Antimicrobial characterization and safety aspects of the bacteriocinogenic Enterococcus hirae F420 isolated from Moroccan raw goat milk.

The F420 strain, isolated from raw goat milk and identified as Enterococcus hirae, was selected because of its strong activity against gram-positive bacteria, including Listeria monocytogenes. Interestingly, the F420 strain lacks the virulence genes and decarboxylase activity of histidine, lysine, and ornithine, and it is susceptible to 11 of 14 tested antibiotics, including vancomycin. The antimicrobial compounds produced by E. hirae F420 strain showed high resistance to heat treatment and to acidic and basic pHs. The MALDI-TOF mass spectrometry analysis coupled with the sequence of peptide and structural gene analysis of one of the purified enterocins showed 100% identity with enterocin P (EntP), previously described in E. faecium strains. The structural gene for EntP is located on a plasmid of 65 kb. Other enterocins with molecular mass higher than 7 kDa were also detected. This is the first report of the production of EntP by E. hirae species naturally occurring in foods. The biotechnological characteristics of the F420 strain and its enterocins indicate their potential for application in the control of L. monocytogenes and other undesirable bacteria in food systems.

Aplicaciones del exopolisacárido producido por la cepa HK30 de Halomonas nitroreducens en la industria farmacéutica / Pharmaceutical applications of the exopolysaccharides produced by Halomonas nitroreducens strain HK30

INTRODUCCIÓN: Una de las líneas de investigación que desarrolla el Grupo Exopolisacáridos Microbianos (BIO 188) es el aislamiento y caracterización de nuevos microorganismos halófilos productores de EPSs con propiedades físicas y químicas competitivas con los ya existentes en la industria. De estos estudios destaca el polímero V2-7 (Halomonas eurihalina F2-7) que origina geles transparentes1, el maurano(H. maura S-30) cuya viscosidad compite con la del xantano2 y los EPSs producidos por Salipigermucescens, Halomonas. anticariensis, H. ventosae, Idiomarina fontislapidosi, I. ramblicola y Alteromonas hispanica que emulsionan diferentes compuestos orgánicos y pueden actuar como biodetoxificadores 3,4. Además, estos polímeros tienen sulfatos en su composición que es una característica inusual e interesante para su aplicación en Medicina y el producido por S. mucescens contiene un carbohidrato llamado fucosa, con interesantes aplicaciones en cosmética y que resulta caro de obtener por síntesis química 5. El grupo de investigación tiene colaboraciones con empresas del sector alimentario, farmacéutico y cosmético para aplicar las propiedades funcionales de sus polisacáridos en estas áreas. OBJETIVO: La búsqueda y selección de bacterias productoras de exopolisacáridos de ambientes hipersalinos situados en Marruecos con propiedades interesantes para la industria(AU)

METODOLOGÍA: Cepas bacterianas y condiciones de cultivo. Las cepas fueron aisladas de ambientes hipersalinos situados en Marruecos (suelos y aguas hipersalinos, alimentos en salazón y las raíces de las plantas marinas). Para producir los EPSs las bacterias se cultivaron en medio MY 7,5 % p/v de sales y se incubaron a 32ºC en agitación (100 rpm) durante 5 días. Producción de exopolisacáridos. Los exopolisacáridos se extrajeron de acuerdo con la metodología descrita por Quesada y col. 6 Caracterización química y determinación de la masa molecular. El contenido en carbohidratos, proteínas, ácidos urónicos, restos acetilos, iones sulfato y fosfato, así como la composición en monosacáridos y la masa molecular de los EPSs purificados se analizó siguiendo la metodología previamente descrita en nuestros trabajos 4. Caracterización física. El estudio de las características reológicas y actividad emulgente se realizó siguiendo la metodología descrita por Mata y col. 3. Estudio de la participación de los EPS en la formación de biofilms. Se hizo mediante tinción con cristal violeta en placas microtiter tras 40 horas de incubación en medios MY y MM al 7,5% (p/v) de NaCl7. Determinación de la actividad floculante. Se prepararon soluciones de caolín y se mezclaron con diferentes concentraciones de EPS 8,9. CONCLUSIÓN /DISCUSIÓN: A partir de una colección de 120 cepas se han seleccionado las 26 mejores productoras de EPSs, que han sido identificadas taxonómicamente. Tras un primer análisis de la productividad y características físicas y químicas de los EPSs, se ha elegido el polímero producido por la cepa HK30 para llevar a cabo un estudio más completo. La cepa HK30, por su mayor rendimiento en la producción de un polímero con actividad viscosizante y emulgente, se considera un excelente candidato de interés biotecnológico con aplicaciones en la industria farmacéutica(AU)

