Hempseed Protein Isolate Research Articles

Acid-induced gelation of thermal co-aggregates from egg white and hempseed protein: Impact of microbial transglutaminase on mechanical and microstructural properties of gels

The thermal co-aggregates from mixed egg white protein (EWP) and hempseed protein isolate (HPI), named mixed EWP/HPI aggregates, was produced by heating the mixture solutions (5.0% w/v) at different EWP/HPI weight ratios (0/100, 75/25, 50/50, 25/75 and 100/0) under alkaline pH. Subsequent, the acid gelation of the thermal aggregates was induced by adding glucono-δ-lactone (GDL). The thermal treatment at alkaline pH produced the aggregates assembled by both disulfide and non-disulfide covalent bonds, and they exhibited low viscosity, and high solubility EWP alone formed gels of high mechanical strength in the presence of GDL, whereas no gels were obtained from the thermal aggregates of HPI alone. The EWP/EWP aggregates of different EWP/HPI ratios formed acid-induced self-supported gels. With increasing HPI content, the elastic modulus, tan δ, creep performance, texture parameters, fracture stress/strain, and microstructural homogeneity of the mixed EWP/HPI gels tended to decrease. Treatment with transglutaminase (TGase) relatively improved the mechanical properties of mixed gels, but not enough to completely off-sets the weakening effects. This research has offered insight into the fabrication of cold-set gels from different protein aggregates and the generated information may provide the opportunity to develop functional food formulations enriched in plant proteins.

The impact of hempseed dehulling on chemical composition, structure properties and aromatic profile of hemp protein isolate

Hempseed protein has attracted increasing attention in both scientific and industrial field because of its high nutritional value. In the current study, the influence of dehulling process on the chemical composition, structure and functional properties of hempseed protein isolate (HPI) was determined by using SDS-PAGE, SEC-HPLC-UV/RI/MALS, CD spectroscopy and DSC. The result demonstrated that dehulling process significantly increased the extraction and protein recovery yields of HPI. Meanwhile, dehulled HPI also exhibited a higher protein purity and Arg/Lys ratio with improved color. No significant impact on protein composition and structure was observed upon seed dehulling, but HPI extracted from the dehulled seeds showed a reduced thermal stability as compared to non-dehulled HPI. The aromatic profile of HPI was determined by HS-SPME-GC-MS, and the data suggested that dehulled HPI accumulated more terpenes and less lipid oxidation volatiles than non-dehulled HPI. These findings indicated that dehulling process greatly enhances the extraction yield with higher protein content and more characteristic aroma of HPI. The current study for the first time provides valuable information about the influence of dehulling processing on the properties of HPI, which will facilitate further research and application of hempseed and its valued added ingredients in both academic and industry field.

Genipin-Aided Protein Cross-linking to Modify Structural and Rheological Properties of Emulsion-Filled Hempseed Protein Hydrogels.

Genipin, a natural electrophilic cross-linker, was applied (5, 10, 20, and 30 mM) to modify hempseed protein isolate (HPI). Genipin treatments resulted in general losses of total sulfhydryls (up to 2.9 nmol/mg) and free amines (up to 77.3 nmol/mg). Surface hydrophobicity decreased by nearly 90% with 30 mM genipin, corresponding to similar tryptophan fluorescence quenching. The genipin treatment converted HPI into highly cross-linked polymers. Hydrogels formed with such polymers when also incorporated with hemp oil emulsions exhibited substantially enhanced gelling ability: up to 3.3- and 2.6-fold increases, respectively, in gel strength and gel elasticity over genipin-untreated protein. The genipin-modified composite gels also exhibited superior water-holding capacity. Microstructural analysis revealed a compact gel network filled with protein-coated oil globules that interacted intimately with the protein matrix when treated with genipin. Such gels remained readily digestible. Hence, genipin-treated hemp protein hydrogels show promise as functional food components.

Comparative Biological Activities Determination of Aqueous Extracts of Hempseed Oil and Hempseed Protein Isolate Production Coproducts

AbstractIndustrial hempseed is an important agricultural crop with increasing potential for food applications. The objective of this study was to determine the different biological activities of hempseed coproducts after hempseed oil extraction and hempseed protein isolate precipitation. Hempseed oil was extracted by n‐hexane and the defatted meal was dried. The hempseed protein isolate was prepared by alkaline pH solubilization followed by isoelectric precipitation. Two coproducts were obtained by freeze drying the precipitate after alkaline solubilization and freeze drying the supernatant after isoelectric precipitation. The coproducts were called hempseed meal after protein isolation (HM‐PI) and hempseed supernatant after protein isolation (HS‐PI). The biological activities of HM‐PI and HS‐PI were determined by measuring their antioxidant properties, antiproliferative properties against two human colon cancer cell lines, and anti‐inflammatory activities in lipopolysaccharide‐induced RAW 264.7 macrophages. The results showed that HS‐PI has better biological activities than HM‐PI, which correlated positively with the concentrations of soluble proteins, soluble polyphenols, tannins, and flavonoids. Our results showed for the first time that coproducts of hempseed processing can be potential sources of food ingredients with health‐promoting properties.

