Alkali-assisted extraction, characterization and encapsulation functionality of enzymatic hydrolysis-resistant prolamin from distilled spirit spent grain.

Curcumin is a natural lipophilic polyphenol that exhibits significant various biological properties such as antioxidant and anti-inflammatory properties following oral administration. However, its uses have shown limitations concerning aqueous solubility, bioavailability and biodegradability that could be improved by prolamin-based nanoparticle. In this study, curcumin was encapsulated into prolamin from sorghum (SOP) and wheat (WHP) and distilled spirit spent grain (DSSGP), which was obtained after microbial proteolysis of the former two cereal grains. All the three prolamins showed clear variation of protein profiles and microstructure as confirmed by electrophoresis analysis, disulfide bond determination and Fourier-transform infrared spectroscopy (FTIR). For curcumin-loaded nanospheres (NPs) fabrication, three prolamin-based NPs shared features of spherical shape, uniform particle size, and smooth surface. The average size ranged from 122 to 193 nm depending on the prolamin variety and curcumin loading. In the experiments in vitro, curcumin showed significantly improved UV/thermal stability. Furthermore, DSSGP was more resistant to enzymatic digestion in vitro, hence achieving the controlled release of curcumin in gastrointestinal tract. Collectively, the results indicated the improved bioavailability and biodegradability of curcumin encapsulated by DSSGP, which would be an innovative potential encapsulant for effective protection and targeted delivery of hydrophobic compounds.

Fabrication and characterization of novel prolamin nanoparticle-filled starch gels incorporating resveratrol.

This study aimed to improve the mechanical properties of wheat starch gels (WSG) and the stability and bioaccessibility of resveratrol (Res) in prolamin nanoparticles. Res-loaded gliadin (Gli), zein, deamidated gliadin (DG) and deamidated zein (DZ) nanoparticles were filled in WSG. The hardness, G' and G'' of WSG were notably increased. It can be attributed to the more ordered and stable structure induced by the interaction of prolamin nanoparticles and starch. The Res retention of nanoparticles and nanoparticle-filled starch gels was at least 24.6 % and 36.0 % higher than free Res upon heating. When exposed to ultraviolet, the Res retention was enhanced by over 6.1 % and 37.5 %. The in-vitro digestion demonstrated that the Res releasing percentage for nanoparticle-filled starch gels was 25.8 %-38.7 % lower than nanoparticles in the simulated stomach, and more Res was released in the simulated intestine. This resulted in a higher bioaccessibility of 82.1 %-93.2 %. The bioaccessibility of Res in Gli/Res/WSG and DG/Res/WSG was greater than that of Zein/Res/WSG and DZ/Res/WSG. More hydrophobic interactions occurred between Res and Gli, DG. The interactions between Res and zein, DZ were mainly hydrogen bonding. The microstructure showed that nanoparticles exhibited dense spherical structures and were uniformly embedded in the pores of starch gels.

Extrusion modification of prolamins from distiller's grains to facilitate the construction of biopolymer films.

BACKGROUND: Distiller's grains (DGs), which are rich in natural ingredients such as prolamins, are often used as low-value feed or discarded directly, resulting in great environmental pollution and resource waste. Prolamins from DGs (PDGs) were found to be a potential material for the construction of biopolymer films due to their good film-forming properties. In this study, extrusion processing was conducted to modify the physicochemical and structural properties of PDGs to facilitate the construction of biopolymer films with superior characteristics. RESULTS: Results indicated that extrusion led to improved solubility (17.91% to 39.95%) and increased disulfide bonds (1.46 to 6.13 µmol g-1) in PDGs. The total and sulfur amino acid contents of extruded PDGs were increased by 13.26% and 38.83%, respectively. New aggregation patterns were formed after extrusion according to the results of scanning electron microscopy, Fourier transform infrared spectroscopy and X-ray diffraction. Extrusion resulted in reduced surface hydrophobicity of PDGs (10 972 to 3632), sufficient evidence for which could be also found from structure analyses of PDGs. Finally, PDGs extruded at 110 °C were found to facilitate the forming of biopolymer films with superior mechanical properties, water resistance and thermal stability. CONCLUSIONS: Physicochemical and structural properties of PDGs were effectively modified by extrusion processing, and extrusion modification of PDGs could be a great way to facilitate the construction of biopolymer films with superior characteristics. It could provide more possibilities to extend the applications of DGs to alleviate the problems of environmental pollution and resource waste. © 2024 Society of Chemical Industry.

