Antioxidant potential of oregano (Oreganum vulgare L.), basil (Ocimum basilicum L.) and thyme (Thymus vulgaris L.): application of oleoresins in vegetable oil

Por razões econômicas e de proteção à saúde, pesquisas têm sido dirigidas para ampliar a estabilidade dos óleos vegetais. Existe uma tendência para a adição de antioxidantes naturais, em particular, um crescente interesse em ervas e especiarias. Desta forma, os objetivos deste trabalho foram avaliar o potencial antioxidante das oleorresinas de orégano, manjericão e tomilho e seu comportamento ao serem aplicadas ao óleo de soja em diferentes concentrações. Numa primeira etapa foi determinada a atividade antioxidante pelo sistema β-caroteno/ácido linoleico e a quantificação de compostos fenólicos totais. Posteriormente, foram adicionadas ao óleo de soja diferentes concentrações das oleorresinas (500 a 3000 mg.kg-1) e analisado seu potencial antioxidante por meio da estabilidade oxidativa utilizando o Rancimat. A concentração de 3000 mg.kg-1das oleorresinas de orégano e tomilho foi a que apresentou melhor estabilidade oxidativa ao óleo de soja, o que as tornam alternativa natural na conservação de óleos vegetais.

Antioxidant potential of oregano extract (Origanum vulgare L.)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Análise de crescimento, trocas gasosas, potencila antioxidante e óleo essencial de Origanum vulgare L. ssp. vulgare

Pós-graduação em Ciências Biológicas (Botânica) - IBB

Estudio molecular de gluteninas de alto y bajo peso molecular en Triticum aestivum ssp. vulgare L. y su relación con la calidad panadera

