Determining the Impact of Genotype × Environment on Oat Protein Isolate Composition Using HPLC and LC-MS Techniques.

The effect of genotype and environment on oat protein composition was analyzed through size exclusion-high-performance liquid chromatography (SE-HPLC) and liquid chromatography-mass spectrometry (LC-MS) to characterize oat protein isolate (OPI) extracted from three genotypes grown at three locations in the Canadian Prairies. SE-HPLC identified four fractions in OPI, including polymeric globulins, avenins, glutelins, and albumins, and smaller proteins. The protein composition was dependent on the environment, rather than the genotype. The proteins identified through LC-MS were grouped into eight categories, including globulins, prolamins/avenins, glutelins, enzymes/albumins, enzyme inhibitors, heat shock proteins, grain softness proteins, and allergenic proteins. Three main globulin protein types were also identified, including the P14812|SSG2-12S seed storage globulin, the Q6UJY8_TRITU-globulin, and the M7ZQM3_TRIUA-Globulin-1 S. Principal component analysis indicated that samples from Manitoba showed a positive association with the M7ZQM3_TRIUA-Globulin-1 S allele and Q6UJY8_TRITU-globulin, while samples from Alberta and Saskatchewan had a negative association with them. The results show that the influence of G × E on oat protein fractions and their relative composition is crucial to understanding genotypes' behavior in response to different environments.

Characterization of barley (Horduem vulgare) lys3 mutants identifies genes under the regulation of the prolamin-box binding transcription factor and elucidates its role in endosperm promoter methylation during grain development.

Barley ranks fourth in global cereal production and is primarily grown for animal feed and malt. Hordeins, the principal barley seed storage proteins, are homologous to wheat gluten and when ingested elicit an immune response in people with Coeliac disease. Risø 1508 is a chemically induced barley mutant with low hordein levels imparted by the lys3.a locus that is reported to be caused by an SNP in the barley prolamin-box binding factor gene (BPBF). Reports suggest the lys3.a locus prevents CG DNA demethylation at the Hor2 (B-hordein) promoter during grain development subsequently causing hypermethylation and inhibiting gene expression. In lys3.a mutants, endosperm-specific ß-amylase (Bmy1) and Hor2 are similarly downregulated during grain development and thus we hypothesize that the inability to demethylate the Bmy1 promoter CG islands is also causing Bmy1 downregulation. We use whole-genome bisulfite sequencing and mRNA-seq on developing endosperms from two lys3.a mutants and a lys3.b mutant to determine all downstream genes affected by lys3 mutations. RNAseq analysis identified 306 differentially expressed genes (DEGs) shared between all mutants and their parents and 185 DEGs shared between both lys3.a mutants and their parents. Global DNA methylation levels and promoter CG DNA methylation levels were not significantly different between the mutants and their parents and thus refute the hypothesis that the lys3.a mutant's phenotype is caused by dysregulation of demethylation during grain development. The majority of DEGs were downregulated (e.g., B- and C-hordeins and Bmy1), but some DEGs were upregulated (e.g., ß-glucosidase, D-hordein) suggesting compensatory effects and potentially explaining the low ß-glucan phenotype observed in lys3.a germplasm. These findings have implications on human health and provide novel insight to barley breeders regarding the use of BPBF transcription factor mutants to create gluten-free barley varieties.

Biopolymer-Assembled Porous Hydrogel Microfibers from Microfluidic Spinning for Wound Healing.

Hydrogels are considered as a promising medical patch for wound healing. Researches in this aspect are focused on improving their compositions and permeability to enhance the effectiveness of wound healing. Here, novel prolamins-assembled porous hydrogel microfibers with the desired merits for treating diabetes wounds are presented. Such microfibers are continuously generated by one-step microfluidic spinning technology with acetic acid solution of prolamins as the continuous phase and deionized water as the dispersed phase. By adjusting the prolamin concentration and flow rates of microfluidics, the porous structure and morphology as well as diameters of microfibers can be well tailored. Owing to their porosity, the resultant microfibers can be employed as flexible delivery systems for wound healing actives, such as bacitracin and vascular endothelial growth factor (VEGF). It is demonstrated that the resultant hydrogel microfibers are with good cell-affinity and effective drug release efficiency, and their woven patches display superior in vivo capability in treating diabetes wounds. Thus, it is believed that the proposed prolamins-assembled porous hydrogel microfibers will show important values in clinic wound treatments.

