Interfacial characteristics and in vitro digestion of emulsion coated by single or mixed natural emulsifiers: lecithin and/or rice glutelin hydrolysates.

BACKGROUND: The interfacial characteristics and in vitro digestion of emulsion were related to emulsifier type. The mean droplet diameter, ζ-potential, microstructure, interfacial tension, Quartz crystal microbalance with dissipation (QCM-D) and in vitro gastrointestinal fate of emulsions stabilized by soybean lecithin, hydrolyzed rice glutelin (HRG) and their mixture were researched. RESULTS: The value of interfacial tension was much more dramatically declined for the sample containing 20 g kg-1 of HRG. For QCM-D, a rigid layer was formed for all the samples after rinsing. The layer thickness was 0.87 ± 0.20, 2.11 ± 0.31 and 2.63 ± 0.22 nm, and adsorbed mass was 87.17 ± 10.31, 210.56 ± 20.12 and 263.09 ± 23.23 ng cm-2 , for HRG, lecithin and HRG/lecithin, respectively, indicating both HRG and lecithin were adsorbed at the oil-water interface. Structural rearrangements at the interface occurred for HRG/lecithin. The kinetics and final amount of lipid digestion depended on emulsifier type: lecithin > HRG/lecithin > HRG. These differences in digestion rate were primarily due to differences in the aggregation state of the emulsifiers. CONCLUSION: The incorporation of lecithin into HRG emulsions had better interfacial properties comparing with HRG emulsion and facilitated lipid digestibility. These results provide important information for the rational design of plant-based functional food. © 2021 Society of Chemical Industry.

Analyses on the binding interaction between rice glutelin and conjugated linoleic acid by multi-spectroscopy and computational docking simulation.

It is an interesting topic to elucidate the interaction among plant proteins and bioactive lipid components. However, there is a shortage of understanding regarding the nature of the interaction between rice protein and conjugated linoleic acid (CLA). In this study, the intrinsic fluorescence intensity of rice glutelin (RG) was quenched upon increasing concentrations of CLA, indicating the occurrence of an interaction between them. Thermodynamic analysis showed that the RG-CLA binding process occurred spontaneously and hydrogen bonds were the primary driving force. Moreover, only one binding site was calculated between RG and CLA by the intrinsic fluorescence data. The surface hydrophobicity of RG was reduced with increasing CLA. Circular dichroism and synchronous fluorescence spectroscopy showed conformational and microenvironmental changes around the chromophores of RG. The α-helical content increased and ß-sheet content declined after the binding reaction. The computational docking program displayed the target site in which CLA and amino acid residues of RG might be linked together. This study provides valuable insights into the nature of the interactions between plant proteins and fatty acids.

The maize α-zein promoter can be utilized as a strong inducer of cellulase enzyme expression in maize kernels.

Expression of recombinant proteins in plants is a technology for producing vaccines, pharmaceuticals and industrial enzymes. For the past several years, we have produced recombinant proteins in maize kernels using only the embryo, primarily driving expression of foreign genes with the maize globulin-1 promoter. Although strong expression is obtained, these lines use only 10-12% of the seed tissue. If strong embryo expression could be combined with strong endosperm expression, much more recombinant protein could be recovered from a set amount of seed biomass. In this study, we tested three endosperm promoters for expression of a cellulase gene. Promoters tested were rice globulin and glutelin promoters and a maize 19 kDa α-zein promoter. The rice promoters were used in two tandem expression constructs as well. Although the rice promoters were active in producing stable amounts of cellulase, the α-zein promoter was by far the most effective: as much as 9% of total soluble protein was recovered from seed of several independent events and plants. One or two inserts were detected by Southern blot in several lines, indicating that copy number did not appear to be responsible for the differences in protein accumulation. Tissue print analysis indicated that expression was primarily in the endosperm.

Structural characterization and binding interaction of rice glutelin fibrils complexing with curcumin.

The rice glutelin (RG), the separated retentate (RGFs) and filtrate (FGFs) fractions from total glutelin fibrils (TGFs) at pH 3.5 were used as carrier for curcumin in this test. The solubility and antioxidant activities of curcumin were improved after binding with protein and fibrils. Compared to other complexes, the RGFs-curcumin complex exhibited a highest curcumin solubility (48.05%) and a superior sustained release property, probably owing to the stable hydrogen bond between the surface groups of fibrils and hydroxyl groups of polyphenols. In addition, thermodynamic parameters revealed that the RG/TGFs/RGFs-curcumin complexes were stabilized by hydrogen bonds and van der Waals forces, whereas FGFs interacted with curcumin through specific electrostatic interaction. Besides, after interacting with curcumin, the fibrils gathered into coarsened and agglutinated fibrillar aggregates, relating to the increment of a-helix and ß-sheet structure. These results suggested that RGFs could be a good alternative for curcumin delivery in food industry.

