Comparative study of factors affecting the recovery of proteins from malt rootlets using pressurized liquids and ultrasounds.

Malt rootlets (MR) are a waste from brewing with high protein content. This work proposes to study the impact of extracting parameters on the recovery of proteins and the characteristics of extracts from MR using ultrasound-assisted extraction (UAE) and pressurized liquid extraction (PLE). A Box-Behnken experimental design was employed to study the effect of extracting parameters on the protein yield, while characterization comprised the study of antioxidant properties, the identification of extracted proteins using high-resolution tandem mass spectrometry, and the evaluation of the co-extraction of phenolic compounds. Protein extraction was promoted at an ultrasounds amplitude of 68%, for 20 min at 52 °C in UAE, while adding 33% ethanol resulted in the highest yield in PLE. While UAE extracted 53 ± 5% of MR proteins, PLE reached a 73 ± 7%, using more sustainable conditions. Significant antioxidant activities were observed in the PLE extract, although undermined by gastrointestinal digestion. Proteomic analysis detected 68 proteins from Hordeum vulgare in the UAE extract and 9 in the PLE extract. Proteins in MR are very different to that from barley grains or brewer's spent grains. PLE also co-extracted phenolic compounds while this was not significant by UAE.

Feasibility of cationic carbosilane dendrimers for sustainable protein sample preparation.

Protein sample preparation is the bottleneck in the analysis of proteins. The aim of this work is to evaluate the feasibility of carbosilane dendrimers functionalized with cationic groups to make easier this step. Anionic carbosilane dendrimers (sulphonate- and carboxylate-terminated) have already demonstrated their interaction with proteins and their potential in protein sample preparation. In this work, interactions between positively charged carbosilane dendrimers and different model proteins were studied when working under different pH conditions, dendrimer concentrations, and dendrimer generations. Amino- and trimethylammonium-terminated carbosilane dendrimers presented, in some cases, weak interactions with proteins. Unlike them, carbosilane dendrimers with terminal dimethylamino groups could interact, in many cases, with proteins and these interactions were affected by the pH, the dendrimer concentration, and the dendrimer generation. Moreover, dendrimer precipitation was observed at all pHs, although just second and fourth generation (2 G and 4 G) dendrimers resulted in the formation of complexes with proteins. Under experimental conditions promoting dendrimer-protein interactions, 2 G dimethylamino-terminated dendrimers were proposed as an alternative to other methods used in analytical chemistry or analysis in which an organic solvent or a resin are required to enrich/purify proteins in a complex sample.

Capillary liquid chromatography-ion trap-mass spectrometry methodology for the simultaneous quantification of four angiotensin-converting enzyme-inhibitory peptides in Prunus seed hydrolysates.

Prunus genus fruit seeds are sources of highly angiotensin-I-converting enzyme (ACE)-inhibitory peptides. The presence of peptides IYSPH, IYTPH, IFSPR, and VAIP seems to be related to this activity but no previous work has demonstrated the direct relationship between the concentration of these peptides and the antihypertensive activity of hydrolysates. This work describes the development of a method for the quantification of these peptides in Prunus seeds hydrolysates based on capillary liquid chromatography-IT-MS/MS. The analytical characteristics of the method were evaluated through the study of the linearity, LOD, LOQ, presence of matrix interferences, precision, and recovery. The developed methodology was applied to the determination of the four peptides in seed hydrolysates from different Prunus genus fruits: peaches (7 varieties), plums (2 varieties), nectarines (3 varieties), apricots (2 varieties), cherry, and paraguayo. Peaches and plums seed hydrolysates yielded the highest concentrations of these peptides while paraguayo one showed the lowest concentrations. A high correlation between peptides concentrations was demonstrated suggesting that the four peptides could be released from the same seed proteins.

Sulfonate-terminated carbosilane dendron-coated nanotubes: a greener point of view in protein sample preparation.

