRE) and E. coli O157:H7. EOs were introduced into Brain Heart Infusion agar (BHI) (15ml) at a concentration of 0.25 to 2% (vol/vol) to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for each pathogen evaluated. Results showed that the most active essential oils against bacteria tested were thyme oil, with MIC90 and MBC90 for the VRA strains of 0.25% and 0.5%, respectively. Eucalyptus, juniper and clove oils were the least potent agent, with MIC90 and MBC90 of 2%. Furthermore, the inhibitory effect of these EO were evaluated against VRE and E. coli O157:H7, experimentally inoculated (10³ cfu/g) in Feta soft cheese and minced beef meat, which was mixed with different concentrations (0.1%, 0.5% and 1%) of the EO and stored at 7 ºC for 14 days. Out of eucalyptus, juniper, mint, rosemary, sage, clove and thyme oils tested against target bacteria sage and thyme showed the best results. Clove and mint did not show any effect on VRE and E. coli O157:H7 in both kinds of studied foods. The addition of thyme oil at concentrations of 0.5 and 1% caused best significant reduction in the growth rate of VRE and E. coli O157:H7 in cheese and meat at 7 ºC. It is concluded that selected plant EOs can act as potent inhibitors of both microorganisms in a food product. The results revealed the potential of thyme oil as a natural preservative in feta soft cheese and minced beef meat against VRE and E. coli O157:H7 contamination.

Capacidad de formación de biopelículas de cepas de Listeria monocytogenes aisladas a partir de queso tierno de origen costarricense / Biofilm formation capacity of Listeria monocytogens strains isolated from soft cheese from Costa Rica

Listeria monocytogenes, además de ser un género capaz de producir una enfermedad infecciosa grave en el hombre, puede formar biopelículas en distintas superficies relacionadas con el ambiente de producción alimentario. Éstas constituyen un serio problema debido a que son una fuente importante y constante de contaminación para los alimentos y el ambiente de producción, además de que las bacterias presentes en ellas poseen una aumentada resistencia hacia agentes físicos y químicos de uso frecuente. En el presente trabajo se estudió la capacidad de formación de biopelícula de cepas de L. monocytogenes previamente aisladas a partir de queso tierno bajo diferentes condiciones de temperatura y cultivo. Se utilizó una técnica de microplaca con diferentes medios de cultivo (CICC, CTS 1:20 y suero de queso) a diferentes temperaturas de incubación (refrigeración, ambiente y 35ºC). La capacidad de formación de biopelícula fue clasificada según la densidad óptica obtenida a 620 nm. Ninguna de las cepas evaluadas fue clasificada como formadora fuerte de biopelicula bajo ninguna de las variables estudiadas, sí se detectaron formadoras débiles y moderadas. Los resultados obtenidos ponen de manifiesto la influencia del contenido de nutrientes en el medio de cultivo sobre la formación de biopelícula, no obstante, el CICC fue el único medio que permitió la expresión de formadores moderados. Por el contrario, el suero de queso resultó poco favorecedor. La formación de biopelícula es un proceso multifactorial, donde el nivel de adsorción depende de gran cantidad de variables y cuyo estudio debe fomentarse, de manera que se desarrollen metodologías que permitan su reducción o eliminación, de manera que las industrias alimentarias aseguren productos inocuos y de buena calidad microbiológica.

Listeria monocytogenes is a bacteria associated with the production of severe infectious disease in human being, but also with the formation of biofilms in different surfaces related to the food production environment. Biofilm represents a serious problem in food industry, since it is a constant and important contamination source and also, bacteria present in it have an increased resistance towards physical and chemical agents of common use. The capacity of biofilm formation of L. monocytogenes strains previously isolated from soft cheese samples from Costa Rica was studied under different temperature and culture conditions. The microplate technique was performed using different culture media (BHIB, TSB 1:20 and cheese serum) and at different incubation temperatures (refrigeration, environmental and 35ºC). Biofilm formation capacity was classified according to the optical density obtained at 620nm. None of the strains evaluated was classified as strong biofilm former under any of the variables studied, nevertheless, weak and moderate formers were detected. The results obtained show the influence of the nutrient content of the culture media used over biofilm formation; BHIB was the only culture media that allowed the expression of moderate biofilm forms, contrary to cheese serum that did not promote biofilm production. Biofilm formation is a multifactorial process, where adsorption level depends on several variables and its study must be promoted in order to develop methodologies that allow its reduction or elimination, so food industries may offer safe food products to consumers.

