Metabolic changes associated with chilling injury tolerance in tomato fruit with hot water pretreatment.

Hot water treatment (HWT) of tomato (Solanum lycopersicum L.) fruit reduces the symptoms of chilling injury (CI). The aim of this study was to identify metabolites associated with HWT-induced CI tolerance in tomato fruit cv. Imperial. Mature green tomatoes with HWT (42°C/5 min) and control were stored under chilling conditions (5°C/20 days) and then ripened (21°C/7 days). Methanol extracts from pericarp were analyzed for total phenolics (TP), antioxidant activity (AoxA), and metabolic profiling by UPLC-DAD-MS and GC-MS. After cold storage and ripening, HWT fruit showed less CI, higher TP, and AoxA than control. It also showed an increased accumulation of phenolics, sugars, and some alkaloids that may be mediated by azelaic acid, glutamine, and tryptophan. The levels of N-feruloyl putrescine, esculeoside AII, and hydroxy-α-tomatine II were reduced. The better metabolic performance of HWT fruit under cold storage was associated with a higher accumulation of several metabolites (e.g., antioxidants and osmolytes) in ripening fruit. PRACTICAL APPLICATION: The identification of metabolites associated with the reduction of chilling injury (CI) symptoms in HWT tomato fruit extends the understanding of the mechanisms involved in CI tolerance. This information provides targets that could be used to develop strategies for preventing CI (e.g., genetic improvement of tomato, direct application of key metabolites). The application of such strategies will increase the economic value and decrease postharvest losses.

Characterization of peptides with antioxidant activity and antidiabetic potential obtained from chickpea (Cicer arietinum L.) protein hydrolyzates.

Alcalase hydrolyzates were prepared from the albumin (AH) and globulin (GH) fractions of eight chickpea (Cicer arietinum L.) genotypes from Mexico and 10 from other countries. Protein content, antioxidant activity (AA) (ABTS, DPPH), and degree of hydrolysis were evaluated and the best genotype was selected by principal component analysis. The hydrolyzates of the chosen genotype were analyzed for its antidiabetic potential measured as inhibition of α-amylase, α-glucosidase, and dipeptidyl peptidase-4 (DPP4). Peptide profiles were obtained by liquid chromatography-mass spectrometry (UPLC-DAD-MS), and the most active peptides were analyzed by molecular docking. The average antioxidant activity of albumin hydrolyzates was higher than that of globulin hydrolyzates. ICC3761 was the selected genotype and peptides purified from the albumin hydrolyzate showed the best antioxidant activity and antidiabetic potential (FEI, FEL, FIE, FKN, FGKG, and MEE). FEI, FEL, and FIE were in the same chromatographic peak and this mixture showed the best ABTS scavenging (78.25%) and DPP4 inhibition (IC50  = 4.20 µg/ml). MEE showed the best DPPH scavenging (47%). FGKG showed the best inhibition of α-amylase (54%) and α-glucosidase (56%) and may be a competitive inhibitor based on in silico-predicted interactions with catalytic amino acids in the active site of both enzymes. These peptides could be used as nutraceutical supplements against diseases related to oxidative stress and diabetes. PRACTICAL APPLICATION: This study showed that chickpea protein hydrolyzates are good sources of peptides with antidiabetic potential, showing high antioxidant activity and inhibition of enzymes related to carbohydrate metabolism and type 2 diabetes. These hydrolyzates could be formulated in functional foods for diabetes.

ADP-Glucose Pyrophosphorylase Is Located in the Plastid and Cytosol in the Pulp of Tropical Banana Fruit (Musa acuminata).

