New bio-strategies for ochratoxin A detoxification using lactic acid bacteria

The occurrence of mycotoxigenic moulds such as Aspergillus, Penicillium and Fusarium in food and feed has an important impact on public health, by the appearance of acute and chronic mycotoxicoses in humans and animals, which is more severe in the developing countries due to lack of food security, poverty and malnutrition. This mould contamination also constitutes a major economic problem due the lost of crop production. A great variety of filamentous fungi is able to produce highly toxic secondary metabolites known as mycotoxins. Most of the mycotoxins are carcinogenic, mutagenic, neurotoxic and immunosuppressive, being ochratoxin A (OTA) one of the most important. OTA is toxic to animals and humans, mainly due to its nephrotoxic properties. Several approaches have been developed for decontamination of mycotoxins in foods, such as, prevention of contamination, biodegradation of mycotoxins-containing food and feed with microorganisms or enzymes and inhibition or absorption of mycotoxin content of consumed food into the digestive tract. Some group of Gram-positive bacteria named lactic acid bacteria (LAB) are able to release some molecules that can influence the mould growth, improving the shelf life of many fermented products and reducing health risks due to exposure to mycotoxins. Some LAB are capable of mycotoxin detoxification. Recently our group was the first to describe the ability of LAB strains to biodegrade OTA, more specifically, Pediococcus parvulus strains isolated from Douro wines. The pathway of this biodegradation was identified previously in other microorganisms. OTA can be degraded through the hydrolysis of the amide bond that links the L-β-phenylalanine molecule to the ochratoxin alpha (OTα) a non toxic compound. It is known that some peptidases from different origins can mediate the hydrolysis reaction like, carboxypeptidase A an enzyme from the bovine pancreas, a commercial lipase and several commercial proteases. So, we wanted to have a better understanding of this OTA degradation process when LAB are involved and identify which molecules where present in this process. For achieving our aim we used some bioinformatics tools (BLAST, CLUSTALX2, CLC Sequence Viewer 7, Finch TV). We also designed specific primers and realized gene specific PCR. The template DNA used came from LAB strains samples of our previous work, and other DNA LAB strains isolated from elderberry fruit, silage, milk and sausages. Through the employment of bioinformatics tools it was possible to identify several proteins belonging to the carboxypeptidase family that participate in the process of OTA degradation, such as serine type D-Ala-D-Ala carboxypeptidase and membrane carboxypeptidase. In conclusions, this work has identified carboxypeptidase proteins being one of the molecules present in the OTA degradation process when LAB are involved.

Bio-detoxification of mycotoxins by lactic acid bacteria from different food matrices

Lactic acid bacteria (LAB) play a key role in the biopreservation of a wide range of fermented food products, such as yogurt, cheese, fermented milks, meat, fish, vegetables (sauerkraut, olives and pickles), certain beer brands, wines and silage, allowing their safe consumption, which gave to these bacteria a GRAS (Generally Recognised as Safe) status. Besides that, the use of LAB in food and feed is a promising strategy to reduce the exposure to dietary mycotoxins, improving their shelf life and reducing health risks, given the unique mycotoxin decontaminating characteristic of some LAB. Mycotoxins present carcinogenic, mutagenic, teratogenic, neurotoxic and immunosuppressive effects over animals and Humans, being the most important ochratoxin A (OTA), aflatoxins (AFB1), trichothecenes, zearalenone (ZEA), fumonisin (FUM) and patulin. In a previous work of our group it was observed OTA biodegradation by some strains of Pediococcus parvulus isolated from Douro wines. So, the aim of this study was to enlarge the screening of the biodetoxification over more mycotoxins besides OTA, including AFB1, and ZEA. This ability was checked in a collection of LAB isolated from vegetable (wine, olives, fruits and silage) and animal (milk and dairy products, sausages) sources. All LAB strains were characterized phenotypically (Gram, catalase) and genotypically. Molecular characterisation of all LAB strains was performed using genomic fingerprinting by MSP- PCR with (GTG)5 and csM13 primers. The identification of the isolates was confirmed by 16S rDNA sequencing. To study the ability of LAB strains to degrade OTA, AFB1 and ZEA, a MRS broth medium was supplemented with 2.0 g/mL of each mycotoxin. For each strain, 2 mL of MRS supplemented with the mycotoxins was inoculated in triplicate with 109 CFU/mL. The culture media and bacterial cells were extracted by the addition of an equal volume of acetonitrile/methanol/acetic acid (78:20:2 v/v/v) to the culture tubes. A 2 mL sample was then collected and filtered into a clean 2 mL vial using PP filters with 0.45 m pores. The samples were preserved at 4 °C until HPLC analysis. Among LAB tested, 10 strains isolated from milk were able to eliminate AFB1, belonging to Lactobacillus casei (7), Lb. paracasei (1), Lb. plantarum (1) and 1 to Leuconostoc mesenteroides. Two strains of Enterococcus faecium and one of Ec. faecalis from sausage eliminated ZEA. Concerning to strains of vegetal origin, one Lb. plantarum isolated from elderberry fruit, one Lb. buchnerii and one Lb. parafarraginis both isolated from silage eliminated ZEA. Other 2 strains of Lb. plantarum from silage were able to degrade both ZEA and OTA, and 1 Lb. buchnerii showed activity over AFB1. These enzymatic activities were also verified genotypically through specific gene PCR and posteriorly confirmed by sequencing analysis. In conclusion, due the ability of some strains of LAB isolated from different sources to eliminate OTA, AFB1 and ZEA one can recognize their potential biotechnological application to reduce the health hazards associated with these mycotoxins. They may be suitable as silage inoculants or as feed additives or even in food industry.

