Soy protein-phlorizin conjugate prepared by tyrosinase catalysis: Identification of covalent binding sites and alterations in protein structure and functionality.

Tyrosinase-catalyzed synthesis of soy 7S/11S-phlorizin conjugates was performed, and the reaction sites, conformation alterations and functional properties of complexes were evaluated using proteomic, in combination with multispectral technologies. Phlorizin was conjugated to 7S/11S primarily via residues of Lys, Cys, His and Arg residues. The phlorizin binding equivalents and decreased contents of free and total sulfhydryl groups and free amino groups confirmed the covalent interaction in the 7S/11S-phlorizin complexes. Conjugation with phlorizin promoted the conversion of α-helix to ß-sheet and ß-turn, with simultaneous transformation of the microenvironments around Trp and Tyr residues to hydrophilic and hydrophobic microenvironments, respectively, and lowering of the surface hydrophobicity of 7S/11S. The DPPH and ABTS radical scavenging abilities and α-glucosidase inhibitory activities of 7S/11S were increased by three-, two- and three-fold after the covalent binding of phlorizin. The study provided an ideal tyrosinase-catalyzed approach to fabricate custom-tailored nutritional soy protein-polyphenol products.

Heat-induced aggregation of subunits/polypeptides of soybean protein: Structural and physicochemical properties.

To reveal the nature of thermal aggregation of soybean protein at subunit level, structure and physicochemical properties of αα'- and ß-subunits isolated from ß-conglycinin, acidic polypeptide, and basic polypeptide from glycinin, as well as ß-conglycinin and glycinin, were characterized before and after heat treatment. The transmission electron microscopy (TEM) images showed that ß-conglycinin, αα'-subunits and acidic polypeptide formed regular thermal aggregates, which exhibited high solubility, high ζ-potential value, and small particle size. While glycinin, ß-subunit, and basic polypeptide aggregated to insoluble clusters with large particle size distribution. The results of size exclusion chromatography and non-reducing electrophoresis showed that the disulfide bond was the important force in stabilizing the protein conformation of thermal aggregates in ß-conglycinin, glycinin, and their isolated subunits/polypeptides but ß-subunit. The results of surface hydrophobicity and intrinsic fluorescence spectra showed that the thermal aggregations of ß-subunit and basic polypeptide were mainly driven by hydrophobic interactions.

Digestion Resistance of Soybean 7S Protein and Its Implications for Reinforcing the Gastric Mucus Barrier.

Previous studies have found that soybean protein, especially soybean 7S protein (ß-conglycinin), exhibits digestion resistance, but the mechanism of digestion resistance and its implications for human health are still unclear. Here, we show that the extracted soybean 7S protein contains both oligomer globulins and amyloid aggregates, while the gastric digested soybean 7S protein only contains amyloid aggregates and thus exhibits digestion resistance. An animal experiment shows that un-digestible soybean 7S protein effectively prevents aspirin-induced acute gastric mucosa damage. The impacts of un-digestible soybean 7S protein on gastric mucus barrier properties are investigated using quartz crystal microbalance with dissipation (QCM-D), Langmuir monolayer, and multiple particle tracking (MPT). Results show that these un-digestible protein aggregates can penetrate into gastric mucus, increase the viscosity and compactness of the mucin layer, and reinforce the gastric mucus barrier properties. The findings are helpful to understand that high consumption of non-fermented soybean foods is associated with a decreased risk of gastric cancer.

Structure, Immunogenicity, and IgE Cross-Reactivity among Walnut and Peanut Vicilin-Buried Peptides.

Vicilin-buried peptides (VBPs) from edible plants are derived from the N-terminal leader sequences (LSs) of seed storage proteins. VBPs are defined by a common α-hairpin fold mediated by conserved CxxxCx(10-14)CxxxC motifs. Here, peanut and walnut VBPs were characterized as potential mediators of both peanut/walnut allergenicity and cross-reactivity despite their low (∼17%) sequence identity. The structures of one peanut (AH1.1) and 3 walnut (JR2.1, JR2.2, JR2.3) VBPs were solved using solution NMR, revealing similar α-hairpin structures stabilized by disulfide bonds with high levels of surface similarity. Peptide microarrays identified several peptide sequences primarily on AH1.1 and JR2.1, which were recognized by peanut-, walnut-, and dual-allergic patient IgE, establishing these peanut and walnut VBPs as potential mediators of allergenicity and cross-reactivity. JR2.2 and JR2.3 displayed extreme resilience against endosomal digestion, potentially hindering epitope generation and likely contributing to their reduced allergic potential.