ADP-glucose pyrophosphorylase (AGPase) is a key enzyme of starch synthesis in seeds, tubers and fruits. UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of sucrose metabolism in the cytosol while alkaline phosphatase (ALP) is a marker enzyme of the amyloplast that keeps the production of ADPG by removing PPi. Unripe banana accumulates starch in the pulp during development, while ripe fruits are characterized by the accumulation of soluble sugars. The aim of the study was to compare starch granule structure, carbohydrate levels, subcellular location and activities of three enzymes: AGPase, UGPase and ALP. Protein extracts from the cytosolic and amyloplastidial fractions were obtained from the pulp of banana fruit at three developmental stages (11, 16 and 21 weeks after flowering) and analyzed by electrophoresis and immunodetection. Protein profiles were similar during ripening, showing a main electrophoretic band at 50-55 kDa. Higher protein content was found in the cytosolic than in the amyloplastidial fraction. Starch granules and ALP activity were enriched in the amyloplast, whereas AGPase showed a subcellular distribution similar to UGPase. Immunoblot analysis also confirmed the presence of AGPase in both cytosol and amyloplast. AGPase activity was higher in the cytosol than in the amyloplast. Both AGPase activity and western blot band intensity were highest at 16 weeks. UGPase activity was highest at 21 weeks. We conclude that cytosolic production of ADP-glucose is not an exclusive feature of cereal endosperms due to plant breeding, but it also occurs in fruits of non-domesticated plants such as tropical banana (Musa acuminata). This work increases our understanding about pyrophosphorylase activities in the pulp of banana fruit.

Anthocyanins of Pithecellobium dulce (Roxb.) Benth. Fruit Associated with High Antioxidant and α-Glucosidase Inhibitory Activities.

Red arils of Pithecellobium dulce fruit, commonly known as guamuchil, show high antioxidant (AOx) and α-glucosidase inhibitory (IαG) activities, which have been mainly associated with the content of unknown anthocyanins. In this study, the AOx (i.e., DPPH and ABTS as Trolox equivalents, µmol TE/g) and IαG (as half-maximal inhibitory concentration, IC50, mg/mL) activities of the anthocyanin-rich fraction (ARF) obtained from red arils were contrasted with those of the methanol extract (ME), and the main ARF anthocyanins were characterized by HPLC-DAD-ESI-MS, GC-MS and 1H-NMR. The AOx and IαG values of the ARF (DPPH = 597.8; ABTS = 884.01; IαG = 0.06) were better than those of the ME (DPPH = 41.5; ABTS = 142.3; IαG = 17.5); remarkably, the ARF IαG value was about 42 times lower than that of acarbose. The main anthocyanins in ARF were pelargonidin 3-O-glucoside and cyanidin 3-O-glucoside. Thus, the consumption of red P. dulce arils could provide health benefits for prevention/treatment of chronic degenerative diseases such as diabetes.

Phenolic profiles and their contribution to the antioxidant activity of selected chickpea genotypes from Mexico and ICRISAT collections.

Chickpea (Cicer arietinum L.) genotypes, nine kabuli from Mexico and 9 desi from other countries, were investigated for their phenolic profiles and antioxidant activity (AA). Phenolics in methanol extracts (ME) were analyzed by ultra-performance liquid chromatography coupled to diode array detection and mass spectrometry (UPLC-DAD-MS), whereas the AA was measured as Trolox equivalents (TE) by ABTS, DPPH and FRAP methods. Twenty phenolic compounds were identified in the ME and their levels showed a great variability among the chickpea genotypes. Phenolic acids and flavonoids were the most abundant compounds in kabuli and desi genotypes, respectively. The AA values (µmol TE/ 100 g dw) by ABTS (278-2417), DPPH (52-1650), and FRAP (41-1181) were mainly associated with the content of sinapic acid hexoside, gallic acid, myricetin, quercetin, catechin, and isorhamnetin, suggesting they are the main compounds responsible for the AA. The sum of the AA obtained for standards of these compounds evaluated at the concentration found in the extracts accounted for 34.3, 69.8, and 47.0% of the AA in the extract by ABTS, DPPH, and FRAP, respectively. In the AA by DPPH, most of the mixtures of these compounds resulted in synergistic interactions. Three desi genotypes with black seeds (ICC 4418, ICC 6306, and ICC 3761) showed the highest AA and flavonoids content, whereas the most promising kabuli genotypes were Surutato 77, Bco. Sin. 92, and Blanoro that showed the highest values of phenolic acids. These genotypes represent good sources of antioxidants for the improvement of nutraceutical properties in chickpea.

Healthy Ready-to-Eat Expanded Snack with High Nutritional and Antioxidant Value Produced from Whole Amarantin Transgenic Maize and Black Common Bean.

