Walnut Protein Research Articles

Walnut pellicle phenolics greatly influence the extraction and structural properties of walnut protein isolates

This study investigated the effects of walnut phenolics and extraction methods on the composition and structural properties of walnut protein isolates (WPIs). Fluorescence quenching experiments showed that walnut phenolics could bind to walnut globulins, albumins, and glutelins with apparent affinity constants of 5.49 × 104 M−1, 1.71 × 104 M−1, and 3.10 × 104 M−1, respectively. However, the UV turbidity and dynamic light scattering (DLS) measurements indicated that phenolics could lead to the severe precipitation of globulins and albumins but not glutelins. The removal of pellicles could significantly increase the yield rate of salt-soluble globulins to approximately 72.8%. Furthermore, salt- and alkaline-extraction methods could produce comparable WPIs yields when using pellicle-free walnut kernels. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and size-exclusive chromatography indicated that the major protein compositions of the salt- and alkaline-extracted WPIs from pellicle-free walnut kernels were similar, while alkaline-extracted WPIs from kernels with pellicles exhibited phenolic-induced protein aggregation. Fourier transform infrared (FTIR) spectroscopy indicated that WPIs produced from kernels with pellicles contained more α-helix and less β-sheet structures than WPIs produced from pellicle-free kernels. These results confirm that walnut pellicle phenolics and the extraction methods could greatly influence the composition and structural properties of WPIs.

Separation, identification and molecular binding mechanism of dipeptidyl peptidase IV inhibitory peptides derived from walnut (Juglans regia L.) protein

Walnut protein was hydrolyzed with different proteases to evaluate the hydrolytic efficiency and dipeptidyl peptidase IV (DPP-IV) inhibitory activity in vitro. All of walnut protein hydrolysates (WPHs) exhibited DPP-IV inhibitory activity and Alcalase-derived hydrolysate (WPH-Alc) with better DPP-IV inhibitory activity of 33.90% (at 0.50 mg/mL) was subsequently separated by ultrafiltration and cation exchange chromatography on a SP Sephadex C-25 column. The results showed that fractions with lower molecular weight and higher basic amino acid residues possessed stronger DPP-IV inhibitory activity. Comparably, the obtained fraction B with the yield of 19.80% had the highest DPP-IV inhibitory activity of 76.19% at 0.25 mg/mL. Moreover, nine novel DPP-IV inhibitory peptides were identified using MALDI-TOF/TOF-MS. Molecular docking revealed the peptides could interact with DPP-IV through hydrogen bonds, salt bridges, hydrophobic interactions, π-cation bonds and π-π bonds. The walnut DPP-IV inhibitory peptides showed better stability with heating treatment, pH treatment, or in vitro gastrointestinal digestion.

Potential anti-inflammatory activity of walnut protein derived peptide leucine-proline-phenylalanine in lipopolysaccharides-irritated RAW264.7 cells

ABSTRACTThe purpose of this study was to explore the anti-inflammatory mechanism of walnut peptide LPF in the LPS-induced RAW264.7 cells model. The walnut peptide LPF down-regulated the phagocytic ability and the production of nitric oxide (NO) induced by LPS-stimulated RAW264.7. Molecular docking analysis showed that the walnut peptide LPF can bind into the active pocket of inducible nitric oxide synthase (iNOS) with high affinity. Further, the mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and tumour necrosis factor-α (TNF-α) were suppressed by the walnut peptide LPF intervention. Later, we validated the under-expression of INOS protein by western blot. The decreased mRNA expression of IκB-α demonstrated the anti-inflammatory effects of the walnut peptide LPF were related to the regulation of the NF-κB signalling pathway. Our results suggest that the walnut peptide LPF has the potential to be developed as a functional food ingredient against inflammation.

Anti-osteoporosis effect and purification of peptides with high calcium-binding capacity from walnut protein hydrolysates.