The Composition, Structure, and Functionalities of Prolamins from Highland Barley.

The composition, structure, and functionalities of prolamins from highland barley were investigated. These parameters were compared with those of the commonly applied prolamins (zein). There are more charged and hydrophilic amino acids in highland barely prolamins than zein. The molecular weight of highland barely prolamins was between 30 and 63 kDa, which was larger than that of zein (20 and 24 kDa). The main secondary structure of highland barely prolamins was ß-turn helices, while α-helical structures were the main secondary structure in zein. The water holding capacity, thermal stability, emulsifying capacity, and stability of prolamins from highland barley were significantly higher than in zein, while the opposite results were observed for oil absorption capacity between the two. The diameter of fibers prepared using highland barely prolamins was almost six times that of zein, while highland barely prolamins formed ribbon structures instead of fibers. Therefore, the results provide guidance for applications of prolamins from highland barley.

Flexible processing technology of coix seed prolamins by combined heat-ultrasound: Effects on their enzymatic hydrolysis characteristics and the hypoglycemic activities of derived peptides.

The self-assembled structures of coix seeds affected the enzymatic efficiency and doesn't facilitate the release of more active peptides. The influence of heating combined with ultrasound pretreatment (HT + US) on the structure, enzymatic properties and hydrolysates (CHPs) of coix seed prolamin was investigated. Results showed that the structural of coix seed prolamins has changed after HT + US, including increased surface hydrophobicity, reduced α-helix and random coil content, and a decrease in particle size. So that, leads to changes in thermodynamic parameters such as an increase in the reaction rate constant and a decrease in activation energy, enthalpy and enthalpy. The fractions of <1000 Da, degree of hydrolysis and α-glucosidase inhibitory were increased in the HT + US group compared to single pretreatment by 0.68%-17.34%, 12.69%-34.43% and 30.00%-53.46%. The peptide content and α-glucosidase inhibitory activity of CHPs could be maintained at 72.21 % and 57.97 % of the initial raw materials after in vitro digestion. Thus, the findings indicate that HT + US provides a feasible and efficient approach to can effectively enhance the enzymatic hydrolysis efficiency and hypoglycaemic efficacy of CHPs.

In situ thermal modification of kafirin using infrared radiations and microwaves.

BACKGROUND: Kafirin is a prolamin protein located in the corneous endosperm of sorghum. The conventional thermal processing of kafirin reduces its solubility, which limits its utilization in the food industry. Therefore, the study was aimed to investigate the effect of in situ thermal modification of kafirin using two different electromagnetic thermal treatments, namely infrared (IR) and microwave (MW) radiation, on the physicochemical, structural, thermal, and antioxidant properties. RESULTS: The results demonstrated that both the thermal modifications improved yield, purity, and solubility of the kafirin with a decrease in hydrophobicity. However, IR-treated samples showed higher solubility (910.67 g kg-1 ) and lower hydrophobicity (387.67). The IR modifications also improved the ratio of α helix/ß sheets to a great extent. The alterations in the disulfide content were concomitant with the improvement in the thermal stability of kafirin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed variations in the band intensities of ß- and γ-kafirin, indicating alterations in the kafirin subunits. Morphological examination of kafirin revealed surface withering and agglomeration. Notably, IR treatment improved the antioxidant activity more efficiently (from 32.11% to 74.05%). CONCLUSION: Although both the IR and MW treatments modified kafirin, the effect seemed to be more pronounced in IR modification. The IR-modified kafirin had better solubility and lesser hydrophobicity than MW-modified kafirin. The physicochemical and structural changes induced by IR treatment improved the biological activity of kafirin, in terms of antioxidant activity. Therefore, it was concluded that the in situ IR modification of kafirin can alter its characteristic properties, improving its potential as a food ingredient. © 2021 Society of Chemical Industry.

Characterization of gliadin, secalin and hordein fractions using analytical techniques.

