Hempseed Protein Isolate Research Articles

Modification of hempseed protein isolate using a novel two-stage method applying high-pressure homogenization coupled with high-intensity ultrasound

Hempseed protein isolate (HPI), a novel plant protein, possesses advantages as an alternative food protein from a nutritional and sustainable perspective. This study investigated HPI modification by examining the effects of high-pressure homogenization combined with high-intensity ultrasound (HPH + HIU) on the physicochemical and functionality of HPI. Firstly, the optimal homogenization pressure (180 MPa) was selected based on the solubility and particle size of HPI. Then, the effect of ultrasonic treatment time (2, 5, and 10 min) was studied at the optimal homogenization pressure. The results showed increased solubility of HPI after all treatments. Particularly, the HPH + HIU2min treatment had a synergistic effect that maximumly increased the solubility of HPI from 6.88 % to 22.89 % at neutral pH. This treatment significantly decreased the HPI’s particle size, β-sheet and total sulfhydryl contents while maximizing the random coil level, intrinsic fluorescence intensity and surface hydrophobicity compared to the single HPH or HIU2min treatments. The protein structure was modified and unfolded, enhancing the water-protein and oil-protein interactions, as reflected in the increase in water and oil absorption, foaming and emulsifying properties. However, extending the ultrasonic time to 5 min for the HPH + HIU treatment increased protein particle size and weakened the functional properties of HPI. Further prolonging the ultrasonic time to 10 min partially loosened the protein aggregates and restored the functional properties of HPI to some extent. The findings indicate a promising application of HPH + HIU as an efficient way for HPI modification to facilitate its broader application in the food industry.

Binding mechanism and structural characteristics of alloyed protein complex for enhanced solubility of hemp seed protein isolate

Despite the numerous health benefits and high digestibility of hemp seed protein isolate (HPI), its low solubility at neutral pH limits its utilization in the food industry. Therefore, we subjected insoluble HPI and soluble mung bean protein isolate (MBPI) to pH co-shifting under extremely alkaline conditions to form an alloyed protein complex (A-HM). At a mass ratio of HPI:MBPI of 50:50, A-HM exhibited the highest solubility (95.30 ± 0.99 %), and also had high resistance to heat treatment. Native PAGE demonstrated the formation of alloyed protein complexes, and particle size analysis revealed that A-HM exhibited small particle sizes and dispersion in water without aggregation of HPI. Owing to their small size, numerous hydrophobic residues and aromatic ring of HPI were exposed on the surface. Hydrophobic interactions predominantly governed the binding force involved in the formation of A-HM. Our findings may enhance HPI applications in the food industry, particularly in plant-based beverages.

Eco-friendly encapsulation: Investigating plant-based protein-alginate shells for efficient delivery and digestion of hemp seed oil encapsulated via supercritical CO2 dispersion

In this study, supercritical carbon dioxide solution-enhanced dispersion (SEDS) was used to encapsulate hemp seed oil (HSO) within matrices of hemp seed protein isolate (HPI), pea protein (PPI) and soy protein (SPI) (0.5 % w/v) in complex with alginate (AL) (0.01 % w/v). The effects of different pH levels (3–9), NaCl concentrations (0–200 mmol/L) and simulated gastrointestinal conditions on HSO release and digestion patterns were analyzed. The findings revealed that SPI/AL microcapsules effectively maintained structural integrity and controlled oil release across diverse pH levels and salt concentrations. During gastrointestinal phases, minimal oil release was observed during oral digestion (<25 % for all samples), while significant (P < 0.05) gastric release occurred in PPI/AL (55.4 %) and SPI/AL (78.1 %) microcapsules. Surprisingly, HPI/AL microcapsules exhibited delayed and sustained release (27.9 %), indicating their potential as ideal wall material for delivering sensitive food and pharmaceutical ingredients to the intestinal stage while minimizing damage in the harsh gastric environment.

