Effects of Queso Blanco cheese containing Bifidobacterium longum KACC 91563 on fecal microbiota, metabolite and serum cytokine in healthy beagle dogs.

The study describes the effects of administration of Queso Blanco cheese containing Bifidobacterium longum on the fecal microbiota, metabolite and serum cytokine in healthy beagle dogs. Twelve healthy beagle dogs were randomly divided in three groups of four dogs each:a control group, not fed with any cheese, and groups fed with Queso Blanco cheese with B. longum KACC 91563 (QCB) or without B. longum (QC) for 8 weeks. Fecal microbiota was analyzed using a culture-based method and 16s rRNA gene sequencing. Serum cytokine levels, activation of natural killer cells, and proliferation of peripheral blood mononuclear cells were determined. SPME-GC-MS method was used to determine the concentrations of short chain fatty acids and indole in dog feces. Administration of QCB for 4 weeks significantly increased the Bifidobacterium. QCB supplementation for 8 weeks reduceds Enterobacteriaceae and Clostridium perfringens (p < 0.05). The abundance of Fusobacterium, Blautia and Collinesella in QCB group were reduced as compared with the control group. Serum TNF-α and IL-6 levels at 8 weeks significantly increased in QCB group as compared with QC group. There was no change in the concentrations of total short chain fatty acids by B. longum at 0 and 4 weeks. At week 8, the acetic acid, propionic acid and butyric acid of the QCB and QC groups were significantly decreased compared to the control group. In conclusion, our results demonstrate that administration of QCB had positive effects on fecal microbiota and immune response in beagle dogs. We suggest that Queso Blanco cheese containing B. longum KACC 91563 could be used as a functional food for companion animals.

Effects of Queso Blanco Cheese Containing Bifidobacterium longum KACC 91563 on the Intestinal Microbiota and Short Chain Fatty Acid in Healthy Companion Dogs.

The effects of Queso Blanco cheese containing Bifidobacterium longum KACC 91563 was studied on the intestinal microbiota and short chain fatty acids (SCFAs) in healthy companion dogs. There were three experimental groups with five healthy dogs each: a control group, not fed with any cheese, and groups fed with Queso Blanco cheese with (QCB) or without B. longum KACC 91563 (QC) for 8 weeks. Fecal samples were collected 5 times before, during, and after feeding with cheese. Intestinal microbiota was analyzed using two non-selective agar plates (BL and TS) and five selective agar plates (BS, NN, LBS, TATAC, and MacConkey). SPME-GC-MS method was applied to confirm SCFAs and indole in dog feces. The six intestinal metabolites such as acetic, propionic, butyric, valeric, isovaleric acid and indole were identified in dog feces. Administration of B. longum KACC 91563 (QCB) for 8 weeks significantly increased the beneficial intestinal bacteria such as Bifidobacterium (8.4±0.55) and reduced harmful bacteria such as Enterobacteriaceae and Clostridium (p<0.05). SCFA such as acetic and propionic acid were significantly higher in the QCB group than in the Control group (p<0.05). In conclusion, this study demonstrates that administration of Queso Blanco cheese containing B. longum KACC 91563 had positive effects on intestinal microbiota and metabolites in companion dogs. These results suggest that Queso Blanco cheese containing B. longum KACC 91563 could be used as a functional food for companion animals and humans.

Physicochemical, Microbial, and Sensory Properties of Queso Blanco Cheese Supplemented with Powdered Microcapsules of Tomato Extracts.

The present study examined the physical, chemical, microbial, and sensory characteristics of Queso Blanco cheese supplemented with powdered microcapsules containing tomato extracts (0.5-2.0%) during storage at 7°C for 60 d. The lactic acid bacterial count and lycopene concentrations in Queso Blanco cheese supplemented with powdered microcapsules were significantly higher than those of the control. In a texture analysis, the gumminess, chewiness, and hardness values for Queso Blanco cheese were significantly higher with increasing concentrations of the powdered microcapsules containing tomato extracts. Total short-chain fatty acids in Queso Blanco cheese supplemented with powdered microcapsules containing tomato extracts were not significantly altered compared to the control. Sensory evaluation scores for the yellowness, tomato taste, and firmness of Queso Blanco cheese were significantly higher after supplementation with powdered microcapsules containing tomato extracts.

