In Vitro Digestibility, Biological Activity, and Physicochemical Characterization of Proteins Extracted from Conventionally and Organically Cultivated Hempseed (Cannabis sativa L.)

2.2. Solubility of the Commercial HPI Sample

Solubility is one of the most important properties of proteins, since it can affect many functional properties, e.g., the surface activity, rheological and hydrodynamic properties. The solubility of proteins is generally at its lowest value at the isoelectric point, where there is no net charge and therefore no repulsion between proteins. The solubility of proteins is thus strongly dependent on pH. The pH of maximum solubility is used for the extraction of proteins from different plant sources.

Therefore, the solubility of the commercial HPI at different pH (3–12) was determined in order to select a suitable pH for protein extraction from OC, CC1 and CC2. The results showed that protein solubility at alkaline pH was higher than under acidic conditions, with the highest (p < 0.05) solubility at pH 12 being equal to 82.2 ± 4.7% herein. The results showed that the solubility of the commercial HPI under neutral pH conditions was low. The protein solubility of HPI was minimum at pH 3–6, while it increased gradually above pH 6 (Figure 1). These data show that the commercial HPI was a typical alkali soluble protein.

The results herein are in agreement with the results obtained by Malomo and Aluko [7] reporting hempseed meal protein solubility in the range of 5–20% when suspended between pH 3 and 9. Tang et al. [11] reported a protein solubility > 90% when the hempseed protein was adjusted to pH ≥ 8. The results herein are also in agreement with the report by Potin et al. [15], who observed low solubility (<20%) of hempseed protein at pH < 9, while reaching solubility values of 30%, 50%, and 70% at pH 10, 11 and 12, respectively. The minimum solubility of defatted hempseed meal protein has been previously reported to be at pH 5–6 [18]. In general, hempseed protein exhibits low solubility at neutral and acidic pH in comparison to other plant proteins, such as soy protein [11,19]. This may be attributed to the aggregation of the acidic and basic subunits of edestin (globulin) at pH values ≤ 7 [20]. The underlying mechanism of solubilization of hempseed protein at alkaline pH may be related to dissociation of the edestin molecules. It has been reported that hempseed globulin has low solubility in the acidic pH range with values of approximately 20%, which increased to 50% at pH 8 and 9, while the solubility of albumin in hempseed ranged from 57% at pH 3 to 84% at pH 8, which is significantly superior to those of the globulin [7]. Some modifications, such as succinylation, acetylation, and enzymatic treatment, have been reported to improve the solubility of hempseed proteins [18].

The highest solubility for the HPI sample was obtained at pH 12. This value was therefore employed for the extraction and solubilization of the hempseed proteins from defatted OC, CC1 and CC2 samples followed by subsequently isoelectric precipitation at pH 4 (see Section 3.4).

2.3. Hempseed Extract Protein Profiles

The spray-dried CC2 protein extract powder contained significantly (p < 0.05) higher protein than the two other samples. The results showed that the spray-dried OC, CC1 and CC2 extracts contained 65.54 ± 1.24, 62.58 ± 1.61, and 70.03 ± 0.94% protein, respectively. The protein profiles of the spray-dried samples were analyzed using RP-UPLC (Figure 2). The protein profiles of all three samples were similar, showing dominant peaks on the chromatograms eluting at retention times of 25.5, 26.5 and 27.0 min. The elution of edestin at a retention time of ~30 min using a similar RP-HPLC gradient as herein was recently reported [21]. Therefore, the peaks appeared on the chromatogram herein at 25–27 min may correspond to the subunits of edestin. The area under the curve for the region associated with edestin for OC, CC1 and CC2 was 77.6%, 67.7%, and 73.4% (as a percentage of the total chromatogram area), respectively. The difference may depend on differences in the growth conditions. A previous study reported that hempseed proteins consist of ~65–75% edestin and 25–35% albumin [22].

2.4. Particle Size Distribution

The results of the particle size analysis of the aqueous reconstituted hempseed protein extracts showed that the smallest particles were associated with the OC sample while the largest was associated with the CC1 sample. The particle sizes for the CC2 sample were in between these two samples. Nevertheless, these values were lower than the mean particle diameter of commercially available HPI with 50% protein, which were recorded to be 200 µm (Cambridge Commodities Ltd., P19311 product specification). The uniformity of the protein extract samples was not significantly different (p > 0.05); however, the CC1 sample had the widest distribution of particle sizes (d(0.9) − d(0.1) = 590.0 µm) (Table 2). Bimodal particle size distributions were observed in all samples (Figure 3). This may be linked to the presence of two major proteins (i.e., albumin and edestin) showing different interaction patterns at pH 7 in aqueous suspension. Lower particle size and a narrower distribution of sizes are desirable for some applications (e.g., emulsification) of food proteins. The specific surface area (SSA) of the particles was not significantly different (p > 0.05).

