Chickpea Seed Flours Improve the Nutritional and the Antioxidant Profiles of Traditional Shortbread Biscuits: Effects of In Vitro Gastrointestinal Digestion.

Functional gluten-free biscuits enriched with commercial and landrace non-commercial chickpea flours were designed and compared with a traditional shortbread biscuit. They were analyzed in sensory attributes, amino acid profile, and antioxidant properties. Subsequently, the biscuits were digested in vitro to evaluate protein hydrolysis, amino acid bioaccessibility, phenolic compounds release, and antioxidant markers. The presence of chickpea flours provided golden color and heightened biscuit hardness and fracturability (especially in non-commercial), increasing crispness and reducing brittleness. The protein hydrolysis was similar among samples (≈15%), except for one of the non-commercial (≈20%). Amino acids such as arginine, phenylalanine, leucine, tyrosine, and lysine exhibited the highest bioaccessibilities. Incorporating chickpea flour improved the antioxidant activity and polyphenol content in undigested samples and bioaccesible fractions, with higher levels of p-coumaric and ferulic acids after digestion, regardless of the chickpea seed. Non-commercial flours increased the presence of resveratrol and/or catechin in the bioaccessible fraction. Antioxidant action assessed in the Caco-2 cell line showed that the protective effect against reactive oxygen species (ROS) generation did not always correlate with the in vitro antioxidant capacity. Our data support that the inclusion of chickpea flours in the formulation of functional biscuits provides the consumer with products of added nutritional value with attractive organoleptic features.

An Updated Review of Soy-Derived Beverages: Nutrition, Processing, and Bioactivity.

The global market for plant-based drinks is experiencing rapid growth driven by consumer demand for more sustainable diets, including vegetarian and vegan options. Soy beverages in particular are gaining popularity among individuals with lactose intolerance and milk protein allergies. They are considered an excellent source of high-quality protein, vitamin B, unsaturated fatty acids, and beneficial phytochemicals such as phytosterols, soy lecithins, and isoflavones. This review presents a comprehensive market survey of fifty-two soy beverages available in Spain and other European countries. The predominant category among those evaluated was calcium and vitamin-fortified drinks, accounting for 60% of the market. This reflects the need to address the nutritional gap compared to cow's milk and meet essential dietary requirements. The review covers the technological aspects of industrial soy milk production, including both traditional methods and innovative processing techniques. Additionally, it analyzes multiple studies and meta-analyses, presenting compelling evidence for the positive effects of soy beverages on various aspects of health. The review specifically examines the contributions of different components found in soy beverages, such as isoflavones, proteins, fiber, and oligosaccharides. Moreover, it explores controversial aspects of soy consumption, including its potential implications for growth, puberty, fertility, feminization, and the thyroid gland.

Combination of three null mutations affecting seed protein accumulation in pea (Pisum sativum L.) impacts positively on digestibility.

The presence of so-called anti-nutritional factors can reduce the bioavailability of nutrients following consumption of seeds which are otherwise an excellent source of proteins, carbohydrates and micronutrients. Among the proteins associated with negative effects on quality in pea (Pisum sativum L.) seeds are lectin, pea albumin 2 (PA2) and trypsin inhibitors (TI). Here we have investigated the impact of these proteins on protein digestibility and amino acid availability, using naturally occurring and derived mutant lines of pea lacking these proteins. The mutations were stacked to generate a triple mutant which was compared with a wild-type progenitor and a line lacking the major seed trypsin inhibitors alone. In vitro digestions following the INFOGEST protocol revealed significant differences in the degree of hydrolysis, protein profile and apparent amino acid availability among the pea variants. Proteins resistant to digestion were identified by MALDI-TOF mass spectrometry and amino acid profiles of digested samples determined. The results indicate that pea seeds lacking certain proteins can be used in the development of novel foods which have improved protein digestibility, and without negative impact on seed protein concentration or yield.

Antioxidant Potential of the Sweet Whey-Based Beverage Colada after the Digestive Process and Relationships with the Lipid and Protein Fractions.