Integrative approach to assessing bioactivity from hempseed protein isolate extracted and dehydrated by different methods: Synergising in silico prediction and in vitro validation

This study demonstrated a comprehensive workflow combining in silico screening and prediction with in vitro validation to investigate the bioactivity of hempseed protein isolate (HPI) extracted and dehydrated using different methods. By adopting an in silico approach, 13 major proteins of HPI were hydrolysed by 20 selected enzymes, leading to the prediction of 20 potential bioactivities. With papain hydrolysis, dipeptidyl peptidase-IV (DPP4) and angiotensin-converting enzyme (ACE) inhibitory activities emerged as having the highest potential. In vitro experiments confirmed these predictions, with DPP4 and ACE inhibitory activities displaying IC50 values of 0.32–0.42 mg/mL and 6.8–9.17 μg/mL, respectively. A strong correlation (r2 = 0.96) was observed between in vitro protein inhibitory results and in silico predicted data. This study demonstrated an effective integrative approach for predicting bioactive peptides in food protein, providing valuable guidance on its processing to create value-added products.

Innovative microencapsulation of hemp seed oil using plant-based biopolymers: A comparative analysis of dehydration techniques on core stability, digestibility and release pattern

This study explores various dehydration methods, including freeze drying (FD), spray drying (SD), and the supercritical carbon dioxide solution-enhanced dispersion (SEDS) process, to encapsulate hemp seed oil (HSO) within a complex matrix of hemp seed protein isolate and alginate. One noteworthy aspect of this study involves the in-depth analysis of the structural characteristics of the delivery systems, highlighting their pivotal role in shaping HSO release patterns. The findings indicate that SEDS microcapsules (SEDS-M), with their smallest particle size and smooth, spherical morphology, effectively preserved structural integrity and provided controlled oil release across diverse pH levels (3–9) and salt concentrations (0–200 mM). During the gastrointestinal phases, minimal oil release occurred during oral digestion (<25%) for all samples, while the significant gastric release occurred in FD (39.8%) and SD microcapsules (33.4%). With their highest storage stability, SEDS-M demonstrated superior delayed and sustained release (27.9%) during gastric transit, making them ideal for protecting sensitive ingredients and ensuring targeted intestinal delivery.

Effects of protein variations by different extraction and dehydration approaches on hempseed protein isolate: Protein pattern, amino acid profiling and label-free proteomics

This study evaluates the effects of alkaline and micellisation extraction methods, alongside freeze-drying and spray-drying, on the protein subunits, amino acid profiles, and proteome data of hempseed protein isolate (HPI). Findings revealed that the extraction methods affect protein profiles more than the drying methods. Micellisation-extracted HPI showed higher albumin, oleosin, and sulphur-containing protein levels than alkaline-extracted HPI. The alkali-extracted undried sample (AU) gave more potentially allergenic proteins, including Hsp70 and triosephosphate isomerase, than its micellization-extracted counterpart (MU). Unique potential allergens were identified, including malate dehydrogenase and enolase in AU, and RuBisCo in MU samples. Both drying processes impacted the HPI proteome and reduced RuBisCo in the micellisation-extracted HPI. These insights highlight the crucial role of method selection in HPI processing for optimising production in the food industry.

An in-depth comparative study of various plant-based protein-alginate complexes in the production of hemp seed oil microcapsules by supercritical carbon dioxide solution-enhanced dispersion

This study focuses on the microencapsulation of hemp seed oil (HSO) using a range of wall materials derived from plant-based proteins including hemp seed protein isolate (HPI), pea protein (PPI) and soy protein (SPI) in complexation with alginate (AL). The microencapsulation process was accomplished through the utilization of supercritical carbon dioxide solution-enhanced dispersion with water as a co-solvent. According to the results, both HPI/AL and PPI/AL microcapsules exhibited comparable and satisfactory properties in terms of water activity (5.26% and 5.33%), particle size (7.86 μm and 9.61 μm) and bulk density (0.363 g/mL and 0.349 g/mL). However, the overall findings highlighted the superiority of HPI/AL complex in producing microcapsules with the highest microencapsulation efficiency (90.56%), a spherical shape free from pores or fissures and an exceptional glass transition temperature. Furthermore, HPI/AL microcapsules displayed stronger antioxidant activities and oxidative stability, enhancing product shelf life significantly when compared to PPI/AL and SPI/AL microcapsules.