Inhibition mechanism of rice glutelin on extruded starch digestion: From the structural properties of starch and enzyme activity.

To increase the anti-digestion ability of extruded rice starch (ERS), the influence of rice glutelin (RG) on digestive and structural characteristics of ERS were investigated. The resistant starch content increased from 4.49 % to 18.08 % as the RG content increased, while the digestion rate and digestion velocity constant were reduced by the incorporation of RG. Morphological observations showed that ERS was adhered and encapsulated by RG, and the specific area of starch granules were decreased after the addition of RG. The results of XRD and FTIR suggested that the long-range and short-range orders of ERS were improved due to the complexation with RG. The thickness of crystalline of ERS was increased while its amorphous region thickness was reduced by the supplementation with RG. The 1H NMR and 13C NMR data revealed that the branching degree and double helix content of ERS was increased by 46.24 % and 52.67 % when RG content reached to 12 %. Additionally, the addition of RG altered the molecular weight and chain length distribution of ERS. The α-amylase activity and glucoamylase activity was inhibited by RG. These results could provide a valuable basis for the application of RG in extruded rice starchy foods with lower glycemic index.

Specific ions effect on aggregation behaviors and structural changes of amyloid fibrils from rice glutelin.

Metal ions have been considered as an important factor on fibrils assembly. Herein, a comprehensive analysis of specific ions effect on fibril formation and structural changes was investigated. The addition of ions (except Zn2+) accelerated the aggregation kinetics of rice glutelin fibrils (RGFs) from 0.93 to 1.28-2.19 h-1. In addition, the fibrillization rate followed the order of NH4+ > Li+ > Na+ > K+ > Cu2+ > Mg2+ > Ca2+ > Zn2+. The highest yield and length of fibrils were observed with Ca2+, probably due to the ionic bridging effect and hydrated capacity of Ca2+. However, Cu2+ reduced the fibrils yield, which was attributable to the fact that Cu2+ disrupted ß-sheet structure and inhibited the transition of monomer to fibrils. The polymorphism of fibrils was observed with different salts, and the light metals presented a superior effect on fibrils formation than heavy metals. Overall, this work will provide a further information into how to tune the structure of RGFs using various ions.

Elucidating the interaction mechanism of rice glutelin and soybean 11S globulin using multi-spectroscopy and molecular dynamics simulation methods.

Rice gluten, as the hydrophobic protein, exhibits restricted application value in hydrophilic food, which may be enhanced through interaction with soybean 11S globulin, characterized by favorable functional properties. This study aims at revealing their interaction mechanism via multi-spectroscopy and molecular dynamics simulation. The formation and structural change of rice glutelin-soybean 11S globulin complexes were detected using fluorescence, ultra-violet and circular dichroism spectra. The addition of 11S globulin increased the contents of α-helix, ß-turn and random coil, but decreased ß-sheet content, and the change in secondary structure was correlated with particle size. Moreover, exposure of hydrophobic groups and formation of disulfide bonds occurred in the complexes. Molecular dynamics simulation verified these experimental results through analyses of root mean square deviation and fluctuation, hydrogen bond, secondary structure, and binding free energy analysis. This study contributes to expounding the interaction mechanism of protein and protein from the molecular level.

Modifying functional properties of food amyloid-based nanostructures from rice glutelin.

Amyloid-based nanostructures from food sources have been received intensive interests recently in material science, biomedicine and especially delivery system. This is due to the ability of protein-based amyloid architecture that proved to be an attractive system to carry drug and nutrition. However, few research focused on the modification of functional properties of different fractions isolated from amyloid fibrils. Hereby, we separated the retentate (RGFs) and filtrate (FGFs) fractions from rice glutelin fibrils (GFs) using centrifugal filtration and then investigated the structural characteristics and functional properties of these fractions. We proved that protein fibrillization would highly improve both emulsifying and antioxidant abilities of protein dispersion. In addition, further processed RGFs with rich ß-sheet structures exhibited a similar functional performance to GFs dispersion. By contrast, FGFs dispersion with less ß- sheet content, lower molecular weight, interestingly re-assembled into spherical aggregates with weaker interaction, exhibiting better antioxidant and emulsifying properties.