Prolamins, alcohol soluble storage proteins of the Triticeae tribe of Gramineae family, are known as gliadin, secalin and hordein in wheat, rye and barley respectively. Prolamins were extracted from fifteen cultivars using DuPont protocol to study their physiochemical, morphological and structural characteristics. SDS-PAGE of prolamins showed well resolved low molecular weight proteins with significant amount of albumin and globulin as cross-contaminant. The ß-sheet (32.72-37.41%) and ß-turn (30.36-37.91%) were found higher in gliadins, while α-helix (20.32-28.95%) and random coil (9.05-10.28%) in hordeins. The high colloidal stability as depicted by zeta-potential was observed in gliadins (23.5-27.0 mV) followed secalins (11.2-16.6 mV) and hordeins (4.1-7.8 mV). Surface morphology by SEM illustrated the globular particle arrangement in gliadins, sheet like arrangement in secalins and stacked flaky particle arrangement in hordeins fraction. TEM studies showed that secalin and hordein fractions were globular in shape while gliadins in addition to globular structure also possessed rod-shaped particle arrangement. XRD pattern of prolamin fractions showed the ordered crystalline domain at 2θ values of 44.1°, 37.8° and 10.4°. The extracted prolamins fractions showed amorphous as well as crystalline structures as revealed by XRD and TEM analysis. Space saving hexagonal molecular symmetry was also observed in TEM molecular arrangement of prolamins which has profound application in development of plant-based polymers and fibres.

Fabrication and Characterisation of a Photo-Responsive, Injectable Nanosystem for Sustained Delivery of Macromolecules.

The demand for biodegradable sustained release carriers with minimally invasive and less frequent administration properties for therapeutic proteins and peptides has increased over the years. The purpose of achieving sustained minimally invasive and site-specific delivery of macromolecules led to the investigation of a photo-responsive delivery system. This research explored a biodegradable prolamin, zein, modified with an azo dye (DHAB) to synthesize photo-responsive azoprolamin (AZP) nanospheres loaded with Immunoglobulin G (IgG). AZP nanospheres were incorporated in a hyaluronic acid (HA) hydrogel to develop a novel injectable photo-responsive nanosystem (HA-NSP) as a potential approach for the treatment of chorio-retinal diseases such as age-related macular degeneration (AMD) and diabetic retinopathy. AZP nanospheres were prepared via coacervation technique, dispersed in HA hydrogel and characterised via infrared spectroscopy (FTIR), X-ray diffraction (XRD) and thermogravimetric analysis (TGA). Size and morphology were studied via scanning electron microscopy (SEM) and dynamic light scattering (DLS), UV spectroscopy for photo-responsiveness. Rheological properties and injectability were investigated, as well as cytotoxicity effect on HRPE cell lines. Particle size obtained was <200 nm and photo-responsiveness to UV = 365 nm by decreasing particle diameter to 94 nm was confirmed by DLS. Encapsulation efficiency of the optimised nanospheres was 85% and IgG was released over 32 days up to 60%. Injectability of HA-NSP was confirmed with maximum force 10 N required and shear-thinning behaviour observed in rheology studies. In vitro cell cytotoxicity effect of both NSPs and HA-NSP showed non-cytotoxicity with relative cell viability of ≥80%. A biocompatible, biodegradable injectable photo-responsive nanosystem for sustained release of macromolecular IgG was successfully developed.

Assessment of arsenic distribution, bioaccessibility and speciation in rice utilizing continuous extraction and in vitro digestion.

Rice, a staple food for half the world's population, easily accumulates arsenic (As). Research on As distribution in rice protein and starch and its relationship with rice As bioaccessibility remains limited. This study investigated As distribution, chemical composition, As bioaccessibility and speciation in rice by continuous extraction and in vitro digestion. Of the total As, 87.5-94.5% was in rice protein and 5.0-9.8% in rice starch. The As amount in different protein fractions decreased as follows: glutelin > globulin > albumin > prolamin. As(V), As(III) and DMA in rice were more bioaccessible in the small intestinal phase than the gastric phase, and almost all As(V) dissolved in the small intestinal phase. Bioaccessible As in gastrointestinal digestive solution and As mass in protein fractions (albumin, globulin, and glutelin) were significantly positively correlated (p < 0.05). These results illuminate the bioaccessibility of As to humans consuming As-contaminated rice and avoid overassessment.

Fabrication of Composite Structures of Lysozyme Fibril-Zein using Antisolvent Precipitation: Effects of Blending and pH Adjustment Sequences.

