Síntesis de glicoconjugados de albúmina sérica bovina y oligosacáridos de quitosano para el análisis de adhesión bacteriana / Synthesis of glycoconjugates of bovine albumin and chitosan oligosaccharides for the analysis of bacterial adhesion / Síntese de glicoconjugados de albumina serica bovina e oligossacarídeos de quitosana para análise de adesão bacteriana

Resumen El aumento de la resistencia a los antibióticos por parte de bacterias patógenas ha motivado la búsqueda de alternativas para disminuir su utilización. Dentro de las opciones propuestas se encuentra la terapia de antiadherencia, en la cual se utilizan moléculas análogas a los glicoepítopes que son reconocidos por las bacterias para impedir la unión de éstas al tejido celular. En este estudio se llevó a cabo la obtención de glicoconjugados por medio de la reacción de Maillard partiendo de albúmina sérica bovina (BSA) y oligosacáridos de quitosano (oligosacáridos sin ultrafiltrar, ultrafiltrados y ultrafiltrados acetilados), en proporción 1:1 (p/p) a tres temperaturas de incubación (50, 60 y 70 °C) por 30 min. La caracterización de los conjugados sintetizados se realizó utilizando electroforesis (SDS-PAGE), espectroscopía de infrarrojo y espectroscopía de fluorescencia. Se realizaron ensayos de reconocimiento usando aglutinina de germen de trigo (WGA) y bacterias [Escherichia coli (K88ac y K88+)]. La caracterización por medio de electroforesis y espectroscopía de infrarrojo evidenció la unión de los oligosacáridos de quitosano a la estructura de la BSA. Además, los ensayos evidenciaron el reconocimiento de las moléculas sintetizadas tanto por la lectina WGA como por las bacterias. Los glicoconjugados sintetizados sin ultrafiltrar ni acetilar mostraron resultados muy favorables en el reconocimiento por ambas bacterias, lo que constituye una ventaja práctica, ya que su implementación a mayor escala reduciría costos de producción

Abstract The increase in antibiotic resistance by pathogenic bacteria has motivated the search for alternatives to reduce the use of antibiotics. Among these alternatives is anti-adhesion therapy, in which molecules that mimic the glycoepitopes that recognise bacteria are used to prevent their binding to cellular tissue. In this study, glycoconjugates were obtained by means of the Maillard reaction starting from bovine serum albumin (BSA) and chitosan oligosaccharides (unfiltered oligosaccharides, ultrafiltered and acetylated ultrafiltered), in a ratio of 1:1 (w/w) at three incubation temperatures (50, 60 and 70 °C) per 30 min. The characterisation was performed using the techniques of electrophoresis (SDS-PAGE), infrared spectroscopy and fluorescence spectroscopy. Recognition assays were performed using wheat germ agglutinin (WGA) and Escherichia coli bacteria (K88ac and K88+). Characterisation by electrophoresis and infrared spectroscopy demonstrated the binding of chitosan oligosaccharides to the structure of BSA. In addition, the tests showed the recognition of the molecules synthesised by both the WGA lectin and the E. coli bacteria. The glycoconjugates synthesised without ultrafiltration or acetylation showed very favourable results in recognition with both bacteria, which is an advantage, since its implementation on a larger scale would reduce production costs.

Resumo O aumento da resistência aos antibióticos por bactérias patogênicas tem motivado a busca de alterna­tivas para reduzir seu uso. Entre essas alternativas está a terapia anti-adesão, na qual são utilizadas moléculas análogas aos glicoepítopos que são reconhecidas pelas bactérias para impedir sua união ao tecido celular. Neste estudo, os glicoconjugados foram obtidos por meio da reação de Maillard a partir de albumina sérica bovina (BSA) e oligossacarídeos de quitosana (oligossacarídeos não ultra­filtrados, ultrafiltrados e acetilados ultrafiltrados), na proporção de 1:1 (p/p) em três temperaturas de incubação (50, 60 e 70 °C) durante 30 min. A caracterização dos conjugados sintetizados foi realizada utilizando a eletroforese (SDS-PAGE), espectroscopia de infravermelho e espectroscopia de fluorescência. Os ensaios de reconhecimento foram realizados utilizando aglutinina de germe de trigo (WGA) e bactérias [Escherichia coli (K88ac e K88+)]. A caracterização por meio de eletroforese e espectroscopia de infravermelho demonstrou a união dos oligossacarídeos de quitosana à estrutura da BSA. Além disso, os testes evidenciaram o reconhecimento das moléculas sintetizadas tanto pela lectina WGA quanto pelas bactérias. Os glicoconjugados sintetizados sem ultrafiltração ou acetilação apresentaram resultados muito favoráveis no reconhecimento por ambas as bactérias, o que é uma vantagem, visto que sua implementação em maior escala reduziria custos de produção.