Understanding effects of glutelin on physicochemical and structural properties of extruded starch and the underlying mechanism.

This work studied effects of different amounts of rice glutelin (RG) on physicochemical and structural properties of extruded rice starch (ERS) and explored the underlying mechanism of interaction between rice starch and RG upon extrusion processing. The results showed that the addition of RG altered the pasting properties, improved the viscoelastic, and increased the water mobility of ERS. The weight loss of ERS decreased from 71.40 % to 62.61 %, while the degradation temperature increased from 290.48 °C to 296.25 °C as the RG content increased from 0 % to 12 %. The complex index of extruded starch-glutelin complexes significantly elevated from 10.40 % to 35.81 % when RG content increased from 6 % to 12 %. Fourier-transform infrared spectra confirmed that RG interacted with starch via Maillard reactions, and the binding strength between RG and starch was enhanced at a higher RG content. Furthermore, results of rheological property and chemical interactions demonstrated that hydrogen bonding, hydrophobic, and electrostatic interaction were formed between RG and starch during extrusion. In summary, the obtained results of this study can further enrich the theory of starch-protein interactions and show the possibility of RG applied in the extruded starchy foods.

Effects of stirring speed ladder on the acid-promoted refolding of rice glutelin.

The effects of stirring speed (0, 250, 500, 750, 1000, 1250, and 1500 rpm) on the rice glutelin hydrocolloids (1 %, w/v) during the acidified process were investigated. As the stirring speed was increased to 750 rpm, the hydration diameter of the rice glutelin was significantly decreased, but higher stirring speeds had no significant effect on size. The highest and lowest solubility were recorded for the samples treated at 750 and 0 rpm stirring speeds, respectively. The surface hydrophobicity and molecular weight increased first and then decreased, both the minimum value was recorded at 750 rpm sample. The principal component analysis (PCA) was employed to detect patterns between changes in various properties (solubility, particle size, ß-sheet content, surface hydrophobicity, and ζ-potential) and stirring treatment. To conclude, the various properties of rice glutelin refold during acidification are drastically affected by employing different stirring speeds. Choosing a suitable stirring speed is important for quality control in protein hydrocolloid production.

Structural evolution, digestibility and inhibition on starch digestion of rice glutelin fibril aggregates as affected by incubation.

Structural evolution, digestibility and inhibition on starch digestion of rice glutelin fibril aggregates (RGFAs) as affected by incubation were investigated. Thioflavin T fluorescence intensity of the RGFAs, incubated for 4-day, reached the maximum values, which ranged from 845.00 ± 23.52 to 873.67 ± 50.30. Transmission electron microscopy (TEM) observed that the samples heated for 2 h (2 h fibril) were self-assembled from small glutelin aggregates and a few protofibrils into mature fibrils, the samples heated for 4-10 h (4-10 h fibril) were elongated into long, branched fibrils, and the longer fibrils of 15 h fibril sample dissociated into short fibrils after 4-day of incubation. Compared to rice glutelin, the RGFAs showed thermal stability and resistance to proteolysis. The fluorescence retention rate of 6 h fibril, after incubation for 4-day, was 8.62 ± 0.61 % after in vitro stomach and pancreas digestion, which was the highest among all of the samples. The RGFAs incubated for 1-day displayed much better inhibition effects on starch digestion. This was the first study to clarify the relationship between incubation and physicochemical/functional properties of protein fibrils, which could help understand the preservation of food protein fibrils and their application.

Effect of microbial transglutaminase on the structural and rheological characteristics and in vitro digestion of rice glutelin-casein blends.

The effect of microbial transglutaminase (MTGase) in cross-linking and modifying of rice glutelin-casein blends was investigated in this work. The maximum MTGase activity on rice glutelin-casein blends were found when its addition was 15 U/g of protein. Compared with the blends without MTGase, the appropriate addition of MTGase significantly affected the microstructure of the cross-linked proteins, resulting in the gradual 'burying' of Trp residue; while the space for hydrogen bonding was more abundant. Secondary structure changes, denoted by the disappearance of the α-helix and the decrement of the ß-sheet structure, was due to the formation of the large loop and random coil structures. The MTGase-catalyzed reaction improves the protease resistance of the blends but not promote the conversion of free sulfhydryl groups (-SH) to disulfide groups (S-S). All the samples evaluated exhibited Bingham flow behavior instead of shear-thinning behavior, and thermally stable fluid properties dominated by elasticity, regardless of MTGase concentration used. Both the storage modulus (G') and loss modulus (G'') gradually decreased with the addition of MTGase. In short, this work demonstrated how the structure, rheology, digestibility of rice glutelin-casein blends are influenced by MTGase concentration.