Antisolvent precipitation is a widely used method to fabricate prolamin-based composites. In the present study, composite structures of lysozyme amyloid fibrils with zein proteins were fabricated using the antisolvent precipitation method by applying different blending and pH adjustment sequences. Globular prolamins were bound to the amyloid fibrils to combine their respective advantages. The dynamic light scattering showed that the composites with a characteristic stabilized behavior (43.60 ± 1.75 mV ∼ 35.20 ± 0.65 mV) were formed at pH 4.0-5.0, in which noncovalent interactions between fibril and particles occurred. Two different structures: fruit tree-like structure and beaded-like structure, were presented in AFM and TEM images due to the different pH adjustment sequences, while blending sequences had negligible effect on the morphology of the composites. A fruit tree-like entity was detected for lysozyme fibril-zein composites, where its "branches" bear zein globular particles. A beaded-like structure was observed for lysozyme fibril-zein composites, where lysozyme fibril was the thread and zein aggregates were the beads. The potential mechanism of this phenomenon can be explained as the fruit tree-like structure being primarily formed through electrostatic interactions while the beaded-like structure is mainly caused by hydrophobic interactions. The composites of fruit tree-like structures hold a more promising stability than those with beaded-like structures. The results of this research would give constructive information for the fabrication of amyloid fibril-prolamin protein composites, which may exhibit the combined advantages of each components and have potential applications in encapsulation and protection of bioactive substances and stabilizing emulsions.

Functional properties and structural changes of rice proteins with anthocyanins complexation.

This study investigated the functional properties and structural changes associated with the complexation of rice protein (RP) with anthocyanins (ACN). Furthermore, fractions (i.e., albumin, globulin, prolamin and glutelin) isolated from RP complexed with anthocyanins were examined. The interactions with ACN altered the structure of RP, leading to an increase in the ß-sheet and spectral shift of the amide Ⅱ band. Additionally, fluorescence spectroscopy suggested that the hydrophobic and hydrogen bonds were the dominant forces in the formation of RP-ACN complexes. It was interesting to find that the RP-ACN particles exhibited the best functional properties at pH 3, likely due to the specific conformational changes upon interaction. In addition, the combination of RP and ACN increased the antioxidant ability of RP. Overall, this research suggested that RP-ACN particles at pH 3 can be designed to form and stabilize mesostructures such as foams and emulsion, which can lead to health benefits.

Protein-based nanoparticles for drug delivery purposes.

Proteins represent a group of biopolymers with interesting properties to be employed as raw materials in the preparation of nanoparticles for drug delivery purposes. Due to the inherent properties of proteins (i.e., biodegradability, amphiphilic properties, etc.) the resulting nanoparticles can be considered as versatility platforms for a variety of applications. Moreover, some proteins possess a GRAS (Generally Recognized as Safe) status or are considered as excipients by different Regulatory Agencies. As result of this, the resulting nanoparticles and potential translation to clinic would be facilitated, compared to other materials (i.e., polymers). This review is focused on the main proteins employed in the preparation of nanoparticles as well as the procedures permitting their transformation into nanoparticles able of accommodating a high variety of bioactive compounds and drugs. Moreover, the review also provides examples of application of nanoparticles prepared from albumins, globulins, prolamins or macromolecules derived from proteins.

Development of Prolamin-Based Composite Nanoparticles for Controlled Release of Sulforaphane.

Sulforaphane (SFN) has been documented to possess anticancer properties. However, its application is limited by instability and poor bioavailability, which could be enhanced by colloidal delivery systems. In this study, prolamins from two cereal grains, i.e., proso millet (MP) and corn (CP), were extracted and used to fabricate nanoparticles for SFN via an anti-solvent process. A secondary layer with a complex of sodium caseinate (NaCas)/propylene glycol alginate (PGA) at an equal mass was deposited to further improve the stability of nanoparticles. Results indicated that composite nanoparticles with NaCas/PGA at 0.3% (w/v) exhibited a spherical shape with high encapsulation efficiency (>80%), small size (150 nm), and highly negative ζ potential (-39 mV). SFN in MP compared to that in CP showed a similar but lower releasing rate under simulated in vitro digestion. Therefore, prolamins from both sources are promising plant source delivery materials to improve stability and achieve controlled release of bioactives.

