Glycated walnut meal peptide­calcium chelates (COS-MMGGED-Ca): Preparation, characterization, and calcium absorption-promoting.

This study isolated a novel peptide MMGGED with strong calcium-binding capacity from defatted walnut meal and synthesized a novel peptide­calcium chelate COS-MMGGED-Ca with high stability via glycation. Structural characterization and computer simulation identified binding sites, while in vitro digestion stability and calcium transport experiments explored the chelate's properties. Results showed that after glycation, COS-MMGGED bound Ca2+ with 88.75 ± 1.75 %, mainly via aspartic and glutamic acids. COS-MMGGED-Ca released Ca2+ steadily (60.27 %), with thermal denaturation temperature increased by 18 °C and 37 °C compared to MMGGED-Ca, indicating good processing performance. Furthermore, COS-MMGGED significantly enhanced Ca2+ transport across Caco-2 monolayers, 1.13-fold and 1.62-fold higher than CaCl2 and MMGGED, respectively, at 240 h. These findings prove glycation enhances structural properties, stability, calcium loading, and transport of peptide­calcium chelates, providing a scientific basis for developing novel efficient calcium supplements and high-value utilization of walnut meal.

Fabrication of ultrafine Himalayan walnut oil Pickering emulsions by ultrasonic emulsification: Techno-functional properties of emulsions and microcapsules.

In present scenario, much of the attention has been put on the production and utilization of Pickering emulsions deciphering enhanced stability and applicability over wide environmental conditions. In this context the present study was carried out to elaborate effect of different wall materials and pH systems on the physicochemical, structural and morphological properties of Himalayan walnut oil Pickering emulsions by ultrasonic emulsification. In this study, concentrated Pickering emulsion of Himalayan walnut oil (HWO) was prepared utilizing soy protein isolate (SPI), maltodextrin (MD) stabilized by pectin at varying concentrations and pH systems (4.0, 7.0). With increase in pectin and SPI concentration and lowering MD, stable emulsions were obtained as deciphered by an Emulsion stability index (ESI) of 100 for 7 days at ambient storage. HWO Pickering emulsions were analysed for particle size measurements (2.13-13.64 µm) and depicted negative zeta potential values (-3.70 to -18.58). Lyophilized HWO microcapsules depicted moderate encapsulation efficiency (44.69-57.63 %) whereas the hygroscopicity values of the microcapsule ranged from (0.21-12.10 %). Thermogravimetric analysis (TGA) of the samples depicted the temperature of maximum degradation rate up to 550 °C whereas XRD spectra depicted amorphous nature of oil microcapsules. FTIR spectra revealed a close association between the SPI-MD-Pectin matrix. SEM analysis revealed stable oil globules entrapped in protein-polysaccharide matrix with no visible cracks and fissures.

The global transcription factor Clp exerts positive regulatory effects in the walnut bacterial black spot pathogen, Xanthomonas arboricola pv. juglandis.

Walnut blight caused by the bacterium Xanthomonas arboricola pv. juglandis (Xaj) is one of the most common diseases of walnut (Juglans spp.), resulting in serious yield decline and significant economic losses. Crp-like protein (Clp) is an important global regulatory transcription factor in bacteria. In this study, we sought to elucidate the role of Clp in the pathogenicity of Xaj strain DW3F3 and the associated regulatory mechanism. The results indicated that clp gene deficiency significantly reduced the pathogenicity of Xaj DW3F3 in walnut without affecting the growth of the bacterium. We found that Clp positively regulates biofilm formation, extracellular polysaccharide production, exoenzyme secretion, and motility of Xaj, which was consistent with the transcript levels of virulence factor-encoding genes. However, overexpression of clp does not enhance the expression of all virulence genes, it may inhibit the expression of a part of virulence factor-related genes. EMSA assay further showed that Clp specifically binds to the promoters of these genes and regulates their expression, and CD spectra test certified that the ligand of Clp was c-di-GMP. Our findings contribute to the in-depth understanding of the pathogenic mechanism of Xaj and highlight the potential of Clp as a drug target for the development of agents to prevent and control walnut diseases.

