Plasmid-Associated Bacteriocin Produced by Pediococcus pentosaceus Isolated from Smoked Salmon: Partial Characterization and Some Aspects of his Mode of Action.

Strain ST3Ha, isolated from commercially available smoked salmon, was identified as Pediococcus pentosaceus based on biochemical and physiological tests and 16S rRNA sequencing. Strain ST3Ha produces a class IIa bacteriocin active against lactic acid bacteria, Listeria monocytogenes and Enterococcus faecalis. The antimicrobial peptide was inactivated by proteolytic enzymes, confirming his proteinaceous nature, but was not affected when treated with α-amylase, SDS, Tween 20, Tween 80, urea, and EDTA. No change in activity was recorded after 2 h at pH values between 2.0 and 9.0 and after treatment at 100 °C for 120 min or 121 °C for 15 min. The mode of action against Listeria ivanovii subsp. ivanovii ATCC 19119 and E. faecalis ATCC 19443 was bactericidal, resulting in cell lyses and enzyme leakage. The highest level of activity (1.6 × 106 AU/mL) was recorded when cells were grown at 37 °C or 30 °C in MRS broth (pH 6.5). Antimicrobial peptide ST3Ha adsorbs at high levels to the sensitive test organisms on strain-specific manner and depending on incubation temperature, environmental pH, and presence of supplemented chemicals. Based on PCR analysis, P. pentosaceus ST3Ha harbor a 1044-bp plasmid-associated fragment corresponding in size to that recorded for pediocin PA-1. Sequencing of the fragment revealed a gene identical to pedB, reported for pediocin PA-1. The combined application of the low levels (below MIC) of ciprofloxacin and bacteriocin ST3Ha results in the synergetic effect in the inhibition of L. ivanovii subsp. ivanovii ATCC 19119. Expressed by P. pentosaceus ST3Ha, bacteriocin was characterized as low cytotoxic, a characteristic relevant for its application in food industry and/or in human and veterinary medical practices.

Pediocin PA-1 production by Pediococcus pentosaceus ET34 using non-detoxified hemicellulose hydrolysate obtained from hydrothermal pretreatment of sugarcane bagasse.

Listeria monocytogenes is one of the foodborne pathogens of most concern for food safety. To limit its presence in foods, bacteriocins have been proposed as natural bio-preservatives. Herein, a bacteriocin was produced on hemicellulose hydrolysate of sugarcane bagasse by Pediococcus pentosaceous ET34, whose genome sequencing revealed an operon with 100% similarity to that of pediocin PA-1. ET34 grown on hydrolysate-containing medium led to an increase in the expression of PA-1 genes and a non-optimized purification step sequence resulted in a yield of 0.8 mg·L-1 of pure pediocin (purity > 95%). Culture conditions were optimized according to a central composite design using temperature and hydrolysate % as independent variables and validated in 3-L Erlenmeyers. Finally, a process for scaled-up implementation by sugar-ethanol industry was proposed, considering green chemistry and biorefinery concepts. This work stands up as an approach addressing a future proper sugarcane bagasse valorisation for pediocin production.

Recently fixed carbon allocation in strawberry plants and concurrent inorganic nitrogen uptake through arbuscular mycorrhizal fungi.

Most crop species form a symbiotic association with arbuscular mycorrhizal (AM) fungi, receiving plant photosynthate and exchanging nutrients from the soil. The plant carbon (C) allocation to AM fungi and the nitrogen feedback are rarely studied together. In this study, a dual (13)CO2 and (15)NH4(15)NO3 pulse labeling experiment was carried out to determine the allocation of recent photosynthates to mycorrhizal hyphae and the translocation of N absorbed by hyphae to strawberry plants. Plants were grown in pots in which a 50 µm mesh net allowed the physical separation of the mycorrhizal hyphae from the roots in one portion of the pot. An inorganic source of (15)N was added to the hyphal compartment at the same time of the (13)CO2 pulse labeling. One and seven days after pulse labeling, the plants were destructively harvested and the amount of the recently fixed carbon (C) and of the absorbed N was determined. (13)C allocated to belowground organs such as roots and mycorrhizal hyphae accounted for an average of 10%, with 4.3% allocated to mycorrhizal hyphae within the first 24h after the pulse labeling. Mycorrhizae absorbed labeled inorganic nitrogen, of which almost 23% was retained in the fungal mycelium. The N uptake was linearly correlated with the (13)C fixed by the plants suggesting a positive correlation between a plant photosynthetic rate and the hyphal absorption capacity.

