Patent Application
20240225040
The present invention relates to gels formed from pulse protein, and methods of making and using such gels.
One important functional property of food proteins are their gelling capacity to provide food texture. Generally, thermal gelation of globular proteins involves unfolding of the protein by heating, which leads to exposure of active side chains Later, unfolded molecules re-arrange and aggregate via disulfide bridges, hydrogen bonds, hydrophobic and/or van der Waals interactions. Finally, aggregates associate to form a three-dimensional gel network (Clark, Kavanagh, & Ross-Murphy, 2001). Plant proteins usually have weaker gelling capacity compared to animal proteins (e.g., collagen, gelatin, and egg protein) because of their lower solubility and higher denaturation temperature (Liang & Tang, 2013).
Pea protein as an alternative for soy protein has attracted growing interest in food industries, due to its high nutritional value and hypoallergenic status. However, high temperature (>95° C.) is required to enable heat-induced gelation and its gelling properties are relatively weak. Currently, pea protein concentrates and isolates are used as food supplements for athlete diets and functional ingredients in food formulations due to their functional properties (Moreno, Dominguez-Timón, Diaz, Pedrosa, Borderias, & Tovar, 2020).
When compared to plant-based protein like soybean protein, which is more commonly used in food formulations, pea legumin is found to show a relatively weaker gelation performance compared to soybean glycinin due to a lower cysteine content, which contributes to the formation of disulfide bonds and a higher minimum gelling concentration was required for pea legumin (O'Kane, Happe, Vereijken, Gruppen, & van Boekel, 2004).
There is a need in the art for methods of forming pea protein gels which may enable wider applications of pulse protein applications in food formulations where gelling and texturization functions are required. It may be advantageous to enable a pulse protein such as pea protein to be used as a gelling ingredient of plant protein-based food products, such as meat analogues and egg alternatives.
This background information is provided merely to provide information believed to be relevant to a basic understanding of the present invention. It is not an admission that any of the foregoing is prior art against any aspect of the claimed invention.
Aspects of the present invention relate to pulse protein gels, and methods of forming such gels. In some embodiments, the pulse protein gels are formed using either cold plasma as a novel non-thermal technique to improve the gelling properties of pea protein, or by pretreatment with a pH-shifting method, or both cold plasma and pH-shifting.
In one aspect, the invention may comprise a method of treating a pulse protein to produce a pulse protein gel, comprising the steps of:
In some embodiments, the pulse protein comprises protein extracted from peas (Pisum sativum), chickpeas, lentils, or beans.
In some embodiments, the invention may comprise one or more of the following features. The pulse protein may be treated by shifting to the alkaline pH, optionally with ACP treatment, either sequentially in either order or simultaneously. The alkaline pH may be between about pH 10.0 to about 12.0. The protein may be held at the alkaline pH for longer than about 1 hour. The protein may be held at the alkaline pH for about 48 hours. The length of ACP treatment may be greater than about 2 minutes and less than about 20 minutes. The protein may be treated by both ACP and pH shift to about pH 12.0. The protein may be treated by both ACP and pH shift with a total treatment time of between about 2 to about 20 minutes.
In some embodiments, the method may comprise the further step of heating the protein to induce gelation, preferably below about 95° C., about 85° C. or about 80° C.
In some embodiments, the method results in a gel having a polymer-like microstructure, having a minimum gel compressive strength of 1.0 kPa, preferably 2.0 kPa, or more preferably 3.0 kPa, which preferably may be achieved without incorporation of an additional cross-linking agent or non-pulse protein.
In another aspect, the invention may comprise a gel formed from pulse protein, said gel having a filamentous, polymer-like microstructure, having been formed from pulse protein treated with atmospheric cold plasma and/or a pH shift to an alkaline pH greater than pH 9.0, preferably about pH 10 to about 12, or an acidic pH lower than pH 5.0, preferably about pH 1.5 to about 3.5, followed by adjusting the pH back to neutral. In some embodiments, the gel has a minimum gel compressive strength of 1.0 kPa, preferably 2.0 kPa, or more preferably 3.0 kPa, after being formed at a gelation temperature below about 95° C., about 85° C. or about 80° C.