Characterization of volatile organic compounds in walnut oil with various oxidation levels using olfactory analysis and HS-SPME-GC/MS.

Walnut oil oxidizes and becomes rancid during storage, that could be significantly affecting flavor and quality. This study aimed to monitor the volatile compounds present in walnut oil during storage, identify the characteristic markers of walnut oil at different oxidation levels, and establish a correlation network analysis based on the relationship between the olfactory analyzer and the characteristic markers to understand their correlation. The results indicated that the oxidation level of walnut oil had a positive correlation with the response of the olfactory analyzer. 219 volatile compounds were identified in walnut oil, with 89 identified as key volatile compounds (VIP >1). Among these, compounds such as (E, E)-2,4-decadienal (6.10%-23.04%)，(E, E)-2,4-heptadienal (2.23%-13.61%)，(E)-2-octenal (0.95%-11.71%)， hexanoic acid (1.63%-4.30%)，1-octen-3-ol (2.53%-19.01%)，(Z)-2-heptenal (5.95%-25.01%)，2,3-dihydro-furan (1.08%-3.20%)，2-pentyl-furan (0.13%-0.54%), pyrazine (0.33%-1.32%), hexanal (24.52%-1.33%)，3-hethylbutylacetate (12.44%-1.29%)， 2-methyl butyl acetate (7.74%-1.56%) and ethenyl hexanoate (4.39%-0.41%) were found to be characteristic volatile compounds in the oxidation process of walnut oil. Furthermore, the correlation network analysis revealed a strong correlation between the olfactory analyzer sensors and the characteristic volatile compounds. The findings of this study can provide valuable data for the development of rapid determination of the oxidation level of walnut oil.

Electron beam irradiation coupled ultrasound-assisted natural deep eutectic solvents extraction: A green and efficient extraction strategy for proanthocyanidin from walnut green husk.

Proanthocyanidin (PAC) is recognized as a potent natural antioxidant that prevents various diseases. As societal awareness increases, eco-friendly and efficient natural product extraction technologies are gaining more attention. In this study, an electron beam irradiation (EBI) coupled with ultrasound-assisted natural deep eutectic solvents (NADES) extraction method was developed to enable the green and highly efficient extraction of PAC from walnut green husk (WGH). NADES, prepared with choline chloride and ethylene glycol, demonstrated excellent extraction capacity and storage stability for PAC. Molecular dynamics simulations elucidated the high compatibility between NADES and PAC, attributed mainly to a higher SASA value (207.85 nm2), a greater number of hydrogen bonds (330.99), an extended hydrogen bonding lifetime (4.54 ps), and lower inter-molecular interaction energy. Based on these findings, the optimal conditions (13 kGy EBI, 42 mL/g liquid-solid ratio, 38 °C extraction temperature, 70 min extraction time) resulted in a maximum PAC extraction yield of 56.34 mg/g. Notably, this yield was 32.93 % higher than that observed in samples not treated with EBI and ultrasound-assisted extraction (UAE). Analysis of tissue morphology, extract functional groups and thermal behavior suggested a possible mechanism for the synergistically enhanced PAC extraction by the EBI-NADES-UAE method. Additionally, the PAC extracted using the NADES by the EBI coupled with ultrasound-assisted method exhibited outstanding antioxidant activity (comparable to Vc), digestive enzyme inhibition (IC50: 17-0.61 mg/mL), and anti-glycation capacity (IC50: 86.49 µg/mL). Overall, this work provided a green and efficient strategy for PAC extraction from WGH, elucidated the extraction mechanism and bioactivities, and offered valuable insights for potential industrial applications.

Flame Retardancy and Thermal Stability of Rigid Polyurethane Foams Filled with Walnut Shells and Mineral Fillers.