Applications of Prolamin-Based Edible Coatings in Food Preservation: A Review.

Foods are susceptible to deterioration and sour due to external environmental influences during production and storage. Coating can form a layer of physical barrier on the surface of foods to achieve the purpose of food preservation. Because of its good barrier properties and biocompatibility, prolamin-based film has been valued as a new green and environment-friendly material in the application of food preservation. Single prolamin-based film has weaknesses of poor toughness and stability, and it is necessary to select appropriate modification methods to improve the performance of film according to the application requirements. The practical application effect of film is not only affected by the raw materials and the properties of the film itself, but also affected by the selection of preparation methods and processing techniques of film-forming liquid. In this review, the properties and selection of prolamins, the forming mechanisms and processes of prolamin-based coatings, the coating techniques, and the modifications of prolamin-based coatings were systematically introduced from the perspective of food coating applications. Moreover, the defects and deficiencies in the research and development of prolamin-based coatings were also reviewed in order to provide a reference for the follow-up research on the application of prolamin-based coatings in food preservation.

Proteins from Blackberry Seeds: Extraction, Osborne Isolate, Characteristics, Functional Properties, and Bioactivities.

Blackberry fruit contains high levels of nutrients and phenolic compounds. Blackberry pomace accounts for 20~30% of its whole fruit during processing and is generally treated as fertilizer. Blackberry pomace has many seeds that contain carbohydrates, polyphenols, flavonoids, pectin, protein, and other bioactive nutrients. However, its functional properties and seed protein compositions have not been reported. We used a single-factor experiment, response surface, and Osborne isolate method to extract protein isolate, albumin, globulin, glutelin, and prolamin from blackberry seeds for the first time and evaluated their characteristics and functional properties. Glutelin and protein isolate showed good water-holding capacity, emulsification, and foaming capacity, while albumin and globulin showed good oil-holding capacity and thermal stability. They were found to have good antioxidant activities that might be good DPPH free radical scavengers, especially prolamin, which has the lowest IC50 value (15.76 µg/mL). Moreover, globulin had the lowest IC50 value of 5.03 µg/mL against Hela cells, 31.82 µg/mL against HepG2 cells, and 77.81 µg/mL against MCF-7 cells and a high selectivity index (SI), which suggested globulin had better anti-cervical, antihepatoma, and anti-breast activity but relatively low cytotoxicity. These seed proteins may have great prospects for the development and application of food and drugs in the future.

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.

Wheat DOF transcription factors TaSAD and WPBF regulate glutenin gene expression in cooperation with SPA.

Grain storage proteins (GSPs) quantity and composition determine the end-use value of wheat flour. GSPs consists of low-molecular-weight glutenins (LMW-GS), high-molecular-weight glutenins (HMW-GS) and gliadins. GSP gene expression is controlled by a complex network of DNA-protein and protein-protein interactions, which coordinate the tissue-specific protein expression during grain development. The regulatory network has been most extensively studied in barley, particularly the two transcription factors (TFs) of the DNA binding with One Finger (DOF) family, barley Prolamin-box Binding Factor (BPBF) and Scutellum and Aleurone-expressed DOF (SAD). They activate hordein synthesis by binding to the Prolamin box, a motif in the hordein promoter. The BPBF ortholog previously identified in wheat, WPBF, has a transcriptional activity in expression of some GSP genes. Here, the wheat ortholog of SAD, named TaSAD, was identified. The binding of TaSAD to GSP gene promoter sequences in vitro and its transcriptional activity in vivo were investigated. In electrophoretic mobility shift assays, recombinant TaSAD and WPBF proteins bound to cis-motifs like those located on HMW-GS and LMW-GS gene promoters known to bind DOF TFs. We showed by transient expression assays in wheat endosperms that TaSAD and WPBF activate GSP gene expression. Moreover, co-bombardment of Storage Protein Activator (SPA) with WPBF or TaSAD had an additive effect on the expression of GSP genes, possibly through conserved cooperative protein-protein interactions.