El trigo blando (Triticum aestivum ssp vulgare L., AABBDD, 2n=6x=42) presenta propiedades viscoélasticas únicas debidas a la presencia en la harina de las prolaminas: gluteninas y gliadinas. Ambos tipos de proteínas forman parte de la red de gluten. Basándose en la movilidad en SDS-PAGE, las gluteninas se clasifican en dos grupos: gluteninas de alto peso molecular (HMW-GS) y gluteninas de bajo peso molecular (LMW-GS). Los genes que codifican para las HMW-GS se encuentran en tres loci del grupo 1 de cromosomas: Glu-A1, Glu-B1 y Glu-D1. Cada locus codifica para uno o dos polipéptidos o subunidades. La variación alélica de las HMW-GS es el principal determinante de de la calidad harino-panadera y ha sido ampliamente estudiado tanto a nivel de proteína como de ADN. El conocimiento de estas proteínas ha contribuido sustancialmente al progreso de los programas de mejora para la calidad del trigo. Comparadas con las HMW-GS, las LMW-GS forman una familia proteica mucho más compleja. La mayoría de los genes LMW se localizan en el grupo 1 de cromosomas en tres loci: Glu-A3, Glu-B3 y Glu-D3 que se encuentran estrechamente ligados a los loci que codifican para gliadinas. El número de copias de estos genes ha sido estimado entre 10-40 en trigo hexaploide, pero el número exacto aún se desconoce debido a la ausencia de un método eficiente para diferenciar los miembros de esta familia multigénica. La nomenclatura de los alelos LMW-GS por electroforesis convencional es complicada, y diferentes autores asignan distintos alelos a la misma variedad lo que dificulta aún más el estudio de esta compleja familia. El uso de marcadores moleculares para la discriminación de genes LMW, aunque es una tarea dificil, puede ser muy útil para los programas de mejora. El objetivo de este trabajo ha sido profundizar en la relación entre las gluteninas y la calidad panadera y desarrollar marcadores moleculares que permitan ayudar en la correcta clasificación de HMW-GS y LMW-GS. Se han obtenido dos poblaciones de líneas avanzadas F4:6 a partir de los cruzamientos entre las variedades ‘Tigre’ x ‘Gazul’ y ‘Fiel’ x ‘Taber’, seleccionándose para los análisis de calidad las líneas homogéneas para HMW-GS, LMW-GS y gliadinas. La determinación alélica de HMW-GS se llevó a cabo por SDS-PAGE, y se complementó con análisis moleculares, desarrollándose un nuevo marcador de PCR para diferenciar entre las subunidades Bx7 y Bx7*del locus Glu-B1. Resumen 2 La determinación alélica para LMW-GS se llevó a cabo mediante SDS-PAGE siguiendo distintas nomenclaturas y utilizando variedades testigo para cada alelo. El resultado no fue concluyente para el locus Glu-B3, así que se recurrió a marcadores moleculares. El ADN de los parentales y de los testigos se amplificó usando cebadores diseñados en regiones conservadas de los genes LMW y fue posteriormente analizado mediante electroforesis capilar. Los patrones de amplificación obtenidos fueron comparados entre las distintas muestras y permitieron establecer una relación con los alelos de LMW-GS. Con este método se pudo aclarar la determinación alélica de este locus para los cuatro parentales La calidad de la harina fue testada mediante porcentaje de contenido en proteína, prueba de sedimentación (SDSS) y alveógrafo de Chopin (parámetros P, L, P/L y W). Los valores fueron analizados en relación a la composición en gluteninas. Las líneas del cruzamiento ‘Fiel’ x ‘Taber’ mostraron una clara influencia del locus Glu-A3 en la variación de los valores de SDSS. Las líneas que llevaban el nuevo alelo Glu-A3b’ presentaron valores significativamente mayores que los de las líneas con el alelo Glu-A3f. En las líneas procedentes del cruzamiento ‘Tigre ’x ‘Gazul’, los loci Glu-B1 y Glu-B3 loci mostraron ambos influencia en los parámetros de calidad. Los resultados indicaron que: para los valores de SDSS y P, las líneas con las HMW-GS Bx7OE+By8 fueron significativamente mejores que las líneas con Bx17+By18; y las líneas que llevaban el alelo Glu-B3ac presentaban valores de P significativamente superiores que las líneas con el alelo Glu-B3ad y significativamente menores para los valores de L . El análisis de los valores de calidad en relación a los fragmentos LMW amplificados, reveló un efecto significativo entre dos fragmentos (2-616 y 2-636) con los valores de P. La presencia del fragmento 2-636 estaba asociada a valores de P mayores. Estos fragmentos fueron clonados y secuenciados, confirmándose que correspondían a genes del locus Glu-B3. El estudio de la secuencia reveló que la diferencia entre ambos se hallaba en algunos SNPs y en una deleción de 21 nucleótidos que en la proteína correspondería a un InDel de un heptapéptido en la región repetida de la proteína. En este trabajo, la utilización de líneas que difieren en el locus Glu-B3 ha permitido el análisis de la influencia de este locus (el peor caracterizado hasta la fecha) en la calidad panadera. Además, se ha validado el uso de marcadores moleculares en