Recently, the influence of the concept of environmental sustainability has increased, which includes environmentally friendly measures related to reducing the consumption of petrochemical fuels and converting post-production feedstocks into raw materials for the synthesis of polymeric materials, the addition of which would improve the performance of the final product. In this regard, the development of bio-based polyurethane foams can be carried out by, among other things, modifying polyurethane foams with vegetable or waste fillers. This paper investigates the possibility of using walnut shells (WS) and the mineral fillers vermiculite (V) and perlite (P) as a flame retardant to increase fire safety and thermal stability at higher temperatures. The effects of the fillers in amounts of 10 wt.% on selected properties of the polyurethane composites, such as rheological properties (dynamic viscosity and processing times), mechanical properties (compressive strength, flexural strength, and hardness), insulating properties (thermal conductivity), and flame retardant properties (e.g., ignition time, limiting oxygen index, and peak heat release) were investigated. It has been shown that polyurethane foams containing fillers have better performance properties compared to unmodified polyurethane foams.

Chemical Characterization and Beneficial Effects of Walnut Oil on a Drosophila melanogaster Model of Parkinson's Disease.

A nutritional approach could be a promising strategy to prevent or decrease the progression of neurodegenerative disorders such as Parkinson's disease (PD). The neuroprotective role of walnut oil (WO) was investigated in Drosophila melanogaster treated with rotenone (Rot), as a PD model, WO, or their combination, and compared to controls. WO reduced mortality and improved locomotor activity impairment after 3 and 7 days, induced by Rot. LC-MS analyses of fatty acid levels in Drosophila heads showed a significant increase in linolenic (ALA) and linoleic acid (LA) both in flies fed with the WO-enriched diet and in those treated with the association of WO with Rot. Flies supplemented with the WO diet showed an increase in brain dopamine (DA) level, while Rot treatment significantly depleted dopamine content; conversely, the association of Rot with WO did not modify DA content compared to controls. The greater intake of ALA and LA in the enriched diet enhanced their levels in Drosophila brain, suggesting a neuroprotective role of polyunsaturated fatty acids against Rot-induced neurotoxicity. The involvement of the dopaminergic system in the improvement of behavioral and biochemical parameters in Drosophila fed with WO is also suggested.

Maturation-induced changes in phenolic forms and their antioxidant activities of walnuts: A dual view from kernel and pellicle.

The phenolic profiles and antioxidant activities during walnut maturation are not well understood. This study used UPLC-MS/MS to evaluate phenolic content in walnuts, including free, esterified, and bound forms, at different maturation stages. Findings showed that free phenolics were predominant, comprising 44.57 % in kernels and 56.54 % in pellicles. In vitro assays showed antioxidant capacity decreased with maturation, with IC50 values of 0.87-84.43 µg/mL in pellicles and 48.51-712.30 µg/mL in kernels. Most monomeric phenols decreased in concentration as the fruit ripened. OPLS-DA identified 5 and 8 maturity-sensitive phenolics (MSPs) in kernels and pellicles, respectively, with fold changes from 2.32 to 1664.72. Pearson correlation analysis showed a significant correlation between MSPs and antioxidant activity (r > 0.75, p < 0.05). Bioinformatics analysis elucidated three key metabolic pathways involved in these changes. This research provides insights into walnut phenolic composition, important for optimizing harvest practices and enhancing nutritional value.

Mechanism of adsorption and targeted degradation of antimicrobial micropollutant sulfamethoxazole in aquatic environments.

FHWSB as an integrated absorptive catalyst, based on Walnut shell biochar (WSB) via hydrochloric acid modification and ferrous chloride impregnation, was prepared, reacted with H2O2 to generate active free radicals •OH and •O2-, which oxidized and degraded about 80% of micro-pollutant sulfamethoxazole (SMX) from water, effectively resolving micro-pollutants' removal being inefficient because of high toxicity, persistence, and bioaccumulation in existed methods. It was clarified the specific degradation pathways and mechanisms of SMX by FHWSB synergistic H2O2 via characterization and analysis assisted DFT calculations. Furthermore, it was found that the toxicity of a series of intermediates produced by SMX degraded continued to decline, consistent with its direction of degradation via toxicological analysis. The work provides a simple and feasible strategy for the effective removal of antibiotic micro-pollutants in aquatic environments.