Improving the solubility and interfacial absorption of hempseed protein via a novel high pressure homogenization-assisted pH-shift strategy

A pH shift treatment aided by high pressure homogenization (HPH) with various pressures (0–120 MPa) was employed to structurally modify hempseed protein isolate (HPI). Compared with individual pH shift or HPH treatment, HPH-assisted pH shift improved the structural flexibility of HPI, as revealed by the increased random coil in protein secondary structure. With the incorporation of HPH into pH shift, the intrinsic fluorescence intensity was remarkably attenuated and redshifted, whereas the surface hydrophobicity was pronouncedly boosted, indicating the extensive unfolding of protein structure. Moreover, the cotreated HPI exhibited a smaller and more homogenous particle size, notably at a higher pressure. Consequently, the solubility was drastically raised by the cooperated treatments, to the maximum value (62.8 %) at 120 MPa. These physicochemical changes in the cotreated HPI facilitated a consolidated interfacial activity. Moreover, the cooperated treatment, especially highly pressured (120 MPa), facilitated the penetration and rearrangement of proteins at the oil–water interface.

A study on pH-mediated variations and differences in the structure and function of hemp seed albumin, globulin, and protein isolate

In this comprehensive study, hemp seed protein isolate (HPI), albumin (ALB), and globulin (GLB) were prepared from hemp seed kernels. ALB was identified as a potential glycoprotein, distinguishing itself with a higher count of free sulfhydryl groups, fewer disulfide bonds, and a smaller molecular weight distribution. Conversely, GLB demonstrated fewer free sulfhydryl groups, a larger number of disulfide bonds, and a larger molecular weight distribution. HPI predominantly consisted of globulin with minor albumin. Varying pH from 3.0 to 9.0 considerably changed the structure and conformation of GLB and HPI but had minimal impact on ALB, according to surface hydrophobic, circular dichroism, and fluorescence spectrum assays. The pH-induced structural difference and variations in ζ-potential and solubility had synergistic effects on the emulsifying properties of GLB and HPI, while ALB's emulsifying properties were primarily associated with changes in ζ-potential and solubility. GLB and HPI demonstrated superior solubility and emulsification ability at pH 3. In contrast, ALB exhibited better functionality at pH 9. Understanding the pH-dependent properties of hemp seed proteins is crucial for optimizing their application in diverse fields.

The physicochemical properties, functionality, and digestibility of hempseed protein isolate as impacted by spray drying and freeze drying

Hempseed protein has gained increasing attention for its sustainability and nourishment. This study aimed to investigate the effects of spray drying and freeze drying on the physicochemical properties, functionality, and digestibility of hempseed protein isolate (HPI). Compared to undried-HPI, both drying techniques altered physicochemical and structural properties. Particularly, protein denaturation temperature increased in freeze-dried HPI (FD-HPI) and spray-dried HPI (SD-HPI) samples (∼90 °C) than in undried-HPI (82.5 °C). Lysine content decreased from 38.26 mg/g in undried-HPI to 35.03 and 33.18 mg/g in FD-HPI and SD-HPI, respectively. Results revealed the loss of 26 and 17 kDa bands after drying. Notably, FD-HPI exhibited higher emulsifying stability and oil-holding capacity than SD-HPI. While both FD-HPI and SD-HPI had higher digestibility than undried-HPI, a 50% reduction in the liberation of free α-amino groups after digestion was found. This study provided information regarding changes in HPI after drying, offering insights for HPI production and application in the food industry.