Formation, structural characteristics, foaming and emulsifying properties of rice glutelin fibrils.

The rice glutelin fibrils (RGFs) were formed under heating at acidic condition, and the optimal condition was achieved at pH 2, 150 mM (ionic strength), 4% (protein concentration), 90 °C and 300 rpm (stirring speed) through the thioflavin T intensity. The atomic force microscopy images showed that the average contour length of RGFs increased from < 100 to 365 nm under the optimal fibrillation. The average particle size of rice glutelin (RG) decreased from 650 to 221 nm after initial heating time. Combining the degraded subunits, it suggested that RG was hydrolyzed to peptides, then these released peptides assembled into the ordered fibrils via intermolecular interactions, accompanying by the structural rearrangement. Additionally, the foaming and emulsifying properties were improved during fibrillation, which could be related to the interfacial properties and structure of RGFs. This work will deepen the understanding of the formation of RGFs and explore their potential application.

Complexation of rice glutelin fibrils with cyanidin-3-O-glucoside at acidic condition: Thermal stability, binding mechanism and structural characterization.

The complexation of rice glutelin fibrils (RGFs) with cyanidin-3-O-glucoside (C3G) at acidic condition was investigated. The RGFs at pH 3.5 had a greatest protective effect on the thermal stability of C3G. The binding of C3G for RGFs was exothermic and driven by hydrophobic and electrostatic interactions. The RGFs exhibited a stronger binding interaction with C3G than rice glutelin (RG), resulting from the exposure of hydrophobic groups and positive charges on the fibrils surface, and thus RGFs exhibited better protective effect on C3G. The interaction with C3G resulted in the rearrangement of polypeptide chain, thereby reducing the ß-sheet content. The larger aggregates were observed in RG/RGFs-C3G complexes due to protein-polyphenols aggregation. It was noteworthy that the pre-formed RGFs were restructured into entangled aggregates due to the interaction. This study proposed a novel protein fibril to protect anthocyanins, expanding the application of anthocyanins as stable and functional ingredients in acidic food systems.

Structural, gelation properties and microstructure of rice glutelin/sugar beet pectin composite gels: Effects of ionic strengths.

In this study, the rice glutelin (RG)/sugar beet pectin (SBP) composite gels were prepared by laccase induced cross-linking and subsequent heat treatment, and the effects of different calcium ion concentrations (0-400 mM) on the gelation, structural properties and microstructure of the RG/SBP composite gels were investigated. The results showed that the addition of 200 mM calcium ion could improve the rheological, textural properties and water holding capacity of the RG/SBP composite gels. The addition of SBP and calcium ions enhanced the hydrophobic interaction between RG molecules, thereby increased the gel properties of RG. The changes in Raman spectroscopy reflected the positive effect of the addition of SBP and calcium ions on the formation of a denser and more homogeneous protein gel, as evidenced by the results of scanning electron microscopy. Overall, SBP and calcium ions could be applied to the plant protein gel systems as gel-strengthening agents.

Effects of anionic polysaccharides on the digestion of fish oil-in-water emulsions stabilized by hydrolyzed rice glutelin.

The effects of two anionic polysaccharides (pectin and xanthan gum) on the in vitro lipid digestibility of fish oil-in-water emulsions stabilized by hydrolyzed rice glutelin (HRG) were determined. The emulsions were passed through a simulated gastrointestinal tract (GIT) consisting of mouth, stomach, and small intestine phases. The physicochemical properties of the colloidal particles in the gastrointestinal fluids (mean particle diameter, ξ-potential, and structural organization) were monitored throughout the GIT. In addition, the kinetics of lipid digestion was characterized in the small intestine phase. Interestingly, the addition of polysaccharide led to a marked increase in both the rate and extent of lipid digestion, with the effect depending on polysaccharide type. For instance, the initial rates of lipid digestion were 8.6 ±â€¯0.6, 13.3 ±â€¯0.4 and 16.1 ±â€¯0.6% free fatty acids (FFA) released min-1 for HRG emulsions containing no polysaccharides, pectin, and xanthan gum, respectively. Similarly, the calculated final extents of lipid digestion were 71.9 ±â€¯3.6, 97.7 ±â€¯3.2, and 100.0 ±â€¯3.8%, respectively. This was probably because the polysaccharides inhibited droplet flocculation, thereby increasing the surface area of lipids exposed to the digestive enzymes. Moreover, the polysaccharides may have interacted with other components involved in the lipid digestion process, such as enzymes, bile salts, and calcium ions. Our results may facilitate the design of plant-based functional foods and beverages that can control lipid digestion in the gastrointestinal tract.