The snack foods market is currently demanding healthier products. A ready-to-eat expanded snack with high nutritional and antioxidant value was developed from a mixture (70:30) of whole amarantin transgenic maize (Zea mays L.) and black common bean (Phaseolus vulgaris L.) by optimizing the extrusion process. Extruder operation conditions were: feed moisture content (FMC, 15-25 %, wet basis), barrel temperature (BT, 120-170 °C), and screw speed (SS, 50-240). The desirability numeric method of the response surface methodology (RSM) was applied as the optimization technique over four response variables [expansion ratio (ER), bulk density (BD), hardness (H), antioxidant activity (AoxA)] to obtain maximum ER and AoxA, and minimum BD, and H values. The best combination of extrusion process variables for producing an optimized expanded snack (OES, healthy snack) were: FMC = 15 %/BT = 157 °C/SS = 238 rpm. The OES had ER = 2.86, BD = 0.119 g/cm (3) , H = 1.818 N, and AoxA = 13,681 µmol Trolox equivalent (TE)/100 g, dry weight. The extrusion conditions used to produce the OES increased the AoxA (ORAC: +18 %, ABTS:+20 %) respect to the unprocessed whole grains mixture. A 50 g portion of OES had higher protein content (7.23 vs 2.32 g), total dietary fiber (7.50 vs 1.97 g), total phenolic content (122 vs 47 mg GAE), and AoxA (6626 vs 763 µmol TE), and lower energy (169 vs 264 kcal) than an expanded commercial snack (ECS = Cheetos™). Because of its high content of quality protein, dietary fiber and phenolics, as well as high AoxA and low energy density, the OES could be used for health promotion and chronic disease prevention and as an alternative to the widely available commercial snacks with high caloric content and low nutritional/nutraceutical value.

Effectiveness of hydrothermal-calcium chloride treatment and chitosan on quality retention and microbial growth during storage of fresh-cut papaya.

Rapid degradation of fresh-cut papaya limits its marketability. Hydrothermal treatments in combination with a calcium dip, applied to whole fruit before slicing, and also the application of chitosan as a coating film, have been found to have very good results in maintaining the quality of fresh-cut fruits. Based on these considerations, the aim of this study was to evaluate the effect of hydrothermal treatment (HT; 49 °C, 25 min) containing calcium chloride (Ca; 1%, w/v) followed by dipping in chitosan (Chit; 1%, w/v, 3 min) on the physical, chemical, and microbial qualities of papaya slices stored at 5 °C for 10 d. Pulp color, firmness, ascorbic acid, total phenolics, ß-carotene, and lycopene were evaluated every 2 d while the microbial quality (mesophilics, psychrophilics, molds, and yeasts) was evaluated every 5 d. Fruit treated with HT-Ca and HT-Ca + Chit showed better color and firmness retention than Control and Chit. Papaya slices treated with HT-Ca + Chit had higher nutritional content and lower microbial growth at the end of storage. The application of the HT-Ca + Chit could be used to reduce deterioration processes, maintaining physical, chemical, and microbial qualities and increasing the shelf life of fresh-cut papaya stored at 5 °C.

Microsatellite-based genetic diversity among accessions of maize landraces from Sinaloa in México.

In the state of Sinaloa México, traditional farmers still cultivate maize accessions with a wide diversity of morphological characteristics, but the gene reservoir maintained in these populations has been poorly studied and it is being lost due to changes in land use and the adoption of hybrid commercial varieties. The aim of this study was to evaluate the genetic diversity of some of these maize populations to contribute to their preservation. Twenty eight accessions were used for the analysis. DNA was extracted from 396 individuals and probed with 20 microsatellites distributed across the maize genome. A total of 121 alleles were obtained (average of 6.1 alleles per locus) and a total genetic diversity of 0.72. The UPGMA-cluster analysis, model-based population structure and principal component analysis revealed three major groups, one formed mainly by accessions of races typical of the Northwestern lowlands (Chapalote, Dulcillo del Noroeste, Tabloncillo Perla, Blando de Sonora and Elotero de Sinaloa) and the other two with accessions mainly from Tabloncillo and Tuxpeño. The high number of alleles per locus and total genetic diversity found in this study demonstrate a broad genetic basis of the accessions of maize landraces from Sinaloa, representing a gene reservoir useful in breeding programs.

Technological properties, antioxidant activity and total phenolic and flavonoid content of pigmented chickpea (Cicer arietinum L.) cultivars.