Application of Edible Films Containing Oregano (Origanum vulgare) Essential Oil on Queso Blanco Cheese Prepared with Flaxseed (Linum usitatissimum) Oil.

Fortification of queso blanco (QB) with flaxseed oil (FO) containing omega-3 polyunsaturated fatty acids may provide a functional food with health benefits such as improved cell, brain, and retina functionality, and protection against cardiovascular and immune-inflammatory diseases. However, QB experiences a short shelf life because of the early development of yeasts and molds and addition of FO may increase susceptibility to lipid oxidation. Oregano essential oil (OEO) is known for its antimicrobial and antioxidant properties, but due to its intense flavor compounds it may not be suitable for direct incorporation into QB. Thus, incorporation of OEO into an edible film prepared with whey protein isolate (WPI) may improve the shelf life of QB. Scanning electron microscopy (SEM) micrographs revealed that FO was successfully retained by the cheese after homogenization. The thiobarbituric-acid-reactive-substances (TBARS) and yeast and mold counts (YMC) of the wrapped cheeses were analyzed during 60 d of refrigerated storage. The oxidation rate increased significantly for nonwrapped QB containing FO (QBFO) during storage, however wrapping with WPI edible films containing OEO (WOF) significantly limited lipid oxidation and prevented growth of yeasts and molds. This study demonstrated the antioxidant and antimicrobial properties of WOF for preservation of QBFO during refrigerated storage.

Detección de Listeria monocytogenes en queso blanco criollo, mediante la reacción en cadena de la polimerasa (PCR) / Detection of Listeria monocytogenes in white cheese by Polymerase Chain Reaction (PCR)

La reacción en cadena de la polimerasa, conocida como PCR, es un método que permite replicar miles de veces, en pocas horas e in vitro, pequeñas cantidades de ADN. La aplicación de métodos rápidos y sensibles, para detectar Listeria monocytogenes en muestras de queso blanco, permitirá un mejor control microbiológico del proceso de producción. Se aplicó PCR a 30 muestras de queso blanco, de una quesera de Valencia Estado Carabobo. Se detectó especificidad y sensibilidad para PCR mediante el empleo de la cepa control Listeria monocytogenes 446. Extracción de ADN según metodología descrita por Torres y col., Marcador de peso molecular de 100 pares de base. Se emplearon: cuatro cebadores del gen hlyA de listeriolisina O; iniciadores L1/U1 para banda 938 pb y LF/LR para banda 750 pb del gen hlyA. Estadístico EpiInfo V6 para concordancia de observaciones en geles, mediante coeficiente Kappa (K). Resultados: 8 de las 30 muestras de queso analizadas, mostraron crecimiento presuntivo de Listeria spp en Agar PALCAM. De las cuales 2 de las muestras no pertenecian al género Listeria; en las 6 restantes las pruebas de confirmación arrojaron que: 2 eran L. monocytogenes, 3 L.ivanovii y 1 L. seeligeri. Mediante PCR 2 muestras resultaron positivas para L. monocytogenes al amplificar la banda 938 pb para Listeria y banda 750 pb para la especie monocytogenes. Se concluye que PCR demostró ser altamente específico y sensible para L. monocytogenes, teniendo ventaja sobre agar PALCAM al evidenciar la presencia especifica del patógeno en un tiempo relativamente corto.

The Polymerase Chain Reaction, known as PCR, is a method to replicate thousands of times within a few hours and in vitro, small amounts of DNA. The application of rapid and sensitive methods to detect Listeria monocytogenes in cheese samples, allow a better microbiological control of the production process. PCR was applied to 30 samples of of white cheese, from Valencia, Carabobo State. It was detected PCR specificity and sensitivity by using the control strain Listeria monocytogenes 446. DNA extraction according to the methodology described by Torres et al., Molecular weight marker 100 base pairs. Were used: four primers hlyA gene of listeriolysin O; L1/U1 primers for 938 bp band and LF / LR 750 bp band hlyA gene. EpiInfo Statistical V6 to match observations in gels, by Kappa coefficient (K). Results: 8 out of 30 cheese samples analyzed showed presumptive growth of Listeria spp in PALCAM Agar. Two of the samples not belonged to the genus Listeria, in the 6 remaining sample confirmation tests showed that: 2 were L. monocytogenes, 3 L. ivanovii and 1 L. seeligeri. In PCR 2 samples were positive for L. monocytogenes by amplify the 938 bp band for Listeria and 750 bp band for the species monocytogenes. We concluded that PCR was highly specific and sensitive to L. monocytogenes, taking advantage of PALCAM agar to detect the presence of the pathogen specifies a relatively short time.