Heating-Aided pH Shifting Modifies Hemp Seed Protein Structure, Cross-Linking, and Emulsifying Properties.

Alkaline pH12 shift treatment performed at varying temperatures (20-80 °C for 1, 5, and 60 min) was applied to structurally modify hemp seed protein isolate (HPI). The solubility of HPI (∼20%) was remarkably improved ( p < 0.05) with elevating the temperature and prolonging the holding time, reaching 97.5% at 80 °C for 60 min. The treated HPI exhibited a strong tendency of forming soluble large aggregates. To limit lysinoalanine (LAL) production, heating was methodically controlled to 60 °C and 5 min, where the LAL content never exceeded 100 mg/100 g of protein and the loss of cysteine and lysine was also minimal. The emulsifying activity of HPI was improved by this mild pH shift-heating combination treatment as a result of the dissociation of protein subunits, unraveling of the tertiary structure, and exposure of hydrophobic groups. Moreover, the emulsion formed by the treated protein maintained a superior stability in particle size and distribution during storage.

New ACE-Inhibitory Peptides from Hemp Seed (Cannabis sativa L.) Proteins.

A hemp seed protein isolate, prepared from defatted hemp seed meals by alkaline solubilization/acid precipitation, was subjected to extensive chemical hydrolysis under acid conditions (6 M HCl). The resulting hydrolysate was fractionated by semipreparative RP-HPLC, and the purified fractions were tested as inhibitors of angiotensin converting enzyme (ACE). Mono- and bidimensional NMR experiments and LC-MS analyses led to the identification of four potentially bioactive peptides, i.e. GVLY, IEE, LGV, and RVR. They were prepared by solid-phase synthesis, and tested for ACE-inhibitory activity. The IC50 values were GVLY 16 ± 1.5 μM, LGV 145 ± 13 μM, and RVR 526 ± 33 μM, confirming that hemp seed may be a valuable source of hypotensive peptides.

In Vitro Acetylcholinesterase‐Inhibitory Properties of Enzymatic Hemp Seed Protein Hydrolysates

AbstractThe aim of this work was to characterize the structural and functional properties of hemp seed protein‐derived acetylcholinesterase (AChE)‐inhibitory enzymatic hydrolysates. Hemp seed protein isolate hydrolysis was performed using six different proteases (pepsin, papain, thermoase, flavourzyme, alcalase and pepsin + pancreatin) at different concentrations (1–4 %). The degree of hydrolysis was directly related to the amount of protease used but had no relationship with AChE‐inhibitory activity. Amino acid composition results showed that the hemp seed protein hydrolysates (HPHs) had high levels of negatively charged amino acids (39.62–40.18 %) as well as arginine. The 1 % pepsin HPH was the most active AChE inhibitor with ~6 µg/mL IC50 value when compared to 8–11.6 µg/mL for the other HPHs. Mass spectrometry analysis showed that most of the peptides in all the hydrolysates were less than 1000 Da in size. However, the pepsin HPHs contained larger‐sized peptides (244–1009 Da) than the papain HPHs (246–758 Da), which in turn was larger than the alcalase HPH (246–607 Da). The higher AChE‐inhibitory effects of the pepsin HPHs may be due to increased synergistic effects from a wider peptide size range when compared to the papain and alcalase HPHs that had narrower ranges. The narrow peptide size range in the alcalase HPH confirms the higher efficiency of this protease in releasing small‐sized peptides from food proteins.

Structural and functional properties of hemp seed protein products.

The effects of pH and protein concentration on some structural and functional properties of hemp seed protein isolate (HPI, 84.15% protein content) and defatted hemp seed protein meal (HPM, 44.32% protein content) were determined. The HPI had minimum protein solubility (PS) at pH 4.0, which increased as pH was decreased or increased. In contrast, the HPM had minimum PS at pH 3.0, which increased at higher pH values. Gel electrophoresis showed that some of the high molecular weight proteins (>45 kDa) present in HPM were not well extracted by the alkali and were absent or present in low ratio in the HPI polypeptide profile. The amino acid composition showed that the isolation process increased the Arg/Lys ratio of HPI (5.52%) when compared to HPM (3.35%). Intrinsic fluorescence and circular dichroism data indicate that the HPI proteins had a well-defined structure at pH 3.0, which was lost as pH value increased. The differences in structural conformation of HPI at different pH values were reflected as better foaming capacity at pH 3.0 when compared to pH 5.0, 7.0, and 9.0. At 10 and 25 mg/mL protein concentrations, emulsions formed by the HPM had smaller oil droplet sizes (higher quality), when compared to the HPI-formed emulsions. In contrast at 50 mg/mL protein concentration, the HPI-formed emulsions had smaller oil droplet sizes (except at pH 3.0). We conclude that the functional properties of hemp seed protein products are dependent on structural conformations as well as protein concentration and pH.