Designing novel ice creams using nut oil emulsion gels based on blueberry pectin and CaCl2 as fat replacers: Insights from physicochemical and sensory properties.

This study aimed to utilize blueberry pectin and calcium chloride to design a gel network structure for loading nut oils (peanut and walnut oil, respectively). The optimization of emulsion gel preparation was conducted through orthogonal experiments, utilizing the oil-holding ratio and gel strength as critical indicators. The emulsion gel was applied to the ice cream production. It was revealed that the peroxide value of the nut oil emulsion gels was significantly lower than that of nut oils. Both nut oil emulsion gel ice creams exhibited higher expansion rates, lower melting rates, and decreased hardness than the nut oil ice creams. Notably, walnut oil emulsion gel ice cream demonstrated a melting rate similar to traditional butter-based ice cream. Emulsion gel ice cream has higher fat globule instability and viscosity. Overall, the comprehensive emulsion gel ice cream indicators were comparable to conventional butter ice cream and notably superior to peanut and walnut oil ice cream. Using emulsion gel as a fat substitute in ice cream was feasible. The implications of these results were significant for advancing the utilization of nut oil emulsion gel within the ice cream industry.

Characterization of ultrasonic-assisted antifungal film loaded with fermented walnut meal on Rosa roxburghii Tratt during near-freezing temperature storage.

Fermented walnut meal (FW) has antifungal activity against Penicillium victoriae, a fungus responsible for Rosa roxbughii Tratt spoilage. This study characterized and applied ultrasonic-assisted antifungal film loaded with FW to preserve R. roxbughii Tratt during near-freezing temperature (NFT). Results showed that O2 and CO2 transmission rates decreased by 80.02% and 29.05%, respectively, and antimicrobial properties were improved with ultrasound at 560 W for 5 min and 1% FW. Fourier transform infrared spectroscopy and X-ray diffraction results revealed ultrasound improved hydrogen bonds and inductive effect via ─NH, ─OH, and C═O bonds. The addition of FW led to the formation of CMCS-GL-FW polymer via C═O bond. Thermogravimetric analysis and transmission electron microscope results demonstrated thermal degradation process was decomposed by ultrasound, and the internal structure of P. victoriae was accelerated by the addition of FW. Compared to the U-CMCS/GL group, the vitamin C content, peroxidase, and catalase activities of U-CMCS/GL/FW were enhanced by 4.24%, 8.52%, and 14.3% during NFT (-0.8 to -0.4°C), respectively. Particularly, the fungal count of the U-CMCS/GL/FW group did not exceed 105 CFU g-1 at the end of storage, and the relative abundance of P. victoriae decreased to 0.007%. Our findings provide an effective route for agricultural waste as natural antifungal compounds in the active packaging industry. PRACTICAL APPLICATION: In this study, the barrier and antimicrobial properties of film were successfully improved by ultrasonic treatment and loaded fermented walnut meal. The ultrasonic-assisted antifungal film loaded with fermented walnut meal effectively delayed the degradation of nutrients and reduced microbial invasion of Rosa roxburghii Tratt. These results provide a theoretical basis for the application of agricultural waste in the food packaging industry.

Green synthesis of copper oxide nanoparticles using walnut shell and their size dependent anticancer effects on breast and colorectal cancer cell lines.

