Quality of lettuce Lactuca sativa (var. Tropicana M1) grown with two low-salinity shrimp effluents.

In a previous study, we investigated the use of shrimp effluents from well water (WW) and diluted seawater (DSW) (both with 2.7 dS m-1 electrical conductivity (EC)), and a hydroponic solution (HS) as the control treatment in greenhouse lettuce production. This new paper completes the previous one by focusing on the quality of lettuce harvested. Compared to the lettuce from the other treatments, WW-lettuce exhibited higher levels of phenolic compounds and a higher antioxidant capacity, mainly in the soluble fraction. The lettuce cultivated with DSW showed no significant difference in total phenolics and flavonoids with respect to the HS lettuce. These results reveal that the functional properties (antioxidant properties, polyphenols and flavonoid content) are even better in the lettuce produced with WW and DSW shrimp effluents. In contrast, agronomical properties (weight, number of leaves and yield) were found to be better in the case of lettuce grown with the hydroponic solution (control).

Mortality in Patients With Chronic Renal Disease Without Health Insurance in Mexico: Opportunities for a National Renal Health Policy.

INTRODUCTION: Despite a systematic increase in the coverage of patients with end-stage renal disease (ESRD) who have received dialytic therapies and transplantation over the past 2 decades, the Mexican health system currently still does not have a program to provide full coverage of ESRD. Our aim was to analyze mortality in patients with ESRD without health insurance. METHODS: This was a prospective cohort study of 850 patients with advanced chronic kidney disease (CKD). Risk factors associated with death were calculated using a Cox's proportional hazards model. We used the statistical package SPSS version 22.0 for data analysis. RESULTS: The mean age of patients was 44.8 ± 17.2 years old. At the time of hospital admission, 87.6% of the population did not have a social security program to cover the cost of renal replacement treatment, and 91.3% of families had an income below US$300 per month. During the 3 years of the study, 28.8% of the cohort patients were enrolled in 1 of Mexico's social security programs. The 3-year mortality rate was of 56.7% among patients without access to health insurance, in contrast to 38.2% of patients who had access to a social security program that provided access to renal replacement therapy (P < 0.001). Risk factor analysis revealed that not having health insurance increased mortality (risk ratio: 2.64, 95% confidence intervals: 1.84-3.79; P = 0.001). CONCLUSION: Mexico needs a coordinated National Kidney Health and Treatment Program. A program of this nature should provide the basis for an appropriate educational and intervention strategy for early detection, prevention, and treatment of patients with advanced chronic kidney disease.

Establishment of an efficient genetic transformation method in Dunaliella tertiolecta mediated by Agrobacterium tumefaciens.

Microalgae are photosynthetic microorganisms widely used for the production of highly valued compounds, and recently they have been shown to be promising as a system for the heterologous expression of proteins. Several transformation methods have been successfully developed, from which the Agrobacterium tumefaciens-mediated method remains the most promising. However, microalgae transformation efficiency by A. tumefaciens is shown to vary depending on several transformation conditions. The present study aimed to establish an efficient genetic transformation system in the green microalgae Dunaliella tertiolecta using the A. tumefaciens method. The parameters assessed were the infection medium, the concentration of the A. tumefaciens and co-culture time. As a preliminary screening, the expression of the gusA gene and the viability of transformed cells were evaluated and used to calculate a novel parameter called Transformation Efficiency Index (TEI). The statistical analysis of TEI values showed five treatments with the highest gusA gene expression. To ensure stable transformation, transformed colonies were cultured on selective medium using hygromycin B and the DNA of resistant colonies were extracted after five subcultures and molecularly analyzed by PCR. Results revealed that treatments which use solid infection medium, A. tumefaciens OD600 = 0.5 and co-culture times of 72 h exhibited the highest percentage of stable gusA expression. Overall, this study established an efficient, optimized A. tumefaciens-mediated genetic transformation of D. tertiolecta, which represents a relatively easy procedure with no expensive equipment required. This simple and efficient protocol opens the possibility for further genetic manipulation of this commercially-important microalgae for biotechnological applications.

Functionomics of NCC mutations in Gitelman syndrome using a novel mammalian cell-based activity assay.

