Walnut is an important woody oil crop. Walnut meal left after oil extraction is rich in high-quality protein, but it is underutilized, causing a waste of resources. Bioactive peptides derived from walnut meal protein have the potential for food development and are a promising ingredient in functional foods owing to their diverse bioactivities such as antioxidant, cardiovascular protective and memory improving activities. The effective utilization of walnut meal protein, for example by developing bioactive peptides is of great significance to realize the comprehensive utilization of walnut meal and to improve the added value of products. This review summarizes previous studies on the preparation, purification, identification and biological activity of walnut peptides in order to provide a theoretical basis for the high-value utilization of walnut meal protein resources and in-depth studies of walnut protein peptides.

Three walnut peptides with different molecular masses were prepared from walnut meal protein by sequential enzymatic hydrolysis and ultrafiltration, and separately glycosylated with five sugars (glucose, sucrose, lactose, maltodextrin and dextran). The reaction conditions were optimized based on emulsifying properties and anti-lipid oxidation capacity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Fourier transform infrared (FTIR) spectroscopy and circular dichroism (CD) spectroscopy were used to characterize the glycosylated peptides, their physicochemical properties were studied, and their emulsion stability was determined by laser confocal microscopy (LCM). The results showed that the optimal conditions for the glycosylation reaction between walnut peptide with molecular mass greater than 30 kDa and maltodextrin were as follows: ratio of walnut peptide to maltodextrin 1:2, reaction time 8 h, reaction temperature 80 ℃, and peptide concentration 15 mg/mL. SDS-PAGE confirmed the formation of a covalent complex between the walnut peptide and maltodextrin through glycosylation reaction. FTIR spectroscopy showed that sugar molecules were connected to walnut peptide molecules by covalent bonds. CD spectroscopy showed that the peptide’s structure changed. The proportion of α-helix increased from 4.85% to 6.43%, the proportion of antiparallelism increased from 30.16% to 35.91%, the proportion of parallelism increased from 3.28% to 4.33%, the proportion of β-turn decreased from 23.21% to 20.77%, and the proportion of random coil decreased from 33.59% to 31.22%. The glycosylation modification did not improve the Fe²⁺ chelating capacity, but increased the solubility, emulsifying capacity to (83.24 ± 1.64) m²/g, emulsion stability to (218.49 ± 3.55) min, water absorption capacity to (3.92 ± 0.36) g/g, oil absorption capacity to (3.17 ± 0.24) g/g, surface hydrophobicity to 251.05 ± 6.91, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging capacity to (86.74 ± 2.14)%, and anti-lipid oxidation capacity to (62.17 ± 3.51)%. The results of LCM showed that the encapsulation capacity of walnut peptides for walnut oil was enhanced after glycosylation, suggesting a potential application for improving the stability of emulsions. The glycosylation modification of walnut peptides provides a new idea for the processing and utilization of walnut meal by-products and provides a reference for the application of glycosylation modification in food development.

In this study, a mixture of ultrasound or microwave pretreated walnut meal protein peptides and CaCl₂ was used for the preparation of walnut peptide-calcium chelate. The effects of different pretreatments on the calcium chelating capacity, structural changes and stability of walnut meal protein peptides were analyzed. The results showed that compared with walnut meal protein peptide-calcium chelate (WPP-Ca), the chelation rates of ultrasound-pretreated walnut meal protein peptide-calcium chelate (UP-WPP-Ca) and microwave-pretreated walnut meal protein peptide-calcium chelate (MP-WPPCa) were enhanced, which indicated that ultrasound and microwave pretreatments improved the calcium-chelating capacity of the peptides effectively. Using ultraviolet-visible (UV-Vis) absorption spectroscopy and Fourier transform infrared (FTIR) spectroscopy, it was found that ultrasound and microwave pretreatments mainly affected the calcium ion binding sites such as amino groups, carbonyl groups, carboxyl groups, amide bonds and carboxylate groups of walnut meal protein peptides. The results of X-ray diffraction (XRD) showed that ultrasound and microwave treatments changed the molecular arrangement of walnut meal protein peptides, thereby making the structure of walnut peptide-calcium chelate more ordered. Fluorescence spectroscopy showed that ultrasonic and microwave treatments promoted the chelation between aromatic amino acids and calcium ions. In addition, UP-WPP-Ca and MP-WPP-Ca showed good stability toward different pH values, temperatures, and gastrointestinal digestion. In short, ultrasonic and microwave pretreatments can improve the calcium-chelating capacity and stability of walnut meal protein peptides, which is of guiding significance for the processing of walnut peptide-calcium chelate and the development of calcium supplements.