Metal oxide nanoparticles(NPs) contain unique properties which have made them attractive agents in cancer treatment. The CuO nanoparticles were green synthesized using walnut shell powder in different calcination temperatures (400°, 500°, 700°, and 900 °C). The CuO nanoparticles are characterized by FTIR, XRD, BET, SEM and DLS analyses. SEM and DLS analyses showed that by increasing the required calcination temperature for synthesizing the NPs, their size was increased. DPPH analysis displayed no significant anti-oxidative properties of the CuO NPs. The MTT analysis showed that all synthesized CuO NPs exhibited cytotoxic effects on MCF-7, HCT-116, and HEK-293 cell lines. Among the CuO NPs, the CuO-900 NPs showed the least cytotoxic effect on the HEK-293 cell line (IC50 = 330.8 µg/ml). Hoechst staining and real-time analysis suggested that the CuO-900 NPs induced apoptosis by elevation of p53 and Bax genes expression levels. Also, the CuO-900 NPs increased the Nrf-2 gene expression level in MCF-7 cells, despite the HCT-116 cells. As can be concluded from the results, the CuO-900 NPs exerted promising cytotoxic effects on breast and colon cancer cells.

Transcriptome analysis of lipid biosynthesis during kernel development in two walnut (Juglans regia L.) varieties of 'Xilin 3' and 'Xiangling'.

BACKGROUND: Walnut is an oilseed tree species and an ecologically important woody tree species that is rich in oil and nutrients. In light of differences in the lipid content, fatty acid composition and key genes expression patterns in different walnut varieties, the key gene regulatory networks for lipid biosynthesis in different varieties of walnuts were intensively investigated. RESULTS: The kernels of two walnut varieties, 'Xilin 3' (X3) and 'Xiangling' (XL) were sampled at 60, 90, and 120 days post-anthesis (DPA) to construct 18 cDNA libraries, and the candidate genes related to oil synthesis were identified via sequencing and expression analysis. A total of 106 differentially expressed genes associated with fatty acid biosynthesis, fatty acid elongation, unsaturated fatty acid biosynthesis, triglyceride assembly, and oil body storage were selected from the transcriptomes. Weighted gene co-expression network analysis (WGCNA), correlation analysis and quantitative validation confirmed the key role of the FAD3 (109002248) gene in lipid synthesis in different varieties. CONCLUSIONS: These results provide valuable resources for future investigations and new insights into genes related to oil accumulation and lipid metabolism in walnut seed kernels. The findings will also aid future molecular studies and ongoing efforts to genetically improve walnut.

Population genetic insights into the conservation of common walnut (Juglans regia) in Central Asia.

The common walnut (Juglans regia) is one of the most economically important nut trees cultivated worldwide. Despite its importance, no comprehensive evaluation of walnut tree population genetics has been undertaken across the range where it originated, Central Asia. In this study, we investigated the genetic diversity and population structure of 1082 individuals from 46 populations across Central Asia. We found moderate genetic diversity of J. regia across Central Asia, with 46 populations clustered into three groups with a weak relationship between genetic and geographic distance. Our findings reveal that the western Himalaya might be the core region of common walnut genetic diversity in Central Asia and that, except for two populations in Gongliu Wild Walnut Valley, humans might have introduced walnut populations to Xinjiang, China. The observed distribution of the genetic landscape has probably been affected by historical climate fluctuation, breeding system, and prolonged anthropogenic activity. We propose the conservation of the core genetic diversity resources in the western Himalaya and pay special attention to populations from Gongliu in Xinjiang. These findings enhance our understanding of the genetic variation throughout the distribution range of J. regia in Central Asia, which will provide a key prerequisite for evidence-based conservation and management.

In vitro digestive properties and the bioactive effect of walnut green husk on human gut microbiota.

Introduction: Walnut green husk (WGH) is a waste byproduct from walnut industry. However, it is not well-known about its bioactive effect on human gut health. Methods: This study conducted in vitro digestion and fermentation experiments to study the bioactive effect of WGH. Results: Microbial fermentation was the primary mechanism to efficiently release phenolics and flavonoids, resulting in more excellent antioxidant capacities (DPPH, ABTS, and FRAP assays), which reached a highest value with 14.82 ± 0.01 mg VcE/g DW, 3.47 ± 0.01 mmol TE/g DW, and 0.96 ± 0.07 mmol FeSO4·7H2O/g DW, respectively. The surface microstructure of WGH became loose and fragmented after microbial fermentation. The analytical results of gut microbiota demonstrated that WGH could significantly increase the relative abundance of Proteobacteria in phylum level and Phascolarctobacterium in genus level while certain pro-inflammatory bacteria (such as Clostridium_sensu_stricto_1, Dorea, Alistipes, and Bilophila) was inhibited. Additionally, 1,373 differential metabolites were identified and enriched in 283 KEGG pathways. Of which some metabolites were significantly upregulated including ferulic acid, chlorogenic acid, umbelliferone, scopolin, muricholic acid, and so forth. Discussion: These results indicated that WGH could have antioxidant and anti-inflammatory activities in the human gut, which could improve the economical value of WGH in the food industry.