2.5. Hempseed Protein Digestibility

3.2. Sample Preparation

Two conventionally grown hempseed (CC1 and CC2) and one organically grown hempseed (OC) samples were provided by Hemptech Ireland Ltd., Wicklow, Ireland. The hemp Cannabis sativa L. (Finola)) was grown under license from the Irish Department of Health (license numbers 5C/134-1-2019, 5C/94-1-2019 and 5C/135-1-2019). The hemp was sown between 25 and 27 May 2019 and CC2 and CC1 were harvested between 11 and 12 October 2019, while the OC crop was harvested on 22 October 2019. The samples were cleaned by physical separation of the whole seeds from any other materials.

In addition, a commercial hempseed protein isolate (HPI, Pulsin Ltd., Gloucester, UK) was purchased from a local health-food store.

3.3. Proximate Analysis and Sample Characteristics

3.4. Extraction of Hempseed Protein

The nitrogen solubility of the commercial HPI at different pH values was firstly assessed in order to select the optimum pH for extraction of the proteins from the hempseeds. For this, the HPI sample was suspended 1:20 (w/v) in dH₂O. The HPI suspension was stirred for 10 min and the pH was then adjusted to pH 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 using HCl (0.5 M) or NaOH (0.5 M) followed by stirring for 30 min at room temperature. An aliquot of the suspension (7 mL) was centrifuged (3000× g at 4 °C for 15 min). The nitrogen content in the supernatant obtained was analyzed (n = 3) using the Kjeldahl procedure. The pH of the supernatant with the highest protein content (maximum solubility, pH 12) was subsequently selected for protein extraction from the hempseed samples.

Protein extraction from the hempseed samples was carried out according to the pH selected from the previous step that indicated the maximum solubility. First, the hempseed samples were de-oiled using a stainless steel laboratory automatic oil press (TTLIFE, Nanjing, Jiangsu, China). The de-oiled samples were subsequently ground using a coffee grinder. As described previously, the ground samples were then suspended in dH₂O (1:20, (w/v)) and stirred at room temperature for 10 min. The pH was then adjusted to that of maximum protein solubility (pH 12). Samples were stirred for 30 min followed by centrifugation (3500× g, 4 °C, 15 min) using a 320R Hettich centrifuge (Tuttlingen, Germany). The pH of the supernatant was then adjusted to pH 4, i.e., the pH corresponding to the isoelectric point of hempseed protein [23] using 0.5 M HCl. The pellet collected after centrifugation (3500× g, 4 °C, 15 min) was resuspended in dH₂O (1:10 (w/v)). The suspension was then dried using a B-290 mini spray dryer (Buchi, Flawil, Switzerland) at an inlet temperature of 150 °C and an outlet temperature of 85 °C. The protein content of each spray-dried powder sample was determined using the Kjeldahl method as outlined earlier.

3.5. Reverse-Phase Ultrahigh-Performance Liquid Chromatography (RP-UPLC)

The protein extracted from the OC and CC hempseeds was analyzed using an Acquity UPLC system (Waters, Dublin, Ireland). Mobile phase A was 0.1% (v/v) TFA and mobile phase B was 80% (v:v) ACN and 0.1% (v/v) TFA. The samples were reconstituted in dH₂O to reach a concentration of 0.5% (w/v). The samples were filtered through 0.2 µm cellulose acetate filters. An aliquot of 10 μL was injected into the Acquity UPLC BEH C18, 130 Å column (2.1 mm × 50 mm × 1.7 mm) equipped with an Acquity BEH C18 (1.7 mm) vanguard precolumn. The flow rate was set on 0.3 mL/min over 51 min with the gradient program as previously detailed by Cermeño et al. [35].

3.6. Particle Size (PS) Analysis of Reconstituted Hempseed Protein Extracts

An aliquot (1 mL) of reconstituted (5% (w/v)) protein extract adjusted at pH 7.0 was pipetted into a Mastersizer 2000 (Malvern Instruments, Malvern, Worcestershire, England) equipped with a Hydro 2000S sample dispersion system (set at a stirring speed of 1000 rpm) interfaced with Mastersizer 2000 software (version 5.61; Malvern Instruments, Malvern, UK). The volume weighted mean (D[4, 3]), the surface weighed mean (D[3, 2]), the absolute deviation from the median (uniformity) as well as the average particle size (d(0.5)), the sizes of particles below which 10% and 90% of the sample lie (d(0.1) and d(0.9), respectively) and the specific surface area (SSA) were recorded. The particle refractive index and the continuous phase refractive index used were 1.52 and 1.33, respectively. The beam length was 2.35 mm and the minimum detection limit was 0.02 µm. Measurement integration time was 12,000 ms. The analysis was performed in triplicate with three measurements during each run.