la determinación alélica de las LMW-GS y su relación con la calidad panadera. Summary 3 Bread wheat (Triticum aestivum ssp vulgare L., AABBDD, 2n=6x=42) flour has unique dough viscoelastic properties conferred by prolamins: glutenins and gliadins. Both types of proteins are cross-linked to form gluten polymers. On the basis of their mobility in SDS-PAGE, glutenins can be classified in two groups: high molecular weight glutenins (HMW-GS) and low molecular weight glutenins (LMW-GS). Genes encoding HMW-GS are located on group 1 chromosomes in three loci: Glu-A1, Glu-B1 and Glu-D1, each one encoding two polypeptides, named subunits. Allelic variation of HMW-GS is the most important determinant for bread making quality, and has been exhaustively studied at protein and DNA level. The knowledge of these proteins has substantially contributed to genetic improvement of bread quality in breeding programs. Compared to HMW-GS, LMW-GS are a much more complex family. Most genes encoded LMW-GS are located on group 1 chromosomes. Glu-A3, Glu-B3 and Glu-D3 loci are closely linked to the gliadin loci. The total gene copy number has been estimated to vary from 10–40 in hexaploid wheat. However, the exact copy number of LMW-GS genes is still unknown, mostly due to lack of efficient methods to distinguish members of this multigene family. Nomenclature of LMW-GS alleles is also unclear, and different authors can assign different alleles to the same variety increasing confusion in the study of this complex family. The use of molecular markers for the discrimination of LMW-GS genes might be very useful in breeding programs, but their wide application is not easy. The objective of this work is to gain insight into the relationship between glutenins and bread quality, and the developing of molecular markers that help in the allele classification of HMW-GS and LMW-GS. Two populations of advanced lines F4:6 were obtained from the cross ‘Tigre’ x ‘Gazul’ and ‘Fiel’ x ‘Taber’. Lines homogeneous for HMW-GS, LMW-GS and gliadins pattern were selected for quality analysis. The allele classification of HMW-GS was performed by SDS-PAGE, and then complemented by PCR analysis. A new PCR marker was developed to undoubtedly differentiate between two similar subunits from Glu-B1 locus, Bx7 and Bx7*. The allele classification of LMW-GS was initially performed by SDS-PAGE following different established nomenclatures and using standard varieties. The results were not completely concluding for Glu-B3 locus, so a molecular marker system was applied. DNA from parental lines and standard varieties was amplified using primers designed in conserved domains of LMW genes and analyzed by capillary electrophoresis. The pattern of amplification products obtained was compared among samples and related to the protein allele classification. It was possible to establish a correspondence between specific amplification products and almost all LMW alleles analyzed. With this method, the allele classification of the four parental lines was clarified. Flour quality of F4:6 advanced lines were tested by protein content, sedimentation test (SDSS) and alveograph (P, L, P/L and W). The values were analyzed in relation to the lines prolamin composition. In the ‘Fiel’ x ‘Taber’ population, Glu-A3 locus showed an influence in SDSS values. Lines carrying new allele Glu-A3b’, presented a significantly higher SDSS value than lines with Glu-A3f allele. In the ‘Tigre ’x ‘Gazul’ population, the Glu-B1 and Glu-B3 loci also showed an effect in quality parameters, in SDSS, and P and L values. Results indicated that: for SDSS and P, lines with Bx7OE+By8 were significantly better than lines with Bx17+By18; lines carrying Glu-B3ac allele had a significantly higher P values than Glu-B3ad allele values. lines with and lower L The analysis of quality parameters and amplified LMW fragments revealed a significant influence of two peaks (2-616 y 2-636) in P values. The presence of 2-636 peak gave higher P values than 2-616. These fragments had been cloned and sequenced and identified as Glu-B3 genes. The sequence analysis revealed that the molecular difference between them was some SNPs and a small deletion of 21 nucleotides that in the protein would produce an InDel of a heptapeptide in the repetitive region. In this work, the analysis of two crosses with differences in Glu-3 composition has made possible to study the influence of LMG-GS in quality parameters. Specifically, the influence of Glu-B3, the most interesting and less studied loci has been possible. The results have shown that Glu-B3 allele composition influences the alveograph parameter P (tenacity). The existence of different molecular variants of Glu-B3 alleles have been assessed by using a molecular marker method. This work supports the use of molecular approaches in the study of the very complex LMW-GS family, and validates their application in the analysis of advanced recombinant lines for quality studies.