Effect of ultrasound pretreatment on the functional and antioxidant properties of fermented and germinated Lupin protein isolates grafted with glucose.

BACKGROUND: This study examined the functional and antioxidant properties of Maillard reaction (MR) products of lupin protein isolate (LPI), fermented (FLPI), and germinated (GLPI) with glucose (G), treated with ultrasound (US) at different power levels (20-40-60-80%) for 15 min. The MR was conducted in a water bath for 180 min at 90 °C. RESULTS: The Trolox-equivalent antioxidant capacity (TEAC) values were found to be 46.79%, 56.43%, and 35.56% for the control (C), 58.99%, 80.17%, and 69.73% for conjugates of LPI-G, FLPI-G, and GLPI-G treated at 80% US, respectively. The maximum 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity of LPI-G, FLPI-G, and GLPI-G was found to be 39.68%, 59.54%, and 48.41%, respectively after 80% US. The FLPI-G sample showed the highest antioxidant activity compared with the samples treated at the same power level for DPPH and TEAC. The Fe-chelating activity of GLPI-G showed significant differences when compared with FLPI-G. The solubility of LPI-G, FLPI-G, and GLPI-G increased with increasing US power. The highest solubility was 74.29% for 80% US-treated GLPI-G. The emulsifying activity index (EAI) increased at 20% US and decreased with further increase in the US power. The EAI and emulsifying stability index (ESI) were negatively affected by the MR and US processes. CONCLUSION: The findings of current study proved that conjugation of LPI with G with the MR and with US pretreatment is an effective method for improving the bio- and techno-functional properties of LPI. It is therefore likely that the properties of plant proteins modified by biochemical and physical treatments may widen their applications in the food industry. © 2021 Society of Chemical Industry.

Soybean ß-Conglycinin: Structure Characteristic, Allergenicity, Plasma Lipid-Controlling, Prevention of Obesity and Non-alcoholic Fatty Liver Disease.

Soybean has become an important world commodity because of its low price, good nutritional value and recognized functional health benefits in recent years. ß-conglycinin is one of the major storage proteins in soybean. It has captured a growing interest recently because of its allergenicity and potential health benefits, which continues to drive the research and commercial development of ß-conglycinin-based food products and ingredients. In this review, the structure, the amino acid composition, extraction methods and electrophoretic pattern of ß-conglycinin are briefly summarized. Studies on ß-conglycinin by allergenicity, plasma lipid-controlling, obesity and nonalcoholic fatty liver disease are highlighted, critically discussing their main shortcomings and challenges and identifying the research gaps. Studies to date have demonstrated the cultivation of ß-conglycinin with health benefits as functional ingredients and foodstuffs. The current research focuses on proteins, mainly challenging the mechanisms of subunit/peptide effects interaction and identifying and characterizing the hidden biological activities in the polypeptide chains. There is much scope for further exploration into various aspects of ß-conglycinin, such as the selection of mutant strains and genetic engineering and prospects on targeted ß-conglycinin exploitation in the nutraceutical area. In addition, the safety evaluation of ß-conglycinin and its stabilized emulsions deserve more attention to food-related applications.

Dietary supplementation of ß-conglycinin, with or without sodium butyrate on the growth, immune response and intestinal health of hybrid grouper.