Effect of sodium tripolyphosphate incorporation on physical, structural, morphological and stability characteristics of zein and gliadin nanoparticles.

With the extensive applications of chemical means in food systems, phosphorylation has become a promising approach to modify the functionalities of proteins. In this study, effects of sodium tripolyphosphate (TPP) on physicochemical properties of gliadin and zein nanoparticles were comprehensively explored by fluorescence spectroscopy analysis, circular dichroism spectrum and Fourier transform infrared analysis. The results suggested that an increase in TPP concentration could affect the particle size and microstructures of gliadin nanoparticles through enhanced repulsion force among nanoparticles. The phosphorylation of gliadin and zein was ascribed to the interactions of phosphate groups, i.e., tryptophan and tyrosine residues, respectively. FTIR analysis revealed that the intermolecular interactions were influenced with the secondary structure altered. More specifically, both PO and PO bonds were incorporated into gliadin and zein molecules when TPP concentration was above 0.3 mg/mL, which could then improve physical stability of prolamin nanoparticles. Moreover, CNP and COP bonds were deduced to be formed only with the existence of gliadin, whose presence nevertheless enhanced the emulsifying property of nanoparticles. These profound findings could therefore expand the application of prolamin in delivery systems.

Speciation of Selenium in Brown Rice Fertilized with Selenite and Effects of Selenium Fertilization on Rice Proteins.

Foliar Selenium (Se) fertilizer has been widely used to accumulate Se in rice to a level that meets the adequate intake level. The Se content in brown rice (Oryza sativa L.) was increased in a dose-dependent manner by the foliar application of sodium selenite as a fertilizer at concentrations of 25, 50, 75, and 100 g Se/ha. Selenite was mainly transformed to organic Se, that is, selenomethionine in rice. Beyond the metabolic capacity of Se in rice, inorganic Se also appeared. In addition, four extractable protein fractions in brown rice were analyzed for Se concentration. The Se concentrations in the glutelin and albumin fractions saturated with increasing Se concentration in the fertilizer compared with those in the globulin and prolamin fractions. The structural analyses by fluorescence spectroscopy, Fourier transform infrared spectrometry, and differential scanning calorimetry suggest that the secondary structure and thermostability of glutelin were altered by the Se treatments. These alterations could be due to the replacements of cysteine and methionine to selenocysteine and selenomethionine, respectively. These findings indicate that foliar fertilization of Se was effective in not only transforming inorganic Se to low-molecular-weight selenometabolites such as selenoamino acids, but also incorporating Se into general rice proteins, such as albumin, globulin glutelin, and prolamin, as selenocysteine and selenomethionine in place of cysteine and methionine, respectively.

Analysis of novel high-molecular-weight prolamins from Leymus multicaulis (Kar. et Kir.) Tzvelev and L. chinensis (Trin. ex Bunge) Tzvelev.

Nine novel high-molecular-weight prolamins (HMW-prolamins) were isolated from Leymus multicaulis and L. chinensis. Based on the structure of the repetitive domains, all nine genes were classified as D-hordeins but not high-molecular-weight glutenin subunits (HMW-GSs) that have been previously isolated in Leymus spp. Four genes, Lmul 1.2, 2.4, 2.7, and Lchi 2.5 were verified by bacterial expression, whereas the other five sequences (1.3 types) were classified as pseudogenes. The four Leymus D-hordein proteins had longer N-termini than those of Hordeum spp. [116/118 vs. 110 amino acid (AA) residues], whereas three (Lmul 1.2, 2.4, and 2.7) contained shorter N-termini than those of the Ps. juncea (116 vs. 118 AA residues). Furthermore, Lmul 1.2 was identified as the smallest D-hordein, and Lmul 1.2 and 2.7 had an additional cysteines. Phylogenetic analysis supported that the nine D-hordeins of Leymus formed two independent clades, with all the 1.3 types clustered with Ps. juncea Ns 1.3, whereas the others were clustered together with the D-hordeins from Hordeum and Ps. juncea and the HMW-GSs from Leymus. Within the clade of four D-hordein genes and HMW-GSs, the HMW-GSs of Leymus formed a separated branch that served as an intermediate between the D-hordeins of Ps. juncea and Leymus. These novel D-hordeins may be potentially utilized in the improvement of food processing properties particularly those relating to extra cysteine residues. The findings of the present study also provide basic information for understanding the HMW-prolamins among Triticeae species, as well as expand the sources of D-hordeins from Hordeum to Leymus.