Development of a barley reference material for gluten analysis.

Celiac disease (CD) can be triggered in susceptible individuals by the consumption of gluten, a complex storage protein mixture present in wheat, rye and barley. There is no specific reference material (RM) available for barley and this leads to inaccurate quantitation of barley gluten in supposedly gluten-free foods. Therefore, the aim was to select representative barley cultivars to establish a new barley RM. The relative protein composition of the 35 barley cultivars averaged 25% albumins and globulins, 11% d-hordeins, 19% C-hordeins, and 45% B/γ-hordeins. The mean gluten and protein content was 7.2 g/100 g and 11.2 g/100 g, respectively. The prolamin/glutelin ratio (1:1) commonly used in ELISAs to calculate the gluten content was found to be inappropriate for barley (1.6 ± 0.6). Eight cultivars suitable as potential RMs were selected to ensure a typical barley protein composition and improve food safety for CD patients.

Difference analysis of the structure and functional properties among coix seed prolamin fractions (α-, ß-, and γ-coixin).

The differences in proteins in structural characteristics are reported to affect their physicochemical and functional properties. In this study, three types of prolamins (γ-, α-, and ß-coixin) derived from coix seed separately distributed among fractions 1-3 extracts. They were studied respecting molecular weight, amino acid composition, secondary structure, microstructure, surface hydrophobicity, solubility, water holding capacity, and oil holding capacity. Results showed that the molecular weights of those three fractions were between 10 and 40 kDa. The secondary structure of those fractions was almost the same, mainly based on ß-sheet and irregular structure. The microstructure of α- and γ-coixin presented an irregular shape, whereas ß-coixin presented a regular spherical shape. Those three fractions exhibited species of abundant essential amino acids with the same amino acid composition but different contents. The ß-coixin fraction had the highest content of hydrophobic amino acids (238.39 mg/g) followed by the α-coixin fraction (235.05 mg/g), whereas the γ-coixin fraction had the lowest content (33.27 mg/g). The γ-coixin fraction has the maximum surface hydrophobicity, whereas the ß-coixin fraction has the highest solubility. In addition, the good amphiphilicity of ß-coixin fraction made it possible to be used as a surfactant. The excellent functional properties of the ß-coixin fraction presented in this research would widen the applications of coix seed prolamins. PRACTICAL APPLICATION: The molecular weights of those three fractions were between 10 and 40 kDa. The secondary structure was almost the same, mainly based on ß-sheet and irregular structure. Those three fractions exhibited species of abundant essential amino acids with the same amino acid composition but different contents. The WHC and OHC of ß-coixin were the best, indicating its potential as a surfactant and forming stable lotion.

Influence on Accumulation Levels and Subcellular Localization of Prolamins by Fusion with the Functional Peptide in Transgenic Rice Seeds.

To exploit the rice seed-based oral vaccine against Sjögren's syndrome, altered peptide ligand of N-terminal 1 (N1-APL7) from its M3 muscarinic acetylcholine receptor (M3R) autoantigen was expressed as fusion protein with the representative four types of rice prolamins (16 kDa, 14 kDa, 13 kDa, and 10 kDa prolamins) under the control of the individual native prolamin promoter. The 10kD:N1-APL7 and 14kD:N1-APL7 accumulated at high levels (287 and 58 µg/grain), respectively, whereas production levels of the remaining ones were remarkably low. Co-expression of these fusion proteins did not enhance the accumulation level of N1-APL7 in an additive manner. Downregulation of endogenous seed storage proteins by RNAi-mediated suppression also did not lead to substantial elevation of the co-expressed prolamin:N1-APL7 products. When transgenic rice seeds were subjected to in vitro proteolysis with pepsin, the 10kD:N1-APL7 was digested more quickly than the endogenous 10 kDa prolamin and the 14kD:N1-APL7 deposited in PB-Is. This difference could be explained by the finding that the 10kD:N1-APL7 was unexpectedly localized in the PB-IIs containing glutelins. These results indicated that not only accumulation level but also subcellular localization of inherent prolamins were highly influenced by the liked N1-APL7 peptide.