Elucidation of synergistic interactions between anionic polysaccharides and hemp seed protein isolate and their functionalities in stabilizing the hemp seed oil-based nanoemulsion

This study aimed to enhance the physical stability of hemp seed oil (HSO) nanoemulsions stabilized by hemp seed protein isolate (HPI)-polysaccharides (carrageenan, alginate, gum arabic and pectin) complexes. Extensive investigations were conducted to evaluate the surface charge characteristics of HPI and polysaccharides, along with an assessment of their binding strength and complex formation. The researchers also examined various influential factors such as polysaccharide type and concentration, nanoemulsion droplet size and charge, environmental stresses, shearing rate, and creaming stability on the HSO nanoemulsions stabilized using HPI-polysaccharide emulsifiers. The results demonstrated that the main influencing parameters were polysaccharide type and concentration. In addition, all HPI (0.5% (w/v)-polysaccharide (0.01% (w/v)) nanoemulsions exhibited significant resistance to droplet aggregation and creaming, even under thermal treatment (≤100 °C). This stability can be attributed to the synergistic effects of the mixed emulsifiers with opposite surface charges, which resulted in nanoemulsions with a positive charge under optimal pH conditions (pH = 3 for carrageenan and 3.5 for other polysaccharides) due to repulsive forces between droplets. Notably, only the HPI-alginate nanoemulsion reveals stability across all salt levels (≤ 500 mM), while the other nanoemulsions remained stable solely at low NaCl concentrations (≤ 200 mM). This exceptional stability can be attributed to the alginate flexibility and formation of a compact complex between HPI at the surface of the nanoemulsion droplets. This study's findings carry significant implications for the food and beverage industries, especially regarding the use of HPI as a functional food ingredient. Furthermore, HPI-alginate nanoemulsions offer great potential as alternative encapsulation materials.

An Investigation into the Mechanism of Alkaline Extraction-Isoelectric Point Precipitation (AE-IEP) of High-Thiol Plant Proteins

Hempseed protein isolate (HPI) has drawn significant attention as a promising source of plant-based protein due to its high nutritional value. The poor functionality (e.g., solubility and emulsifying properties) of HPI has impeded its food application for years. This study provides important new information on hempseed protein extraction, which may provide further insights into the extraction of other high-thiol-based plant proteins to make valuable plant-based products with improved functional properties. In this study, HPI was produced from hempseed meals using the AE-IEP method. The underlying mechanisms and extraction kinetics were investigated under different experimental conditions (pH 9.0–12.0, temperature 24–70 °C, and time 0–120 min). The results suggested that disulphide bond formation is an inevitable side reaction during hempseed protein extraction and that the protein yield and the free -SH content can be influenced by different extraction conditions. A high solution pH and temperature, and long extraction time result in increased protein yield but incur the formation of more intermolecular disulphide bonds, which might be the reason for the poor functionality of the HPI. For instance, it was particularly observable that the protein solubility of HPI products reduced when the extraction pH was increased. The emulsifying properties and surface tension data demonstrated that the functionality of the extracted hempseed protein was significantly reduced at longer extraction times. A response surface methodology (RSM) optimization model was used to determine the conditions that could maximise HPI functionality. However, a three-fold reduction in protein yield must be sacrificed to obtain the protein with this high functionality.

Bromelain Hydrolysis Modified the Functionality, Antioxidant, and Anti-Inflammatory Properties of Hempseed (Cannabis sativa L.) Protein Isolated at pH 12

Hempseed (Cannabis sativa L.) is currently being investigated for its health-promoting properties acting as antioxidant and anti-inflammatory agents. Here, we present a modified protein isolation method of producing hempseed protein isolate from industrial hempseed hearts (HPI-12) using pH 12. In addition, bromelain was used to hydrolyze HPI-12 (BHPI-12) to determine the effect on functionality and bioactivity. HPI-12 is a rich source of essential amino acids with low protein solubility, effective in reducing 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals without oxygen radical antioxidant capacity (ORAC). Further, HPI-12 reduced pro-inflammatory IL-6 and TNF-α cytokines in lipopolysaccharide-induced inflammation in THP-1 macrophages. Upon hydrolysis, protein solubility was improved accompanied by modified water- and oil-holding capacities. BHPI-12 was more effective at quenching DPPH radicals with improved ORAC. BHPI-12 showed an improved ability to reduce IL-6. We report for the first time the ability of both HPI-12 and BHPI-12 as potential functional food ingredients with antioxidant and anti-inflammatory activity.

The Impact of High-Intensity Ultrasound-Assisted Extraction on the Structural and Functional Properties of Hempseed Protein Isolate (HPI).