The Microbial Exopolysaccharide Research Group (BIO 188) has been carrying out a wide search aimed at isolating and characterizing exopolysac charides produced by halophilic microorganisms with different physical and chemical properties of interest to industry. As a result of these studies, we selected and characterized the polymer V2-7 (Halomonas eurihalina F2-7) that has emulsifying activity and jellifying properties at acid pH1; the mauran, which has a viscosifying activity similar to that of xanthan gum2 and the EPSs produced by Salipiger mucosus, Halomonas anticariensis, H.ventosae, Idiomarina fontislapidosi, I. ramblicola and Alteromonas hispanica that emulsify different organic compounds 3,4. In addition, all of these polymers have sulphate groups in their composition, anunusual and interesting feature for their application in medicine. More over, the fucose-rich EPS of Salipiger mucosus has applications in the fields of medicine and cosmetics. The chemical orenzymatic hydrolysis of fucose-rich polysaccharides offers a new efficient way to process fucose 5. The Research Group BIO 188 has established collaborations with several companies related to the food, pharmaceutical and cosmetics industries in order to test the functional properties of our halophilicexopolysaccharides in these areas. AIM: Searching and selection of exopolysaccharide-producing bacteria from hypersaline environments located in Morocco with interesting properties to industry(AU)

MATERIAL AND METHODS: Bacterial strains and culture conditions. The strains were isolated from hypersaline environments in Morocco. Bacteria were cultured in MY with 7.5% (w/v) of salts and incubated at 32 °C with shaking(100 rpm) for 5 days. Production of exopolysaccharides. The exopolysaccharides were isolated according to the methodology described by Quesada et al. 6 Chemical characterization and molecular weight determination. The content of carbohydrates, proteins, uronic acids, acetyl residues, sulphates, phosphates, monosaccharide composition, and molecular weight of purified EPSs were analyzed following the methodology previously described [4]. Physical characterization. The study of the rheological properties and emulsifying activity was carried out following the methodology described by Mata et al. 3 Production of biofilms. The production of biofilms were tested by crystal violet staining in microtiter plates after 40 hours incubation in the media MM and MY 7.5% (w/v) 7. Determination of flocculating activity. Kaolin solutions were prepared and mixed with different concentrations of EPS8,9(AU)

Functional and ultrastructural changes in Pseudomonas aeruginosa and Staphylococcus aureus cells induced by Cinnamomum verum essential oil.

AIMS: To study cellular damage induced by Cinnamomum verum essential oil in Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213. METHODS AND RESULTS: The effect of cinnamon bark essential oil on these two strains was evaluated by plate counts, potassium leakage, flow cytometry and transmission electron microscopy (TEM). Exposure to this oil induced alterations in the bacterial membrane of Ps. aeruginosa, which led to the collapse of membrane potential, as demonstrated by bis-oxonol staining, and loss of membrane-selective permeability, as indicated by efflux of K(+) and propidium iodide accumulation. Thus, respiratory activity was inhibited, leading to cell death. In Staph. aureus, cells treated with the oil entered a viable but noncultivable (VNC) state. The oil initially caused a considerable decrease in the metabolic activity and in the replication capacity of these bacterial cells. The loss of membrane integrity appeared later, as indicated by bis-oxonol and Propidium iodide (PI) staining. Data provided by TEM showed various structural effects in response to cinnamon essential oil. In Ps. aeruginosa cells, coagulated cytoplasmic material was observed, and intracellular material was seen in the surrounding environment, while oil-treated Staph. aureus showed fibres extending from the cell surface. CONCLUSIONS: Cinnamon essential oil damages the cellular membrane of Ps. aeruginosa, which leads to cell death. There is evidence of VNC Staph. aureus after exposure to the oil. SIGNIFICANCE AND IMPACT OF THE STUDY: Cinnamon essential oil shows effective antimicrobial activity and health benefits and is therefore considered a potential food additive. To use this oil as a natural food preservative, especially in combination with other preservation methods, a thorough understanding of the mechanism through which this oil exerts its antibacterial action is required.

Investigation of functional and morphological changes in Pseudomonas aeruginosa and Staphylococcus aureus cells induced by Origanum compactum essential oil.

