Standardization of in vitro digestibility and DIAAS method based on the static INFOGEST protocol

Resumen de la conferencia invitada presentada en el 7th International Conference on Food Digestion. Cork, Irlanda. 3-5 Mayo (2022

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

[EN] 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.We thank Dr Espen Granum for critically reading the manuscript, Maria Isabel Gaspar Vidal and Elena Sanchez Romero for technical assistance, the Instrumental Technical Service at EEZ-CSIC for DNA sequencing and ICP-OES mineral analysis and Michael O'Shea for proofreading the text. In addition, we thank Dr Ana P. Ortega who assisted in preliminary experiments. This work was supported by ERDF-cofinanced grants, AGL2010-17090 and AGL2013-41733-R (A.B.), AGL2015-64991-C3-3-R (V.M.) and AGL2014-56675-R (M.J.A.) from the Spanish "Ministerio de Economia, Industria y Competitividad'; CVI-7558, Proyecto de Excelencia, from Junta de Andalucia (A.B); and the Australian Research Council (ARC) for Centre of Excellence (CE14010008) and Future Fellowship (FT130100709) funding (M.G.). N.J-P. was supported by an FPI program BES-2011-046096 and her stay in M.G.'s lab by a short-stay EEBB-I-14-08682, both from the Spanish from "Ministerio de Economia Industria y Competitividad'. The authors have no conflict of interest to declare.Jaime-Perez, N.; Pineda Chaza, BJ.; García Sogo, B.; Atarés Huerta, A.; Athman, A.; Byrt, CS.; Olias, R.... (2017). The sodium transporter encoded by the HKT1;2 gene modulates sodium/potassium homeostasis in tomato shoots under salinity. Plant Cell & Environment. 40(5):658-671. https://doi.org/10.1111/pce.12883S65867140

Standardization of in vitro digestibility and DIAAS method based on the static INFOGEST protocol

Background: The FAO recommends the digestible indispensable amino acid score (DIAAS) as the measure for protein quality, for which the true ileal digestibility needs to be assessed in humans or pigs. However, due to high costs and ethical concerns, the FAO strongly encourages as well the development of validated in vitro methods, which complement the in vivo experiments. Method: Recently, an in vitro workflow, based on the validated static INFOGEST protocol, was developed and compared towards in vivo data. In parallel to the validation with in vivo data, the repeatability and reproducibility of the in vitro protocol were tested in an international ring trial (RT) with the aim to establish an international ISO standard method within the International Dairy Federation (IDF). Five different dairy products (skim milk powder, whole milk powder, whey protein isolate, yoghurt, and cheese) were analyzed in 32 different laboratories from 18 different countries, across 4 continents. Results: in vitro protein digestibilities based on Nitrogen, free R-NH2, and total amino acids as well as DIAAS values were calculated and compared to in vivo data, where available. Conclusion: The in vitro method is suited for quantification of digestibility and will be further implemented to other food matricesinfo:eu-repo/semantics/publishedVersio

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

Nutritional and functional properties of Bowman-Birk inhibitors from legumes in food and feed

Resumen del poster presentado en el II Congreso de Investigación PTS. Granada, 9-11 febrero (2022)Este trabajo ha sido financiado por los proyectos AGL2017-83772-R y P20_00242 del Ministerio de Ciencia e Innovación y Junta de Andalucía, respectivament

RESEARCH ARTICLE 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 ani-mal 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 muta-tions 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 tryp-sin / 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

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 35kb 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

Impact of mutations on enzyme inhibition.

<p>Trypsin (TIU, <b>a, c</b>) and chymotrypsin (CIU, <b>b, d</b>) inhibitory units per mg of meal (<b>a, b</b>) or per mg of protein (<b>c, d</b>) of three TILLING mutants (C77Y, S85F, E109K) and their corresponding wild-type pea lines. For each plot, significant differences (p < 0.01) between wild-type and mutant lines within each pair are denoted (a, b, as appropriate on bars in each chart).</p

Identification of TI1 and TI2 diagnostic peptides by mass spectrometry.

<p>Identification of TI proteins eluted by cation-exchange chromatography (P, peaks 1–4) of wild-type seed extracts, as shown in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134634#pone.0134634.g003" target="_blank">3</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134634#pone.0134634.g005" target="_blank">5</a>. The peptides obtained for P1 and P2 corresponded to the amino acid sequence for TI2 (UniProt accession: Q41066), whereas those deduced for P3 and P4 corresponded to the amino acid sequence for TI1 (UniProt accession: Q41065). The sequences of the inhibitory domains are underlined. The amino acid residues that distinguish TI1 and TI2 proteins are shown in bold. Lys (K) and Leu (L) or Tyr (Y) at position P1 (*) determine specificity for trypsin and chymotrypsin, respectively.</p><p>Identification of TI1 and TI2 diagnostic peptides by mass spectrometry.</p