Reduction or removal of solvents and reagents in protein sample preparation is a requirement. Dendrimers can strongly interact with proteins and have great potential as a greener alternative to conventional methods used in protein sample preparation. This work proposes the use of single-walled carbon nanotubes (SWCNTs) functionalized with carbosilane dendrons with sulfonate groups for protein sample preparation and shows the successful application of the proposed methodology to extract proteins from a complex matrix. SEM images of nanotubes and mixtures of nanotubes and proteins were taken. Moreover, intrinsic fluorescence intensity of proteins was monitored to observe the most significant interactions at increasing dendron generations under neutral and basic pHs. Different conditions for the disruption of interactions between proteins and nanotubes after protein extraction and different concentrations of the disrupting reagent and the nanotube were also tried. Compatibility of extraction and disrupting conditions with the enzymatic digestion of proteins for obtaining bioactive peptides was also studied. Finally, sulfonate-terminated carbosilane dendron-coated SWCNTs enabled the extraction of proteins from a complex sample without using non-environmentally friendly solvents that were required so far. Graphical Abstract Green protein extraction from a complex sample employing carbosilane dendron coated nanotubes.

Factors affecting interactions between sulphonate-terminated dendrimers and proteins: A three case study.

This work proposes a deep study on the interactions between sulphonate-terminated carbosilane dendrimers and proteins. Three different proteins with different molecular weights and isoelectric points were employed and different pHs, dendrimer concentrations and generations were tested. Variations in fluorescence intensity and emission wavelength were used as protein-dendrimer interaction probes. Interaction between dendrimers and proteins greatly depended on the protein itself and pH. Other important issues were the dendrimer concentration and generation. Protein-dendrimer interactions were favored under acidic working conditions when proteins were positively charged. Moreover, in general, high dendrimer generations promoted these interactions. Modeling of protein-dendrimer interactions allowed to understand the different behaviors observed for every protein.

Proof of concept of a "greener" protein purification/enrichment method based on carboxylate-terminated carbosilane dendrimer-protein interactions.

Protein sample preparation is a critical and an unsustainable step since it involves the use of tedious methods that usually require high amount of solvents. The development of new materials offers additional opportunities in protein sample preparation. This work explores, for the first time, the potential application of carboxylate-terminated carbosilane dendrimers to the purification/enrichment of proteins. Studies on dendrimer binding to proteins, based on protein fluorescence intensity and emission wavelengths measurements, demonstrated the interaction between carboxylate-terminated carbosilane dendrimers and proteins at all tested pH levels. Interactions were greatly affected by the protein itself, pH, and dendrimer concentration and generation. Especially interesting was the interaction at acidic pH since it resulted in a significant protein precipitation. Dendrimer-protein interactions were modeled observing stable complexes for all proteins. Carboxylate-terminated carbosilane dendrimers at acidic pH were successfully used in the purification/enrichment of proteins extracted from a complex sample. Graphical Abstract Images showing the growing turbidity of solutions containing a mixture of proteins (lysozyme, myoglobin, and BSA) at different protein:dendrimer ratios (1:0, 1:1, 1:8, and 1:20) at acidic pH and SDS-PAGE profiles of the corresponsing supernatants. Comparison of SDS-PAGE profiles for the pellets obtained during the purification of proteins present in a complex sample using a conventional "no-clean" method based on acetone precipitation and the proposed "greener" method using carboxylate-terminated carbosilane dendrimer at a 1:20 protein:dendrimer ratio.

Identification of plum and peach seed proteins by nLC-MS/MS via combinatorial peptide ligand libraries.