Investigation on the binding interaction between rice glutelin and epigallocatechin-3-gallate using spectroscopic and molecular docking simulation.

The interaction between plant protein and polyphenol is a topic of considerable interest. However, there is relatively little understanding about the interaction between rice protein and epigallocatechin-3-gallate (EGCG). The spectroscopy and computational docking program were used to investigate the potential interaction between rice glutelin (RG) and EGCG. It was found that the intrinsic fluorescence of RG could be quenched by EGCG, which indicated interaction occurred between them. Thermodynamic analysis elucidated that the interaction process between RG and EGCG happened spontaneously with hydrogen bond as the primary driving force. The ANS-fluorescence indicated that the surface hydrophobicity of RG reduced with the increasing of EGCG. Circular dichroism spectra and synchronous fluorescence gave further information for the conformational and microenvironmental changes of RG. Particularly, the α-helix structure reduced and random coil structure increased after the binding interaction. Furthermore, the computational docking program exhibited target sites in which the amino acid residues of RG and EGCG might be bound together.

The effects of phosphorylation modification on the structure, interactions and rheological properties of rice glutelin during heat treatment.

Rice glutelin (RG) and phosphorylated rice glutelin (PPRG) were treated with heating for different time (15, 30, and 45 min), the effects of phosphorylation modification on the structure, interactions and rheological properties of rice glutelin during heat treatment were investigated. The results showed that the turbidity of PPRG samples were higher than those of RG samples after heating. Particle size distribution showed that the protein aggregates with particle size of 1000-1500 nm were formed after heating for 45 min. Changes in protein structure indicated that the protein unfolded after heating for a short time, and aggregated when heating time extended to 45 min. In addition, the microstructure of PPRG sample became tight when heated for 45 min. Rheological analysis showed that phosphorylation modification and heat treatment improved RG viscoelasticity. These results suggest that phosphorylation modification improves thermal aggregation of RG, which will facilitate the application of RG in food industry.

Effects of sodium tripolyphosphate modification on the structural, functional, and rheological properties of rice glutelin.

In this study, rice glutelin isolated from glutinous rice was modified by sodium tripolyphosphate at different sodium tripolyphosphate concentrations (1%, 4%, 7%). The structural, functional and rheological properties of phosphorylated rice glutelin were investigated. The results indicated that phosphorylated rice glutelin samples showed small particle size and high absolute zeta potential values. Phosphorylation reaction was occurred between -OH and -NH2 groups in rice glutelin and sodium tripolyphosphate, as confirmed by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. The particle surface of phosphorylated rice glutelin samples became very rough and exhibited some small convex and concave holes. Moreover, the solubility, emulsifying and foaming properties, thermal stability and apparent viscosity of phosphorylated rice glutelin samples were significantly increased. These results suggest that the solubility, emulsification and rheological properties of rice glutelin are significantly improved after phosphorylation, which will be beneficial to expand the application of rice glutelin in food industry.

Effect of selenite on organic selenium speciation and selenium bioaccessibility in rice grains of two Se-enriched rice cultivars.

The accumulation of organic selenium (Se) in grains is helpful in understanding its bioaccessibility in various Se-enriched rice cultivars after soil selenite treatments. In the present study conducted with two rice cultivars, the organic Se as well as the glutelin-derived Se and the selenomethionine (SeMet) percentage in grains increased with soil Se levels. The available Se that was detected in the forms of SeMet (∼40%) and selenite (∼12%) accounted for more than 50% of the total Se in grains, and no significant differences were observed between the two rice cultivars (P > 0.05). Simulated gastrointestinal digestion showed that the digested grain SeMet increased with the soil Se treatment levels, whereas there were minimal changes in the percentages of selenite in the grain. Furthermore, approximately half of the available SeMet was derived from the grain glutelin. These results suggest that the non-glutelin-derived Se in rice should be further studied.