Calidad sanitaria en queso artesanal tipo “telita”. Upata, estado Bolívar, Venezuela / Sanitary conditions of hand-made “telita” type cheese in Upata, Bolivar State, Venezuela

En este trabajo se investigaron microorganismos indicadores de calidad sanitaria en queso artesanal tipo “telita” de Upata, municipio Piar, estado Bolívar. Se analizaron 60 muestras y se investigaron estafilococos coagulasa positivos (Staphylococcus aureus) según Norma Venezolana COVENIN 1292-89 como indicador de manipulación; bacterias coliformes según Norma Venezolana        COVENIN 1104-96 y presencia de Escherichia coli como indicador de contaminación fecal. Todos los crecimientos bacterianos correspondieron a estafilococos coagulasa negativos con recuentos de hasta 10(4) diluciones decimales. Coliformes totales mostraron recuentos de hasta ≤10(5) NMP/g y coliformes fecales en concentración ≤10(4) NMP/g. Escherichia coli estuvo presente en 43,3% de los quesos. Se concluyó que el queso artesanal tipo “telita” que se expende en los alrededores de Upata, estado Bolívar, evidencia fallas en la manipulación e higiene posterior a su elaboración; y por no cumplir con los criterios que establece el Reglamento Centroamericano de Criterios Microbiológicos de los Alimentos Procesados, se considera un producto que podría representar un alto riesgo microbiológico para el consumidor.

This study investigated microorganisms indicators of sanitary conditions in hand-made “telita” type cheese in Upata, Piar Municipality, Bolivar State. Sixty cheese samples were analyzed, and coagulase-positive staphylococci (Staphylococcus aureus) were investigated according to Venezuelan COVENIN Regulation 1292-89 as indicator of manipulation, coliform bacteria according to the Venezuelan COVENIN Regulation 1104-96 and presence of Escherichia coli as fecal contamination indicator. All bacteria growths corresponded to coagulase-negative staphylococci with counts up to 10(4) decimal dilutions. Total coliforms showed counts of up to ≤10(5) NMP/g, and fecal coliforms in concentrations of ≤10(4) NMP/g. Escherichia coli, appeared in 43.3% of samples. It was concluded that the hand-made “telita” type cheese that is sold in the surroundings of Upata, Bolivar State, shows evidences of faulty manipulation and hygienic conditions after its production , and that it does not fulfill the criteria established by the Central American Regulation of Microbiological Criteria For Processed Foods, and is considered as a product which could represent a high microbiological risk for consumers.

Comparación del empleo de nisina y cultivos de lactococcus lactis subsp. lactis para la biopreservación de queso blanco / Comparation of nisin and lactococcus lactis subsp. lactis culturues for biopreservation of white cheese