We investigated the effects of low and high doses of ß-conglycinin and the ameliorative effects of sodium butyrate (based on high-dose ß-conglycinin) on the growth performance, serum immunity, distal intestinal histopathology, and gene, protein expression related to intestinal health in hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂). The results revealed that the instantaneous growth rate (IGR) of grouper significantly increased, decreased, and increased in the low-dose ß-conglycinin (bL), high-level ß-conglycinin (bH) and high-level ß-conglycinin plus sodium butyrate (bH-NaB), respectively. The feed coefficient ratio (FCR) was significantly increased in the bH and bH-NaB, serum levels of IFN-γ, IL-1ß, and TNF-α were upregulated in the bH. The intestinal diameter/fold height ratio was significantly increased in the bH. Furthermore, there were increases in nitric oxide (NO), total nitric oxide synthase (total NOS), and peroxynitrite anion (ONOO-) in the bH, and decreases in total NOS and ONOO- in the bH-NaB. In the distal intestine, IL-1ß and TGF-ß1 mRNA levels were downregulated and upregulated, respective in the bL. The mRNA levels of TNF-α and IL-6 were upregulated in the bH, and downregulated in the bH-NaB, respectively. Occludin, claudin3 and ZO-3 mRNA levels were upregulated in the bL, downregulated in the bH and then upregulated in the bH-NaB. No significant differences were observed in the mRNA levels of IFN-γ and jam4. And the p-PI3K p85Tyr458/total PI3K p85 value was significantly increased in the bH and then decreased in the bH-NaB, and the total Akt value was significantly increased in the bH. These indicate ß-conglycinin has a regulatory effect on serum immunity and affect distal intestinal development by modulating distal intestinal injury-related parameters. Within the distal intestinal tract, low- and high-dose ß-conglycinin differentially affect immune responses and tight junctions in the distal intestine, which eventually manifests as a reduction in growth performance. Supplementing feed with sodium butyrate might represent an effective approach for enhancing serum immunity, and protects the intestines from damage caused by high-dose ß-conglycinin.

Plant asparaginyl endopeptidases and their structural determinants of function.

Asparaginyl endopeptidases (AEPs) are versatile enzymes that in biological systems are involved in producing three different catalytic outcomes for proteins, namely (i) routine cleavage by bond hydrolysis, (ii) peptide maturation, including macrocyclisation by a cleavage-coupled intramolecular transpeptidation and (iii) circular permutation involving separate cleavage and transpeptidation reactions resulting in a major reshuffling of protein sequence. AEPs differ in their preference for cleavage or transpeptidation reactions, catalytic efficiency, and preference for asparagine or aspartate target residues. We look at structural analyses of various AEPs that have laid the groundwork for identifying important determinants of AEP function in recent years, with much of the research impetus arising from the potential biotechnological and pharmaceutical applications.

Digestive characteristics and peptide release from wheat embryo proteins in vitro.

Due to the scarcity of the data on digestion and metabolism of wheat embryo proteins WEP, a simulated gastrointestinal digestion (SGID) scheme in vitro was utilized to explain the protein hydrolysis and biological activity of WEP during the digestion process. WEP had a certain degree of resistance to gastric digestion, especially the protein with a molecular weight of 50 kDa. In all the samples, no visually intact protein band emerged in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) during the intestinal phase, which was consistent with a gradually increasing content of released free amino acids. Moreover, the resistant digestion peptides (the amino acid sequences were ISQFXX and GTVX) were identified at the end of the gastrointestinal digestion (GID) product by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Although the complete protein in the sample was degraded, the antioxidant activity was not negatively affected, rather it showed an increasing trend and maintained a higher level of activity. The amount of the ß-sheet gradually increased as that of the α-helix declined, the random coil decreased, whereas no obvious change was noticed in ß-turn content. The results provide a better understanding for optimal selection of peptide candidates for designing protein products in the food processing industry as well as for WEP digestion and metabolism in the human body.

Impact of Soy ß-Conglycinin Peptides on PCSK9 Protein Expression in HepG2 Cells.

BACKGROUND: Dyslipidaemias, particularly elevated plasma low-density lipoprotein cholesterol (LDL-C) levels, are major risk factors for cardiovascular disease (CVD). Besides pharmacological approaches, a nutritional strategy for CVD prevention has gained increasing attention. Among functional foods, the hypocholesterolemic properties of soy are driven by a stimulation of LDL-receptor (LDL-R) activity. AIM: To characterize the effect of two soy peptides, namely, ß-conglycinin-derived YVVNPDNDEN and YVVNPDNNEN on the expression of proprotein convertase subtilisin/kexin type 9 (PCSK9), one of the key-regulators of the LDL-R. METHODS: PCSK9 promoter activity (luciferase assay), PCSK9 protein expression (WB) and secretion (ELISA), PCSK9 interaction with LDL-R (binding assay) and human HepG2 cells were the objects of this investigation. RESULTS: Treatment with YVVNPDNNEN peptide has led to a rise in PCSK9 gene expression (90.8%) and transcriptional activity (86.4%), and to a decrement in PCSK9 intracellular and secreted protein (-42.9%) levels. YVVNPDNNEN peptide reduced the protein expression of transcriptional factor HNF1α. Most changes driven by YVVNPDNDEN peptide were not statistically significant. Neither peptide inhibited the PCSK9-LDLR interaction. CONCLUSIONS: Although sharing a common effect on LDL-R levels through the inhibition of 3-hydroxy-3-methylglutaryl CoA reductase activity, only the YVVNPDNNEN peptide has an additional mechanism via the downregulation of PCSK9 protein levels.

