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.

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.

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.

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.

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.

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.

The identification and expression analysis of walnut Acyl-ACP thioesterases.

Walnuts (Juglans regia L.), renowned for their nutritional potency, are a rich source of unsaturated fatty acids. Their regular intake plays a pivotal role in health maintenance and recuperation from a myriad of ailments. Fatty acyl-acyl carrier protein thioesterases, which orchestrate the hydrolysis of acyl-ACP thioester bonds, thereby yielding fatty acids of varying chain lengths, are instrumental in augmenting plant fatty acid content and modulating the balance between saturated and unsaturated fatty acids. Despite some investigative efforts into the synthesis and metabolic pathways of fatty acids in walnuts, our comprehension of Fat in walnuts remains rudimentary. This research undertook a comprehensive characterization of the JrFat family, predicated on the complete genome sequence of walnuts, leading to the identification of 8 JrFat genes and an exploration of their protein physicochemical properties. Utilizing Arabidopsis and soybean Fat genes as outgroups, JrFat genes can be categorized into 5 distinct subgroups, three of which encompass a pair of homologous gene pairs. These genes have demonstrated remarkable conservation throughout the evolutionary process, with highly analogous conserved base sequences. The promoter region of JrFats genes predominantly harbors light response and plant hormone response regulatory elements, with no discernible disparity in promoter elements among different JrFats. Predictive analyses indicate that JrFats proteins engage extensively with walnut fatty acid synthesis and metabolism-associated proteins. qRT-PCR analysis reveals an initial surge in the expression of JrFats during the development of walnut kernels, which either stabilizes or diminishes following the hard core period. Homologous gene pairs exhibit analogous expression patterns, and the expression trajectory of JrFats aligns with the dynamic accumulation of fatty acids in kernels. The expression of JrFatA2 exhibits a strong correlation with the content of Alpha-linolenic acid, while the expression of JrFatB2 is inversely correlated with the content of two saturated fatty acids. Collectively, these findings enrich our understanding of fatty acid synthesis and metabolism in walnuts and furnish gene resources for enhancing the content and ratio of fatty acids in walnuts.

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.

Preparation and Properties of Walnut Protein Isolate-Whey Protein Isolate Nanoparticles Stabilizing High Internal Phase Pickering Emulsions.

To enhance the functional properties of walnut protein isolate (WalPI), hydrophilic whey protein isolate (WPI) was selected to formulate WalPI-WPI nanoparticles (nano-WalPI-WPI) via a pH cycling technique. These nano-WalPI-WPI particles were subsequently employed to stabilize high internal phase Pickering emulsions (HIPEs). By adjusting the mass ratio of WalPI to WPI from 9:1 to 1:1, the resultant nano-WalPI-WPI exhibited sizes ranging from 70.98 to 124.57 nm, with a polydispersity index of less than 0.326. When the mass ratio of WalPI to WPI was 7:3, there were significant enhancements in various functional properties: the solubility, denaturation peak temperature, emulsifying activity index, and emulsifying stability index increased by 6.09 times, 0.54 °C, 318.94 m2/g, and 552.95 min, respectively, and the surface hydrophobicity decreased by 59.23%, compared with that of WalPI nanoparticles (nano-WalPI), with the best overall performance. The nano-WalPI-WPI were held together by hydrophobic interactions, hydrogen bonding, and electrostatic forces, which preserved the intact primary structure and improved resistance to structural changes during the neutralization process. The HIPEs stabilized by nano-WalPI-WPI exhibited an average droplet size of less than 30 µm, with droplets uniformly dispersed and maintaining an intact spherical structure, demonstrating superior storage stability. All HIPEs exhibited pseudoplastic behavior with good thixotropic properties. This study provides a theoretical foundation for enhancing the functional properties of hydrophobic proteins and introduces a novel approach for constructing emulsion systems stabilized by composite proteins as emulsifiers.

Walnut meal improves meat quality by modulating intestinal microbes in white feather broilers.