Physicochemical and Functional Properties of Black Walnut and Sycamore Syrups.

Historically, tree sap has been used globally for medicinal purposes, in fermented beverages, and for syrup production. Maple tree sap is notably concentrated into syrup and is valued as a natural sweetener rich in phenolic compounds and minerals compared to refined sugar. Recently, syrups from other trees like black walnut (Juglans nigra) and sycamore (Platanus occidentalis) have gained popularity, yet their properties are not well understood scientifically. To address this gap, we collected sycamore, black walnut, and maple syrup samples and analyzed their physicochemical and functional properties. Our findings showed significant differences among the syrups in pH, browning intensity, and water activity (p < 0.05). Sycamore syrup had the highest total phenolic content, followed by black walnut and maple syrups. Both black walnut and sycamore syrups exhibited similar antioxidant activity, significantly higher than maple syrup (p < 0.05). High-resolution mass spectrometry identified 54 phenolic acids and 22 flavonoids in these syrups, including Acetylsalicylic acid, 3,5-Dihydroxybenzoic acid, and syringic acid, known for their antioxidant and anti-inflammatory properties. Additionally, sycamore syrups and most black walnut syrups displayed varying degrees of antimicrobial activity against Gram-positive and/or Gram-negative microorganisms. This study offers insights into the properties and potential health benefits of these specialty tree syrups.

Colletotrichum species (Glomerellales, Glomerellaceae) causing walnut anthracnose in China.

Colletotrichum species can function as plant pathogens, saprobes or endophytes on a wide variety of plant hosts and are considered amongst the ten most significant genera of plant pathogens globally. China contributes almost half the walnut production in the world. However, Colletotrichum species occurring on walnut remain largely unresolved in China. To explore the Colletotrichum species found on walnut in China, 470 walnut fruit or leaf samples with anthracnose were collected from 14 main walnut-producing regions across seven provinces. A total of 165 Colletotrichum strains were isolated from these samples. The Colletotrichum isolates were identified, based on morphological characteristics and sequence analyses of ACT, CHS-1, GAPDH, ITS and TUB2. Twelve species, including 11 known Colletotrichum species (C.boninense, C.citrulli, C.fioriniae, C.fructicola, C.godetiae, C.juglandicola, C.karsti, C.mengyinense, C.pandanicola, C.peakense and C.siamense) and a novel species (C.chinensis sp. nov.) were identified. The species distribution revealed regional prevalence as follows: C.mengyinense was the most dominant species in Gansu, C.mengyinense and C.siamense in Shandong, C.chinensis in Beijing, C.pandanicola in Shaanxi and C.godetiae in Yunnan. Colletotrichumsiamense was the sole species isolated in Sichuan and Xinjiang Provinces. Koch's postulates were fulfilled, demonstrating that all 12 species cause anthracnose on walnut. This is the first report of C.boninense, C.citrulli and C.karsti as pathogens of walnut anthracnose worldwide.

Effectiveness of Green Cupric Oxide Nanoparticles for Walnut Storage Pest Management.