La principal enfermedad transmitida por alimentos en Venezuela es la intoxicación estafilocóccica y es el queso blanco el principal alimento involucrado. La nisina es una bacteriocina capaz de frenar el crecimiento de microorganismos Gram positivos, entre ellos Staphylococcus aureus. Una de las limitaciones de su uso es su alto costo. El empleo de cultivos iniciadores productores de bacteriocinas, representa una alternativapara elaboración de quesos con mejor calidad microbiológica. El objetivo del presente estudio fue evaluar el efecto biopreservador del Lactococcus lactis subsp. lactis (ATCC 11454) y compararlo con la adición de nisina a la leche. Para ello se elaboraron cinco tipos de quesos con leche pasteurizada: control; con L. lactis; con L. lactis y S. aureus; con S. aureus; con S. aureus y nisina. Las determinaciones microbiológicas y físico-químicas se realizaron de acuerdo a las normas COVENIN. La concentración de nisina se determinó mediante la técnica de difusión en agar. Los datos se analizaron utilizando el análisis de varianza y se probaron las medias por el método de losmínimos cuadrados. Se encontró producción de nisina por parte del cultivo iniciador, pero en bajas concentraciones. En los quesos elaborados con leche a la que se le inoculó S. aureus (103 ufc/mL) no se encontró efecto inhibidor del cultivo sobre este microorganismo patógeno. Sin embargo, los quesos elaborados con Lactococcus sin inoculación de Staphylococcus y los quesos elaborados con nisina, presentaron escaso crecimiento de estafilococos y de bacterias coliformes. La nisina resultó ser más eficiente para lograr biopreservación de los quesos blancos, aunque la utilización del cultivo iniciador productor de la bacteriocina, permitió obtener quesos con bajo conteo de patógenos, cuando la carga microbiana inicial en la leche era escasa.

The main disease transmitted by foods in Venezuela is the staphylococci poisoning, and it is white cheeses the most involved food. The nisin is a bacteriocin able to inhibit the growthof Gram positive microorganisms; among them, Staphylococcus aureus. One of the limitations of its use is its high cost. The use of bacteriocin producing starter cultures represents an alternative for milk industry. The objective of the present study was to evaluate the biopreservating effect of the Lactococcus lactis subsp. lactis (ATCC 11454) and to compare it with the addition of nisin to milk. Five types of cheese from pasteurized milk were elaborated: control; addition of L. lactis; addition of L. lactis and S. aureus; addition of S. aureus; addition of nisinaand S. aureus. Both, microbiological and physical-chemical determinations were made according to COVENIN. The nisinconcentration was measured using the agar diffusion technique. The data was analyzed with an analysis of variance and testing the means by the method of least squares means. The starter cultures produced nisin, but in low amounts. In cheeses elaborated with milk inoculated whit S. aureus (103 ufc/mL), no inhibiting effect of the culture on this pathogenic microorganismwas observed. Nevertheless, cheeses elaborated with both, Lactococcus without inoculation of Staphylococcus and with nisin, presented little growth of Staphylococcus and coliforms. Nisin was more efficient to obtain biopreservation of white cheeses, although the use of producing bacteriocin starter culture, allowed to obtain cheeses with low counts of pathogens, if the initial contamination of the milk was low.

Estafilococos oxacilino resistentes en queso blanco fabricado en el estado Anzoátegui, Venezuela / Oxacyllin resistant Staphylococcus in white cheese manufactured at the Anzoategui State, Venezuela

El objetivo de esta investigación fue detectar especies estafilocócicas oxacilino resistentes en quesos blanco duro y blando, fabricados artesanalmente en diversos municipios de la zona norte del estado Anzoátegui. Las cepas de Staphylococcus fueron aisladas e identificadas empleando métodos convencionales y se confirmaron las especies coagulasa negativas mediante galerías API 32 Staph (BioMérieux); la susceptibilidad a oxacilina y otros antibióticos por el método de difusión en disco y la presencia del gen mecA, por prueba de látex MRSA Slidex (BioMérieux) que pone de manifiesto la proteína PBP2a. De 130 quesos evaluados, se aislaron 171 cepas estafilocócicas, 26 (15,2%) resistentes a oxacilina (61,5% con presencia del gen), distribuidas como sigue: 14 cepas de S. epidermidis (8,2%), 6 de S. capitis (3,4%), 2 de S. aureus (1,2%), 2 de S. hominis (1,2%), 1 de S. warneri (0,6%) y 1 de S. saprophyticus (0,6%). La presencia del gen que codifica para la proteína PBP2a se detectó tanto en cepas con homorresistencia (76,9%) como con heterorresistencia (23,1%). S. haemolitycus fue 100% sensible a oxacilina. La presencia de cepas estafilocócicas oxacilino resistentes en el queso blanco, puede representar un riesgo para salud pública ya que podría servir como fuente de diseminación de éstas y acrecentar el problema de la resistencia.