Improving the number of amino acids and unsaturated fatty acids in the diet is a good way to raise the quality of the meat. Currently, most research on the quality of broiler meat focuses on genetic traits; nevertheless, it is unclear how meat quality is regulated. This experiment was conducted to investigate the effects of different supplemental levels of walnut meal (WM) on growth performance, amino acid and fatty acid composition, microbial composition, and meat quality of white feather broilers. 1 week old white feather broilers (n = 120; Body weight 83.76 ± 2.32 g), were randomly divided into 3 treatments and 4 replicates. Walnut meal of basic diet (CK), 5 %(WM-L) and 10 %(WM-H) were added to the diets of white feather broilers, respectively. The results showed that walnut meal could increase L* 24 h (24 h brightness) of breast muscle of white feathered broilers (p < 0.05). The amount of essential amino acids (e.g., isoleucine, methionine, leucine, tryptophan, and phenylalanine), umami amino taste acids (glutamic acid), and PUFA/SFA (polyunsaturated fatty acid) (n-3PUFA and n-6 PUFA) in breast muscle increased as the dose was increased. Furthermore, walnut meal regulated amino acid flavour metabolism by increasing the relative abundance of Bacteroides, bifidobacterium, and enterococcus faecalis, according to 16S rRNA sequencing and functional prediction analysis. The correlation showed that amino acid and fatty acid composition was one of the key factors affecting pH value, meat color and tenderness of chicken. In conclusion, dietary addition of walnut meal can increase the content of essential amino acids and unsaturated fatty acids and the relative abundance of beneficial bacteria of broilers, which is of great significance for improving meat quality of white feather broilers.

Evaluation of the growth performance, meat quality, and gut microbiota of broilers fed diets containing walnut green husk extract.

This study was conducted to evaluate the effects of walnut green husk extract (WGHe) on the growth performance, meat quality, antioxidative status, gut morphology, and microbiota diversity of broilers. A total of 216 one-day-old broilers were divided into 4 groups, each consisting of 9 replicates (6 birds per replicate) as follows: 1) control group, basal diet; 2) antibiotic group, basal diet supplemented with enduracidin and colistin sulfate; 3) low-dose group, basal diet supplemented with 5.0 g/kg WGHe; and 4) high-dose group, basal diet supplemented with 10.0 g/kg WGHe. The results revealed that the percentage of abdominal fat decreased, and the ratio of the duodenal villus length to crypt depth (V/C), as well as the α-diversity of the ileal microbiota, increased with 10.0 g/kg WGHe supplementation (P < 0.05). The shear force of the breast muscle and plasma malondialdehyde (MDA) concentration decreased, whereas the plasma peroxidase (POD) activity, Trolox equivalent antioxidant capacity (TEAC), and jejunal villus length increased in response to WGHe supplementation (P < 0.05). Compared with the antibiotic diet, the addition of 5.0 g/kg WGHe resulted in a significant increase in the relative abundances of Candidatus Arthromitus, Eubacterium coprostanoligenes, and Ruminococcaceae UCG-014 (P < 0.01). Furthermore, the addition of 10.0 g/kg WGHe increased the relative abundances of Candidatus Arthromitus and Lachnoclostridium, whereas the relative abundance of unidentified Chloroplast decreased (P < 0.05). In conclusion, dietary supplementation with 10.0 g/kg WGHe is advantageous for intestinal health, meat quality, and antioxidant status in broilers, suggesting its potential as a functional additive in poultry production.

Caveolin Regulates the Transport Mechanism of the Walnut-Derived Peptide EVSGPGYSPN to Penetrate the Blood-Brain Barrier.

Bioactive peptides, derived from short protein fragments, are recognized for their neuroprotective properties and potential therapeutic applications in treating central nervous system (CNS) diseases. However, a significant challenge for these peptides is their ability to penetrate the blood-brain barrier (BBB). EVSGPGYSPN (EV-10) peptide, a walnut-derived peptide, has demonstrated promising neuroprotective effects in vivo. This study aimed to investigate the transportability of EV-10 across the BBB, explore its capacity to penetrate this barrier, and elucidate the regulatory mechanisms underlying peptide-induced cellular internalization and transport pathways within the BBB. The results indicated that at a concentration of 100 µM and osmotic time of 4 h, the apparent permeability coefficient of EV-10 was Papp = 8.52166 ± 0.58 × 10-6 cm/s. The penetration efficiency of EV-10 was influenced by time, concentration, and temperature. Utilizing Western blot analysis, immunofluorescence, and flow cytometry, in conjunction with the caveolin (Cav)-specific inhibitor M-ß-CD, we confirmed that EV-10 undergoes transcellular transport through a Cav-dependent endocytosis pathway. Notably, the tight junction proteins ZO-1, occludin, and claudin-5 were not disrupted by EV-10. Throughout its transport, EV-10 was localized within the mitochondria, Golgi apparatus, endoplasmic reticulum, lysosomes, endosomes, and cell membranes. Moreover, Cav-1 overexpression facilitated the release of EV-10 from lysosomes. Evidence of EV-10 accumulation was observed in mouse brains using brain slice scans. This study is the first to demonstrate that Cav-1 can facilitate the targeted delivery of walnut-derived peptide to the brain, laying a foundation for the development of functional foods aimed at CNS disease intervention.