Gitelman syndrome (GS) is an autosomal recessive salt-wasting tubular disorder resulting from loss-of-function mutations in the thiazide-sensitive NaCl cotransporter (NCC). Functional analysis of these mutations has been limited to the use of Xenopus laevis oocytes. The aim of the present study was, therefore, to analyze the functional consequences of NCC mutations in a mammalian cell-based assay, followed by analysis of mutated NCC protein expression as well as glycosylation and phosphorylation profiles using human embryonic kidney (HEK) 293 cells. NCC activity was assessed with a novel assay based on thiazide-sensitive iodide uptake in HEK293 cells expressing wild-type or mutant NCC (N59I, R83W, I360T, C421Y, G463R, G731R, L859P, or R861C). All mutations caused a significantly lower NCC activity. Immunoblot analysis of the HEK293 cells revealed that 1) all NCC mutants have decreased NCC protein expression; 2) mutant N59I, R83W, I360T, C421Y, G463R, and L859P have decreased NCC abundance at the plasma membrane; 3) mutants C421Y and L859P display impaired NCC glycosylation; and 4) mutants N59I, R83W, C421Y, C731R, and L859P show affected NCC phosphorylation. In conclusion, we developed a mammalian cell-based assay in which NCC activity assessment together with a profiling of mutated protein processing aid our understanding of the pathogenic mechanism of the NCC mutations.

Effects of outdoor cultures on the growth and lipid production of Phaeodactylum tricornutum using closed photobioreactors.

One of the principal challenges for large scale production of microalgae is the high costs of biomass production. Aiming for minimize this problem, microalgal biodiesel production should focus on outdoors cultures, using available solar light and allowing lower energy cost process. Testing species that proved to be common and easy to culture may be a good approach in this process. The present work reports indoor-outdoor cultures of Phaeodactylum tricornutum using different bioreactors types, using cell growth, biochemical composition, and the profiles of the fatty acids produced as the parameters to test the optimization processes. The results show that the use of outdoor cultures is a good choice to obtain P. tricornutum biomass with a good potential for biodiesel production. The microalgae produced reached better growth efficiency, major lipid content and showed an increment in the percentage of saturated fatty acids (required on the biodiesel production) respect indoor cultures. These results are important to show the relevance of using outdoor cultures as a way to improve the efficiency and the energetic balance of the biodiesel production with P. tricornutum algae.

Enhancement of nutritional properties, and antioxidant and antihypertensive potential of black common bean seeds by optimizing the solid state bioconversion process.

The aim of this work was to obtain a functional flour with enhanced nutritional properties, and antioxidant and antihypertensive potential from black bean seeds by optimizing the solid state bioconversion (SSB) process using a Rhizopus oligoporus strain. Response surface methodology was applied as optimization technique. A central composite experimental design with two factors [fermentation temperature (FT) = 30-40 °C/fermentation time (Ft) = 6-108 h] and five levels was used (13 treatments). The bioprocessed cotyledons from each treatment were dried, milled, and blended with its previously dried-milled seed coats. The best combination FT/Ft of SSB to obtain the functional flour was 38 °C/100 h. SSB increased the calculated protein efficiency ratio (from 1.59 to 2.40), antioxidant activity (from 13 948 to 22 733 µmol ET/100 g, dw), total phenolic compounds (TPC) (from 190 to 432 mg EGA/100 g, dw) and antihypertensive potential (IC(50) from 95.57 to 0.0321 µg/mL). SSB is an effective strategy to improve the TPC of common beans for enhanced functionality.

Valorisation of biodiesel production wastes: Anaerobic digestion of residual Tetraselmis suecica biomass and co-digestion with glycerol.

One of the principal opportunity areas in the development of the microalgal biodiesel industry is the energy recovery from the solid microalgal biomass residues to optimise the fuel production. This work reports the cumulative methane yields reached from the anaerobic digestion of the solid microalgal biomass residues using different types of inocula, reporting also the improvement of biogas production using the co-digestion of microalgal biomass with glycerol. Results demonstrate that the solid microalgal biomass residues showed better biogas production using a mesophilic inoculum, reaching almost two-fold higher methane production than under thermophilic conditions. Furthermore, the solid microalgal biomass residues methane production rate showed an increase from 173.78 ± 9.57 to 438.46 ± 40.50 mL of methane per gram of volatile solids, when the co-digestion with glycerol was performed. These results are crucial to improve the energy balance of the biodiesel production from Tetraselmis suecica, as well as proposing an alternative way to treat the wastes derived from the microalgae biodiesel production.

Histopathological changes in third-instar and adult Anastrepha ludens (Diptera: Tephritidae) after in vitro heat treatment.