Efecto de la temperatura de refrigeración y calentamiento de fórmulas lácteas Infantiles en el crecimiento de cronobacter sakazakii / Effect of temperature cooling and heating of powered infant formula on the growth of cronobacter sakazakii

Cronobacter sakazakii (C. sakazakii) es un microorganismo perteneciente a la familia Enterobacteriaceae asociado con patologías como meningitis y septicemia en recién nacidos. Las fórmulas lácteas infantiles (FLI) han sido reconocidas como el vehículo más importante de transmisión de C. sakazakii, por condiciones inadecuadas durante su producción o preparación. El objetivo de la presente investigación fue evaluar el efecto de la temperatura de refrigeración y el calentamiento de las FLI en el crecimiento de C. sakazakii. Las muestras de FLI reconstituidas se contaminaron con 102, 104 y 106 UFC/mL de C. sakazakii y se refrigeraron durante cuatro horas a 4°C, 6°C y 10°C, posterioriormente, se calentaron en baño hidrotérmico hasta 37°C. Las muestras fueron sembradas al tiempo “0” (postrefrigeración) y luego del calentamiento hasta 37°C en placas con agar cromogénico y se incubaron a 35°C por 24h. Para el análisis de los datos se utilizó la prueba de Wilcoxon. Según los datos obtenidos no hubo variación significativa en la población de C. sakazakii en las FLI luego de 4 horas en refrigeración a 4°C, 6°C y 10°C; sin embargo, al calentar las fórmulas hasta 37°C, hubo en todos los casos una reducción de un ciclo logarítmico en la población de C. sakazakii con un valor de p = 0.0056 y Z= 2.752. Se concluye que el calentamiento a 37°C post-refrigeracion de las muestras, disminuye la población de C. sakazakii en FLI (p<0,05)(AU)

Cronobacter sakazakii (C. sakazakii) is a microorganism belonging to the family Enterobacteriaceae associated with diseases such as meningitis and sepsis in newborns. The powdered infant formula (PIF) have been recognized as the most important vehicle of transmission of C. sakazakii, by inadequate conditions during production or during its preparation. The aim of this investigation was to assess the effect of the cooling temperature and the heating of PIF in the growth of C. sakazakii. For this, PIF reconstituted samples were contaminated with 102, 104 and 106 CFU / mL of C. sakazakii and refrigerated for four hours at 4 ° C, 6 ° C or 10 ° C after this hydrothermal bath heated to 37 ° C. Samples were cultured at time “0” (after- cooling) and then heating to 37 ° C in chromogenic agar plates and incubated at 35 ° C for 24h . For data analysis we used the Wilcoxon test. According to the data there was no significant variation in the population of C. sakazakii in PIF after 4 hours in the refrigerator at 4 ° C, 6°C and 10 ° C , however , by heating the formulas to 37 ° C , in all cases there was a reduction of one log cycle in population C. sakazakii with a value of p = 0.0056 and Z = 2.752 . We conclude that heating at 37°C post- cooling of the samples, decreases the population of C. sakazakii in FLI ( p < 0,05 )(AU)

Characterisation of an antiviral pediocin-like bacteriocin produced by Enterococcus faecium.

The bacteriocin-producing strain Enterococcus faecium ST5Ha was isolated from smoked salmon and identified by biomolecular techniques. Ent. faecium ST5Ha produces a pediocin-like bacteriocin with activity against several lactic acid bacteria, Listeria spp. and some other human and food pathogens, and remarkably against HSV-1 virus. Bacteriocin ST5Ha was produced at high levels in MRS broth at 30 degrees C and 37 degrees C, reaching a maximum production of 1.0 x 10(9) AU/ml, checked against Listeria ivanovii ATCC19119 as target strain and surrogate of pathogenic strain Listeria monocytogenes. The molecular weight of bacteriocin ST5Ha was estimated to be 4.5 kDa according to tricine-SDS-PAGE data. Ent. faecium ST5Ha harbors a 1.044 kb chromosomal DNA fragment fitting in size to that of pediocin PA-1/AcH. In addition, the sequencing of bacteriocin ST5Ha gene indicated 99% of DNA homology to pediocin PA-1/AcH. The combined application of low levels (below MIC) of ciprofloxacin and bacteriocin ST5Ha resulted in a synergetic effect in the inhibition of target strain L. ivanovii ATCC19119. Bacteriocin ST5Ha displayed antiviral activity against HSV-1, an important human pathogen, with a selectivity index of 173. To the best of our knowledge, this is the first report on Ent. faecium as a potential producer of pediocin-like bacteriocin with antiviral activity.

Growth control of Listeria innocua 2030c on vacuum-packaged cold-smoked salmon by lactic acid bacteria.

