Lutein, a natural pigment with multiple beneficial bioactivities, faces limitations in food processing due to its instability. In this study, we constructed four modified walnut protein isolate (WNPI) based emulsions as emulsion-based delivery systems (EBDS) for lutein fortification. The modification treatments enhanced the encapsulation efficiency of the WNPI-based EBDS on lutein. The modified WNPI-based EBDS exhibited improved storage and digestive stability, as well as increased lutein delivery capability in simulated gastrointestinal conditions. After in vitro digestion, the lutein retention in the modified WNPI-based EBDS was higher than in the untreated WNPI-based EBDS, with a maximum retention of 49.67 ± 1.10 % achieved after ultrasonic modification. Furthermore, the modified WNPI-based EBDS exhibited an elevated lutein bioaccessibility, reaching a maximum value of 40.49 ± 1.29 % after ultrasonic modification, nearly twice as high as the untreated WNPI-based EBDS. Molecular docking analysis indicated a robust affinity between WNPI and lutein, involving hydrogen bonds and hydrophobic interactions. Collectively, this study broadens WNPI\'s application and provides a foundation for fortifying other fat-soluble bioactive substances.

Walnut isolate protein (WPI)-epigallocatechin gallate (EGCG) conjugates can be employed to creat food-grade delivery systems for preserving bioactive compounds. In this study, WPI-EGCG nanoparticles (WENPs) were developed for encapsulating lycopene (LYC) using the ultrasound-assisted method. The results indicated successful loading of LYC into these WENPs, forming the WENPs/LYC (cylinder with 200-300 nm in length and 14.81-30.05 nm in diameter). Encapsulating LYC in WENPs led to a notable decrease in release rate and improved stability in terms of thermal, ultraviolet (UV), and storage conditions compared to free LYC. Simultaneously, WENPs/LYC exhibited a synergistic and significantly higher antioxidant activity with an EC50 value of 23.98 μg/mL in HepG2 cells compared to free LYC\'s 31.54 μg/mL. Treatment with WENPs/LYC led to a dose-dependent restoration of intracellular antioxidant enzyme activities (SOD, CAT, and GSH-Px) and inhibition of intracellular malondialdehyde (MDA) formation. Furthermore, transcriptome analysis indicated that enrichment in glutathione metabolism and peroxisome processes following WENPs/LYC addition. Quantitative real-time reverse transcription PCR (qRT-PCR) verified the expression levels of related genes involved in the antioxidant resistance pathway of WENPs/LYC on AAPH-induced oxidative stress. This study offers novel perspectives into the antioxidant resistance pathway of WENPs/LYC, holding significant potential in food industry.

Angiotensin I-converting enzyme (ACE)-inhibiting peptides were isolated from walnut protein isolate (WPI) using ultrasound-assisted extraction. This study aimed to assess the impact of ultrasonic pretreatment on the physicochemical properties of WPI. The optimal extraction conditions for WPI were determined as a 15-min ultrasonic treatment at 400 W. Subsequently, the hydrolysate exhibiting the highest in vitro ACE-inhibiting activity underwent further processing and separation steps, including ultrafiltration, ion exchange chromatography, liquid chromatography-tandem mass spectrometry, ADMET screening, and molecular docking. As a result of this comprehensive process, two previously unidentified ACE-inhibiting peptides, namely Tyr-Ile-Gln (YIQ) and Ile-Tyr-Gln (IYQ), were identified. In addition, a novel peptide, Ile-Lys-Gln (IKQ), was synthesized, demonstrating superior ACE-inhibiting activity and temperature stability. In silico analysis estimated an in vivo utilization rate of 21.7% for IKQ. These peptides were observed to inhibit ACE through an anti-competitive mechanism, with molecular docking simulations suggesting an interaction mechanism involving hydrogen bonding. Notably, both IYQ and IKQ peptides exhibited no discernible toxicity to HUVECs cells and promoted nitric oxide (NO) generation. These findings underscore the potential of ultrasonicated WPI in the separation of ACE-inhibiting peptides and their utility in the development of novel ACE inhibitors for functional food applications.