The purpose of this study was to detect oxacyllin resistant Staphylococcus species in hard and soft white cheese manufactured by artisans at various municipalities of the northern area of Anzoátegui State. The Staphylococcus strains were isolated and identified using conventional methods and coagulase negative species were confirmed through API 32 Staph (BioMérieux) galleries; susceptibility to oxacyllin and other antibiotics was determined by the disk diffusion method and presence of the mecA gene with the MRSA Slidex (BioMérieux) latex test that identifies PBP2a protein. From 130 cheeses evaluated, we isolated 171 Staphylococcus strains, 26 of which (15,2%) were oxacyllin-resistant (61,5% with presence of the gene), distributed as follows: 14, S. epidermidis strains (8,2%); 6, S. capitis (3,4%); 2, S. aureus (1,2%); 2, S. hominis (1,2%); 1, S. warneri (0.6%); and 1, S. saprophyticus (0.6%). Presence of the protein PBP2a codifying gene was detected in both homoresistant (76.9%) and heteroresistant (23.1%) strains. S. haemolitycus was 100% oxacyllin sensitive. Presence of oxacyllin resistant strains in white cheese can represent a public health risk since it could serve as a dissemination source, increasing the resistance problem.

Efecto de la nisina sobre la microflora patógena del queso blanco artesanal tipo"telita" elaborado en una quesera de Upata, Estado Bolívar, Venezuela / Effect of nisin over the pathogenic microflora of artisan "telita" type white cheese produced at a cheese factory in Upata, Bolivar State, Venezuela

Resumen Se evaluó el efecto inhibitorio de dos concentraciones de nisina (10,0 y 16,7 mg/kg) sobre la población de Staphylococcus aureus, coliformes totales, Escherichia coli, Salmonella spp y Listeria monocytogenes en queso blanco tipo "telita" elaborado en una quesera de Upata, Estado Bolívar. El número de S. aureus, coliformes totales y Escherichia coli se determinó mediante la siembra en películas secas rehidratables Petrifilm 3M; siendo los recuentos de S. aureus significativamente menores (p < 0,05) en las muestras de queso "telita" con las dos concentraciones de nisina ensayadas con respecto a las muestras control. No se detectó Salmonella spp ni Listeria monocytogenes en ninguna de las muestras analizadas Se encontró que las dos concentraciones de nisina adicionadas al queso "telita" ejercieron un efecto inhibitorio sobre la población de S. aureus presente como microflora contaminante en las mismas.

Abstract The inhibitory effect of two nisin concentrations (10.0 and 16.7 mg/kg) over the Staphylococcus aureus, total coliforms, Escherichia coli, Salmonella spp and Listeria monocytogenes population was evaluated in "telita" type white cheese produced at a cheese factory in Upata, Bolivar State. The number of S. aureus, total coliforms and Escherichia coli was determined by inoculation in Petrifilm 3M dry rehydratable films. The S. aureus counts were significantly smaller (p<0.05) in the "telita" cheese samples with the two nisin concentrations tested as compared with the control samples. No Salmonella spp or Listeria monocytogenes were detected in any of the samples analyzed. It was determined that the two nisin concentrations added to the "telita" cheese exerted an inhibitory effect over the S. aureus present in them as contaminant microflora.

Comparación de los métodos PetrifilmTM coliformes y Número Más Probable (NMP) para la determinación de coliformes fecales en muestras de queso blanco / Comparison study between the coliform PetrifilmTM and the Most Probable Number methods for the assessment of fecal coliforms in white cheese

Los coliformes fecales son un grupo importante de microorganismos indicadores de inocuidad en alimentos, constituido principalmente por Escherichia coli, el cual es considerado como indicador de contaminación reciente de origen fecal, por ello la importancia de investigar su presencia y determinar rápidamente el nivel poblacional en alimentos. Este trabajo tiene como objetivo comparar el método tradicional Número Más Probable (NMP) para la determinación de coliformes fecales, según Norma Venezolana Covenin Nº 1104-96 con el método de placas rehidratables PetrifilmTM 3M coliformes incubadas en baño de agua circulante (PBA) a 45 ± 0,2°C por 24 ± 2 horas y en estufa con aire circulante 44 ± 1°C por 24 ± 2 horas (PES) de acuerdo con lo recomendado por la Asociación Francesa de Normalización (AFNOR) y la Corporación 3M. Se analizaron un total de 42 muestras de queso blanco aplicando ambas metodologías, dispensando simultáneamente diluciones seriadas de las muestras. Para el análisis estadístico se realizó una correlación de muestras relacionadas (SPS versión 7,5), obteniéndose los siguientes resultados r = 0,952 entre NMP y PES y r = 0,944 entre NMP y PBA; lo que indica una buena correlación positiva entre ambas metodologías en sus diferentes modalidades para la determinación de coliformes fecales en muestras de queso blanco. Se concluye que las placas PetrifilmTM coliformes incubadas a la temperatura óptima de crecimiento de dichos microorganismos es un método alternativo, rápido y confiable para la determinación del nivel de coliformes fecales en queso blanco.