The Mexican fruit fly, Anastrepha ludens Loew (Diptera: Tephritidae), is one of the most harmful pests of mango causing direct damage by oviposition on the fruit pulp. Mango for export is subjected to hydrothermal treatment as a quarantine method for the control of this pest, but exposure to heat for long periods of time reduces considerably the quality and shelf-life of treated fruit. The aim of this work was to study morphological changes of third-instar larvae and adults of A. ludens after in vitro exposure to high temperature at sublethal times. A heating block system was used to expose larvae at 46.1°C for 19.6 and 12.9 min, producing 94.6 and 70% mortality, respectively. Treated larvae were processed for optical microscopy. A fraction of surviving treated larvae was separated into containers with artificial diet to allow development into adults. Adult sexual organs were dissected and processed for transmission electron microscopy analysis. Results showed that 94.6% of the treated larvae died at 46.1°C for 19.6 min and none of the surviving larvae eclosed to adulthood, as they developed as malformed puparia. For the in vitro treatment at 46.1°C during 12.9 min, 70% of the treated larvae died and only 3.75% reached the adult stage, but ultrastructural damage in the male testes and in the female ovaries was observed. Additionally, 11.1% of the adult flies from the in vitro treatment also showed wing malformation and were incapable of flying. The analysis showed that surviving flies were unable to reproduce.

Increasing the antioxidant activity, total phenolic and flavonoid contents by optimizing the germination conditions of amaranth seeds.

The aim of this study was to optimize the germination conditions of amaranth seeds that would maximize the antioxidant activity (AoxA), total phenolic (TPC), and flavonoid (TFC) contents. To optimize the germination bioprocess, response surface methodology was applied over three response variables (AoxA, TPC, TFC). A central composite rotable experimental design with two factors [germination temperature (GT), 20-45 ºC; germination time (Gt), 14-120 h] in five levels was used; 13 treatments were generated. The amaranth seeds were soaked in distilled water (25 °C/6 h) before germination. The sprouts from each treatment were dried (50 °C/8 h), cooled, and ground to obtain germinated amaranth flours (GAF). The best combination of germination bioprocess variables for producing optimized GAF with the highest AoxA [21.56 mmol trolox equivalent (TE)/100 g sample, dw], TPC [247.63 mg gallic acid equivalent (GAE)/100 g sample, dw], and TFC [81.39 mg catechin equivalent (CAE)/100 g sample, dw] was GT = 30 ºC/Gt = 78 h. The germination bioprocess increased AoxA, TPC, and TFC in 300-470, 829, and 213%, respectively. The germination is an effective strategy to increase the TPC and TFC of amaranth seeds for enhancing functionality with improved antioxidant activity.

Solid-state bioconversion of chickpea (Cicer arietinum L.) by Rhizopus oligosporus to improve total phenolic content, antioxidant activity and hypoglycemic functionality.

The objective of this investigation was to study the effect of time during solid state bioconversion (SSB) on total phenolic content (TPC), antioxidant activity (AoxA), and inhibitory properties against α-amylase and α-glucosidase of chickpea. Chickpea cotyledons were inoculated with a suspension of Rhizopus oligosporus and incubated at 35 °C for 24, 36, 48, 60, 72, 84, 96 and 108 h. The best time to produce bioprocessed chickpea (added with seed coats) flour with the highest AoxA was 108 h. SSB substantially increased TPC and AoxA of chickpea extracts in 2.78 and 1.80-1.94 times, respectively. At 36 and 96 h of fermentation, the SSB process improved in vitro α-amylase and α-glucosidase inhibition (AI and GI indexes) activities of chickpea extracts in 83 and 370%, respectively. SSB is a good strategy to enhance health-linked functionality of chickpea, due to improved TPC, AoxA and content of strong natural inhibitors of enzymes associated with diabetes.

High antioxidant activity mixture of extruded whole quality protein maize and common bean flours for production of a nutraceutical beverage elaborated with a traditional Mexican formulation.

The objective of this study was to determine the best combination of extrusion process variables for the production of whole quality protein maize (EQPMF) and common bean (ECBF) flours to prepare a high antioxidant activity mixture (EQPMF + ECBF) suitable to produce a nutraceutical beverage with high acceptability elaborated with a traditional Mexican formulation. Processing conditions were obtained from a factorial combination of barrel temperature (BT = 120-170 °C) and screw speed (SS = 120-200 rpm). Response surface methodology was applied to obtain maximum values for antioxidant activity (A ( ox ) A) of the flour mixture (EQPMF + ECBF) and acceptability (A) of the nutraceutical beverage. The best combinations of extrusion process variables for EQPMF and ECBF to prepare an optimized mixture (60%EQPMF + 40%ECBF) were BT = 98 °C/SS = 218 rpm and BT = 105 °C/SS = 83 rpm, respectively. The optimized mixture had A ( ox ) A = 14,320 µmol Trolox equivalent (TE)/100 g sample dry weight (dw) and a calculated protein efficiency ratio (C-PER) of 2.17. A 200 ml portion of a beverage prepared with 25 g of the optimized flour mixture had A ( ox ) A = 3,222 µmol TE, and A = 89 (level of satisfaction "I like it extremely"). This nutraceutical beverage could be used as an alternative to beverages with low nutritional/nutraceutical value, such as those prepared with water, simple sugars, artificial flavoring and colorants, which are widely offered in the market.