Walnut yield and quality are often affected by beetle infestations, particularly those caused by Carpophilus truncatus (Murray) (Nitidulidae) and Oryzaephilus mercator (L.) (Silvanidae). Beetle damage exposes walnuts to microbial food spoilers such as Fusarium species. Insecticides currently used for beetle control are environmentally unfriendly. This work explored a green synthesis approach for copper oxide nanoparticles (CuO-NPs) in a basic medium at 30°C by hydrolates, aqueous extracts obtained from Lippia integrifolia and Pimpinella anisum, denoted as CuO-I and CuO-A, respectively. Characterization through XRD, FT-IR, Raman, UV-visible absorbance, and AFM techniques indicated that CuO-A and CuO-I have a size ranging from 2-10 nm in height. The antifungal assay showed that both have a similar efficacy (MID = 320 µg), 3-fold stronger than CuO- NPs obtained in absence of hydrolates (denoted CuO-W) (MID = 960 µg), with the broadest inhibitory halos (ID = 126-128 mm) observed for CuO-A. Insecticidal activity of CuO-NPs showed a concentration-dependent behavior, with CuO-I showing an effect comparable to that of diatomaceous earth. SEM images confirmed the adhesion of nanoparticles to insect surfaces, which could induce oxygen deprivation and disruption of metabolic processes. Both CuO-A and CuO-I are promising for their use in integrated pest control in walnut storage.

Quality characteristics and fibrous structure formation mechanism of walnut protein and wheat gluten meat analogues during high-moisture extrusion cooking process.

Blending two or more materials to create better high-moisture meat analogues has been actively studied in the food science and technology field. Walnut protein is a high-quality plant-based protein resource, yet its full potential remains underexploited. Thus, this study focused on exploring the quality characteristics and fibrous structure formation mechanism of walnut protein (WP) and wheat gluten (WG) meat analogues during high-moisture extrusion cooking process. Results showed that the optimized WP and WG-blended high-moisture meat analogues exhibited a more pronounced anisotropic and oriented fibrous structure. The blending of WP and WG can protect the molecular chains from the thermal transition, and promote the aggregation of protein molecules mainly by enhancing the interaction between hydrophobic interactions and hydrogen bonds, increasing the apparent viscosity and forming protein subunits with larger molecular weights (>100 kDa) to stabilize the newly formed conformation. Additionally, the content of α-helix was the highest among the secondary structures. This study provides a theoretical basis for the application of WG and WP to produce HMMAs with rich fibrous structures.

JrPPO1/2 play distinct roles in regulating walnut fruit browning by different spatiotemporal expression and enzymatic characteristics.

Polyphenol oxidase (PPO) activity drives walnut fruit browning, but the roles of its only two-family genes, JrPPO1 and JrPPO2, remain unclear. This study explores the spatiotemporal expression and enzymatic characteristics of JrPPO1 and JrPPO2 in walnut. Treatment with the PPO activator CuSO4 and H2O2 accelerated fruit browning and up-regulated JrPPO1/2 expression, whereas treatment with the PPO inhibitor ascorbic acid delayed browning, down-regulating JrPPO1 and up-regulating JrPPO2 expression. Compared to mJrPPO1, mJrPPO2 can exhibited better enzyme activity at higher temperatures (47 °C) and in more acidic environments (pH 4.25). mJrPPO2 exhibited a higher substrate specificity over mJrPPO1, and the preferred substrates are catechol, chlorogenic acid, and epicatechin. Additionally, mJrPPO2 adapted better to low concentration of oxygen (as low as 1.0% O2) and slightly elevated CO2 levels compared to mJrPPO1. Subcellular localization and spatiotemporal expression patterns showed that JrPPO1 is only expressed in green tissues and located in chloroplasts, while JrPPO2 is also located in chloroplasts, partly associated with membranes, and is expressed in both green and non-green tissues. Silencing JrPPO1/2 with virus-induced gene silencing (VIGS) reduced fruit browning, maintained higher total phenols, and decreased MDA production. Notably, silencing JrPPO1 had a greater impact on browning than JrPPO2, indicating JrPPO1's greater contribution to PPO activity and fruit browning in walnut fruits. Consequently, JrPPO1 can be effectively regulated both at the molecular level and by manipulating environmental conditions, to achieve the objective of controlling fruit browning.