This study investigated the effects of the interaction of walnut protein isolate (WPI) with epigallocatechin gallate (EGCG), chlorogenic acid (CLA), (+)-catechin (CA), and ellagic acid (EA) on the structural and functional properties of proteins. The results for polyphenol binding equivalents and content of free amino and sulfhydryl groups as well as those from sodium dodecyl sulfate‒polyacrylamide gel electrophoresis confirmed the covalent interaction between WPI and the polyphenols. The binding capacities of the WPI-polyphenol mixtures and conjugates were as follows: WPI-EGCG > WPI-CLA > WPI-CA > WPI-EA. Fourier transform infrared spectroscopy (FTIR) and fluorescence spectrum analysis identified changes in the protein structure. The conjugation process obviously increased the polyphenols\' antioxidant properties and the surface hydrophobicity was substantially reduced. WPI-EGCG conjugates had the best functional properties, followed by WPI-CLA, WPI-CA, and WPI-EA. Lycopene (LYC) was loaded into nanocarriers by WPI-EGCG self-assembly. These results indicated that WPI-polyphenol conjugates can be utilized to develop food-grade delivery systems to protect chemically lipophilic bioactive compounds.
UNASSIGNED:
UNASSIGNED: The online version contains supplementary material available at 10.1007/s13197-023-05768-2.

BACKGROUND: Walnut kernels are high in polyphenols (PPs), which cause low protein solubility, limiting the use of walnut protein in the food industry. To obtain the best technical parameters of the dephenolization treatment, the defatted walnut powder was dephenolized using ultrasound-assisted ethanol extraction (UAE), and the response surface optimization was performed on the basis of single factor. On this basis, the effects of dephenolization on the solubility, emulsifying properties and foaming properties of walnut protein isolates (WPIs) were compared to those of defatted walnut powder without dephenolization.
RESULTS: The results showed that PP extraction in the UAE could significantly increase PP yield. The optimal process parameters were as follows: 51% (v/v) ethanol concentration, 140 W ultrasound power, 10 min extraction time, 30 °C ultrasound temperature, and a material-liquid ratio of 1:30 (w/v). The results revealed that the UAE dephenolization treatment significantly improved the functionality of WPI and that the functionality of the dephenolized WPI by UAE was superior to that of the protein without dephenolization, and that the functionality of both walnut proteins was the worst at pH 5, with solubility of 5.31% and 4.86%, emulsifying activity index (EAI) of 24.95 and 19.91 m2 /g, and foaming capacity (FC) of 3.66% and 2.94%, respectively; and the best at pH 11, with solubility of 82.35% and 73.55%, EAI of 46.35 and 37.28 m2 /g, and FC of 35.85% and 18.87%, respectively.
CONCLUSIONS: The study found that dephenolization by UAE can significantly improve the functionality of WPI, and this method should be promoted and used in walnut and walnut protein processing industries. © 2023 Society of Chemical Industry.

Alkaline-extracted walnut protein isolates showed relatively poor solubility and emulsifying properties in many previous studies. However, whether they can be used as potential emulsifiers to stabilize high internal phase emulsions (HIPEs) remains unknown. Herein, walnut protein isolates were prepared by alkaline extraction from walnut kernels with or without pellicles (named PAWPI and AWPI, respectively). PAWPI conjugated with pellicle polyphenols showed improved solubility and higher antioxidant capacity than AWPI. HIPEs were fabricated via a one-step method using AWPI or PAWPI as the sole protein emulsifier. HIPEs (oil fraction of 0.8, with 0.1% β-carotene) could be stabilized by PAWPI at a relatively low concentration of 0.2% (w/v), while at least 1% (w/v) AWPI was required to effectively stabilize HIPEs. HIPEs stabilized by PAWPI had smaller oil droplet sizes than those stabilized by AWPI. Rheological analysis indicated that PAWPI-stabilized HIPEs showed higher viscosity and better viscoelasticity than AWPI-stabilized HIPEs. Large-amplitude oscillation shearing analysis suggested that PAWPI-stabilized HIPEs were stiffer but more brittle than AWPI-stabilized HIPEs. Moreover, both PAWPI- and AWPI-stabilized HIPEs exhibited good storage stability and were relatively stable against heat treatment and ionic strength. PAWPI-stabilized HIPEs showed a higher protective capacity for encapsulated β-carotene than AWPI-stabilized HIPEs. In addition, PAWPI-stabilized HIPEs showed good 3D printability and could be used as a promising edible ink.