Physicochemical characterization of antioxidant film based on ternary blend of chitosan and Tulsi-Ajwain essential oil for preserving walnut.

This study focuses on changes in the physiochemical properties of chitosan film when incorporated with a blend of essential oils of Tulsi and Ajwain. The essential oil blend-loaded films showed a decrement in transparency. Tulsi essential oil decreased the moisture content, swelling capacity, and water solubility. However, adding Ajwain along with Tulsi essential oil led to a significant increase in these properties. Meanwhile, the water vapor transmission rate didn't change significantly due to non-polar constituents in Tulsi essential oil, except when only Ajwain essential oil was present. The mechanical properties showed that the tensile strength of films increased with the addition of Tulsi essential oil (14.95 MPa to 31.27 MPa) but decreased further with increasing Ajwain oil concentration in films (32.13 MPa to 15.89 MPa). On the other hand, an increment in percent elongation at break (8.26 % to 24.02 %) was observed due to the excellent plasticization effect of Ajwain essential oil. Antioxidant activity was observed for the Tulsi essential oil-containing films and increased significantly with adding Ajwain essential oil. Finally, walnuts were packed in the active film. The active film showed better antioxidant activity against the oxidation of oil in walnuts, which the FTIR of the packed product confirmed.

A new approach for artificial pollination in walnut trees: AirPoll.

Pollination is the first step in the plant's fruit development. Therefore, fruit setting does not occur without pollination. Some problems encountered in natural pollination cause pollination not to be achieved as desired and cause significant losses in yield and fruit quality. Artificial pollination applications with drones are the best way to solve these problems. In this study, the AirPoll artificial pollination machine, which performs artificial pollination through the air using drone technology, was developed and the operating success of the machine was tested in walnut gardens. In the experiment gardens, female flowers on 18 branches of 5 trees each in the artificially pollinated area with a drone and in the control area were marked with colored strings. Control trees were selected from a distance that would not be possible to transport pollen with a drone. As a result of the study carried out in 2020 and 2021, the average fruit setting rate in trees pollinated by drone was determined as 94.61%. In control trees, 32.33% fruit setting was achieved. Thus, it was determined that the productivity increase in artificial pollination with AirPoll was 62.28%. In addition, in the study, Computational Fluid Dynamics (CFD) simulation analysis was performed using ANSYS Fluent 2024 R1 software to predict the downward air flow and pollen distribution in the walnut tree crown. The analysis was carried out in 680 iterations using drone propellers at a rotation speed of 4500 rpm, 4 m/s airflow and a k-w viscous model. In the analysis, it was observed that the pollen was distributed homogeneously with the determined height and the created artificial pollination environment. Based on the results obtained from the simulations, a convergence criterion of 5e-3 for continuity and 1e-6 for speed, k, w was determined. Considering all the results, the ease of use of the developed AirPoll artificial pollination machine and the successful results obtained in field trials reveal the effectiveness of the AirPoll artificial pollination machine.

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.

Protective effect of walnut active peptide against dextran sulfate sodium-induced colitis in mice based on untargeted metabolomics.