The walnut protein hydrolysate (WPH) was prepared via simulated gastrointestinal digestion. The degree of hydrolysis (DH), amino acid composition, and relative molecular weight distribution of WPH were analyzed. The results showed that the DH of WPH was 11.6%, WPH was rich in Glu and Pro, and the relative average molecular weight of 572 Da accounted for 59.78%. The effects of WPH on osteoporosis were evaluated using a model of retinoic acid-induced osteoporosis rat. Treatment with WPH effectively increased the serum calcium and phosphorus contents, alleviated calcium loss, and reduced tartrate-resistant acid phosphate and alkaline phosphatase activities and bone gla protein content. WPH treatment significantly improved the biomechanical properties of the bone and increased the value of bone mineral density. In addition, WPH treatment improved the bone microstructure. WPH was isolated and purified by Sephadex G-25 gel filtration chromatography and semi-preparative reversed-phase high-performance liquid chromatography. A fraction with high calcium-binding activity was obtained and 15 peptides were identified.

The necessity of enzymatically hydrolyzing walnut protein to exert antihypertensive activity based on in vitro simulated digestion and in vivo verification.

Since not all proteins are suitable for preparing bioactive peptides by enzymatic degradation, the purpose of this study is to evaluate the necessity of walnut protein (WP) enzymolysis to exert its potential antihypertensive activity. Five proteases were used to hydrolyze WP to produce WP hydrolysate (WPH) enzymatically. The angiotensin-I-converting enzyme (ACE) inhibitory activity of WP and WPH before and after simulated digestion in vitro was measured, and the antihypertensive effect was evaluated in vivo. The results showed that after simulated digestion in vitro, the ACE inhibitory activity of WP digests (44.85%) was not significantly different from that of WPH digests (p > 0.05). In vivo experimental results showed that both WP and WPH had significant blood pressure lowering effects in the acute and long-term administrative experiments. The mechanism of its antihypertensive activities was regulating the balance of the renin-angiotensin-aldosterone system and the kallikrein-kinin system by inhibiting ACE activities in tissues and regulating the level of endothelium-derived vasoconstrictor factors and relaxing factors in serum. It seems unnecessary to carry out enzymatic hydrolysis to produce walnut peptides with antihypertensive activity.

Purification and identification of dual-enzyme hydrolysates obtained from defatted walnut and its antioxidant effects on d-galactose-induced aging mice

The purpose of this study was to explore the antioxidant activity in vivo of dual-enzyme hydrolysates obtained from defatted walnut, and to purify and identify it. The walnut protein hydrolysates (WPH) were obtained through two-step enzymatic hydrolysis of alkaline protease and trypsin. The effects of WPH on the oxidative stress-related biochemical indexes of d-galactose-treated mice were investigated. Next, WPH was passed through a Sephadex G-25 gel chromatography, anion exchange chromatography and Sephadex G-15 gel chromatography. Subsequently, peptides in the high antioxidant activity fraction were identified by RP-nano-HPLC–MS/MS. Finally, 14 potential peptides of length 11 AAs to 21 AAs with antioxidant activity were identified from WPH.

Nutritional and sensory evaluation of wheat-maize cookies enriched with African walnut (Tetracarpidium conophorum) seed protein isolate

The study examined the nutritional composition and sensory attributes of cookie. In this study, blends of commercial wheat (CWF), yellow maize flours (YMF) and African walnut protein isolate (AWPI) were optimized for cookie production. The cookies were studied in a completely randomized design and assessment of the proximate composition was conducted. The best cookie samples with high protein and fiber, low fat and highest rating in sensory attributes were selected for the determination of vitamins and mineral composition. The result revealed that the control cookie was highest in moisture, fat and carbohydrate but lower in protein, ash and fibre. Enriched cookie 36.69g CWF, 39.57g YMF, 23.74g AWPI and 42.86g CWF, 42.86g YMF, 14.29g AWPI were highest in protein and fibre, respectively while 55.56g CWF, 33.33g YMF, 11.11g AWPI was low in fat. Overall acceptability was highest in control cookie followed by enriched cookie 55.56g CWF, 33.33g YMF, 11.11g AWPI. The vitamin composition of the selected enriched samples show that sample 42.86g CWF, 42.86g YMF, 14.29g AWPI had the highest value of 2.53 mg/100 g in ascorbic acid; sample 55.56g CWF, 33.33g YMF, 11.11g AWPI scored the highest value of 0.14, 0.07 and 1.33 mg /100 g in thiamine, riboflavin and niacin respectively, while sample 36.69g CWF, 39.57g YMF, 23.74g AWPI scored highest in vitamin A (276.67 mg/100g). The cookie sample 36.69g CWF, 39.57g YMF, 23.74g AWPI shows the highest values of 130.00, 0.043, 9.77, 333.33 and 85.00 for calcium, manganese, iron, sodium and phosphorus, respectively. The study recommends the use of flour proportion in sample 55.56g CWF, 33.33g YMF, 11.11g AWPI as an improvement of the nutritional and sensory attributes of enriched cookie.