Proteolytic activity of selected commercial Lactobacillus helveticus strains on soy protein isolates.

Soy protein isolates were fermented by three commercial Lactobacillus helveticus strains for a maximum of seven days to investigate the resulting proteolysis. The proteolytic activity of the most active strain (LH88) was further analyzed (LC-MS/MS and GC-MS) and it was shown that the ß-conglycinin α subunit 1, ß-conglycinin α' subunit, glycinin G1, and 2S albumin were specifically degraded. Peptigram analysis and visualization of the crystal structure showed that the hydrolysis sites of ß-conglycinin α subunit, α' subunit, and the glycinin G1 were located on the surface of the molecule or at the mobile disordered region, hence being highly accessible for the proteinase of LH88. The proteins were partially further degraded to free amino acids, and subsequently catabolized to volatile compounds. However, most of the proteins remained native, even after seven days of fermentation, thus additional modification of protein structure or adjustment of fermentation conditions are required for effective generation of flavor compounds.

Defining the Familial Fold of the Vicilin-Buried Peptide Family.

Plants and their seeds have been shown to be a rich source of cystine-stabilized peptides. Recently a new family of plant seed peptides whose sequences are buried within precursors for seed storage vicilins was identified. Members of this Vicilin-Buried Peptide (VBP) family are found in distantly related plant species including the monocot date palm, as well as dicotyledonous species like pumpkin and sesame. Genetic evidence for their widespread occurrence indicates that they are of ancient origin. Limited structural studies have been conducted on VBP family members, but two members have been shown to adopt a helical hairpin fold. We present an extensive characterization of VBPs using solution NMR spectroscopy, to better understand their structural features. Four peptides were produced by solid phase peptide synthesis and shown to favor a helix-loop-helix hairpin fold, as a result of the I-IV/II-III ladderlike connectivity of their disulfide bonds. Interhelical interactions, including hydrophobic contacts and salt bridges, are critical for the fold stability and control the angle at which the antiparallel α-helices interface. Activities reported for VBPs include trypsin inhibitory activity and inhibition of ribosomal function; however, their diverse structural features despite a common fold suggest that additional bioactivities yet to be revealed are likely.

Investigation on the interaction of heated soy proteins with anthocyanins from cornelian cherry fruits.

The interaction between preheated soy proteins and anthocyanins from cornelian cherries was evaluated using a spectroscopic approach and molecular modeling. Structural changes of glycinin, ß-conglycinin and soy protein isolate were investigated based on spectra of native and heat treated proteins in the presence of anthocyanins rich extracts from fresh cornelian cherry fruits. The fluorescence maximum emission in the presence of anthocyanins showed significant red shifts when compared with individual proteins, indicating the change of polarity in the surroundings of Trp residues from soy proteins toward more hydrophilic, which were attributed to protein-polyphenols interactions. Soy proteins interacted with cornelian cherries anthocyanins mainly through a static quenching mechanism. Glycinin presented a better affinity toward anthocyanins as revealed by the binding constant. The in silico approach was further employed to provide single molecule level details on the interaction between the main soy proteins and anthocyanins prevailing in cornelian cherry extracts. The docking results are consistent with the fluorescence spectroscopy data indicating better affinity of glycinin for cyanidin 3-glucoside and cyanidin 3-rutinoside, compared to the ß-conglycinin. These findings deliver important insights for efficient development of microencapsulated powders based on soy proteins and anthocyanins from cornelian cherries, from the perspectives of obtaining value-added ingredients.