Chickpeas are rich sources of highly nutritious protein and dietary fibre; the health benefits of consuming legumes such as antioxidant activity (AoxA) could be effective for the expansion of their food uses. The technological properties and antioxidant potential of five pigmented chickpea cultivars were evaluated. Protein content of the grains varied from 24.9 to 27.4 g/100 g sample (dw). The cooking time (CT) of the whole grains ranged from 90.5 to 218.5 min; the lowest CT corresponded to Black ICC3761 cultivar. The total phenolic content (TPC) and AoxA [oxygen radical absorbance capacity (ORAC) value] varied from 1.23 to 1.51 mg GAE/g sample (dw) and from 5011 to 5756 µmol TE/100 g sample (dw), respectively; Red ICC13124 showed the highest ORAC value. The differences in technological properties and AoxA among cultivars could be used in chickpea breeding programmes. Chickpea cultivars could contribute significantly to the management and/or prevention of degenerative diseases associated with free radical damage.

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.

Characterization of monofunctional aspartate kinase genes in maize and their relationship with free amino acid content in the endosperm.

A quantitative trait locus has previously been identified in maize (Zea mays L.) that influences the level of free amino acids in the endosperm, especially those from the aspartate pathway: lysine, threonine, methionine, leucine, and isoleucine. Because this locus occurs in a region of the genome containing ask2, a monofunctional aspartate kinase, the nature of the monofunctional aspartate kinase genes in the parental inbreds, Oh545o2 and Oh51Ao2, was investigated. Two genes, Ask1 and Ask2 were isolated, and Ask2 was mapped to the ask2 locus. Nucleotide sequence analysis of the Ask2 alleles from Oh545o2 and Oh51Ao2 showed they differ by one amino acid. Both alleles complemented a yeast aspartate kinase mutant, hom3, and based on the growth of the yeast mutant it appeared that Ask2-Oh545o2 produces an enzyme with greater total activity than that encoded by the Oh51Ao2 allele. The results suggest that the higher level of free amino acids derived from the aspartate pathway in Oh545o2 endosperm results from a single amino acid change in the ASK2 enzyme that has pleiotropic effects on its activity.

Cytoskeletal proteins are coordinately increased in maize genotypes with high levels of eEF1A.

The opaque2 (o2) mutation increases the Lys content of maize (Zea mays) endosperm by reducing the synthesis of zein storage proteins and increasing the accumulation of other types of cellular proteins. Elongation factor 1A (eEF1A) is one of these proteins, and its concentration is highly correlated with the amount of other Lys-containing proteins in the endosperm. We investigated the basis for this relationship by comparing patterns of protein accumulation and gene expression between a high (Oh51Ao2) and a low (Oh545o2) eEF1A inbred, as well as between high and low eEF1A recombinant inbred lines obtained from their cross. The content of alpha-zein and several cytoskeletal proteins was measured in high and low eEF1A inbred lines, and the levels of these proteins were found to correlate with that of eEF1A. To extend this analysis, we used an endosperm expressed sequence tag microarray to examine steady-state levels of RNA transcripts in developing endosperm of these genotypes. We identified about 120 genes coordinately regulated in association with eEF1A content. These genes encode proteins involved in several biological structures and processes, including the actin cytoskeleton, the endoplasmic reticulum, and the protein synthesis apparatus. Thus, higher levels of eEF1A in o2 mutants may be related to a more extensive cytoskeletal network surrounding the rough endoplasmic reticulum and increased synthesis of cytoskeleton-associated proteins, all of which contribute significantly to the Lys content of the endosperm.

eEF1A isoforms change in abundance and actin-binding activity during maize endosperm development.

Eukaryotic elongation factor 1A (eEF1A) appears to be a multifunctional protein because several biochemical activities have been described for this protein, in addition to its role in protein synthesis. In maize (Zea mays) endosperm, the synthesis of eEF1A is increased in o2 (opaque2) mutants, and its concentration is highly correlated with the protein-bound lysine content. To understand the basis of this relationship, we purified eEF1A isoforms from developing endosperm and investigated their accumulation and their functional and structural properties. Formation of three isoforms appears to be developmentally regulated and independent of the o2 mutation, although one isoform predominated in one high lysine o2 inbred. The purified proteins differ in their ability to bind F-actin in vitro, suggesting that they are functionally distinct. However, they share similar aminoacyl-tRNA-binding activities. Tandem mass spectrometry revealed that each isoform is composed of the four same gene products, which are modified posttranslationally by methylation and phosphorylation. The chemical differences that account for their different actin-binding activities could not be determined.