Five bacteriocin-producing lactic acid bacteria (LAB): Enterococcus faecium ET05, Lactobacillus curvatus ET06, L. curvatus ET30, L. deldrueckii ET32 and Pediococcus acidilactici ET34, selected by their capacity for growth and producing inhibition in vitro at high salt-on-water content, low temperature and anaerobic atmosphere, conditions simulating cold-smoked fish, were inoculated onto salmon fillets, in co-culture with Listeria innocua 2030c, and cold-smoked processed (dry salted for 6 h; drying for 6 h; smoke for 2 h). The finished product was then packed under vacuum and stored at 5 degrees C. Enumeration of LAB and L. innocua was performed during storage. Results showed that strain E. faecium ET05 was the best biopreservative candidate for controlling L. innocua growth in vacuum-packaged cold-smoked salmon (CSS) processed under the salting/drying/smoking parameters referred above. L. curvatus ET30 and L. delbrueckii ET32 also showed a good biopreservation potential for CSS although they were less effective than the former. L. curvatus ET06 and P. acidilactici ET34 showed a bacteriostatic mode of action against the target bacteria in vitro as well as when inoculated into the salmon fillets. This study describes a potential application of five different LAB in the biopreservation of Listeria in CSS.

Could modifications of processing parameters enhance the growth and selection of lactic acid bacteria in cold-smoked salmon to improve preservation by natural means?

Several smoking conditions were examined with the objective of enhancing the numbers of lactic acid bacteria (LAB) by natural means in vacuum-packaged cold-smoked salmon during 21 days of storage at 5 degrees C. Three combinations of salting, drying, and smoking were used: (i) dry salting x time of salting (2 or 6 h); (ii) wet salting (6 h) x dry salting (6 h) x with or without sugar; and (iii) wet salting (6 h) x dry salting (6 h) x different times of smoking (2 or 6 h of drying and 2 or 6 h of smoking). Two batches were processed for each set of conditions. Determinations of pH and salt content in the water phase were carried out for products in each treatment. Microbiological analyses (total viable count, total LAB, Lactobacillus spp., and Enterobacteriaceae) also were conducted at the beginning of storage (t0) and after 21 days of refrigerated storage (tl). There were differential increases in total LAB and lactobacilli during the storage period according to the treatment performed. The most effective treatment to enhance LAB growth was 6 h of dry salting with sugar, 6 h of drying, and 2 h of smoking. These salting-drying-smoking conditions also selected the LAB as the dominant flora at the end of the storage period. The LAB promoted by these processing parameters seem to be potentially useful protective cultures because of their anti-Listeria activity. From the results of this research, we conclude that it is possible to enhance the growth of LAB in general and that of inhibitory strains in particular by suitable choices of processing parameters.

Anti-listerial inhibitory lactic acid bacteria isolated from commercial cold smoked salmon.

The natural microflora of cold-smoked fish at the end of shelf-life are lactic acid bacteria (LAB). Some of these display a capacity to inhibit spoilage as well as several strains of pathogenic micro-organisms, e.g. Listeria monocytogenes which is isolated frequently from cold-smoked salmon (CSS). Eight batches of sliced vacuum-packed CSS from Norway, Scotland and Spain were collected at retail. Packs were stored at 5 degrees C and examined for chemical and microbiological characteristics, at purchase date and at expiration date. pH, water activity and salt content were similar to available data on lightly preserved fish products. There was a consistent pattern in the development of the microflora on CSS; the initial level of LAB was low on freshly produced CSS (10(2) cfu g(-1)); however, storage in vacuum packaging at refrigeration temperature was elective for LAB. At the end of the stated shelf-life these micro-organisms, represented mainly by Lactobacillus spp., attained ca.10(7) cfu g(-1) while Enterobacteriaceae counts were consistently lower (10(5) cfu g(-1)), which indicates the ability of LAB to grow and compete with few carbohydrates available and in the presence of moderate salt concentrations. L. monocytogenes was not found in any sample. Forty-one percent of LAB strains isolated exhibited inhibitory capacity against Listeria innocua, in a plate assay. A majority of the inhibitory effects were non-bacteriocinogenic, but nevertheless were very competitive cultures which may provide an additional hurdle for improved preservation by natural means.