Effect of a Prolonged-Release System of Urea on Nitrogen Losses and Microbial Population Changes in Two Types of Agricultural Soil.

Urea is the nitrogen-containing fertilizer most used in agricultural fields; however, the nutrient given by the urea is lost into the environment. The aim of this research was to determine the effect of two soil textures by applying a prolonged-release system of urea (PRSU) on the N losses. This research shows an important decrease of the nitrate and ammonium losses from 24.91 to 87.94%. Also, the microbiological population increases after the application of the PRSU. It was concluded that both soil textures presented the same loss-reduction pattern, where the N from the nitrates and ammonium was reduced in the leachates, increasing the quality of the soil and the microbial population in both soil textures after the PRSU application.

Cápsulas de polvo de arándano como propuesta nutracéutica para mejorar la bioaccesibilidad de compuestos fenólicos / Blueberry Powder capsules as a nutraceutical proposal to improve bioaccessibility of phenolic compounds. Introduction

El arándano (Vaccinium corymbosum L.) posee un alto contenido de compuestos fenólicos los cuales han sido estudiados principalmente por su actividad antioxidante, antiobesogénica, antiinflamatoria, entre otras. Objetivo. Evaluar el efecto de la digestión gastrointestinal in vitro sobre la bioaccesibilidad de compuestos fenólicos y actividad antioxidante de una formulación nutracéutica de arándano (cápsula), comparado con arándano fresco y polvo. Materiales y métodos. Se obtuvieron extractos metanólicos de muestras de arándano fresco y liofilizado y se determinó su contenido de fenoles, flavonoides y antocianinas totales, así como también actividad antioxidante. Se llevó a cabo un ensayo de simulación de digestión gastrointestinal para evaluar la bioaccesibilidad de los compuestos fenólicos presentes en las muestras. Resultados. Los resultados mostraron que la digestión gástrica de arándano en polvo y en cápsula promovió una mayor bioaccesibilidad de fenoles (42% y 40%), flavonoides (52% y 33%) y antocianinas (45% y 40%) comparado con digestos de arándano fresco. Posterior a la digestión intestinal, la bioaccesibilidad de fenoles (63%) y flavonoides (67%) fue mayor en la cápsula de arándano comparada con su contraparte arándano en polvo. Las condiciones de digestión intestinal afectaron negativamente la bioaccesibilidad de las antocianinas independientemente del tipo de muestra evaluada. Conclusión. Las condiciones de digestión gástrica promueven una mayor estabilidad de los compuestos fenólicos en arándano en polvo y en cápsula lo que pudiera ser relevante para el mantenimiento de un ambiente antioxidante a este nivel. Las condiciones de digestión intestinal afectaron de manera particular a los compuestos fenólicos de arándano fresco y polvo, pero no a la cápsula, lo que puede sugerir que el encapsulamiento protegió de las condiciones alcalinas a los fenoles presentes. Se sugieren estudios posteriores sobre absorción in vitro de los componentes remanentes en intestino y sus posibles efectos sobre biomarcadores de estrés oxidativo en modelos in vivo(AU)

Blueberry (Vaccinium corymbosum L.) has a high content of phenolic compounds which have been studied mainly for their antioxidant, antiobesogenic, anti-inflammatory activity, among others. Objetive. The objective of the present study was to evaluate the effect of in vitro gastrointestinal digestion on the bioaccessibility of phenolic compounds and antioxidant activity of a nutraceutical formulation of blueberry (capsule), compared to fresh and powder blueberry. Materials and methods. Methanolic extracts of fresh and lyophilized blueberry were obtained and determined its total phenols, flavonoids, anthocyanins content, as well as antioxidant activity. A gastrointestinal digestion simulation test also was carried out to assess the bioaccessibility of the phenolic compounds found in samples. Results. The results showed that gastric digestion of powder and capsule blueberry promoted greater bioaccessibility of phenols (42% and 40%), flavonoids (52% and 33%) and anthocyanins (45% and 40%), compared to fresh blueberry digests. After intestinal digestion, the bioaccessibility of phenols (63%) and flavonoids (67%) was higher in the blueberry capsule compared to its powdered blueberry counterpart. The intestinal digestion conditions negatively affected the bioaccessibility of anthocyanins regardless of the type of sample evaluated. Conclusion. Gastric digestion conditions promote greater stability of phenolic compounds in powdered and capsule blueberries, which could be relevant for the maintenance of an antioxidant environment at this level. The intestinal digestion conditions particularly affected the phenolic compounds of fresh and lyophilized blueberry, but not the capsule, which may suggest that encapsulation protected the phenols present from alkaline conditions. Further studies on in vitro absorption of the remaining components in the intestine and their possible effects on oxidative stress biomarkers in in vivo models are suggested(AU)

The Effect of Nixtamalization Extrusion Process and Tortillas Making on the Stability of Anthocyanins from Blue Corn through the Kinetic and Thermodynamic Parameters.