Limited hydrolysis of dehulled walnut (Juglans regia L.) proteins using trypsin: Functional properties and structural characteristics

The effect of limited trypsin hydrolysis on the functional properties and structural characteristics of dehulled walnut proteins (DWP) were studied. The degree of hydrolysis (DH) of DWP was set at 1, 3 and 7.1%. Solubility was higher in all hydrolysates than the native protein, 7.1% DH showed the best protein solubility at 74.8% at pH 7. All hydrolysates showed better functional properties than DWP, including emulsifying properties, foaming capacity and stability, but no improvement of water absorption capacity was observed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the reduction in the molecular weight and the presence of protein bands having an MW < 30 kDa. The protein hydrolysates with higher DH showed higher surface hydrophobicity and intrinsic fluorescence intensity. Intrinsic fluorescence indicated that changes occurred in the tertiary conformation of DWP with enzymatic hydrolysis. Emulsions prepared with hydrolysates were more stable with NaCl addition (0–50 mM) and thermal processing (30–50 °C). The above results indicated that limited enzymatic hydrolysis is a promising approach to improving DWP's functional properties and highlighting the potential use of DWP hydrolysates in the food industry.

Characterizations of a Food Decapeptide Chelating with Zn(II)

Walnut proteins and peptides have been reported to have great zinc-carrying activity. In this study, the decapeptide EPNGLLLPQY (WP-10) derived from walnut protein has been prepared to figure out zinc binding mechanisms and structure–activity relationships. The space-conformation of this peptide has converted after being chelated with Zn(II). As to secondary structure, the β-sheet structure of the peptide turned into α-helix and β-turn structure. According to the atomic absorption spectra analysis, one peptide could bind with one Zn(II) via four different binding sites. The signals of different groups in infrared spectroscopy have shifted before and after chelation, and the major functional groups of this peptide involved in chelation are –NH2 and –C=O. GLU1, PRO2, ASP3, GLU4, and LEU6, and the molecular docking proved their participation in the chelating with Zn(II).

Separation and identification of ACE inhibitory peptides from defatted walnut meal

The peptide obtained from walnut protein enzymatic hydrolysate had high ACE inhibitory activity. Through centrifugation, ultrafiltration, gel chromatography and high performance liquid chromatography (HPLC), the walnut oligo-peptide, which was denoted as P4-c with high ACE inhibitory activity was obtained. Three novel ACE inhibitory peptides, GVVPHN, EHSLDPLK and KTLLNFGPN, were identified from P4-c by high-resolution ion mobility mass spectrometry (IM-MS). The three novel ACE inhibitory peptides possessed good ACE inhibitory activity and the fraction GVVPHN had high ACE inhibitory activity with 27.3 µmol L−1 of IC50. The molar percentage of hydrophobic amino acids in the peptide GVVPHN was 50% while the hydrophobic amino acid contents of EHSLDPLK and KTLLNFGPN were 37.5% and 44.4%, respectively, which indicates ACE inhibitory activities were hydrophobicity of amino acid-dependent.