UNLABELLED: Plum (Prunus domestica L.) and peach (Prunus persica (L.) Batsch) seed proteins are a source of bioactive peptides. These seeds, though, are usual residues produced during canning and beverage preparation that, in most cases, are irreversibly lost. The recovery and identification of these proteins might be of importance in human nutrition. This work employs the combinatorial peptide ligand libraries (CPLLs) technology as a tool to reduce the proteins dynamic concentration range. The most suitable extraction and CPLL capture conditions have been obtained and applied for the comprehensive identification of seed proteins. The analysis of recovered species by nLC-MS/MS has allowed the identification of 141 and 97 unique gene products from plum and peach seeds, respectively. It was possible to identify 16 proteins belonging to the Prunus genus. Moreover, a high number of histones and seed storage proteins were identified. Additionally, 21 and 14 bioactive peptides previously identified were found within protein sequences in plum and peach seeds, respectively. SIGNIFICANCE: Plums and peaches seeds are cheap sources of proteins that are irretrievably lost after canning and beverage production. Although this kind of residues has been used in animal feed or production of biofuel, they are usually incinerated or sent to landfills, wasting their huge potential. In order to exploit this, it is important to comprehensively study proteins present in plum and peach seeds. Nevertheless, since proteomics analysis is in most cases handicapped by the presence of high-abundance proteins masking the detection of the low-abundance ones, it is important to overcome this challenge. In this sense, combinatorial peptide ligand libraries (CPLLs) have been used in this work to reduce the dynamic protein concentration range to enable the identification of a higher amount of proteins than employing conventional methods. In this work, the better extracting conditions have been optimized and up to 141 and 97 unique gene products from plum and peach seeds have been found, respectively. Moreover, 21 and 14 peptides previously identified as bioactive peptides were ascertained within protein sequences in plum and peach seeds, respectively. For that reason, this research takes the first step in the recovery of these valuable proteins and in the extraction of bioactive peptides, which could be successfully adopted in human nutrition.

HPLC-Q-TOF-MS identification of antioxidant and antihypertensive peptides recovered from cherry (Prunus cerasus L.) subproducts.

The processing of fruits, such as cherries, is characterized by generating a lot of waste material such as fruit stones, skins, etc. To contribute to environmental sustainability, it is necessary to recover these residues. Cherry stones contain seeds with a significant amount of proteins that are underused and undervalued. The aim of this work was to extract cherry seed proteins, to evaluate the presence of bioactive peptides, and to identify them by mass spectrometry. The digestion of cherry seed proteins was optimized, and three different enzymes were employed: Alcalase, Thermolysin, and Flavourzyme. Peptide extracts obtained by the digestion of the cherry seed protein isolate with Alcalase and Thermolysin yielded the highest antioxidant and antihypertensive capacities. Ultrafiltration of hydrolysates allowed obtaining fractions with high antioxidant and antihypertensive capabilities. HPLC-Q-TOF-MS together with bioinformatics tools enabled one to identify peptides in these fractions.

Food hypersensitivity in mexican adults at 18 to 50 years of age: a questionnaire survey.

PURPOSE: There is limited epidemiological evidence of food hypersensitivity (FH) in the adult population. We aimed to determine the prevalence of FH in Mexican adults, their clinical features and to establish common food involved in its appearance. METHODS: We designed a cross-sectional study using a fixed quota sampling; 1,126 subjects answered a structured survey to gather information related to FH. RESULTS: The prevalence of FH in adults was 16.7% (95% CI, 14.5% to 18.8%), without statistical significant differences related to gender (women, 17.5% and men, 15.9%) or residential location. The most common clinical manifestations in adults with FH were oral allergy syndrome (70 of 1,126) and urticaria (55 of 1,126). According to category, fruits and vegetables were the most frequent foods to trigger FH (6.12%) and were individually related to shrimp (4.0%), and cow milk (1.5%). Adults under age 25 had a higher frequency of FH (OR, 1.39; 95% CI, 1.01 to 1.91, P <0.001). Personal history of any atopic disease was significantly associated with FH (P <0.0001). CONCLUSIONS: The prevalence of FH is relatively high in Mexican adults, and FH is significantly associated with atopic diseases.