Influencia de gluteninas LMW en la fuerza del gluten de variedades locales españolas de trigo panadero (Triticum aestivum ssp. vulgare L.)

Las prolaminas son las principales proteínas del endospermo de los trigos panaderos que están relacionadas con la calidad panadera. El objetivo de este trabajo es caracterizar gluteninas LMW según la nomenclatura de Liu et al y relacionarlas con la fuerza panadera

S-Adenosyl-l-Methionine:l-Methionine S-Methyltransferase from Germinating Barley1 : Purification and Localization

S-Adenosyl-l-methionine:l-methionine S-methyltransferase (MMT) catalyzes the synthesis of S-methyl-l-methionine (SMM) from l-methionine and S-adenosyl-l-methionine. SMM content increases during barley (Hordeum vulgare L.) germination. Elucidating the role of this compound is important from both a fundamental and a technological standpoint, because SMM is the precursor of dimethylsulfide, a biogenic source of atmospheric S and an undesired component in beer. We present a simple purification scheme for the MMT from barley consisting of 10% to 25% polyethylene glycol fractionation, anion-exchange chromatography on diethylaminoethyl-Sepharose, and affinity chromatography on adenosine-agarose. A final activity yield of 23% and a 2765-fold purification factor were obtained. After digestion of the protein with protease, the amino acid sequence of a major peptide was determined and used to produce a synthetic peptide. A polyclonal antibody was raised against this synthetic peptide conjugated to activated keyhole limpet hemocyanin. The antibody recognized the 115-kD denatured MMT protein and native MMT. During barley germination, both the specific activity and the amount of MMT protein increased. MMT-specific activity was found to be higher in the root and shoot than in the endosperm. MMT could be localized by an immunohistochemical approach in the shoot, scutellum, and aleurone cells but not in the root or endosperm (including aleurone).

Atividade antimicrobiana de nisina e óleo essencial de orégano (Origanum vulgare L.) sobre Listeria monocytogenes isolada de alimentos

Não consta resumo na publicação.

Endophytic colonization of barley (Hordeum vulgare) roots by the nematophagous fungus Pochonia chlamydosporia reveals plant growth promotion and a general defense and stress transcriptomic response

Plant crop yields are negatively conditioned by a large set of biotic and abiotic factors. An alternative to mitigate these adverse effects is the use of fungal biological control agents and endophytes. The egg-parasitic fungus Pochonia chlamydosporia has been traditionally studied because of its potential as a biological control agent of plant-parasitic nematodes. This fungus can also act as an endophyte in monocot and dicot plants, and has been shown to promote plant growth in different agronomic crops. An Affymetrix 22K Barley GeneChip was used in this work to analyze the barley root transcriptomic response to P. chlamydosporia root colonization. Functional gene ontology (GO) and gene set enrichment analyses showed that genes involved in stress response were enriched in the barley transcriptome under endophytism. An 87.5 % of the probesets identified within the abiotic stress response group encoded heat shock proteins. Additionally, we found in our transcriptomic analysis an up-regulation of genes implicated in the biosynthesis of plant hormones, such as auxin, ethylene and jasmonic acid. Along with these, we detected induction of brassinosteroid insensitive 1-associated receptor kinase 1 (BR1) and other genes related to effector-triggered immunity (ETI) and pattern-triggered immunity (PTI). Our study supports at the molecular level the growth-promoting effect observed in plants endophytically colonized by P. chlamydosporia, which opens the door to further studies addressing the capacity of this fungus to mitigate the negative effects of biotic and abiotic factors on plant crops.

Effect of different doses of post-emergence-applied iodosulfuron on weed control and grain yield of malt barley (Hordeum distichum L.), under Mediterranean conditions

A study was carried out over a two year period (2009/2010 and 2012/2013) on an experimental farm in the Alentejo region (Beja), in southern Portugal where rainfed malt barley (Hordeum distichum L.) is sown at the end of autumn or beginning of winter (November– December). The aim of this experiment was to study the efficiency of the herbicide iodosulfuron-methyl-sodium to control post-emergence broadleaved weeds in this cereal crop. The malt barley crop was established using no-till farming. This technology provides the necessary machine bearing capacity of the soil to assure the post-emergence application of herbicides at two diferente weed development stages. The herbicide iodosulfuron-methyl-sodium was applied at three doses (5.0, 7.5, and 10.0 g a. i. · ha–1) and at two different broadleaved weed development stages (3 to 4 and 6 to 7 pairs of leaves), that also corresponded to two diferente crop development stages (beginning of tillering and complete tillering). The results indicated that early herbicide application timing provided a significantly higher efficiency for all the applied herbicide doses, but this better weed control was not reflected in a higher crop grain yield. The lack of a higher crop grain yield was probably due to a crop phytotoxicity of the herbicide, when used at an early application timing.