The blue corn-based products are considered functional foods due to their high concentration of anthocyanins. The aim of this study was to estimate the kinetic and thermodynamic parameters of the thermal degradation of anthocyanins from extruded nixtamalized corn products. A comparative study of anthocyanins thermal stability in these matrices in a buffer solution (pH 2.5) was investigated at different temperatures (60, 75 or 90 °C). Results showed the mechanism of anthocyanins degradation followed first-order reaction kinetics. The values of the reaction rate constant (k) were found to be in a range of 0.027-0.037 h-1 at 60 °C, 0.107-0.113 h-1 at 75 °C and 0.340-0.354 h-1 at 90 °C. The higher the k value was, the shorter the half-life time and D-value. The activation energy (Ea) and z-values were in the range of 75.1-89.2 kJ/mol and 28.8-35.1 °C, respectively. The coefficient Q10 indicated the reaction rate approximately doubles with every 10 °C temperature increase. ∆H, ∆S and ∆G indicated the degradation of anthocyanins was an endothermic and nonspontaneous reaction. Even the major susceptibility of the anthocyanins in extruded nixtamalized corn products at the time-temperature combination applied, there was not difference between flour and tortilla, this imply that most of the anthocyanins were degraded during the nixtamalization extrusion process and no significative further degradation occur in the cooking step. This study provides and advance in the knowledge on the effect of nixtamalization extrusion process and tortillas making on the stability of anthocyanins from blue corn. However, further studies are needed.

Physicochemical, Rheological, and Morphological Characteristics of Products from Traditional and Extrusion Nixtamalization Processes and Their Relation to Starch.

The aim of this study was to compare the physicochemical, rheological, and morphological characteristics of corn, nixtamalized flour, masa, and tortillas from the traditional nixtamalization process (TNP) and the extrusion nixtamalization process (ENP) and their relationship with starch. The traditional and extrusion processes were carried out using the same variety of corn. From both processes, samples of ground corn, nixtamalized flour, masa, and tortillas were obtained. The extrusion process produced corn flour with particle sizes smaller (particle size index, PSI = 51) than that of flour produced by the traditional nixtamalization process (PSI = 44). Masa from the TNP showed higher modulus of elasticity (G') and viscosity (G ″) values than that off masa from the ENP. Furthermore, in a temperature sweep test, masa from the TNP showed a peak in G' and G ″, while the masa from the ENP did not display these peaks. The ENP-produced tortillas had higher resistant starch contents and comparable firmness and rollability to those from the TNP but lower quality parameter values. A comparison of the products' physicochemical properties obtained by the two processes shows the importance of controlling the damage to starch during the milling and extrusion processes to obtain tortillas of better quality. For the first time, we propose the measurement of the viscoelastic parameters G' and G ″ in temperature sweep mode to monitor changes in the degree of starch damage.

Effect of Extrusion Processing Conditions on the Phenolic Compound Content and Antioxidant Capacity of Sorghum (Sorghum bicolor (L.) Moench) Bran.

Sorghum is a cereal with little use in human diet; however, this grain can provide several nutrients and, additionally, has a high content of phenolic compounds concentrated in bran, which could be beneficial to human health due to its high antioxidant capacity. However, these bioactive compounds are bound within the cell wall matrix; it is necessary to release these compounds to take advantage of their antioxidant properties. The extrusion process increases the accessibility of bound phenolic compounds, breaking their bonds from the bran matrix. The aim of this study was to determine the optimal extrusion conditions for maximizing the phenolic compound content and antioxidant capacity of sorghum bran extrudate. The extrusion process factors evaluated were feed moisture (FM) from 25 to 35% and the fourth extrusion zone temperature (T) in the range of 140-180 °C. Analysis of variance and response surface analysis were used in the evaluation. The prediction coefficient, (FM)2, (T)2 and their interaction (FM)(T) significantly affected the free total phenolic compounds. The antioxidant capacity of the free total phenolic compounds was significantly affected by (FM)2 and (T)2. The optimal extrusion conditions were FM = 30% and T = 160 °C, which provided free total phenolic compounds with a value of 7428.95 µg GAE/g (predicted value: 7810.90 µg GAE/g) and antioxidant capacity with a value of 14.12 µmol TE/g (predicted value: 14.85 µmol TE/g). Results confirmed that extrusion process optimization was useful to increase the content of phenolic compounds and improved the antioxidant capacity of sorghum bran.

Nano alterations of membrane structure on both γ-irradiated and stored human erythrocytes.