Whey-based beverages could be an effective way of reusing a by-product of th cheese industry, mitigating environmental hazards and, at the same time, profiting a useful food with high nutritional and antioxidant properties. In this study, a traditional Ecuadorian beverage (Colada) was prepared combining sweet whey, Maracuyá and barley. Antioxidant properties before and after an in vitro digestion using the INFOGEST method were determined, and relationships with intestinal transformations of the lipid and protein fractions were analyzed. The digestive process had a positive effect on antioxidant properties based on increased values of ABTS and FRAP located in the bioaccessible fraction (BF), together with strong increments of total polyphenols. Moreover, pretreatment of Caco-2 cells with the BF of Colada significantly reduced ROS generation (p < 0.001) measured by the dichlorofluorescein assay. Substantial changes of the fatty acid profile occurred during digestion, such as a fall of saturated fatty acids and a rise of polyunsaturated. The protein profile, examined by SDS-PAGE and exclusion molecular chromatography in the BF, showed that the major part of the proteins were digested in the intestinal phase. Analysis of NanoLC-MS/MS revealed 18 antioxidant peptides originated from whey proteins, but also 16 peptides from barley with potential antioxidant properties. In conclusion, combining sweet whey with Maracuyá and barley constitutes an excellent nutritional beverage with a strong antioxidant potential.

A Pea (Pisum sativum L.) Seed Vicilins Hydrolysate Exhibits PPARγ Ligand Activity and Modulates Adipocyte Differentiation in a 3T3-L1 Cell Culture Model.

Legume consumption has been reported to induce beneficial effects on obesity-associated metabolic disorders, but the underlying mechanisms have not been fully clarified. In the current work, pea (Pisum sativum L.) seed meal proteins (albumins, legumins and vicilins) were isolated, submitted to a simulated gastrointestinal digestion, and the effects of their hydrolysates (pea albumins hydrolysates (PAH), pea legumins hydrolysates (PLH) and pea vicilin hydrolysates (PVH), respectively) on 3T3-L1 murine pre-adipocytes were investigated. The pea vicilin hydrolysate (PVH), but not native pea vicilins, increased lipid accumulation during adipocyte differentiation. PVH also increased the mRNA expression levels of the adipocyte fatty acid-binding protein (aP2) and decreased that of pre-adipocyte factor-1 (Pref-1) (a pre-adipocyte marker gene), suggesting that PVH promotes adipocyte differentiation. Moreover, PVH induced adiponectin and insulin-responsive glucose transporter 4 (GLUT4) and stimulated glucose uptake. The expression levels of peroxisome proliferator-activated receptor γ (PPARγ), a key regulator of adipocyte differentiation, were up-regulated in 3T3-L1 cells treated with PVH during adipocyte differentiation. Finally, PVH exhibited PPARγ ligand activity. Lactalbumin or other pea hydrolysates (PAH, PLH) did not exhibit such effects. These findings show that PVH stimulates adipocyte differentiation via, at least in part, the up-regulation of PPARγ expression levels and ligand activity. These effects of PVH might be relevant in the context of the beneficial health effects of legume consumption in obesity-associated metabolic disorders.

Glycation affects differently the main soybean Bowman-Birk isoinhibitors, IBB1 and IBBD2, altering their antiproliferative properties against HT29 colon cancer cells.

Naturally-occurring serine protease inhibitors of the Bowman-Birk family, particularly abundant in legume seeds, exert their potential chemopreventive and/or therapeutic properties via protease inhibition. Processing of legume seeds, including soybeans, has been proposed as a major cause for their loss of bioactivity due to glycation. In order to assess how glycation affected the protease inhibitory activities of major soybean Bowman-Birk isoinhibitors (BBI) and their antiproliferative properties, IBB1 and IBBD2 were purified and subjected to glycation under controlled conditions using glucose at high temperature. Both soybean isoinhibitors showed remarkable heat stability. In the presence of glucose, IBBD2 lost most of its trypsin inhibitory activity while IBB1 maintains similar trypsin and chymotrypsin inhibitory activities as in the absence of sugar. Glycation patterns of both BBI proteins were assessed by MALDI-TOF spectrometry. Our results show that the glycation process affects IBBD2, losing partially its antiproliferative activity against HT29 colon cancer cells, while glycated-IBB1 was unaffected.

The sodium transporter encoded by the HKT1;2 gene modulates sodium/potassium homeostasis in tomato shoots under salinity.