Influence of oxygen conditions on bacterial interactions within biofilms related with cystic fibrosis

Dissertação de mestrado em Bioengineering

Sistemática filogenética y biología reproductiva con énfasis en solanum y capsicum (solanaceae). Systematics, phylogeny and reproductive biology with emphasis in solanum and capsicum (solanaceae).

Muchas respuestas a preguntas básicas sobre relaciones evolutivas, ubicación sistemática y evolución de caracteres morfológicos y ecológicos pueden ser obtenidas a través de las reconstrucciones filogenéticas. Sobre este contexto se pretende encarar en este proyecto estudios de filogenia molecular, revisiones sistemáticas, biología reproductiva y citogenética en Solanáceas americanas. Se intentará resolver la delimitación específica de Solanum sect. Solanum y Geminata, y Capsicum, y establecer relaciones filogenéticas en estos grupos. Se harán revisiones analizándose caracteres vegetativos y reproductivos críticos para evaluar su variabilidad y definir su valor taxonómico; para los estudios moleculares se utilizarán los marcadores ndhF, trnT-L, trnL-F y waxy. En base a los resultados se propondrán agrupamientos y relaciones de parentesco. Además, se hará un estudio cariosistemático para caracterizar y circunscribir especies en Solanum y miembros de la tribu Physaleae, y hasta variedades y/o cultivares en Capsicum, mediante técnicas clásicas y de bandeos de fluorescencia y AgNOR e hibridación in situ fluorescente (FISH). A nivel reproductivo, se estudiará la ecofisiología en las estructuras masculinas y su incidencia en la fructificación en Capscium baccatum. El desarrollo de esta temática comprende experiencias in vivo (a campo y en laboratorio) así como estudios histológicos y químicos.Se espera avanzar en la resolución de algunos problemas: 1) la complicada delimitación de especies de los taxones en estudio; 2) las relaciones filogenéticas en algunos de ellos; 3) la falta de conocimiento de la organización genómica; 4) el origen de las especies cultivadas de Capsicum. En cuanto a la biología reproductiva, para C. baccatum se pretende avanzar en el conocimiento de variables de relevancia en la reproducción, en especial los efectos del ambiente.

Impacto de las prácticas agrícolas en los recursos hídricos de la provincia de Córdoba: aislamiento y caracterización de bacterias degradadoras de pesticidas para su utilización en procesos de biorremediación. Impact of agricultural activities on aquatic environments of Córdoba: isolation and characterization of bacteria with pesticide removal capacities for bioremediation treatments.