Inflammatory bowel disease (IBD) is a chronic condition characterized by inflammation of the digestive tract, whose exact cause remains unknown, and its prevalence is on the rise. This study investigated the effects of a walnut-derived peptide LPLLR (LP-5) on intestinal inflammation and metabolism in IBD mice. Metabolomics revealed that LP-5 regulated the levels of metabolites, such as thalsimidine, fumagillin, and geniposide, and LP-5 could regulate several signaling pathways, such as protein digestion and absorption, aminoacyl-tRNA biosynthesis, and ABC transporters. Additionally, LP-5 alleviated dextran sulfate sodium (DSS)-induced colitis by modulating autophagy and inflammasome pathways. Western blotting demonstrated that LP-5 reduced the expressions of NLRP3, Caspase-1, ASC and IL-1ß, and increased the expressions of Beclin-1 and LC3-II/LC3-I, corresponding to activation of the AMPK/mTOR/ULK1 pathway. These findings suggested that LP-5 activated autophagy in vivo to suppress inflammation and modulate metabolic substances, highlighting potential implications for gut health and the development of functional foods containing LP-5.

Discovering the Ellagitannin Landscape of Dried Walnut Shells by Liquid Chromatography with Tandem Mass Spectrometry.

Walnut shells, often discarded as waste, hold hidden potential as a source of ellagitannins (ETs), compounds known for their promising antioxidant properties and health benefits. This study employed reversed-phase liquid chromatography (RPLC) coupled with Orbitrap-based high-resolution mass spectrometry (HRMS) via electrospray ionization (ESI) in negative polarity to investigate the ET profile in extracts of dried powdered walnut shells. Several compounds belonging to various ET families were successfully identified as deprotonated molecules ([M - H]-) and characterized, including mono-, di-, tri-, tetra-, and pentagalloyl glucopyranoses, as well as ETs containing the hexahydroxydiphenoyl (HHDP) group. Characteristic product ions were identified in HR tandem MS spectra and employed to recognize the ET landscape. Analysis revealed a complex picture with more than 10 isomers identified in some cases. However, the structural similarity and limitations in MS/MS data hindered the definitive identification of all isomers. Characterization of ETs featuring HHDP groups also remained challenging. Despite these restraints, the estimated total content of ETs suggests potential application in the food, pharmaceutical, and cosmetic industries of those extracts. These findings indicate that walnut shells can be considered a sustainable source of health-promoting compounds, contributing to a greener economy.

An innovative walnut cracker: Self-grading and multi-station extrusion mechanism with high efficiency.

The walnut cracking process is the most critical and delicate step for achieving high-quality kernels. The traditional method for cracking (manually) is labor-intensive, time-consuming, and tedious. The existing cracking approaches are low production efficiency and serious walnut kernel breakage. Increasing cracking efficiency with minimum kernel breakage has been a challenging issue in the preliminary processing of walnuts. Therefore, this study develops an innovative walnut cracker with self-grading and multi-station extrusion, combined with theoretical investigation and experiment verification. First, a statistical analysis of walnut physical properties was conducted, including dimensions, shell thickness as well as shape characteristics. The mechanical properties of walnut cracking were examined by a series of experiments. Based on mechanical theory, a grading mechanism was designed for preliminary processing before walnut cracking. Then a shaftless screw conveying mechanism and an extrusion cracking mechanism were developed. To evaluate the cracker's performance, a comprehensive examination was carried out. The experiments yielded impressive results, with a grading rate of 87.3%, a shell-breaking rate of 91.50%, and a kernel-exposed rate of 84.72%. These outcomes signify a substantial improvement in production efficiency while minimizing kernel breakage. The developed walnut cracker plays a crucial role in walnut processing and kernel extraction, thereby elevating economic value. PRACTICAL APPLICATION: A self-grading multi-station extrusion walnut cracker is developed, which includes a grading mechanism with a shaftless screw conveyor and a grid-type trommel screen for conveying and classifying walnuts. This cracker can adapt to different walnut varieties by changing the gap-adjusting guide to control the breaking gap. Compared to similar extrusion-type walnut crackers, the developed cracker not only incorporates preliminary classification but also exhibits superior performance. HIGHLIGHTS: A novel multi-station extrusion mechanism for walnuts cracking is developed. The cracker can accommodate various walnut sizes for self-grading and screening. The design with semi-arc plates converts extrusion force into alternating stress. The shell-breaking rate and kernel-exposed rate achieves 91.50% and 84.72%.