Electrospray Production of Curcumin-walnut Protein Nanoparticles

Electrospray technique was applied to produce nanoparticles (NPs) from walnut protein isolate (WPI) in order to encapsulate curcumin. The optimized condition for the production of the WPI NPs was WPI concentration of 3.5%, a solvent mixture of deionized water: 2-propanol (2:1, v/v), electrospraying at a fixed distance, voltage and flow rate including 17.5 cm, 20 kV and 0.09 mL/h, respectively. The ratio of curcumin to WPI was 1:100 (w/w), and the encapsulation efficiency was around 61.5%. The addition of curcumin to WPI solution affected the size, form and surface properties of the electrosprayed NPs. FESEM, HRTEM, AFM, XRD, FT-IR and fluorescence spectroscopy, were applied for the characterization of the produced NPs via electrospraying. The in vitro study revealed that release of curcumin was limited in the stomach due to the protective effect of WPI NPs. In the small intestine, WPI particles were hydrolyzed and curcumin was released. Furthermore, the antioxidant activity of curcumin-WPI NPs after in vitro digestion was significantly higher than WPI NPs due to the bioactive peptides released from WPI NPs. It can be concluded that WPI NPs could act as a unique food-grade carrier to boost the solubility of curcumin.

CHELATION of WALNUT PROTEIN PEPTIDE with CALCIUM and CALCIUM ABSORPTION PROMOTION in VIVO

In this study, walnut meal was used to prepare walnut protein. Papain was used to hydrolyze walnut protein to obtain active protein peptides. Fourier transform infrared spectroscopy(FTIR), scanning electron microscopy(SEM), and energy dispersive spectroscopy(EDS) were used to study the chelating ability of walnut protein peptides and Ca2+ and complete structural characterization. A Caco-2 monolayer model and a rat calcium deficiency model were established to study the calcium absorption capacity and increase bone mineral density(BMD) of walnut protein peptide chelated calcium. The results showed that the appearance and internal structure of walnut peptides had changed after chelated with calcium. Infrared spectroscopy analysis indicated that walnut peptides may participate in the coordination of Ca2+ to form calcium chelating peptides through groups such as -COOH, -OH, and -NH2. Caco-2 monolayer model experiments show that walnut peptides-Ca can promote the absorption and transport of calcium by regulating the TRPV6 signaling pathway. Calcium deficient rat model experiments show that the combination of walnut peptides and calcium can effectively increase BMD and improve bone formation, thereby preventing calcium deficiency. Walnut protein peptide chelated calcium could be a potential new product for calcium supplements.

Characterization and antioxidant properties of Manchurian walnut meal hydrolysates after calcium chelation

A Manchurian walnut protein hydrolysates-calcium (MWPH-Ca) was prepared its characteristics and antioxidant activities after in vitro simulated gastrointestinal digestion were investigated. The optimal conditions for the chelation reaction were peptide to calcium ratio of 3:1, pH of 8, chelation time of 40 min, and chelation temperature of 45 °C, and the final chelation rate was 67.2%. The peptides were agglomerated after chelation, as the particle size increased. The FTIR results showed that the main sites of chelation are amino nitrogen atoms, carboxyl groups and the CO of the peptide bond which has a negative dipole. The MWPH-Ca generally maintained good solubility in the gastrointestinal environment, which could improve the calcium absorption. In addition, the MWPH-Ca still retained antioxidant activity and was stronger than that of the MWPH after simulated digestion.

Effect of Peroxyl‐Radicals‐Induced Oxidative Modification in the Physicochemical and Emulsifying Properties of Walnut Protein

AbstractIn this paper, the effects of peroxyl radical oxidation on the physicochemical and functional properties of walnut protein were investigated. Walnut protein isolate (WPI) containing 2,2′‐azobis(2‐amidinopropane) dihydrochloride (AAPH) was oxidatively stressed under aerobic conditions in peroxyl radical‐generating media. Incubation of walnut protein with increasing concentration of AAPH resulted in gradual carbonyl generation and free sulfhydryl group degradation. The results of surface hydrophobicity implied that oxidation leads to protein aggregation thus decreasing protein solubility in an AAPH concentration‐dependent manner (P &lt; 0.05). Emulsifying properties exhibited a significant increase (P &lt; 0.05) at AAPH concentrations up to 0.2 mM and higher AAPH concentrations reduced the emulsifying capacity of WPI. These results indicate that appropriate oxidation is helpful to improve the emulsifying properties of protein, and with an increase of oxidation degree, emulsifying functional will damage.