Los sistemas de agua dulce constituyen fuentes vitales para el desarrollo de la vida. Entre otras causas, el excesivo y poco controlado uso de pesticidas por las prácticas agrícolas modernas ha contribuido con la degradación de estos ecosistemas en la provincia de Córdoba (Secretaría de Ambiente, 2008). Los pesticidas son compuestos tóxicos y químicamente estables en el ambiente. Diversas especies microbianas presentan la capacidad para mineralizar dichos compuestos, siendo uno de los mecanismos de descomposición más importante. En el presente trabajo se pretende (1) Detectar y cuantificar principios activos de pesticidas en diferentes aguas ambientales de la región centro-sur de la provincia de Córdoba; (2) Aislar y caracterizar especies bacterianas a partir de muestras de aguas superficiales y subterráneas de la región que demuestren ser eficientes en la biodegradación de diferentes pesticidas. Para ello, se estudiará la presencia de 10 pesticidas organoclorados y organofosforados (lindano, 2,4D, DDT, p,p-DDE, �-endosulfán, �-endosulfán, clorpirifós, dimetoato) y herbicidas (atrazina) en muestras de agua superficiales y subterráneas (8 - 12m de profundidad) de la región agrícola centro-sur de Córdoba. Se establecerán diferentes puntos de muestreo en las principales cuencas hidrográficas: Río Tercero y Embalse de Río Tercero, Canal Desviador de Bell Ville, Laguna La Salada, Río Saladillo, Río Carcarañá y Río Cuarto. La determinación de pesticidas se realizará mediante Cromatografía de Gases (GC). Conjuntamente, se realizará el aislamiento de microorganismos a partir de las muestras de agua suplementadas con diferentes concentraciones de pesticidas (López et al., 2005) y se procederá a la caracterización morfológica y bioquímica (Lechevalier, 1989). Se realizarán curvas de crecimiento (DO600) y se determinará la viabilidad celular mediante el método de recuento en placa. El potencial catabólico de cada aislamiento se determinará analizando la concentración residual de pesticidas en el sobrenadante de los cultivos (Benimeli et al, 2003) y mediante ensayos de resting-cell (Hernández et al, 2008). Finalmente se realizará la caracterización genética (Weisburg, 1991) de los aislamientos que demuestren una mayor eficiencia en la biodegradación de pesticidas. El presente estudio pretende abordar una temática prioritaria como es la contaminación ambiental de ecosistemas acuáticos de la provincia de Córdoba. la detección de principios activos de pesticidas sería, por lo tanto, un indicador de contaminación de origen antropológica y brindaría información respecto al deterioro de la calidad del agua. Por otra parte, el aislamiento de bacterias adaptadas a las condiciones ecológicas de la región y capaces de metabolizar eficientemente diferentes pesticidas como fuentes de carbono y energía sería beneficioso para su utilización en futuros ensayos de biorremediación.

Effects of selected post harvest processes on brown crab (Cancerpagurus L.) and European lobster (Homarus gammarus L.) from Irish Crustacean Fisheries

The crustacean fishery is important to the socio economics of rural and island communities around Ireland; with brown crab (Cancer pagurus) and European lobster (.Homarus gammarus) being the most valuable shellfish species. Brown crab and lobster are marketed live with the majority being exported from Ireland to southern Europe. Post capture processes used in Ireland are very subjective but promote fresh, live products. Common practices used in the crustacean fishery include nicking of brown crab and long term storage of lobster. This study showed that nicking resulted in elevated mean lactate levels of 17.90% (StDev ± 1.74) and elevated mean glucose levels of 120.55 % (StDev ± 0.26) with mean circulating bacteria levels 9 times greater in nicked crab. Nicking resulted in 96.3% increase in tissue necrosis and a subsequent reduction in product quality. These factors possibly compromise the host’s defense system, which may ultimately reduce the animal’s ability to cope with additional stressors caused by post-harvest processes. Long term storage allows lobster to be stored until the market is less saturated and prices are higher. This investigation found that some lobsters contracted bacterial biofilms as a result of long term storage. Bacteria isolated from biofilms were identified as Arcobacter and Campylobacterales with identity and alignment scores of 80% andd 88% respectively.

Age, growth and reproductive biology of Plaice, (Pleuronectes platessa L.) in Irish waters, 2003-2005