Eficácia e seletividade do iodossulfurão-metilo-sódio +mesossulfurão-metilo na cevada dística (Hordeum distichum L.)

Nos anos agrícolas de 2012/2013 e 2013/2014, realizaram-se na Herdade Experimental da Almocreva (Beja) quatro ensaios de campo (dois em cada ano) com o objetivo de estudar a eficácia e seletividade na cultura da cevada dística do herbicida iodossulfurão-metilo-sódio (50 g L-1) + mesossulfurão-metilo (7,5 g L-1). Em cada um dos ensaios, aplicaram-se três doses (5,0 + 0,75; 7,5 + 1,125 e 10,0 + 1,5 g s.a ha-1) do herbicida, em duas fases distintas do desenvolvimento das infestantes e da cultura. No ano de 2012/2013, o herbicida foi aplicado quando as infestantes se encontravam na fase de desenvolvimento de três a quatro pares de folhas e, a cultura, no início do afilhamento, a que correspondeu o estádio 20 da escala de Zadoks (primeira época de aplicação). A segunda época de aplicação realizou-se quando as infestantes estavam na fase de desenvolvimento de seis a sete pares de folhas e, a cultura, no afilhamento completo (estádio 29 da escala de Zadoks). No ano de 2013/2014, a primeira época de aplicação teve lugar quando as infestantes estavam na fase de desenvolvimento de cinco a seis pares de folhas e, a cultura, na fase de colmo principal (estádio 24 da escala de Zadoks). A segunda época de aplicação efetuou-se quando as infestantes estavam na fase de desenvolvimento de sete a oito pares de folhas e, a cultura, no 3º nó visível (estádio 33 da escala de Zadoks). Os resultados obtidos demonstraram, que a antecipação da aplicação para fases mais precoces do desenvolvimento das infestantes e da cultura não conduziu a um aumento da eficácia em relação a aplicações mais tardias, para todas as doses de herbicida. Com as doses recomendadas (7,5 + 1,125 e 10,0 + 1,5 g s.a ha-1) a eficácia foi maior em ambos os anos de ensaios e épocas de aplicação, relativamente à dose inferior (5,0 + 0,75 g s.a ha-1), mas a interação doses x épocas não foi significativa. No ano de 2012/2013 e contrariamente ao sucedido no ano de 2013/2014, verificou-se uma redução significativa da produtividade da cultura com a antecipação da aplicação, consequência da fitotoxidade causada pelo herbicida, quando esta se encontrava no início do afilhamento.

Evaluación de la eficacia antibacteriana in vitro de un colutorio de aceite esencial de Origanum vulgare L. (Oregano) sobre Sterptococcos mutans