Comparison of the generation of α-glucosidase inhibitory peptides derived from prolamins of raw and cooked foxtail millet: In vitro activity, de novo sequencing, and in silico docking.

Foxtail millet prolamin has been demonstrated to have anti-diabetic effects. In this study, we compared the generation of anti-α-glucosidase peptides derived from prolamins of raw and cooked foxtail millet (PRFM and PCFM). PRFM and PCFM hydrolysates (PRFMH and PCFMH) both exhibited α-glucosidase inhibitory activity. After ultrafiltration according to molecular weight (Mw), the fraction with Mw < 3 kDa in PCFMH (PCFMH<3) showed higher α-glucosidase inhibitory activity than that in PRFMH (PRFMH<3). The composition of α-glucosidase inhibitory peptides identified by de novo sequencing in PCFMH<3 and PRFMH<3 was compared by virtual screening, combining biological activity, net charge, grand average of hydropathicity (GRAVY), and key hydrophobic amino acids (Met, Pro, Phe, and Leu). We found that the proportion of peptides with excellent α-glucosidase binding force in PCFMH<3 was higher than in PRFMH<3. Overall, cooking may positively affect the generation of peptides that perform well in inhibiting α-glucosidase derived from foxtail millet prolamin.

Fabrication of foxtail millet prolamin/caseinate/chitosan hydrochloride composite nanoparticles using antisolvent and pH-driven methods for curcumin delivery.

Co-assembled foxtail millet (FP)-sodium casein (NaCas) nanocomplex and NaCas coated FP nanoparticles (NPs) were produced by using pH-cycle and anti-solvent methods, respectively. Subsequently, the effects of chitosan hydrochloride (CHC) coating on the particle size, surface charge and physicochemical stability of the two different FP/NaCas nanoparticles (NPs) were evaluated. With the addition of CHC, the particle size of NaCas coated FP NPs and co-assembled FP-NaCas nanocomplex significantly increased from 128.3 nm and 69.5 nm to 183.5 nm and 113.8 nm, respectively. The stability of the two kinds of CHC coated FP-based NPs to different pH values and varying ionic strengths was different due to their different NP structures. Using different fabrication formulations, co-assembled FP-NaCas NPs entrapped curcumin in relatively hydrophilic microenvironment and showed higher curcumin retention rate in comparison with NaCas coated FP NPs in terms of long-term storage stability. The results revealed that the produced CHC coated FP/NaCas nanocomplexes could be very beneficial in entrapping and delivering bioactive substances.

3D-Printed Prolamin Scaffolds for Cell-Based Meat Culture.

Cultivating meat from muscle stem cells in vitro requires 3D edible scaffolds as the supporting matrix. Electrohydrodynamic (EHD) printing is an emerging 3D-printing technology for fabricating ultrafine fibrous scaffolds with high precision microstructures for biomedical applications. However, edible EHD-printed scaffolds remain scarce in cultured meat (CM) production partly due to special requirements with regard to the printability of ink. Here, hordein or secalin is mixed, which are cereal prolamins extracted from barley or rye, with zein to produce pure prolamin-based inks, which exhibit favorable printability similar to common polycaprolactone ink. Zein/hordein and zein/secalin scaffolds with highly ordered tessellated structures are successfully fabricated after optimizing printing conditions. The prolamin scaffolds demonstrated good water stability and in vitro degradability due to the porous fiber surface, which is spontaneously generated by culturing muscle cells for 1 week. Moreover, mouse skeletal myoblasts (C2C12) and porcine skeletal muscle satellite cells (PSCs) can adhere and proliferate on the fibrous matrix, and a CM slice is produced by culturing PSCs on prolamin scaffolds with high tissue similarity. The upregulation of myogenic proteins shows that the differentiation process is triggered in the 3D culture, demonstrating the great potential of prolamin scaffolds in CM production.