Angiotensin-converting enzyme inhibitory and antioxidative activities and functional characterization of protein hydrolysates of hard-to-cook chickpeas.

BACKGROUND: The potential use of hard-to-cook (hardened) chickpeas to obtain value-added functional food ingredients was evaluated. For that purpose, some nutraceutical and functional attributes of several chickpea protein hydrolysates (CPHs) prepared from both fresh and hard-to-cook grains were evaluated. RESULTS: All the CPHs prepared from both fresh and hard-to-cook grains, with the enzymes alcalase, pancreatin and papain, showed high angiotensin converting enzyme inhibitory (ACE-I) activity with IC50 values ranging from 0.101 to 37.33 µg mL⁻¹; similarly, high levels of antioxidant activity (around 18.17-95.61 µmol Trolox equivalent antioxidant capacity µg⁻¹ CPH) were obtained through both the 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) methods. Regarding functional characterization of the CPHs, oil absorption values ranged from 1.91 to 2.20 mL oil g⁻¹ CPH, with water solubility almost 100% from pH 7 to 10. CONCLUSION: The high antioxidant and ACE-I activities as well as the good functional properties of the CPH prepared from both fresh and hard-to-cook grains, suggest its use in food formulations with value added in human health.

Endoplasmic reticulum-retention C-terminal sequence enhances production of an 11S seed globulin from Amaranthus hypochondriacus in Pichia pastoris.

The methylotrophic yeast Pichia pastoris was used to express an 11S seed globulin from Amaranthus hypochondriacus. Three different plasmids were tested for expression of amarantin. One of them, which included the untranslated regions (UTR) of the full cDNA, failed to express the amarantin under tested conditions, whereas the other plasmids, one without UTR and the other similar but including the endoplasmic reticulum-retention signal KDEL, were able to express the proamarantin in P. pastoris. After 48 h of induction, KDEL-proamarantin had accumulated quite significantly compared to unmodified proamarantin. Different solubilization patterns were also obtained from both proamarantin versions; only soluble protein was obtained from the system that included the KDEL retrieval signal. Protein fractionation was carried out by differential precipitation with ammonium sulfate, and proamarantin purification was performed using an HPLC ion exchange column. The endoplasmic reticulum-retention C-terminal sequence (KDEL retrieval signal), not commonly employed in this heterologous expression system, can therefore be used to enhance accumulation of recalcitrant protein in P. pastoris. The results obtained here also suggest that this expression system is suitable for expression and evaluation of engineered seed globulin proteins.

One-step purification and structural characterization of a recombinant His-tag 11S globulin expressed in transgenic tobacco.

Amarantin, an 11S globulin, is one of the most important storage proteins of amaranth seeds, with relevant nutritional-functional and nutraceutical characteristics. Its cDNA was cloned in-frame with a sequence encoding a polyhistidine tag and expressed under the direction of a 35S promoter in transgenic tobacco seeds. The presence of a (His)(6) tag on the polypeptide permitted a high-yield single-step purification using immobilized metal-ion affinity chromatography and rapid characterization. Purified His-tag amarantin accounted for up to 5% of total soluble seed protein. Biochemical characterization indicated that purified His-tag amarantin migrated with the expected molecular weight (53 kDa) and was correctly processed into an acidic polypeptide (32 kDa) with isoelectric point (pI) of 5.58 and a basic polypeptide (21 kDa) with pI of 9.24, linked by a disulfide bridge. Moreover, His-tag amarantin was assembled into both homo- and hetero-hexameric 11S structures. These results show that the His tag did not change the biochemical and physicochemical properties of amarantin. The strategy presented here for rapid and high-yield expression and purification procedure should facilitate structure-function studies for this nutritional protein.

Safety assessment by in vitro digestibility and allergenicity of genetically modified maize with an amaranth 11S globulin.

Prospective testing for allergenicity of proteins obtained from sources with no prior history of causing allergy has been difficult to perform. Thus, the objective of this work was to assess the food safety of genetically modified maize with an amaranth globulin protein termed amarantin. Transgenic maize lines evaluated showed, in relation to nontransgenic, 4-35% more protein and 0-44% higher contents of specific essential amino acids. Individual sequence analysis with known amino acid sequences, reported as allergens, showed that none of these IgE elicitors were identified in amarantin. Amarantin was digested within the first 15 min by Simulated Gastric Fluid treatment as observed by Western blot. Expressed amarantin did not induce important levels of specific IgE antibodies in BALB/c mice, as analyzed by ELISA. We conclude that the transgenic maize with amarantin is not an important allergenicity inducer, just as nontransgenic maize.