con el objetivo de evaluar la eficacia antibacteriana in vitro de dos formulaciones de un colutorio de aceite esencial de orégano sobre Streptococcus mutans (S. mutans) el principal agente causal de la placa bacteriana dental, para determinar dicha actividad, primero se realizó la determinación de la concentración inhibitoria mínima (CIM) del aceite esencial de orégano (AEO) sobre S. mutans mediante el método de microdilución en caldo y se obtuvo una CIM de 0.0025 %, esa concentración o una mayor podría ser útil en una forma farmacéutica con propósito antibacteriano. se realizó la determinación de la aptitud de los métodos de recuento microbiano que permitió indicar la metodología adecuada para evaluar la biocarga microbiana en los colutorios. Esta prueba indicó que el polisorbato 80 al 1% es un agente neutralizante idóneo para recuperar el S. mutans si se encuentra presente en los colutorios de AEO al 0.05 % y 0.10 %, y la mezcla de tiosulfato de sodio al 0.3 %, lecitina al 0.5% y polisorbato 80 al 1 % es la indicada para neutralizar las propiedades antimicrobianas del colutorio de clorhexidina al 0.12 % ylograr una recuperación adecuada del microorganismo en estudio cuando estuviera presente. Finalmente, se realizó la determinación de la eficacia antibacteriana de los colutorios de AEO, el vehículo de estos y un colutorio comercial de clorhexidina sobre S. mutans, los resultados reflejaron que el porcentaje máximo alcanzado de reducción para los colutorios de AEO al 0.05% y 0.10 % fue de 48.280 % a los 15 minutos y de 78.535 % en 1 minuto respectivamente, dichas reducciones fueron mayores que la que presento el vehículo, la cual fue de 29.292 % a los 15 minutos. El colutorio de clorhexidina al 0.12 % fue el que produjo la máxima reducción del microorganismo de estudio, la cual fue de99.996% en 6 minutos. Todos los ensayos se realizaron en el laboratorio del Centro de Investigación y Desarrollo en Salud, durante el periodo de julio a septiembre de 2015. Se concluyó que, ningún colutorio alcanzó el 99.999 % de reducción especificado por la AOAC para germicidas y sanitizantes, se observaron reducciones que sugieren buenas perspectivas de desarrollo para este tipo de formulaciones que incluyen activos de origen natural, por lo tanto se recomienda la necesidad de realizar más estudios sobre la composición y diferentes actividades del aceite esencial de orégano que pueden aprovecharse en futuras formulaciones.

A new semi-dwarfing gene identified by molecular mapping of quantitative trait loci in barley.

Semi-dwarfing genes have been widely used in spring barley (Hordeum vulgare L.) breeding programs in many parts of the world, but the success in developing barley cultivars with semi-dwarfing genes has been limited in North America. Exploiting new semi-dwarfing genes may help in solving this dilemma. A recombinant inbred line population was developed by crossing ZAU 7, a semi-dwarf cultivar from China, to ND16092, a tall breeding line from North Dakota. To identify quantitative trait loci (QTL) controlling plant height, a linkage map comprised of 111 molecular markers was constructed. Simple interval mapping was performed for each of the eight environments. A consistent QTL for plant height was found on chromosome 7HL. This QTL is not associated with maturity and rachis internode length. We suggest the provisional name Qph-7H for this QTL. Qph-7H from ZAU 7 reduced plant height to about 3/4 of normal; thus, Qph-7H is considered a semi-dwarfing gene. Other QTLs for plant height were found, but their expression was variable across the eight environments tested.

Genetic and Malt Quality Diversity of East Asian Two-Rowed Barley Accessions in Relationship to North American Malting Barley.

Because of epidemics of Fusarium head blight (FHB; caused by Fusarium graminearum Schwabe [teleomorph Gibberella zeae (Schwein.) Petch]) in the northern Great Plains of the United States and Canada in the past two decades, malting barley breeders have been forced to use nonadapted barley (Hordeum vulgare L.) accessions as sources of FHB resistance. Many of the resistant accessions are from East Asia, and limited information is available on their genetic diversity and malt quality. The objectives of this study were to determine the genetic diversity among 30 East Asian accessions and two North American cultivars. Genetic diversity was based on 49 simple-sequence repeat markers. All accessions were tested for barley grain brightness; protein content; 1,000-kernel weight; malting loss; fine-grind malt extract; content of plump kernels, free amino nitrogen, soluble protein, and wort beta-glucan; the Kolbach index (i.e., the ratio of malt soluble protein to malt total protein); a-amylase activity; diastatic power; won color; and wort viscosity. A few accessions had equal quality compared with Harrington and Conlon barley for individual traits but not for all. Qing 2, Mokkei 93-78, and Nitakia 48 could be excellent sources for increased malt extract; Nitakia 48 is a possible source for low wort viscosity; and Mokkei 93-78 and Nitakia 48 are putative sources of low beta-glucan content. The cluster analyses also implied that the malt quality of an accession cannot be predicted based on the country where it was developed.