3.7. Simulated Gastrointestinal Digestion (SGID) of Hempseed Protein Extracts

SGID was carried out on the hempseed protein samples according to Walsh et al., [36]. The samples (100 mL, 2% (w/v) protein) were incubated in a water bath (37 °C, 30 min). The pH was adjusted to pH 2 using 1 M HCl. Following addition of pepsin (2.5% (w/v) protein), gastric digestion took place for 90 min before the sample was heated to 90 °C for 20 min, deactivating the enzyme. The pH of the sample was then adjusted to pH 7.5 using 1 M NaOH. Intestinal digestion occurred with the addition of Corolase PP (1% (w/v) protein) and incubation for 150 min at 37 °C before deactivation. The pH values were kept constant throughout each digestion phase using a pH stat (Metrohm 902 Titrando pH-STAT, Herisau, Switzerland). The samples containing digested protein were then freeze-dried (Labconco benchtop freeze-dryer, Kansas City, MO, USA). Therefore, three samples were obtained from each hempseed protein extract: a sample prior to digestion, a sample that had undergone simulated gastric digestion and a sample that had undergone simulated gastric and intestinal digestion. These were subsequently characterized for their degree of hydrolysis (DH%), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel-permeation high-performance liquid chromatography (GP-HPLC) profiles, in vitro antioxidant activity, and lipase-inhibitory activity.

3.8. Degree of Hydrolysis (DH%)

The DH was determined according to the method described by Nongonierma et al., [37], where samples at 5% (w/v) protein were diluted in 1% (w/v) SDS to reach a concentration of 0.1% (w/v). Samples were heated for 30 min at 50 °C. The 5% (w/v) TNBS stock solution was diluted to 0.05% (w/v) using a 1:1 solution of dH₂O and sodium phosphate buffer (0.2125 M, pH 8.2). An aliquot of 10 μL from each sample was then added to a 96-well microplate (Sarstetd, Dublin, Ireland) along with 160 μL of TNBS in each well. The absorbance was measured at 50 °C every 5 min during 1 h at λ = 350 nm using a microplate reader (BioTek Synergy HT reader, BioTek, Winooski, VT, USA). Leucine at various concentrations (0, 2, 5, 7, 14, 21, 28 and 56 mg/mL in 1% (w/v) SDS) was used as a standard.

3.9. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) of Hempseed Protein Extracts

SDS-PAGE analysis was used to obtain the profiles of each hempseed protein sample. Samples (~4 mg/mL on a protein basis) were mixed 1:1 with a mixture of 2-mercaptoethanol and Laemmli buffer (1:19) in Eppendorf tubes. The tubes were shaken for 5 min at 200 rpm and incubated at 95 °C using a DX-100/DX-100R Dry Block Thermo-Shaker (IRIS/analytical, London, UK). An aliquot of 5 µL of a protein marker standard and 10 µL hempseed protein samples (~20 µg protein) was pipetted into each lane. The electrophoresis apparatus (Power PAC 1000, Bio-Rad, Hercules, CA, USA) was set at 150 V and 15–50 mA for 90 min.

3.10. Gel-Permeation High-Performance Liquid Chromatography (GP-HPLC) Analysis

Protein suspensions containing 0.25% (w/v) hempseed protein dissolved in mobile phase (30% ACN, 0.1% TFA) were prepared for GP-HPLC analysis. Samples were then filtered into Eppendorf tubes using 0.2 μm polytetrafluoroethylene syringe filters and 200 μL of each sample was transferred into HPLC sample vials. Separation was performed by isocratic elution on a TSK G2000 SW separating column (600 mm length × 7.5 mm ID) fitted with a TSKGEL SW guard column (75 mm length × 7.5 mm ID—Tosoh Bioscience, Tokyo, Japan) over 51 min. The absorbance of the eluent was monitored at 214 nm and separation was carried out at a flow rate of 1 mL/min.

3.11. ABTS^● Scavenging Activity Assay

Hempseed proteins and their digested samples (21 mg on a protein basis) were diluted to 1 mL with phosphate-buffered saline (PBS, pH 7.4) and centrifuged at 3500× g at 4 °C for 5 min. ABTS^● was diluted to an absorbance of ~0.7 at λ = 734 nm. An aliquot (200 μL) of this was added to 10 μL of samples. PBS was used as a blank. All samples including the blank were applied to a microplate and the absorbance recorded over 6 min, with shaking at 30 °C using a BioTek Synergy HT microplate reader. The scavenging by the samples of the ABTS^● was calculated according to Re et al. [38].

3.12. Lipase-Inhibitory Activity

The lipase-inhibitory activity assay was performed according to Amigo-Benavent et al. [39]. The assay buffer used was 13 mM Tris–HCl (pH 8.0) containing 150 mM NaCl and 1.3 mM CaCl₂. Porcine pancreatic lipase was dissolved in assay buffer (1 mg/mL). The substrate was a solution of 4-methylumbelliferyl oleate (4-MUO) dissolved in DMSO to reach a concentration of 1 mM. Samples (1 mg/mL on a protein basis) were dissolved in DMSO. The assay was performed by addition of preheated (30 °C) lipase (25 µL, 50 mU/mL) to 50 µL of substrate, 25 µL test sample and 100 µL assay buffer in a microplate followed by shake-incubation at 30 °C for 30 min. Afterwards, the fluorescence was measured at excitation and emission wavelengths of 360 and 460 nm, respectively, using a BioTek Synergy HT microplate reader. The positive control was Orlistat^® which was prepared similarly to the test samples. The percentage of lipase inhibition of samples at a concentration of 1 mg/mL (on a protein basis) was reported.

L.G. was in receipt of an Erasmus-funded internship via AgroSup Dijon—Université Bourgogne Franche-Comté, France.