Reverse-phase HPLC Separation of Hemp Seed (Cannabis sativa L.) Protein Hydrolysate Produced Peptide Fractions with Enhanced Antioxidant Capacity

Hemp seed protein hydrolysate (HPH) was produced through simulated gastrointestinal tract (GIT) digestion of hemp seed protein isolate followed by partial purification and separation into eight peptide fractions by reverse-phase (RP)-HPLC. The peptide fractions exhibited higher oxygen radical absorbance capacity as well as scavenging of 2,2-diphenyl-1-picrylhydrazyl, superoxide and hydroxyl radicals when compared to HPH. Radical scavenging activities of the fractionated peptides increased as content of hydrophobic amino acids or elution time was increased, with the exception of hydroxyl radical scavenging that showed decreased trend. Glutathione (GSH), HPH and the RP-HPLC peptide fractions possessed low ferric ion reducing ability but all had strong (>60%) metal chelating activities. Inhibition of linoleic acid oxidation by some of the HPH peptide fractions was higher at 1mg/ml when compared to that observed at 0.1mg/ml peptide concentration. Peptide separation resulted in higher concentration of some hydrophobic amino acids (especially proline, leucine and isoleucine) in the fractions (mainly F5 and F8) when compared to HPH. The elution time-dependent increased concentrations of the hydrophobic amino acids coupled with decreased levels of positively charged amino acids may have been responsible for the significantly higher (p < 0.05) antioxidant properties observed for some of the peptide fractions when compared to the unfractionated HPH. In conclusion, the antioxidant activity of HPH after simulated GIT digestion is mainly influenced by the amino acid composition of some of its peptides.

Kinetics of Enzyme Inhibition and Antihypertensive Effects of Hemp Seed (Cannabis sativa L.) Protein Hydrolysates

AbstractThe aim of this study was to determine the antihypertensive effects of enzymatic hemp seed protein hydrolysate (HPH) and its peptide fractions. Hemp seed protein isolate was digested by the sequential action of pepsin and pancreatin to mimic gastrointestinal digestion in human beings. The resultant HPH was separated by membrane ultrafiltration into peptide fractions with different sizes (&lt;1 and 1–3 kDa). The HPH led to significantly higher (P &lt; 0.05) in vitro inhibition of the activities of angiotensin I‐converting enzyme (ACE) and renin, the two main enzymes involved in abnormal blood pressure elevation (hypertension). Kinetic studies showed that HPH and peptide fractions inhibited renin and ACE activities in a mixed‐type pattern, indicating binding to areas other than the active site. Oral administration of HPH (200 mg/kg body weight) to spontaneously hypertensive rats led to significant reductions (P &lt; 0.05) in systolic blood pressure (SBP) that reached a maximum of −30 mmHg after 8 h. In contrast, the hypotensive effects of peptide fractions (&lt;1 and 1–3 kDa) had a maximum value of about −15 mmHg after 6–8 h post oral administration. The results suggest a synergistic antihypertensive effect of the peptides present within HPH; this effect was reduced significantly (P &lt; 0.05) upon separation into peptide fractions.

Hempseed protein derived antioxidative peptides: Purification, identification and protection from hydrogen peroxide-induced apoptosis in PC12 cells

Hempseeds have not been utilised to any significant extent by the industrial food markets. A novel antioxidant peptide derived from hempseed by-product was investigated. Hempseed protein isolate was prepared by alkaline extraction and subsequent isoelectric precipitation. Its enzymatic hydrolysate, obtained by Alcalase® treatment, was subjected to DA201-C macroporous absorption resin, with simultaneous desalting and concentrating of hydrophobic fragments with improved free radical-scavenging activities. The active fraction was further separated by gel filtration and reversed-phase high performance liquid chromatography to obtain two purified peptides; the peptides were characterised as NHAV and HVRETALV, by MALDI-TOF/TOF MS/MS. The protective effects of the purified peptides on hydrogen peroxide-induced cell apoptosis were investigated in rat pheochromocytoma line PC12 cells. The peptides, at a concentration of 10 μg/ml, possessed protective effects ( P < 0.05) against cell death and oxidative apoptosis. The findings are significant for the discovery and development of natural antioxidants from hempseed by-products.