Comparison of formation of visco-elastic masses and their properties between zeins and kafirins.

Zeins of differing sub-class composition much more readily formed visco-elastic masses in water or acetic acid solutions than equivalent kafirin preparations. Visco-elastic masses could be formed from both zein and kafirin preparations by coacervation from glacial acetic acid. Dissolving the prolamins in glacial acetic acid apparently enabled protonation and complete solvation. Stress-relaxation analysis of coacervated zein and kafirin visco-elastic masses showed they were initially soft. With storage, they became much firmer. Zein masses exhibited predominantly viscous flow properties, whereas kafirin masses were more elastic. The γ-sub-class is apparently necessary for the retention of visco-elastic mass softness with kafirin and zein, and for elastic recovery of kafirin. Generally, regardless of water or acetic acid treatment, all the zein preparations had similar FTIR spectra, with greater α-helical conformation, than the kafirin preparations which were also similar to each other. Kafirin visco-elastic masses have a much higher elastic character than zein masses.

Chromatographic and mass spectrometry analysis of wheat flour prolamins, the causative compounds of celiac disease.

Immunogenic gluten peptides trigger Celiac Disease (CD), an adaptive immune response in genetically predisposed individuals. Given the structural similarity between all gluten proteins their individual CD influence is not clear. Hence, the extraction, separation and characterization of wheat gluten proteins have become relevant to measure their individual potential immunoreactivity. Wheat proteins were extracted from commercial wheat flour and further isolated by preparative HPLC. The resulting richest gliadin sub-fractions were characterized by nano-LC-MS/MS following a shotgun proteomic approach in order to identify the prolamins in the original commercial wheat flour. It was found that the gliadin extract was additionally composed of glutenins and avenin-like proteins. Accurate prolamin identification has emerged as a need to delve deep into the influence of each fraction on the onset of celiac disease. After protein characterization, the immunoreactivity towards the main epitope related to CD was verified by ELISA and western blotting for several different gluten fractions.

Food for thought: Selecting the right enzyme for the digestion of gluten.

Gluten describes a complex mixture of proteins found in wheat, rye, barley and oats that pose a health risk to people affected by conditions such as coeliac disease and non-coeliac gluten sensitivity. Complete digestion of gluten proteins is of critical importance during quantitative analysis. To this end, chymotrypsin was investigated for its ability to efficiently and reproducibly digest specific classes of gluten in barley. Using proteomics a chymotryptic peptide marker panel was elucidated and subjected to relative quantification using LC-MRM-MS. Thorough investigation of peptide markers revealed robust and reproducible quantification with CVs <15% was possible, however a greater proportion of non-specific cleavage variants were observed relative to trypsin. The selected peptide markers were assessed to ensure their efficient liberation from their parent proteins. While trypsin remains the preferred enzyme for quantification of the avenin-like A proteins, the B-, D- and γ-hordeins, chymotrypsin was the enzyme of choice for the C-hordeins.

Characterization of globulin storage proteins of a low prolamin cereal species in relation to celiac disease.

Brachypodium distachyon, a small annual grass with seed storage globulins as primary protein reserves was used in our study to analyse the toxic nature of non-prolamin seed storage proteins related to celiac disease. The main storage proteins of B. distachyon are the 7S globulin type proteins and the 11S, 12S seed storage globulins similar to oat and rice. Immunoblot analyses using serum samples from celiac disease patients were carried out followed by the identification of immune-responsive proteins using mass spectrometry. Serum samples from celiac patients on a gluten-free diet, from patients with Crohn's disease and healthy subjects, were used as controls. The identified proteins with intense serum-IgA reactivity belong to the 7S and 11-12S seed globulin family. Structure prediction and epitope predictions analyses confirmed the presence of celiac disease-related linear B cell epitope homologs and the presence of peptide regions with strong HLA-DQ8 and DQ2 binding capabilities. These results highlight that both MHC-II presentation and B cell response may be developed not only to prolamins but also to seed storage globulins. This is the first study of the non-prolamin type seed storage proteins of Brachypodium from the aspect of the celiac disease.