Expression, purification and thermal stability evaluation of an engineered amaranth protein expressed in Escherichia coli

Background: The acidic subunit of amarantin (AAC)-the predominant amaranth seed storage protein-has functional potential and its third variable region (VR) has been modified with antihypertensive peptides to improve this potential. Here, we modified the C-terminal in the fourth VR of AAC by inserting four VY antihypertensive peptides. This modified protein (AACM.4) was expressed in Escherichia coli. In addition, we also recombinantly expressed other derivatives of the amarantin protein. These include: unmodified amarantin acidic subunit (AAC); amarantin acidic subunit modified at the third VR with four VY peptides (AACM.3); and amarantin acidic subunit doubly modified, in the third VR with four VY peptides and in the fourth VR with the RIPP peptide (AACM.3.4). Results: E. coli BL21-CodonPlus (DE3)-RIL was the most favorable strain for the expression of proteins. After 6 h of induction, it showed the best recombinant protein titer. The AAC and AACM.4 were obtained at higher titers (0.56 g/L) while proteins modified in the third VR showed lower titers: 0.44 g/L and 0.33 g/L for AACM.3 and AACM.3.4, respectively. As these AAC variants were mostly expressed in an insoluble form, we applied a refolding protocol. This made it possible to obtain all proteins in soluble form. Modification of the VR 4 improves the thermal stability of amarantin acidic subunit; AAC manifested melting temperature (Tm) at 34°C and AACM.4 at 37.2°C. The AACM.3 and AACM.3.4 did not show transition curves. Conclusions: Modifications to the third VR affect the thermal stability of amarantin acidic subunit.

Callus culture development of two varieties of Tagetes erecta and carotenoid production

Background The properties of natural pigments, such as antioxidants, functional, medical, and nutraceutical, have demonstrated the advantages of these natural compounds over synthetic ones. Some products are accepted only when they are pigmented with natural, food-quality colorants: for example poultry products (manly marigold flower extracts). Carotenoids such as β-carotene, β-criptoxanthin and lutein are very attractive as natural food colorants due to their antioxidant and pro-vitamin activities which provide additional value to the target products. Marigold (Tagetes erecta) is an Asteraceous ornamental plant native to Mexico, and it is also important as a carotenoid source for industrial and medicinal purposes but nowadays its production is destined mainly for ornamental purposes. Results Friable callus of T. erecta yellow flower (YF) and white flower (WF) varieties was induced from leaf explants on Murashige and Skoog (MS) medium supplemented with 9.0 µM 4-dichlorophenoxyacetic acid (2,4-D) and 8.8 µM benzyladenine (BA). Calluses developed from both varieties were different in pigmentation. Extract characterization from callus cultures was carried out by high-performance liquid chromatography (HPLC). This analytical process detected several carotenoids; the main pigments in extracts from YF callus were lutein and zeaxanthin, whereas in the extracts of the WF callus the main pigments were lutein, zeaxanthin, β-cryptoxanthin and β-carotene. Callus cultures of T. erecta accumulated pigments even after several rounds of subculture. Conclusions WF callus appeared to be a suitable candidate as a source of different carotenoids, and tested varieties could represent an alternative for further studies about in vitro pigment production.

Efecto de la nixtamalización sobre las características moleculares del almidón de variedades pigmentadas de maíz / Effect of the nixtamalización on the molecular characteristics of the starch of pigmented varieties of corn

Se estudiaron los almidones de dos maíces pigmentados (negro y azul) y uno blanco como referencia, antes y después del proceso de nixtalmalización. El contenido de amilosa aparente fue mayor en los almidones aislados después de la nixtamalización, debido a que el grupo acilo de las antocianinas pudiera estar interaccionando con la amilosa y la nixtamalización rompe este complejo aumentando su concentración. En el almidón aislado de maíz negro nixtamalizado se encontró un color más oscuro. El proceso de nixtalamalización produjo una disminución en la concentración de antocianinas que fue mayor en el caso del maíz azul, debido que a pesar de antocianinas de este maíz se encuentran en el endospermo, quedaron más exúestas al ser sensibilizadas las proteínas que forman parte de la subaleurona y parte de ellas se perdieron durante la nixtamalización. El patrón de Rx en los almidines del maíz sin nixtamalizar y nixtamalizado fue tipo A, con ligeros cambios en la forma y tamaño de los picos. El ordenamiento de corto rango mostró un rearreglo en la estructura cristalina de los almidones de los maíces nixtamalizados azul y blanco, no así en el negro que presentó una disminución en el componente cristalino después de la nixtamalización. Los almodones de los maíces pigmentados nixtamalizados mostraron mayor temperatura de gelatinización, debido al proceso de "anillado", que estabilizó la estructura del polisacárido. La información obtenida puede ser de utilidad en el procesamiento y almacenamiento de los productos elaborados con estos maíces, cuyas antocianinas son consideradas como compuestos nutracéuticos