Hempseed protein has become a promising candidate as a future alternative protein source due to its high nutritional value. In the current study, hempseed protein isolate (HPI) was obtained using ultrasonic-assisted extraction with the aim to improve the functionality of HPI via protein structure modification. The solubility of HPI could be improved twofold under 20 kHz ultrasound processing compared to conventional alkaline extraction-isoelectric point precipitation. The protein solubility was gradually enhanced as the ultrasonic power improved, whereas excessive ultrasound intensity would cause a decline in protein solubility. Ultrasonic processing was found to have beneficial effects on the other functionalities of the extracted HPI, such as emulsifying and foaming properties. This improvement can be ascribed to the physical effects of acoustic cavitation that changed the secondary and tertiary structures of the protein to enhance surface hydrophobicity and decrease the particle size of the extracted protein aggregates. In addition, more available thiols were observed in US-treated samples, which could be another reason for improved functionality. However, the results of this study also revealed that prolonged high-power ultrasound exposure may eventually have a detrimental impact on HPI functional properties due to protein aggregation. Overall, this study suggests that high intensity ultrasound can enhance the functionality of HPI, which may ultimately improve its value in HPI-based food products.

The effects of germination on the composition and functional properties of hemp seed protein isolate

The effects of germination on the functional properties of hemp seed protein isolates was evaluated in this study. Significant seed storage protein degradation was observed after three days of germination, while protein surface charge was altered after one day of germination. Protein solubility increased in neutral to slightly acidic pH, with 5-day germination increasing hemp seed protein solubility from 10.3% to 21.8% at pH 7.0. Water holding capacity increased from 1.03 g H 2 O/g isolate to 1.34 g H 2 O/g isolate while oil holding capacity stayed relatively constant. Germinated protein isolates demonstrated higher foaming capacity (69.2%–111.2%) and foaming stability (53.6%–75.1%) than non-germinated protein isolates. Five-day germinated hemp seed protein isolate was the only adequate emulsifier in this study, capable of stabilizing emulsion droplets down to 2.90 μm in diameter. Hemp seed protein formed self-supporting gels at minimum of 2 or 3 w/v %, and germination increased the hardness of hemp seed protein gels (38.02 g–49.12 g for 10 w/v % gels). These findings indicate the ability to leverage germination as a clean-label process for improving the functional properties of hemp seed protein. • Hemp seed storage proteins are degraded after three days of germination. • Germinated hemp seed protein isolates are more soluble near a neutral pH. • Germination improves hemp protein WHC, foaming, emulsifying, and gelling properties. • LGC for hemp protein at pH 7 was determined at 2–3 w/v %.

Physicochemical and Antioxidant Properties of Industrial Hemp Seed Protein Isolate Treated by High-Intensity Ultrasound.

Ultrasound is one of the non-thermal, green, and novel technologies used to functionalize plant proteins. We recently determined the optimum conditions of high-intensity ultrasound (HIUS) treatment for maximum solubility and investigated the functional properties of hemp seed protein isolate (HSPI) under the optimal conditions. In this study, we analyzed changes in primary, secondary, and tertiary structures, physical microstructures, thermal stability, and antioxidant capacity of ultrasound-applied hemp protein isolate (HSPI-HIUS). The free SH group content (+59%) and zeta potential (+25%) increased upon ultrasound treatment. The electrophoretic protein patterns of HSPI showed no significant change after HIUS treatment. The FTIR spectrum revealed the wavenumber shifts in Amid 1 and 2 regions of protein. The denaturation temperature and the ratio of β-structure increased after sonication. Antioxidant properties of hemp seed protein isolates were increased by 38% by ultrasound treatment. The obtained data in this study showed that HIUS treatment would be promising for improving the functional, physicochemical, and antioxidant properties of HSPI.