Interactions between kafirin and pickle-like odorants in soy sauce flavor Baijiu: Aroma profile change and binding mechanism.

The pickle-like odor, which was caused by the excess volatile sulfur compounds (VSCs), is an undesirable odor in soy sauce flavor Baijiu (SSB). The aim of this study was to explore the suppressing effect of kafirin, the internal prolamin of the raw material of Baijiu, on the pickle-like odor of SSB. The instrumental analysis (comprehensive two-dimensional gas chromatography, ultraviolet and visible spectrophotometry), human perceptions (aroma profile, odor thresholds) and in silico analysis methods (molecular docking) were combined to detect the changes of the pickle-like odor. After the addition of kafirin, the aroma profile of the pickle-like SSB changed and approached to that of the normal SSB. The volatility and the odor thresholds of VSCs decreased 22.05%-64.28% and increased 87.29%-232.57%, respectively. In conclusion, kafirin exhibited a significant suppressing effect on the pickle-like off-odor of SSB, and this effect could be used to reduce the off-odor of SSB and SSB-derived alcoholic beverages.

Nitrogen-dependent binding of the transcription factor PBF1 contributes to the balance of protein and carbohydrate storage in maize endosperm.

Fluctuations in nitrogen (N) availability influence protein and starch levels in maize (Zea mays) seeds, yet the underlying mechanism is not well understood. Here, we report that N limitation impacted the expression of many key genes in N and carbon (C) metabolism in the developing endosperm of maize. Notably, the promoter regions of those genes were enriched for P-box sequences, the binding motif of the transcription factor prolamin-box binding factor 1 (PBF1). Loss of PBF1 altered accumulation of starch and proteins in endosperm. Under different N conditions, PBF1 protein levels remained stable but PBF1 bound different sets of target genes, especially genes related to the biosynthesis and accumulation of N and C storage products. Upon N-starvation, the absence of PBF1 from the promoters of some zein genes coincided with their reduced expression, suggesting that PBF1 promotes zein accumulation in the endosperm. In addition, PBF1 repressed the expression of sugary1 (Su1) and starch branching enzyme 2b (Sbe2b) under normal N supply, suggesting that, under N-deficiency, PBF1 redirects the flow of C skeletons for zein toward the formation of C compounds. Overall, our study demonstrates that PBF1 modulates C and N metabolism during endosperm development in an N-dependent manner.

Characterization of rye flours and their potential as reference material for gluten analysis.

The safety of gluten-free products relies on accurate gluten analysis, most commonly using ELISA. These test kits are calibrated to gliadins or wheat gluten, because there is no reference material (RM) for rye. Our aim was to select representative samples out of 32 rye cultivars for use as RM. All cultivars were characterized by RP-HPLC, gel permeation HPLC and R5 and G12 ELISA. The protein and gluten content ranged from 5.5 to 11.2 g/100 g and 3.0 to 7.8 g/100 g, respectively. The average protein distribution was 40% albumins/globulins, 23% γ-75k-secalins, 17% γ-40k-secalins, 14% ω-secalins and 6% high-molecular-weight-secalins. The mean prolamin/glutelin ratio was 4.4 for rye and this translates to an estimated conversion factor from rye prolamins to gluten of 1.2, instead of the usual factor of 2. Seven cultivars were selected for RM production based on cluster analysis, geographical origin and availability to comprehensively cover the diversity of rye.

Ascorbic acid-loaded gliadin nanoparticles as a novel nutraceutical formulation.