Efecto de la temperatura de almacenamiento sobre los cambios post-mortem y frescura en híbridos de Cachama (Colossoma macropomum x Piaractus brachypomus cultivaodos) / Effect of storage temperature on post-mortem changes and freshness of cultured hybrids of cachama (Colossoma macropomum x Piaractus brachypomus)

Con la finalidad de evaluar la influencia de la temperatura de almacenamiento sobre los cambios post mortem en híbridos de cachama (colossoma macropomum x piaractus brachypomus) cultivados, se almacenaron 80 ejemplares de pescado, bajo tres temperaturas: 0ºC (agua mas hielo) y 27ñ3ºC (ambiente). Durante el almacenamiento se evaluó el desarrollo de rigor mortis (IR), concentración de adenosina trifosfato (ATP), adenosina difosfato (ADP), adenosina monosfato (AMP), inosina monosfato (IMP), inosina (HxR), hipoxantina (Hx) por HPLC e índice de frescura (valor K). En los pescados almacenados a 0ºC se presentó una contradicción violenta, con valores de IR máximo de 100 por ciento en 2 horas, mientras que a 10ºC la máxima contracción se presentó después de 10 horas y a 27ñ3ºC, el máximo valor de IR fue de 82,97 por ciento después de 24 horas. La tasa de hidrólisis de ATP fue mayor durante las primeras 24 horas en los pescados almacenados a 10ºC, seguido por los almacenados a 27ñ3ºC y por los almacenados a 0ºC. El valor K, se incrementó más lentamente en los pescados a 0ºC que en las otras temperaturas. En todas las temperaturas se observó una correlación positiva significativa (p<0.05) de los valores K con el tiempo de almacenamiento (r =0,931; r =,0945). Los híbridos de cachama permanecieron muy frescos hasta 14 días a 0ºC, hasta 7 días a 10ºC y hasta 8 horas a 27ñ3ºC.

Estabilidad de híbridos de Bagre pseudoplatystoma fasciatum x pimelodus blochii y Pseudoplatystoma fasciatum x perrunichthys perruno almacenados en hielo / Stability of pseudoplatystoma fasciatum x pimelodus blonchii and pseudoplatystoma fasciatum x perrunichthys perruno catfish hybrids stored on ice

Híbridos de bagre provenientes de los cruces P. fasciatum x P. blochii y P. fasciatum x P. perruno, designados como "chorrosco" y "perruno" respectivamente, cultivados, caracterizados mediante análisis proximal y rendimiento porcentual, fueron almacenados en hielo por 19 días evaluándoles la frescura mediante análisis químico (ph, nitrógeno básico volátil), microbiológico (contaje de aeróbios mesófilos y psicrófilos) y sensorial. El contenido graso del bagre "chorrosco" fue aproximadamente el doble al del bagre "perruno", el rendimiento porcentual de ambos híbridos fue similar. Los resultados obtenidos durante el almacenamiento sugieren que la evaluación sensorial es el indicador más útil para predecir el tiempo de vida útil de ambos híbridos y que el tiempo límite de aceptabilidad corresponde con el 10mo. día de almacenamiento en hielo para el bagre "perruno" y entre el 10mo y 12vo día para el bagre "chorrosco" , después comienza un rápido deterioro. Las determinaciones de ph y nitrógeno básico volátil dan información sobre el estado de frescura de ambos híbridos. Para el 7mo día de almacenamiento en hielo el ph alcanzó valores de 7.3 y 7.5 para "chorrosco" y "perruno" respectivamente; los valores de nitrógeno básico volátil se incrementaron de 15.49 mg/100g a 33.5 mg/100g para el bagre "chorrosco" y de 9.62 mg/100g a 30.38 mg/100g en el bagre "perruno", estos incrementos en los valores de ph y de NBVT coincidieron con el inicio de pérdida de la frescura de ambos híbridos según la escala de criterios utilizada en la evaluación sensorial. En el límite de aceptabilidad los contajes de microorganismos psicrófilos y mesófilos fueron del orden de 10 elevado a la cinco UFC/g en el caso del bagre "perruno" y de 10 elevado a la cinco UFC/g y 10 elevado a la tres UFC/g respectivamente, en el caso del bagre "chorrosco"

Control de la actividad proteolítica en ensilado de pescado / Proteolitic activity control in fish silage

Se elaboró un ensilado microbiano a partir de una mezcla de pescado de bajo valor comercial y la adición de 15 por ciento de melaza, almacenándolo a 35ºC. Se realizaron experiencias para observar el efecto de la adición de una preparación comercial de bromelina (0,20-0,90 por ciento) sobre el proceso de obtención del ensilado. Estas concentraciones fueron calculadas en base a los resultados obtenidos en las pruebas bioquímicas realizadas en este trabajo. La licuefacción del ensilado se evaluó por medio de determinaciónes de consistencia (cm recorridos en 30 s) y de nitrógeno no proteíco. Los resultados indican que la adición de bromelina (0,7 por ciento ó más) acorta considerablemente el tiempo de licuefacción del producto obteniéndose la mayor actividad proteolítica en las primeras 3 horas de almacenamiento. Tales ensilados alcanzaron su licuefacción máxima en 12 horas en comparación con ensilados a los que no se les adicionó bromelina los cuales se licuaron en 15 días. Por lo tanto, se recomienda la inclusión de la preparación de bromelia comercial para la obtención de los ensilados