Fecal coliform belong to an important group of sanitary quality indicator microorganisms in food, mainly constituted for Escherichia coli, considerated as indicators of recent fecal contamination, that is why it is very important to investigate their presence and to detect their population in food rapidly. The objetive of this study was to compare the Most Probable Number (NPM) method for fecal coliform determination, according to Venezuelan standard COVENIN Nº 1104-96, with the coliform PetrifilmTM 3M TM plates, incubated in circulating thermostatically - controlled water bath (PBA) at 45 ± 0,2 ° C for 24 ± 2 hours and in a circulating air incubator at 44 ± 1° C for 24 ± 2 hours (PES), according to the recommendation of Association Francoise of Normalization, Paris (AFNOR) and 3M TM corporation. Forty-two white cheese samples were analyzed using both methods mentioned above. They were dispensed at the same time with decimal dilutions of the samples. Data generated were subjected to correlation of related samples (SPS 7.5 version) and the correlation coefficients (r) were obtained; r = 0.952 NMP and PES; r = 0.944 NMP and PBA. It is interesting to observe a good correlation between the methodologies in their different forms for fecal coliform determination in white cheese. Coliform PetrifilmTM plates incubated at the optimal temperature of coliform fecal culture represent a rapid alternative and reliable method for the assessment of fecal coliform population in white cheese.

Effects of Acid Type and Alta™2341 on Listeria monocytogenes in a Queso Blanco Type of Cheese.

The behavior of Listeria monocytogenes was evaluated during storage of a queso blanco type of cheese produced with acidulants (citric, malic, or acetic acids) and a commercial lactic acid bacterium fermentation product, ALTA™2341 (ALTA). The cheese was prepared by direct acidification (final pH 5.2), with and without 0.6% ALTA, inoculated with 106 CFU/g of L. monocytogenes , and stored at 4 or 20°C for 42 and 7 days, respectively. Levels of L. monocytogenes increased in cheese coagulated with citric or malic acids and stored at 4°C, but decreased slightly in cheese coagulated with acetic acid. At 20°C, counts of L. monocytogenes increased in cheeses acidified with citric or malic acid, but counts did not increase appreciably in cheese acidified with acetic acid. When cheese was stored at 4°C, the presence of 0.6% ALTA resulted in lower counts of L. monocytogenes compared with counts in cheese that did not contain ALTA. However, at 20°C populations of L. monocytogenes increased in cheese containing ALTA regardless of acid type. Additional studies compared the effects of acetic acid, alone or in combination with 0.6 or 2.5% ALTA, against low (102 CFU/g) and high (106 CFU/g) inoculum levels. When inoculum levels were low, pathogen counts decreased by > 1.1 log10 CFU/g in all formulations at 4°C. After 7 days at 20°C, pathogen counts increased in the queso blanco type of cheese prepared with acetic acid alone. In contrast, in the presence of 0.6 or 2.5% ALTA, 7-day counts were less than the initial inoculum. With high inoculum levels at 4°C, counts of L. monocytogenes were less than the initial inoculum in the acetic acid-coagulated queso blanco type of cheese with or without ALTA. At 20°C, counts increased in the queso blanco type of cheese prepared with 0.6% ALTA, but decreased appreciably in cheese prepared with 2.5% ALTA. These results demonstrate that acetic acid is significantly more effective than malic or citric acids for controlling L. monocytogenes in queso blanco, and that inclusion of ALTA can provide added protection against the pathogen.