BACKGROUND: Walnut proteins display poor solubility and dispersity under acidic pH conditions, which limits their application in acidic beverages and foods. This study aimed to fabricate stable nanocomplexes between phosphorylated walnut protein (PWPI) and chitosan (CS) in an acidic pH and to investigate the encapsulation capacity of the complexes.
RESULTS: The PWPI/CS nanocomplexes prepared at a mass ratio of 2:1 showed small Z-average sizes (approximately 285 nm at pH 5.5 and 222 nm at pH 3.5) with a narrow particle distribution (polydispersity index <0.3). Caffeic acid phenethyl ester (CAPE) can be effectively encapsulated into PWPI/CS with improved solubility. Circular dichroism analysis indicated that PWPI/CS and CAPE-loaded PWPI/CS (PWPI/CS-CAPE) had reduced α-helical content and increased β-sheet content. Fourier transform infrared spectroscopy analysis further identified the different driving forces for the complexation of PWPI and CS at pH 3.5 and 5.5 and confirmed the successful encapsulation of CAPE. The rheological results revealed that the PWPI/CS and PWPI/CS-CAPE formed at pH 3.5 (PWPI/CS-CAPE-3.5) had a higher apparent viscosity and better viscoelasticity than the complexes formed at pH 5.5. The PWPI/CS-CAPE-3.5 also showed good stability under heat treatment, salt treatment, and long-term storage. The PWPI/CS-CAPE complexes showed controlled release of CAPE.
CONCLUSIONS: Walnut protein and chitosan nanocomplexes prepared at acidic pH levels were stable and promising carriers for CAPE, which could expand the application of walnut proteins in the food industry. © 2023 Society of Chemical Industry.

Walnut protein isolate (WPI) is a nutritious protein with poor solubility, which severely limits its application. In this study, composite nanoparticles were prepared from WPI and soy protein isolate (SPI) using the pH-cycle technology. The WPI solubility increased from 12.64 to 88.53% with a WPI: SPI ratio increased from 1: 0.01 to 1: 1. Morphological and structural analyses illustrated that interaction forces with hydrogen bonding as the main effect jointly drive the binding of WPI to SPI and that protein co-folding occurs during the neutralization process, resulting in a hydrophilic rigid structure. In addition, the interfacial characterization showed that the composite nanoparticle with a large surface charge enhanced the affinity with water molecules, prevented protein aggregation, and protected the new hydrophilic structure from damage. All these parameters helped to maintain the stability of the composite nanoparticles in a neutral environment. Amino acid analysis, emulsification capacity, foaming, and stability analysis showed that the prepared WPI-based nanoparticles exhibited good nutritional and functional properties. Overall, this study could provide a technical reference for the value-added use of WPI and an alternative strategy for delivering natural food ingredients.

Walnut protein is a kind of natural, high-quality plant protein resource. However, its high content of gluten, strong hydrophobicity and poor gelation ability have greatly limited its development and utilization in gel products. It was found in this experiment that ultrasonic power combined with transglutaminase (TGase) had a significant effect on the gel properties of the walnut protein isolate (WNPI)-κ-carrageenan (KC) complex. The results showed that the gel strength of the WNPI-KC complex first increased and then decreased with the increase in ultrasonic power (0-400 W). WNPI-KC composite gel had the best texture properties, rheological properties, water-holding capacity (99.41 ± 0.76%), swelling ratio (2.31 ± 0.29%) and thermal stability (83.22 °C) following 200 W ultrasonic pretreatment. At this time, the gel network was more uniform and much denser, and the water molecules were more tightly bound. Further, 200 W ultrasonic pretreatment could promote the transformation of α-helices to β-folds in protein molecules, improve the fluorescence intensity, increase the content of free sulfhydryl groups and enhance the intermolecular forces. The experimental results could provide technical support for the development of walnut protein gel food.

Alkaline-extracted walnut protein isolate (AWPI) shows poor solubility in aqueous solutions, resulting in relatively low emulsion capacity. This work investigated the influence of ellagic acid (EA) or epigallocatechin-3-gallate (EGCG) conjugation on the solubility and emulsifying properties of AWPI. The increase in polyphenol content and decrease in free amino and thiol groups of walnut proteins confirmed successful conjugation. AWPI polyphenol conjugates showed significantly reduced surface hydrophobicity, greatly enhanced surface charge, and consequently improved solubility. Circular dichroism (CD) results indicated that the polyphenol-conjugated AWPI contained relatively higher α-helical and lower β-sheet contents than AWPI. The antioxidant capacity of the polyphenol-conjugated AWPI was significantly enhanced. Additionally, polyphenol conjugation resulted in decreased mean particle sizes and increased surface charges of the walnut protein-covered oil droplets. The emulsions stabilized by AWPI-polyphenol conjugates were relatively more stable over a range of pH values (7.0-11.0) and thermal treatments (25 °C-90 °C). Moreover, they exhibited greater storage stability than those stabilized by unmodified AWPI.