Excessive soil salinity diminishes crop yield and quality. In a previous study in tomato, we identified two closely linked genes encoding HKT1-like transporters, HKT1;1 and HKT1;2, as candidate genes for a major quantitative trait locus (kc7.1) related to shoot Na+ /K+ homeostasis - a major salt tolerance trait - using two populations of recombinant inbred lines (RILs). Here, we determine the effectiveness of these genes in conferring improved salt tolerance by using two near-isogenic lines (NILs) that were homozygous for either the Solanum lycopersicum allele (NIL17) or for the Solanum cheesmaniae allele (NIL14) at both HKT1 loci; transgenic lines derived from these NILs in which each HKT1;1 and HKT1;2 had been silenced by stable transformation were also used. Silencing of ScHKT1;2 and SlHKT1;2 altered the leaf Na+ /K+ ratio and caused hypersensitivity to salinity in plants cultivated under transpiring conditions, whereas silencing SlHKT1;1/ScHKT1;1 had a lesser effect. These results indicate that HKT1;2 has the more significant role in Na+ homeostasis and salinity tolerance in tomato.

Eliminating anti-nutritional plant food proteins: the case of seed protease inhibitors in pea.

Several classes of seed proteins limit the utilisation of plant proteins in human and farm animal diets, while plant foods have much to offer to the sustainable intensification of food/feed production and to human health. Reduction or removal of these proteins could greatly enhance seed protein quality and various strategies have been used to try to achieve this with limited success. We investigated whether seed protease inhibitor mutations could be exploited to enhance seed quality, availing of induced mutant and natural Pisum germplasm collections to identify mutants, whilst acquiring an understanding of the impact of mutations on activity. A mutant (TILLING) resource developed in Pisum sativum L. (pea) and a large germplasm collection representing Pisum diversity were investigated as sources of mutations that reduce or abolish the activity of the major protease inhibitor (Bowman-Birk) class of seed protein. Of three missense mutations, predicted to affect activity of the mature trypsin / chymotrypsin inhibitor TI1 protein, a C77Y substitution in the mature mutant inhibitor abolished inhibitor activity, consistent with an absolute requirement for the disulphide bond C77-C92 for function in the native inhibitor. Two further classes of mutation (S85F, E109K) resulted in less dramatic changes to isoform or overall inhibitory activity. The alternative strategy to reduce anti-nutrients, by targeted screening of Pisum germplasm, successfully identified a single accession (Pisum elatius) as a double null mutant for the two closely linked genes encoding the TI1 and TI2 seed protease inhibitors. The P. elatius mutant has extremely low seed protease inhibitory activity and introgression of the mutation into cultivated germplasm has been achieved. The study provides new insights into structure-function relationships for protease inhibitors which impact on pea seed quality. The induced and natural germplasm variants identified provide immediate potential for either halving or abolishing the corresponding inhibitory activity, along with associated molecular markers for breeding programmes. The potential for making large changes to plant protein profiles for improved and sustainable food production through diversity is illustrated. The strategy employed here to reduce anti-nutritional proteins in seeds may be extended to allergens and other seed proteins with negative nutritional effects. Additionally, the novel variants described for pea will assist future studies of the biological role and health-related properties of so-called anti-nutrients.

Two closely linked tomato HKT coding genes are positional candidates for the major tomato QTL involved in Na+ /K+ homeostasis.

The location of major quantitative trait loci (QTL) contributing to stem and leaf [Na(+) ] and [K(+) ] was previously reported in chromosome 7 using two connected populations of recombinant inbred lines (RILs) of tomato. HKT1;1 and HKT1;2, two tomato Na(+) -selective class I-HKT transporters, were found to be closely linked, where the maximum logarithm of odds (LOD) score for these QTLs located. When a chromosome 7 linkage map based on 278 single-nucleotide polymorphisms (SNPs) was used, the maximum LOD score position was only 35 kb from HKT1;1 and HKT1;2. Their expression patterns and phenotypic effects were further investigated in two near-isogenic lines (NILs): 157-14 (double homozygote for the cheesmaniae alleles) and 157-17 (double homozygote for the lycopersicum alleles). The expression pattern for the HKT1;1 and HKT1;2 alleles was complex, possibly because of differences in their promoter sequences. High salinity had very little effect on root dry and fresh weight and consequently on the plant dry weight of NIL 157-14 in comparison with 157-17. A significant difference between NILs was also found for [K(+) ] and the [Na(+) ]/[K(+) ] ratio in leaf and stem but not for [Na(+) ] arising a disagreement with the corresponding RIL population. Their association with leaf [Na(+) ] and salt tolerance in tomato is also discussed.