Construction of hemp seed protein isolate-phosphatidylcholine stablized oleogel-in-water gel system and its effect on structural properties and oxidation stability

Rice bran wax was added to hemp seed oil (HSO) to prepare oleogel, and hemp seed protein isolate (HPI)-phosphatidylcholine (PC) was used as the emulsifier to obtain an oleogel-in-water (Og/W) gel system. The effect of HPI concentration on the construction of gel system was studied. Microscopic observations found that the oil droplets were encapsulated by the emulsifier. X-ray diffraction and Fourier transform infrared spectroscopy analysis showed that the increase in HPI concentration promoted the interaction between PC and protein, but didn’t affect the crystal structure of gel. Thermogravimetric analysis showed that when the HPI concentration was 8 %, the sample formed a dense gel network and had good thermal stability. At this time, the oil holding capacity of gel was 98.81 ± 0.08 %, and the gel hardness was 109.55 ± 1.74 g. After 30 days of storage, the retention rate of Δ9-THC reached 96.3 %, and the peroxide value was 4.98 mmol/kg.

Application of ultrasound treatment to improve the technofunctional properties of hemp protein isolate

The hemp seed protein isolate (HPI) was extracted from defatted hemp seed meal using alkaline extraction (pH = 10.0) acid precipitation (pH = 4.3) method. The effects of ultrasound treatment on its solubility, emulsifying properties, particle size, microstructure, molecular subunits, secondary and tertiary structures, and surface hydrophobicity were determined. Solubility and emulsifying properties of HPI were significantly improved after ultrasound treatment (20 kHz) at 400 W for 12 min. The solubility, emulsion activity, and emulsion stability indices increased by 3.7- 1.7- and 1.5-fold, respectively. The improvement in functional properties correlated with reduced particle size, increased surface hydrophobicity and the alteration in secondary and tertiary structure of HPI. The solubility and emulsifying properties of the ultrasound treated HPI were better than native pea protein isolate but still not as good as those of native soy protein isolate for which further research is needed.

Physicochemical and Functional Properties of 2S, 7S, and 11S Enriched Hemp Seed Protein Fractions.

The hemp seed contains protein fractions that could serve as useful ingredients for food product development. However, utilization of hemp seed protein fractions in the food industry can only be successful if there is sufficient information on their levels and functional properties. Therefore, this work provides a comparative evaluation of the structural and functional properties of hemp seed protein isolate (HPI) and fractions that contain 2S, 7S, or 11S proteins. HPI and protein fractions were isolated at pH values of least solubility. Results showed that the dominant protein was 11S, with a yield of 72.70 ± 2.30%, while 7S and 2S had values of 1.29 ± 0.11% and 3.92 ± 0.15%, respectively. The 2S contained significantly (p < 0.05) higher contents of sulfhydryl groups at 3.69 µmol/g when compared to 7S (1.51 µmol/g), 11S (1.55 µmol/g), and HPI (1.97 µmol/g). The in vitro protein digestibility of the 2S (72.54 ± 0.52%) was significantly (p < 0.05) lower than those of the other isolated proteins. The intrinsic fluorescence showed that the 11S had a more rigid structure at pH 3.0, which was lost at higher pH values. We conclude that the 2S fraction has superior solubility, foaming capacity, and emulsifying activity when compared to the 7S, 11S, and HPI.

Modification of hemp seed protein isolate (Cannabis sativa L.) by high-intensity ultrasound treatment. Part 1: Functional properties

High-intensity ultrasound (HIUS) is one of the physical methods to modify protein functionality. This study aims to improve the functional properties of hemp seed protein isolate (HSPI) based on the solubility and particle size using the Response Surface Methodology. The acoustic intensity of 37 W/cm2 and protein concentration of 6.9% for 7.8 min were optimum process conditions that increased HSPI solubility (78%). The functional properties of ultrasound treated isolate (HSPI-HIUS) under the optimal conditions were compared with untreated HSPI. The results revealed that the emulsification, oil absorption, and foaming properties of HSPI-HIUS significantly enhanced after HIUS, whereas gelling concentration significantly decreased. Enhanced functional properties in HSPI-HIUS have been associated with structural changes based on surface hydrophobicity, particle size, and fluorescence intensity. This study indicated that HIUS can increase the functionality and prevalence of hemp seed protein isolate in food products.