Binding of benzoic acid and anions within the cupin domains of the vicilin protein canavalin from jack bean (Canavalia ensiformis): Crystal structures.

X-ray intensities extending to 1.4 Å resolution were collected on the P63 hexagonal crystal form of canavalin, and extended to 1.9 Å for the orthorhombic C2221 crystals. Structure determination of a new crystal form of canavalin having space group P212121 is reported as well. Both the N and C terminal cupin domains contained identifiable ligands. For hexagonal crystals, in the cavity of the C terminal cupin, a molecule of benzoic acid was found, bound through carboxyl oxygens to Histidine 297, asparagine 284 and Arginine 376. The benzene ring was immersed in a cluster of at least 8 hydrophobic amino acid side chains. The N terminal cupin contained a molecule of citrate. Benzoic acid was also found to be present in the C terminal cupins of in the C2221 and P212121 crystal forms. In rhombohedral crystals, the C terminal cupin domain appeared to be occupied by a phosphate ion, but this was ambiguous. In cubic crystals, both domains were vacant. The N terminal cupin domains of canavalin in the P212121 and rhombohedral crystals were also vacant, but the N terminal cupin domain of the C2221 crystals contained a ligand whose identity is uncertain, but which has been modeled as HEPES buffer. A possible physiological role for the ligands and their complexes with canavalin is considered.

GPA5 Encodes a Rab5a Effector Required for Post-Golgi Trafficking of Rice Storage Proteins.

Dense vesicles (DVs) are vesicular carriers, unique to plants, that mediate post-Golgi trafficking of storage proteins to protein storage vacuoles (PSVs) in seeds. However, the molecular mechanisms regulating the directional targeting of DVs to PSVs remain elusive. Here, we show that the rice (Oryza sativa) glutelin precursor accumulation5 (gpa5) mutant is defective in directional targeting of DVs to PSVs, resulting in discharge of its cargo proteins into the extracellular space. Molecular cloning revealed that GPA5 encodes a plant-unique phox-homology domain-containing protein homologous to Arabidopsis (Arabidopsis thaliana) ENDOSOMAL RAB EFFECTOR WITH PX-DOMAIN. We show that GPA5 is a membrane-associated protein capable of forming homodimers and that it is specifically localized to DVs in developing endosperm. Colocalization, biochemical, and genetic evidence demonstrates that GPA5 acts in concert with Rab5a and VPS9a to regulate DV-mediated post-Golgi trafficking to PSVs. Furthermore, we demonstrated that GPA5 physically interacts with a class C core vacuole/endosome tethering complex and a seed plant-specific VAMP727-containing R-soluble N-ethylmaleimide sensitive factor attachment protein receptor complex. Collectively, our results suggest that GPA5 functions as a plant-specific effector of Rab5a required for mediating tethering and membrane fusion of DVs with PSVs in rice endosperm.

Binding interaction between ß-conglycinin/glycinin and cyanidin-3-O-glucoside in acidic media assessed by multi-spectroscopic and thermodynamic techniques.

The noncovalent binding mechanisms between cyanidin-3-O-glucoside (C3G) and two main soy protein fractions: ß-conglycinin (7S) and glycinin (11S) at pH 3.0 were investigated and compared. C3G modified the secondary structure of two fractions by increasing α-helix and random coil content while decreasing ß-sheet content. The binding of C3G also altered the tertiary structure and microenvironment of two fractions, demonstrated by synchronous and three-dimensional fluorescence spectra. Additionally, C3G binding reduced the surface hydrophobicity and thermostability of both 7S and 11S. Moreover, the fluorescence quenching results showed that the binding of C3G to two fractions were spontaneous complexation processes driven by electrostatic forces. The number of C3G bound per protein molecule (n) was near 1. The binding constant (Ka) was 2.41 (±0.42) × 104 M-1 for 11S and 0.81 (±0.01) × 104 M-1 for 7S at 298 K. 11S showed a stronger binding ability for C3G than 7S. These findings contribute to the knowledge of interactions between soy protein fractions and dietary polyphenols under acidic condition, and are beneficial for the application of soy protein-based products in foods.

Effects of divergent ultrasound pretreatment on the structure of watermelon seed protein and the antioxidant activity of its hydrolysates.