Involvement of SlSOS2 in tomato salt tolerance.

The Ca(2+)-dependent SOS pathway has emerged as a key mechanism in the homeostasis of Na(+) and K(+) under saline conditions. We recently identified and functionally characterized by complementation studies in yeast and Arabidopsis the gene encoding the calcineurin-interacting protein kinase of the SOS pathway in tomato, SlSOS2.(1) We also show evidences on the biotechnological potential of SlSOS2 conferring salt tolerance to transgenic tomato. The increased salinity tolerance of SlSOS2 overexpressing plants is associated with higher sodium content in stems and leaves. SlSOS2 overexpression upregulates the Na(+)/H(+) exchange at the plasma membrane (SlSOS1) and K(+), Na(+)/H(+) antiport at the endosomal and vacuolar compartments (LeNHX2 and LeNHX4). Therefore, SlSOS2 seems to be involved in tomato salinity tolerance through regulation of Na(+) extrusion from the root, active loading of Na(+) into the xylem and Na(+) and K(+) compartmentalization.

Overexpression of SlSOS2 (SlCIPK24) confers salt tolerance to transgenic tomato.

The Ca(2+)-dependent SOS pathway has emerged as a key mechanism in the homeostasis of Na(+) and K(+) under saline conditions. We have identified and functionally characterized the gene encoding the calcineurin-interacting protein kinase of the SOS pathway in tomato, SlSOS2. On the basis of protein sequence similarity and complementation studies in yeast and Arabidopsis, it can be concluded that SlSOS2 is the functional tomato homolog of Arabidopsis AtSOS2 and that SlSOS2 operates in a tomato SOS signal transduction pathway. The biotechnological potential of SlSOS2 to provide salt tolerance was evaluated by gene overexpression in tomato (Solanum lycopersicum L. cv. MicroTom). The better salt tolerance of transgenic plants relative to non-transformed tomato was shown by their faster relative growth rate, earlier flowering and higher fruit production when grown with NaCl. The increased salinity tolerance of SlSOS2-overexpressing plants was associated with higher sodium content in stems and leaves and with the induction and up-regulation of the plasma membrane Na(+)/H(+) (SlSOS1) and endosomal-vacuolar K(+), Na(+)/H(+) (LeNHX2 and LeNHX4) antiporters, responsible for Na(+) extrusion out of the root, active loading of Na(+) into the xylem, and Na(+) and K(+) compartmentalization.

The Na(+)/H(+) exchanger SOS1 controls extrusion and distribution of Na(+) in tomato plants under salinity conditions.

Maintaining a high K(+)/Na(+) ratio in the cell cytosol, along with the transport processes implicated in the xylem and phloem loading/unloading of Na(+) in plants (long-distance transport) are key aspects in plant salt tolerance. The Ca(2+)-dependent SOS pathway regulating Na(+) and K(+) homeostasis and long-distance Na(+) transport has been reported in Arabidopsis. However, Arabidopsis might not be the best model to analyze the involvement of the SOS pathway in long-distance Na(+) transport due to the very short stem of these plants which do not allow a precise dissection of the relative content of Na(+) in stem versus leaf. This separation would be critical to assess the role of SOS1 in xylem loading/unloading, Na(+) export by roots, retention in stems and the differential distribution/accumulation in old leaves. Towards this goal, tomato might represent a superior model due to its anatomical structure and agricultural significance. We recently demonstrated the key role played by the plasma membrane Na(+)/H(+) antiporter SlSOS1 in salt tolerance in tomato by maintaining ion homeostasis under salinity stress and in the partitioning of Na(+) in plant organs.

The plasma membrane Na+/H+ antiporter SOS1 is essential for salt tolerance in tomato and affects the partitioning of Na+ between plant organs.