Ascorbic acid (AA) is one of the foremost antioxidants. Unfortunately, its sensitivity to different external stimuli such as light, heat and oxygen are concrete limitations for its use. Various approaches have been investigated in order to circumvent this problem and enhance the stability of the active compound, besides promoting its use for different applications. In this investigation, AA was encapsulated in a vegetal protein-based matrix made up of gliadin, the prolamin obtained from wheat kernels, with the aim of proposing a novel nutraceutical formulation. The nanosystems were characterized by an average diameter of < 200 nm and a negative surface charge of âˆ¼ -40 mV. The samples were not destabilized after incubation at different temperatures (up to 70 °C) or after the pasteurization procedure. Suitable stability was also observed in NaCl 100 mM, as well as after cryodesiccation when 10 % w/v of mannose was used. The gliadin nanoparticles showed the ability to retain high amounts of AA, promoting its prolonged release in PBS and under simulated gastrointestinal conditions. The nanosystems enhanced the antioxidant features of the compound as compared to its free form and preserved its chemical stability following UV exposition. The results demonstrate the potential application of the investigated nanoparticles as a novel nutraceutical formulation or as food fortificants.

Hierarchically Porous Films Architectured by Self-Assembly of Prolamins at the Air-Liquid Interface.

Artificial recapitulation of hierarchically porous films gained great interest due to their versatile functionalities and applications. However, the development of novel eco-friendly and nontoxic biopolymer-based porous films is still limited by the time-consuming fabrication processes and toxic organic reagents involved. Here, we reported a novel approach to rapidly (within 5 s) fabricate biopolymer-based hierarchically porous films via inducing the laterally occurring interfacial self-assembly of prolamins at the air-liquid interface during an antisolvent dripping procedure. The as-prepared films exhibited a hierarchically porous microstructure (with sizes of about 500 nm to 5 µm) with location-graded and Janus features. The formation mechanism involved the solvent gradient controlled self-assembly of prolamin into an anisotropic defect structure in longitudinal and lateral directions. Accordingly, the macroscopic morphologies together with the porosity and pore size could be precisely tuned by solvents and operating parameters in a convenient way. Furthermore, alcohol-soluble but water-insoluble bioactive compounds could be incorporated simultaneously via a one-step loading procedure, which endowed films with large loading efficiency and sustained release features suitable for controlled release applications. The effect of the curcumin-loaded porous film on skin wound healing, as one of the potential applications of this novel material, was then investigated in vivo in a full-thickness wound model, wherein satisfying wound healing effects were achieved through multitarget and multipathway mechanisms. This pioneering work offers a novel strategy for the rapid architecture of biopolymer-based hierarchically porous film with versatile application potentials.

Single/co-encapsulation capacity and physicochemical stability of zein and foxtail millet prolamin nanoparticles.

In the last few decades, zein has been extensively studied owing to its wide commercial availability and the ability to self-assemble into nanosphere structure to encapsulate biologically active substances for targeted delivery. This work emphasized on comparing the encapsulation efficiency of hydrophobic active biomolecules and the physicochemical stability of composite nanoparticles (NPs) made up of zein- and foxtail millet prolamin (FP) -caseinate. Puerarin, resveratrol, diosmetin, and curcumin with various LogP values were selected as model drugs to study the single/co-encapsulation capacity, storage stability, and in vitro release profiles. Both LogP values (polarity) and specific structure are the main factors affecting the encapsulation efficiency. FP-based NPs could entrap more resveratrol, which may be related to the lower hydrophobic amino acid content of FP in comparison with that of zein. Co-encapsulation, in vitro release and long-term storage stability experiments confirmed that the model drugs were encapsulated in different NP regions mediated by polarity. Moreover, co-encapsulation changed the environment of curcumin from relatively polar microenvironment to hydrophobic regions. These hydrophobic regions retained significantly more curcumin during long-term storage stability. Overall, our results suggest that the hydrophobic amino acid composition of prolamin affects the encapsulation capacity. Various bioactives were encapsulated in the prolamin-based NPs via polarity mediation, and co-encapsulation could effectively retain the active molecules during storage.