Robust peroxidase from Bacillus mojavensis TH309: Immobilization on walnut shell hydrochar and evaluation of its potential in dye decolorization.

Peroxidases have received considerable attention as a cost-effective and environmentally friendly catalyst for bioremediation. Their rapid activity loss under harsh environmental conditions and inability to be used repetitively limit their exploitation in real-world wastewater treatment. First, a peroxidase was produced extracellularly by Bacillus mojavensis TH309 and purified 8.12-fold with a final yield of 47.10 % using Sephadex G-100 superfine resin. The pure peroxidase (BmPer) possessed a relatively low molecular weight of ∼21 kDa and was active against L-DOPA on acrylamide gel after electrophoresis. BmPer was immobilized by adsorption functionalized walnut shell hydrochar (WsH) with 61.99 ± 1.34 % efficiency and 37.07 ± 4.16 % activity loss. BmPer and its immobilized form (WsH-BmPer) exhibited maximum activity at 50 °C and pH 9. WsH-BmPer exhibited 3.23-, 2.37-, 1.65-, and 2.25-fold longer half-life than BmPer at 50, 60, 70, and 80 °C, respectively. Immobilization significantly enhanced the stability of the enzyme under acidic conditions. BmPer and WsH-BmPer showed maximal activity in the presence of 1 % salt and retained more than 85 % of their activity even after pre-incubation with 2.5 M salt for 60 min at 50 °C. Their catalytic efficiency was significantly stimulated by pre-incubation with Triton X-100 (1 mM), Tween20 (1 mM), and Mg2+ (1 and 10 mM). Immobilization strongly reduced the loss of activity caused by inhibitors including Ba2+, Hg2+, and Cu2+. Moreover, both forms of the enzyme were compatible with solvents. The Michaelis constant (Km) values of BmPer and WsH-BmPer were 0.88 and 2.66 mM for 2,4 DCP, respectively. WsH-BmPer peroxidase maintained about 82 % and 85 % of its activity when stored at 4 °C for 30 days and reused for up to 10 cycles, respectively. Furthermore, it decolorized Cibacron red (CR), Poly R-478 (PR), Remazol Brilliant Blue R (RBBR), and Methyl red (MR) dyes by 60.13 %, 91.34 %, 86.41 %, and 50.51 % within 60 min, respectively.

Preparation, characterization and microencapsulation of walnut (Juglans regia L.) peptides-zinc chelate.

In this research, a novel kind of walnut (Juglans regia L.) peptides-zinc (Zn-WPs) chelate was obtained using the mass ratio of the walnut peptides (WPs) to ZnSO4.7H2O of 3.5:1 at pH 8.5 and 50°C for 84 min, with the chelation rate of 84.5%. In comparison to walnut peptides (WPs), the contents of aspartic acid and glutamic acid in Zn-WPs chelate are approximately 27%, indicating that hydrophilic amino acids predominantly bind with walnut peptides. Following chelation with zinc ions, the ultraviolet-visible (UV) characteristic absorption peak shifted from 213 nm to 210 nm, while the average particle size of the chelate increased to 8.0 ± 0.14 µm, presenting a loose spherical structure under scanning electron microscopy. These findings suggest the formation of new substances. Fourier-transform infrared spectroscopy (FTIR) revealed carboxyl, amino, and peptide bonds as the chelation sites of WPs and zinc. The IC50 of walnut peptides-zinc (Zn-WPs) chelate is 2.91 mg/mL, indicative of a favorable DPPH radical scavenging rate. Furthermore, Zn-WPs chelate microcapsules were produced via the spray drying method, achieving an encapsulation rate of 75.67 ± 0.83% under optimal conditions. These microcapsules demonstrate robust stability across diverse environmental conditions. This study underscores the potential of Zn-WPs and its chelate microcapsules to enhance stability and bioactivity under varying circumstances. PRACTICAL APPLICATION: In this study, a new walnut peptide-zinc (Zn-WPs) chelate was prepared. The presence of zinc ions changes the structure and properties of walnut peptides and improves its stability. The production of Zn-WPs chelate microcapsules enables Zn-WPs to have strong in vitro stability under different pH and simulated gastrointestinal digestion conditions. These results provide novel insights for developing the walnut peptides as bioactive ingredients in functional foods.