We have identified a plasma membrane Na(+)/H(+) antiporter gene from tomato (Solanum lycopersicum), SlSOS1, and used heterologous expression in yeast to confirm that SlSOS1 was the functional homolog of AtSOS1. Using post-transcriptional gene silencing, we evaluated the role played by SlSOS1 in long-distance Na(+) transport and salt tolerance of tomato. Tomato was used because of its anatomical structure, more complex than that of Arabidopsis, and its agricultural significance. Transgenic tomato plants with reduced expression of SlSOS1 exhibited reduced growth rate compared to wild-type (WT) plants in saline conditions. This sensitivity correlated with higher accumulation of Na(+) in leaves and roots, but lower contents in stems of silenced plants under salt stress. Differential distribution of Na(+) and lower net Na(+) flux were observed in the xylem sap in the suppressed plants. In addition, K(+) concentration was lower in roots of silenced plants than in WT. Our results demonstrate that SlSOS1 antiporter is not only essential in maintaining ion homeostasis under salinity, but also critical for the partitioning of Na(+) between plant organs. The ability of tomato plants to retain Na(+) in the stems, thus preventing Na(+) from reaching the photosynthetic tissues, is largely dependent on the function of SlSOS1.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of proteins in dry-cured hams: data registration and multivariate analysis across multiple gels.

This study investigates whether dry-cured hams from two European countries can be distinguished using SDS-PAGE. Thirty-seven commercial hams (19 Spanish, 18 French) were used in the study. Four protein fractions were extracted from each sample, with sufficient material prepared to allow each fraction to be analysed in triplicate lanes. The complete extraction process was carried out in duplicate. The 24 specimens originating from each ham sample were randomly allocated to different lane positions and gels, as were at least two reference lanes (for reference proteins). In total, 118 gels were prepared. Mathematical routines were developed using a matrix language to process the gel image files. Procedures were written to carry out 'within-gel' image correction, lane extraction and normalization, 'between-gel' data registration and linear discriminant analysis (LDA) of each fraction's data to establish whether the provenance could be systematically distinguished. The between-gel registration was carried out using a genetic algorithm (GA). Feature selection was also performed using a GA, to pass subsets of features to the LDA routine. Cross-validated classification success rates were 84, 91, 81 and 85%, respectively, for the four fractions. We conclude that SDS-PAGE can be conducted in a sufficiently quantitative manner and can potentially verify the provenance of regional speciality dry-cured hams.

Biosynthesis of strawberry aroma compounds through amino acid metabolism.

The fate of amino acids in relation to aroma biogenesis was studied in strawberries using the in vitro growth approach. This fruit presented differences in the level of metabolization for different amino acids. Incubations of strawberries with L-isoleucine gave rise to an increase of fourteen compounds in this fruit aroma, either not detected previously or constituents of strawberry aroma. However, L-valine incubations did not provide a significant change in this fruit aroma. Strawberry feeding with L-isoleucine resulted in a 7-fold increase in the sum of 2-methylbutanoate esters, and a double production of 2-methylbutyl esters compared to those of control fruits. Around 94% of the ester increase corresponded to 2-methylbutanoates, with ethyl 2-methylbutanoate being the most representative compound (92%). On the other hand, among the 2-methylbutyl esters, comprising around 6% of total aroma volatiles increase, 2-methylbutyl acetate was the major compound (95%) arising from L-isoleucine strawberry feeding. The role of enzymatic activities within the amino acid metabolic pathway in strawberry fruits is discussed.

Catalytic properties of alcohol acyltransferase in different strawberry species and cultivars.

The substrate specificity of alcohol acyltransferase (AAT) enzymes from different strawberry varieties was studied. Proteins with AAT activity from fruits of Fragaria x ananassa Duch. cv. Oso Grande were purified to apparent homogeneity and used for kinetic studies with different straight-chain alcohols and acyl-CoAs. K(m) values obtained for Oso Grande enzyme with six different alcohols, using acetyl-CoA as cosubstrate, decreased with increasing length of the alcohol chain. In similar experiments the increase in the acyl-CoA carbon chain was also found to be correlated with a higher substrate specificity. Heptanol (K(m) = 0.73 mM) and hexanoyl-CoA (K(m) = 0.41 mM) were the best substrates for Oso Grande AAT. Comparative catalytic studies were carried out with AAT partially purified extracts from the wild type Fragaria vesca and five commercial strawberry varieties: Tudnew, Carisma, Camarosa, Sweet Charlie, and Eris. The specificities of these enzymes toward five selected alcohols and acyl-CoAs reflected interesting cultivar differences.