PURPOSE: Storage and ionizing radiation of human red blood cells (RBC) produce alterations on RBC membranes and modify their normal shape and functionality. We investigated early morphological and biochemical changes in RBC due to those stressing agents at the nanoscale level and their impact on blood quality. MATERIALS AND METHODS: Whole blood samples from healthy donors were γ-irradiated with 15, 25, 35, and 50 Gy. Non-irradiated and non-stored RBC were used as control samples. Irradiated blood samples were stored separately at 4 °C and analyzed immediately and after 5 and 13 d. Atomic force microscopy (AFM), osmotic fragility and Raman spectroscopy were used to detect morphological and biochemical changes. RESULTS: RBC function is challenged by both irradiation and storage. The storage procedure caused nanometric variations over the surface of RBC membrane for both irradiated and non-irradiated cells. The membrane of RBC became more fragile, while the biochemical fingerprint of hemoglobin (Hb) remained unaltered. CONCLUSIONS: Our work shows that the irradiation procedure leads to an increase in the number and size of nanovesicles along with the dose. The functionality of RBC can be affected from changes in the roughness, becoming more fragile and susceptible to breakage.

Adhesion of enterotoxigenic Escherichia coli strains to neoglycans synthesised with prebiotic galactooligosaccharides.

Enterotoxigenic (ETEC) Escherichia coli (E. coli) causes traveller's diarrhoea and high mortality among baby animals. ETEC adhesion is mediated by lectins (adhesins) that bind to glycoconjugates on the surface of host cells. Glycans that compete for adhesion could be used for disease prevention. Neoglycans of porcine albumin (PSA) that were conjugated with prebiotic galactooligosaccharides (GOS) were synthesised using the Maillard reaction. PSA glycation was confirmed by a reduction in the number of available free amino groups, decreased tryptophan intrinsic fluorescence, increased molecular mass and Ricinus communis lectin recognition. The adhesion of four ETEC strains (E. coli H10407, CFA(+), K99 and K88) to PSA-GOS was examined by an enzyme-linked lectin assay. E. coli K88 bound to PSA-GOS with greater affinity (P<0.05) than did E. coli H10407, CFA(+) and K99. In addition, PSA-GOS partially inhibited the adherence of the K88 strain to intestinal mucins. Pig ETEC strain was unable to ferment galactooligosaccharide-neoglycans. These results suggest that neoglycans obtained by the Maillard reaction may serve in the prophylaxis of ETEC K88 diarrhoea.

Characterization of bovine serum albumin glycated with glucose, galactose and lactose.

The non-enzymatic reaction between reducing sugars and proteins, known as glycation, has received increased attention from nutritional and medical research. In addition, there is a large interest in obtaining glycoconjugates of pure well-characterized oligosaccharides for biological research. In this study, glycation of bovine serum albumin (BSA) by d-glucose, d-galactose and d-lactose under dry-heat at 60 degrees C for 30, 60, 120, 180 or 240 min was assessed and the glycated products studied in order to establish their biological recognition by lectins. BSA glycation was monitored using gel electrophoresis, determination of available amino groups and lectin binding assays. The BSA molecular mass increase and glycation sites were investigated by mass spectrometry and through digestion with trypsin and chymotrypsin. Depending on time and type of sugar, differences in BSA conjugation were achieved. Modified BSA revealed reduction of amino groups' availability and slower migration through SDS/PAGE. d-galactose was more reactive than d-glucose or d-lactose, leading to the coupling of 10, 3 and 1 sugar residues, respectively, after 120 minutes of reaction. BSA lysines (K) were the preferred modified amino acids; both K256 and K420 appeared the most available for conjugation. Only BSA-lactose showed biological recognition by specific lectins.

[Characterization of cookies made with deseeded grape pomace]. / Caracterización de galletas elaboradas con cascarilla de orujo de uva.

Characterization of cookies made with deseeded grape pomace. The objective of this study was to evaluate deseeded grape pomace as a potential ingredient to elaborate some food products. Cookies were made with flour containing four levels of deseeded grape pomace (0, 5, 7.5 and 10%). Moisture, protein, ashes, fat, tannins and dietary fiber contents were determined in both the deseeded of grape pomace and the cookies. Besides, color, sensorial acceptability and biological evaluations of Net Protein Ratio (NPR), Apparent Digestibility (AD) and True Digestibility (TD) of Protein were determined to the cookies. Substantial amounts of dietary fiber and ashes were found in both the deseeded grape pomace and the cookies. Total dietary fiber increased while adding more deseeded grape pomace. Cookies were well accepted as observed in the sensory evaluation, showing no significative differences among the four levels of deseeded grape pomace addition. The addition of deseeded grape pomace imparted a darker color to the cookies. The dark color was greater in the samples containing more fiber which was indicated by the lower L color value. Regarding nutritional analysis, the higher the deseeded grape pomace addition, the lower the NPR, AD, and TD values. The NPR was affected in greater degree, although these differences were not significant. It is possible to use deseeded grape pomace as an ingredient to make high fiber cookies with acceptable sensorial attributes.