Resistant starch and in vitro starch digestibility of cooked "ayocote" bean Pphaseolus Coccineous)

Se estudió la composición química y digestibilidad in vitro del frijol "ayocote" cocido (Phaseolus coccineous). El contenido de proteína y cenizas fue de 20,46 y 1,39%, respectivamente, los cuales se encuentran entre los más bajos reportados para el género Phaseolus. En el caso de los lípidos su contenido fue relativamente alto (3,31%). Los valores de almidón disponible (AD) disminuyeron con el tiempo de almacenamiento, variando desde 37,93% (muestra recién cocinada) hasta 32,18% (muestra almacenada por 96h). El comportamiento inverso se encontró para el contenido de almidón resistente (AR). Los valores estuvieron entre 2,24% (muestra control) y 3,49% (muestra almacenada por 96h), lo cual representa un incremento del 56% en el contenido de AR. El almidón resistente asociado a la fibra dietética (ARAF) presentó un comportamiento similar, ya que los valores se incrementaron con el tiempo de almacenamiento. La velocidad de a-amilólisis disminuyó con el tiempo de almacenamiento; por ejemplo, las muestras almacenadas por 96h mostraron una velocidad de digestión lenta. El índice glucémico estimado estuvo entre 62,9 y 59,7%, sugiriendo que el almidón del frijol "ayocote" libera muy lentamente la glucosa cuando es hidrolizado por las enzimas digestivas.

"Ayocote" beans (Phaseolus coccineous) were cooked and studied regarding their chemical composition and in vitro starch digestibility. Protein and ash contents were 20.46 and 1.39%, respectively, which are among the lowest levels for seeds of the Phaseolus genus. On the contrary, lipid content was relatively high (3.31%). Available starch (AS) values decreased with storage at 4ºC, changing from 37.93 (freshly cooked "control" seeds) to 32.18% (seeds stored for 96h). An inverse pattern was found for resistant starch (RS) content. RS ranged between 2.24 and 3.49% for the control and 96h-stored samples, which represents a 56% increase in the RS content. Resistant starch associated to dietary fiber (RSAF) had a similar behavior, as its values increased with the storage time. The a-amylolysis rate decreased with storage, i.e. long-stored (96h) cooked samples exhibited slower starch digestion features. The predicted glycemic index ranged between 62.9 and 59.7%, suggesting slow glucose-release features for starch in "ayocote" beans.

Estudou-se a composição química e digestibilidade in vitro do feijão "ayocote" cozido (Phaseolus coccineous). O conteúdo de proteína e cinzas foi de 20,46 e 1,39%, respectivamente, os quais se encontram entre os mais baixos reportados para o gênero Phaseolus. No caso dos lípidos seu conteúdo foi relativamente alto (3,31%). Os valores de amido disponível (AD) diminuíram com o tempo de armazenamento, variando desde 37,93% (amostra recém cozida) até 32,18% (amostra armazenada por 96h). O comportamento inverso encontrou-se para o conteúdo de amido resistente (AR). Os valores estiveram entre 2,24% (amostra controle) e 3,49% (amostra armazenada por 96h), o qual representa um incremento de 56% no conteúdo de AR. O amido resistente associado à fibra dietética (ARAF) apresentou um comportamento similar, já que os valores se incrementaram com o tempo de armazenamento. A velocidade de a-amilólisis diminuiu com o tempo de armazenamento; por exemplo, as amostras armazenadas por 96h mostraram uma velocidade de digestão lenta. O índice glicêmico estimado esteve entre 62,9 e 59,7%, sugerindo que o amido do feijão "ayocote" libera muito lentamente a glicose quando é hidrolisada pelas enzimas digestivas.

Functional properties of corn, banana and potato starch blends

Potato, corn and banana starches were blended in various combinations and ratios. Stability and clarity, freeze-thaw stability, water retention capacity and apparent viscosity of the pastes (prepared with a hydrothermic treatment using 100 C for 30 min) were evaluated. In general, the samples stored at room temperature (28 C) presented stability as well as low retrogradation rate. However, in the samples stored at 4 ÆC the propagation and maturation of crystals in the amylopectin component were favored, increasing sample retrogradation. A synergistic effect was shown in some starch blends. The blends had poor stability to freeze-thaw cycle, but a high synergestic effect was presented in water retention capacity. Potato: banana and corn: potato blends showed a synergistic effect in the apparent viscosity and in general, starch blends had stability during the 30-min test.