AIMS: Evaluation of the cellular effects of Origanum compactum essential oil on Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213. METHODS AND RESULTS: The damage induced by O. compactum essential oil on these two strains has been studied using different techniques: plate count, potassium leakage, flow cytometry (FC) and transmission electron microscopy (TEM). The results showed that oil treatment led to reduction of cells viability and dissipated potassium ion gradients. Flow cytometric analysis showed that oil treatment promoted the accumulation of bis-oxonol and the membrane-impermeable nucleic acid stain propidium iodide (PI), indicating the loss of membrane potential and permeability. The ability to reduce 5-cyano-2,3-ditolyl tetrazolium chloride was inhibited. Unlike in Ps. aeruginosa, membrane potential and membrane permeability in Staph. aureus cells were affected by oil concentration and contact time. Finally, TEM showed various structural effects. Mesosome-like structures were seen in oil-treated Staph. aureus cells whereas in Ps. aeruginosa, coagulated cytoplasmic material and liberation of membrane vesicles were observed, and intracellular material was seen in the surrounding environment. Both FC and TEM revealed that the effects in Ps. aeruginosa were greater than in Staph. aureus. CONCLUSIONS: Oregano essential oil induces membrane damage showed by the leakage of potassium and uptake of PI and bis-oxonol. Ultrastructural alterations and the loss of cell viability were observed. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the mode of antibacterial effect of the oil studied is of a great interest in it further application as natural preservative in food or pharmaceutical industries.

Investigation of the mutagenic and antimutagenic effects of Origanum compactum essential oil and some of its constituents.

In the present study, the chemical composition of Origanum compactum essential oil was determined by gas chromatography and mass spectrometry, and its mutagenic and antimutagenic activities were investigated by the somatic mutation and recombination test (SMART) in Drosophila melanogaster. No significant increase in the number of somatic mutations was observed with the essential oil tested using both the standard (ST) and high bio-activation (HB) cross. In order to investigate the antimutagenic effect of the essential oil, we have tested the effect on the indirect-acting mutagen urethane (URE), as well as the direct-acting mutagen methyl methanesulfonate (MMS). O. compactum essential oil showed a strong inhibitory effect against URE-induced mutagenicity, especially with the HB cross. However, only a weak inhibitory effect on the mutagenicity induced by MMS was observed. These results suggest that the detected antimutagenicity could be mediated by an inhibitory effect on metabolic activation. The essential oil was fractionated to identify the components responsible of the suppressing effect detected. Seven fractions were obtained: two of them showed the most potent inhibitory effect against URE-induced mutagenicity and were further fractionated. The sub-fractions obtained from the second chromatographic fractionation were tested for their antimutagenic activity, together with carvacrol and thymol. The highest antimutagenic effect obtained with the sub-fractions was similar to the effect of the crude essential oil, as well as to the effect of carvacrol alone. These results suggest the absence of a synergic antimutagenic effect between the components of O. compactum essential oil and indicate that carvacrol was the most active oil component.

Enterococcus faecium F58, a bacteriocinogenic strain naturally occurring in Jben, a soft, farmhouse goat's cheese made in Morocco.

AIMS: Characterization of Ent F-58 produced by Enterococcus faecium strain F58 isolated from Jben, a soft, farmhouse goat's cheese manufactured without starter cultures. METHODS AND RESULTS: E. faecium strain F58 was isolated because of its broad inhibitory spectrum, including activity against food-borne pathogenic and spoilage bacteria. The antimicrobial substance was produced during the growth phase, with maximum production after 16-20 h of incubation at 30 degrees C, and was stable over a wide pH range (4-8) and at high temperatures (5 min at 100 degrees C). The enterocin was purified to homogeneity using cation exchange and hydrophobic interaction on C-18 and reverse-phase high-performance liquid chromatography. The activity was eluted as two individual active fractions (F-58A and F-58B) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis showed masses of 5210.5 and 5234.3 Da respectively. Both peptides were partially sequenced by Edman degradation, and amino-acid sequencing revealed high similarity with enterocin L50 (I). PCR-amplified fragments containing the structural genes for F-58 A and B were located in a 22-kb plasmid harboured by this strain. We verified that it also holds the structural gene for P-like enterocin. CONCLUSION: E. faecium strain F58 from Jben cheese, a producer of enterocin L50, exerts an inhibitory effect against strains of genera such as Listeria, Staphylococcus, Clostridium, Brochothrix and Bacillus. Enterocin was characterized according to its functional and biological properties, purification to homogeneity and an analysis of its amino acid and genetic sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: E. faecium strain F58 is a newly discovered producer of enterocin L50, the biotechnological characteristics of which indicate its potential for application as a protective agent against pathogens and spoilage bacteria in foods.