In the present study, the antioxidant hydrolysates obtained from watermelon seed protein (WSP) after divergent ultrasound and ultrafiltration treatment were studied. The results showed that the slit divergent ultrasound (SDU, 20/28 KHz) pretreatment had considerable influence on the structure and enzymatic efficiency of WSP. Besides, compared with hydrolysates without ultrasonic and ultrafiltration treatment, watermelon protein hydrolysates with molecular weight <1 kDa (WSPHs-I) showed the highest antioxidant activities and could protect RAW 264.7 cells from H2O2-induced oxidative stress damage via activating the Nrf2/HO-1 pathway. Interestingly, WSPHs-I had good stability against oxidation at temperature under 100 °C or in the acidic or neutral condition and still exhibited strong antioxidant activity after simulated gastrointestinal digestion. Taken together, SDU pretreatment could significantly increase the antioxidant activities and stability of WSPHs by improving the structure and facilitating enzymolysis of the WSP.

Almond (Prunus dulcis) Allergen Pru du 8, the First Member of a New Family of Food Allergens.

An almond allergen with two known short peptide sequences was reported as the almond 2S albumin but was later suspected to be almond vicilin. However, this allergen was not designated by the World Health Organization/International Union of Immunological Societies. This study aimed to determine the true identity of this elusive almond allergen. cDNAs were synthesized from total RNA of the Nonpareil almond. The complete sequence of the previously reported almond allergen was determined from its coding sequence. The deduced protein was produced recombinantly and was confirmed to be a food allergen by testing with 18 almond-allergic sera. The allergen is a potential cysteine-rich antimicrobial protein with characteristic C[X]3C-[X]10-12-C[X]3C motifs of the hairpinin antimicrobial protein. This first member of a novel family of food allergens was named Pru du 8. The signature motif of the hairpinin antimicrobial protein can be found in the N-terminal region of some vicilin allergens (e.g., Ara h 1). It can also be found in the signal peptide of other vicilin allergens (e.g., Car i 2). In many species, however, vicilins do not contain such a motif, indicating that the presence of the signature motifs of the hairpinin antimicrobial protein in vicilins might be a result of translocation during evolution.

Shotgun proteomic analysis of quinoa seeds reveals novel lysine-rich seed storage globulins.

Quinoa seeds have high protein content and an exceptional balance of amino acids, with higher contents of lysine, methionine and cysteine than common cereals. To date, only three globulins, all of which have a content of lysine mass that does not exceed 3.8%, have been identified in quinoa. To address the protein present in quinoa seeds, TCA/Acetone protein extraction was performed using four different quinoa seed genotypes with contrasting edaphoclimatic origins. Proteins were identified and analyzed using label-free shotgun proteomics followed by in silico analysis, using the three published quinoa genomes. This analysis allowed us to identify sixteen globulins, thirteen of which are novel: nine legumin-like proteins and seven vicilin-like proteins. Seven of the novel proteins contain 7.5% or more of lysine mass, justifying the high content of lysine repeatedly reported in quinoa seeds. No significant differences were found between the four genotypes here analyzed.

Cocoa Bean Proteins-Characterization, Changes and Modifications due to Ripening and Post-Harvest Processing.

The protein fractions of cocoa have been implicated influencing both the bioactive potential and sensory properties of cocoa and cocoa products. The objective of the present review is to show the impact of different stages of cultivation and processing with regard to the changes induced in the protein fractions. Special focus has been laid on the major seed storage proteins throughout the different stages of processing. The study starts with classical introduction of the extraction and the characterization methods used, while addressing classification approaches of cocoa proteins evolved during the timeline. The changes in protein composition during ripening and maturation of cocoa seeds, together with the possible modifications during the post-harvest processing (fermentation, drying, and roasting), have been documented. Finally, the bioactive potential arising directly or indirectly from cocoa proteins has been elucidated. The "state of the art" suggests that exploration of other potentially bioactive components in cocoa needs to be undertaken, while considering the complexity of reaction products occurring during the roasting phase of the post-harvest processing. Finally, the utilization of partially processed cocoa beans (e.g., fermented, conciliatory thermal treatment) can be recommended, providing a large reservoir of bioactive potentials arising from the protein components that could be instrumented in functionalizing foods.