The current study presents data on age and growth for plaice (Pleuronectes platessa L.) sampled between November 2003 and February 2005 in ICES areas Via (northwest coast of Ireland), Vila (Irish Sea), Vllg (Celtic Sea), VDj (southwest coast of Ireland) and VHb (west coast of Ireland), and data on the reproductive biology and maturity of plaice in ICES area Vllb (west coast of Ireland). This is the first detailed account of the biology of plaice for some of these areas. It is intended that this study will improve understanding of the life cycle of plaice and help fisheries scientists to better predict the effect of fishing effort on Irish plaice stocks. The overall length range found for plaice was 9-51.99cm TL, with a length range of 9-5 lcm TL for females and 9-40cm for males. In all ICES areas the length range for female fish was larger than for male fish. The age range of plaice sampled during this study was 1 to 16 years. In all ICES areas females had a greater range in ages and fish in the larger age groups. From analysis of length and age data it was concluded that there was a significant difference (P=0.000) in growth rate of males and females between ICES areas sampled in March 2004. The highest rate of fishing mortality was determined for ICES area Via (F=1.06) and the lowest for ICES area Vila (F=0.56). In each ICES area male and female plaice have fully recruited to the population by age 4, with the exception of females in ICES area Via, for which a tr value of 5 years was determined. Length at first maturity (L50%) was determined to be 23cm and 21cm for males and females respectively. Age at first maturity (A50%) was determined to be 3 years for both males and females. It was found that males and females in ICES areas Vllb, Vila and Via are well above the length and age at first maturity when they are recruited to the fishery. In ICES area Vllb female plaice spawn from November to March, with peak spawning occurring in February, and male plaice spawn from November to April, with peak spawning occurring in November. Spawning females had an age range of 2 to 10 years and spawning males had an age range of 2 to 7 years. From the oocyte length frequency distributions, it was determined that the plaice is a determinate batch spawner. During this investigation a total of 177 ovaries and 127 testes were staged using both macroscopic and histological criteria. The overall percentage of maturity stages which compared favorably between the two assessment methods was 22.03% for female plaice and 37.80% for male plaice. In general, the findings of this study indicate that there was a very poor match between the macroscopic and histological assessment methods. Given that the histological determination of these stages is based on the observation of a distinct set of developmental features, it is expected that it would be more accurate to use histologically assessed gonads to calculate the annual percentage maturity assessment. The biology of plaice in the areas studied is compared with previous studies of plaice in Irish and European waters.

A manual of human physiology, including histology and microscopical anatomy, with special reference to the requirements of practical medicine by Dr. L. Landois ... Tr. from the 4th German ed. With additions by William Stirling ...

v. 1

Non root feeding with two sources of zinc on Coffea arábica L. 'mundo novo'(B.Rodr.) Choussy

A trial was carried out on an eight old coffee plantation with visible zinc problems. The plantation was situated nearly the city of Jaú (22º30'S, 48º30'W). State of São Paulo, Brazil. The soil is classified as medium texture Oxisol of low base saturation (Latossol Vermelho Amarelo - fase arenosa). The pulverization program started in november 1977, followed in march and July 1978 (heavy harvest) and ended in march and July 1979 (light harvest). Is should be mentioned that a well reconized characteristic of arábica coffe is its habit of biennial bearing, a very heavy harvest is most often followed by a light load the next year. The following treatments and amounts of chemicals per cova hole (4 trees) were tested in accordance with a random block design: 1. 1 g of zinc (zinc sulphate, 0.5%) 2. 3 g of nitrogen (urea, 1.3%) 3. 1 g of zinc + 3 g of nitrogen (zinc sulphate 0.5% + urea 1.3%) 4. 0.25 g, 0.50 g, 1.00 g, 2.00 g of zinc plus 0.75 g, 1.50 g, 3.00 g and 6.00 of nitrogen (correspondent to NZN* 15-0-0-5 as 0.75%, 1-5%, 3.0% and 6.0% by v/v). Foliar absorption data were obtained by collecting the 3rd and 4th pairs of the coffee leaves and analysed them for N, P, K, Ca, Mg, S, B, Cu, Fe, Mn, and Zn. The main results may be summarized as follows: 1. The maximum calculated yields of clean coffee were obtained by the applications of 5.84 1 of NZN (1.13%) per hectare. 2. The applications of zinc sulphate (0.5%) and urea (1.3%) together or separate did not affected the coffee bean production. 3. The applications of 15.0 1 of NZN per hectare reduced the coffee yields. 4. Leaf damages and burning symptoms were observed by the applications of urea (1.3%) plus zinc sulphate (0.5%) and larger doses than 7.5 1 of NZN per hectare. 5. Leaf tissue analysis show that the concentrations of the elements were affecred by the age of the leaves and by the yields of the coffee trees. 6. The applications of increasing doses of NZN causes an increase in the concentration of zinc, manganese and boron in the leaves and decreased the concentration in calcium and potassium the leaves. 7. The concentration of zinc in the leaves associated with the heavy harvest, in July, was 70.0 ppm.

Austral Hepaticae. with a monograph of the genus in temperate Australasia and commentary on extra-Australasian taxa / John J. Engel, G.L. Smith Merrill.

n.s. no.44(2004)