Physicochemical and bio-functional properties of walnut proteins as affected by trypsin-mediated hydrolysis

The walnut proteins were trypsin-hydrolyzed for 0, 15, 30, 60 and 120 min and the effects of hydrolysis on the structural, functional, antioxidant, bioactive-loading, and angiotensin converting enzyme (ACE)-inhibitory properties of walnut protein were studied. The trypsin-mediated hydrolysis of walnut proteins was monitored using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an O-phthaldialdehyde spectrophotometric assay. The unfolding of walnut protein structure during the hydrolysis process was confirmed using intrinsic fluorescence spectroscopy which increased the surface hydrophobicity of protein. The solubility of walnut protein was increased from 17 to 63% after 2 h of trypsin-mediated hydrolysis. The walnut protein hydrolysates showed higher surface activity compared to the native non-hydrolyzed protein, which significantly improved their foaming and emulsifying properties. The hydrolysates also showed significant enhanced antioxidant activity, namely, ABTS radical quenching activity and reducing power due to the release of active antioxidant sequences during the enzymatic hydrolysis. The ability of walnut proteins to inhibit the activity of ACE and their capacity to load hydrophobic bioactive molecules including curcumin and quercetin were also considerably improved by treating with trypsin. The results of this study suggested that the trypsin-mediated hydrolysis could be used as an efficient technology to produce bioactive multi-functional hydrolysates from walnut proteins.

Anti-diabetic effect by walnut (Juglans mandshurica Maxim.)-derived peptide LPLLR through inhibiting α-glucosidase and α-amylase, and alleviating insulin resistance of hepatic HepG2 cells

Abstract In the present study, a novel pentapeptide LPLLR (Leu-Pro-Leu-Leu-Arg) was purified and identified from walnut protein hydrolysates (3–10 kDa) by SEC, RP-HPLC, and LC-MS/MS. Its inhibitory actions against α-glucosidase and α-amylase were evaluated. Then its beneficial effects on glucose metabolism and the underlying molecular mechanism were determined in high glucose-induced insulin resistant (IR) hepatic HepG2 cells. Results demonstrated that LPLLR could inhibit both α-glucosidase and α-amylase, and the inhibition rate reached maximum of 50.12% and 39.08%, respectively, at concentration of 2000 μM. Moreover, LPLLR (100 and 200 μM) elevated the phosphorylation levels of IRS-1, PI3K, Akt, AMPK and GSK3β, as well as the expression levels of GS and GLUT4, while reduced the expression levels of G6Pase and PEPCK. These data revealed the mechanism of LPLLR in improving hepatic IR, which increased glycogen synthesis and glucose uptake, as well as decreased gluconeogenesis via activating the IRS-1/PI3K/Akt and AMPK signal pathways.

Updated population minimal eliciting dose distributions for use in risk assessment of 14 priority food allergens

Food allergy and allergen management are important global public health issues. In 2011, the first iteration of our allergen threshold database (ATDB) was established based on individual NOAELs and LOAELs from oral food challenge in roughly 1750 allergic individuals. Population minimal eliciting dose (EDp) distributions based on this dataset were published for 11 allergenic foods in 2014. Systematic data collection has continued (2011–2018) and the dataset now contains over 3400 data points. The current study provides new and updated EDp values for 14 allergenic foods and incorporates a newly developed Stacked Model Averaging statistical method for interval-censored data. ED01 and ED05 values, the doses at which 1%, and respectively 5%, of the respective allergic population would be predicted to experience any objective allergic reaction were determined. The 14 allergenic foods were cashew, celery, egg, fish, hazelnut, lupine, milk, mustard, peanut, sesame, shrimp (for crustacean shellfish), soy, walnut, and wheat. Updated ED01 estimates ranged between 0.03 mg for walnut protein and 26.2 mg for shrimp protein. ED05 estimates ranged between 0.4 mg for mustard protein and 280 mg for shrimp protein. The ED01 and ED05 values presented here are valuable in the risk assessment and subsequent risk management of allergenic foods.