Qualidade sanitária de queijo prato, comercializado em supermercados de pequeno e médio porte na cidade do Recife, PE / Quality cheese, sold in supermarkets in small and medium businesses in the city of Recife, PE

O presente trabalho avaliou as condições higiênico-sanitárias do queijo Prato fatiado comercializado nos supermercados de pequeno e médio porte na cidade do Recife. Avaliou-se os padrões de contagem para Escherichia coli e Staphylococcus aureus estabelecidos pela ANVISA (Agência Nacional de Vigilância Sanitária) e de coliformes totais, bactérias mesófilas aeróbias, bolores e leveduras. Os supermercados foram selecionados a partir do cadastro da TeleLista e, caracterizados como de pequeno ou de médio porte pelo número de caixas registradoras, sendo os de até três caixas classificados como pequeno porte e os de quatro a dez como de médio porte. Foram selecionados aleatoriamente 16 estabelecimentos de pequeno porte e 14 de médio porte. Os queijos inteiros foram avaliados in locus em relação à aparência, presença ou ausência de embalagem, selo do S.I.F.( Serviço de Inspeção Federal), prazo de validade e temperatura do produto inteiro e do local de exposição dos fatiados. As análises microbiológicas foram realizadas utilizando-se placas de petrifilmTM 3M, aprovadas pela AOAC (Official Methods of Analysis - Aprovados e Certificados) e recomendadas pelo Ministério da Agricultura e do Abastecimento/DIPOA/ DOL Os resultados obtidos mostraram que todos os queijosinteiros apresentavam aparência normal em relação às características organolépticas; duas amostrasnão continham a embalagem; em 28 amostras constava o registro do S.LF.e dentre estas, duas estavamfora do prazo de validade. As temperaturas de armazenamento estiveram fora da recomendada emnove supermercados de pequeno porte e seis em supermercados de médio porte. Das 16 amostras analisadas em supermercados, nove se encontravam fora dos padrões da ANVISA. Portanto, as condições higiênico-sanitárias em que os queijos foram manipulados e armazenados mostraram-se deficientes, propiciando condições favoráveis à deterioração dos queijos e a proliferação de patógenos.(AU)

The sanitary quality of soft cheese sliced and sold in small and medium sized supermarkets in the city of Recifewas evaluated in relation to Escherichia coli and Staphylococcus aureus counts permitted by ANVISA (NationalAgency of Sanitary Vigilance), total coliforms, aerobic mesophilic bacteria, moulds, and yeasts. The supermarketswere selected from a telephone directory (TeleLista) and classified as small or medium-sized according to the numberof cashiers: those with up to three cashiers were considered small-sized and those with four to ten cashiers wereconsidered medium-sized. Sixteen small-sized and fourteen medium-sized supermarkets were selected randomly.The whole cheeses were evaluated in locu in relation to appearance, use or absence of packaging, SIF (Federal lnspection Service) stamp, expiration date, and temperature of the whole product and of the area where the slicedproducts were exposed. The microbiological analysis were carried out using PetrifilmTM 3M plaques, approved bythe Official Methods of Analysis and the Brazilian Ministry of Agriculture and Supply. The results obtainedshowed that all of the whole cheeses had normal appearance in relation to sensory characteristics; two samples wereunpackaged; 28 samples had the SIF stamp; and two samples had expired. In nine small-sized and six medium sizedsupermarkets the storage temperatures were outside of the recommended range. Of the sixteen samples analyzedfrom the small-sized supermarkets, nine were not in agreement with ANVISA's recommendations, and ofthe fourteen samples from the medium sized establishments, five were not in agreement with ANVISA' s recommendations. Therefore, the sanitary conditions of the places where the cheeses were handled and stored were poor, offering favorable conditions for the cheese's deterioration and pathogen proliferation.(AU)