Bioactive Peptides from Walnut Residue Protein.

Walnut residue is a kind of high-quality plant protein resource. The bioactive peptide prepared from walnut residue has excellent health care functions such as antioxidation and antihypertensive activity, but at present, walnut residue is often regarded as waste or low value feed, fertilizer and other materials. The uneconomical use of walnut residue has hindered the development of the walnut industry to some extent. Effective utilization of walnut residue protein to develop bioactive peptides and other products is of great significance to realize the comprehensive utilization of walnut residue, improve the added value of by-products, and change the current low utilization rate of walnut residue. In this paper, the preparation, purification and structure identification of walnut protein bioactive peptides are reviewed, and different functional walnut active peptides (WBPs) are introduced. The potential effects of these bioactivities on human health and their different uses in food, medicine and other industries are discussed. The purpose is to provide reference information for the effective utilization of walnut residue resources and the development of walnut industry.

Inhibitory Effects of Walnut (Juglans regia) Peptides on Neuroinflammation and Oxidative Stress in Lipopolysaccharide-Induced Cognitive Impairment Mice.

Increasing level of inflammation and oxidative stress could lead to memory impairment. The purpose of this study was to determine the neuroprotective effects of walnut peptides against memory deficits induced by lipopolysaccharide (LPS) in mice and further to explore the underlying anti-inflammatory mechanisms against LPS-elicited inflammation in BV-2 cells. Results showed that walnut protein hydrolysate (WPH) and its low-molecular-weight fraction (WPHL) could ameliorate the memory deficits induced by LPS via normalizing the inflammatory response and oxidative stress in brain, especially WPHL. Furthermore, 18 peptides with anti-inflammatory activities on LPS-activated BV-2 cells were identified from WPHL and it was found that Trp, Gly, and Leu residues in peptides might contribute to the anti-inflammation. Meanwhile, the strong anti-inflammatory effects of LPF, GVYY, and APTLW might be related to their hydrophobic and aromatic amino acid residues as well. LPF, GVYY, and APTLW could reduce the content of proinflammatory mediators and cytokines by downregulating related enzyme expressions and mRNA expressions. Additionally, ROS and mitochondria homeostasis might also contribute to their anti-inflammatory effects.

Walnut protein hydrolysates, rich with peptide fragments of WSREEQEREE and ADIYTEEAGR ameliorate UV-induced photoaging through inhibition of the NF-κB/MMP-1 signaling pathway in female rats.

Skin photoaging is a complicated pathological process, and the imbalance of inflammatory regulation is associated highly with photoaging progression. Previously, prepared walnut protein hydrolysates (WPH), rich with peptide fragments of WSREEQEREE and ADIYTEEAGR demonstrated desirable photoprotection. However, it remains unclear if the photoprotection is mediated by the targeted inhibition of the NF-κB signaling pathway. Herein, we examined the regulation of WPH on inflammatory cytokine expression, and elucidated the modulation of the NF-κB/MMP-1 signaling pathway by WPH in a photoaging SD rat model. WPH significantly reduced the expression level of inflammatory cytokines IL-1β and IL-6, but significantly increased the level of IL-2 (all P < 0.05). Furthermore, WPH dramatically inhibited the activation of the NF-κB signaling pathway by mitigating the phosphorylation of IκB and p-65 proteins in a dose-dependent manner. The histopathological results indicated that WPH predominately attenuated epidermal hyperplasia, reduced the inflammatory filtration, and promoted collagen deposition in the photoaging skin tissue. Furthermore, WPH significantly stimulated the expression of TGF-β and procollagen type I, and inhibited the MMP-1 activities (all P < 0.05). Overall, the underlying mechanism of WPH ameliorating skin photoaging may be attributed to the synergistic modulation via reversing the inflammatory imbalance, suppressing the activation of the NF-κB signal pathway, stimulating procollagen type I synthesis, and inhibiting MMP-1 activities. According to these results, it can be concluded that WPH has the potential as an anti-photoaging agent in functional foods.