In another aspect, the invention may comprise a food additive comprising pulse protein which has been treated with atmospheric cold plasma and/or a pH shift to an alkaline pH greater than pH 9.0, preferably about pH 10 to about 12, or an acidic pH lower than pH 5.0, preferably about pH 1.5 to about 3.5, followed by adjusting the pH back to neutral. In some embodiments, the additive may comprise a powder or a liquid suspension.
Aspects of the present invention relate to methods of forming pulse protein gels using a non-thermal plasma, referred to herein as atmospheric cold plasma (ACP), as a novel non-thermal technique to improve the gelling properties of pulse protein, or by pretreatment with a pH-shifting method, or both ACP and pH shifting. The gels may be formed without the use of other cross-linking agents or other non-pulse proteins.
The pulse protein gels described herein may allow wider applications of pulse protein as an alternative to animal or soy protein in food additives, such as to provide gel-like food texture for meat analogues and egg replacers. Furthermore, treated pulse protein powder may be conveniently incorporated into many food formulations.
In some embodiments, the pulse protein may comprise protein extracted from peas such as yellow or green peas (Pisum sativum); chickpeas; lentils such as red or green lentils; beans such as black beans, red kidney beans, Adzuki beans, black gram beans, navy beans, or broad beans (Faba beans); or any other pulses. Those skilled in the art would recognize a pulse as being defined or recognized by the United Nations Food and Agriculture Organization.
As used herein, a “gel” is a semi-solid comprising a substantially dilute cross-linked system, which exhibits no flow when in the steady-state, although the liquid phase may still diffuse through this system. Typically, globular proteins form 2 types of gels, particulate gels and filamentous gels. The preferred gels described herein are different from typical protein gel microstructures. They exhibit a “polymer-like microstructure” which means the gel network is highly cross-linked, in like manner to polysaccharides or synthetic polymers.
While native pea protein concentrate (PPC) (12 wt. %) does not form a gel under 90° C., ACP-treated PPC show satisfactory gelling properties when heated at a temperature between about 70° and 99° C. The gels exhibit homogeneous three-dimensional network structure with interconnected macropores, and those prepared at higher temperatures, eg. 80 and 90° C., possess good mechanical strength and viscoelasticity, as well as high water holding capacity. ACP treatment contributed to the formation of protein fibrillar aggregates, and significantly reduced the PPC denaturation temperature, leading to protein unfolding at reduced temperature of about 80-90° C. ACP treatment increases the protein surface hydrophobicity and exposes free sulfhydryl groups but decreases total free sulfhydryl groups indicating increased disulfide bonds, which allowed the formation of gels with improved mechanical properties.
Heat-induced gels from pulse protein may also be formed by pre-treatment with a pH-shifting method. Exposing the protein to an acidic or alkaline pH causes the formation of a three-dimensional (3D) coagulum, and the coagulum maintains its structure when pH is adjusted back to neutral (less than about 8, or greater than about 6). Heating the pH-shifted protein above about 70° C., for example at 92° C., led to gels of dense cross-linked polymer-like network with thin connective walls and a small pore size, e.g. 1-2 μm. Such uniform polymer-like gel network microstructure is believed to be related to a high level of protein unfolding during pH-shifting treatment, which made the protein chain flexible and created more active sites to promote molecular interactions including hydrogen bonds and hydrophobic interactions when the protein was neutralized and heated.
The pulse protein gels formed by the methods described herein exhibit excellent mechanical properties and water holding content (WHC) comparable to gels made from some animal proteins or soy protein.
The pulse protein may be supplied as a powder. In one example pea protein powder (for example, 53.1% protein, 33.7% carbohydrate, 2.0% fat, 5.3% ash, and 5.9% moisture) was provided by AGT Foods Research & Innovation Centre (Saskatoon, SK, Canada). Pea protein concentrate (PPC) may be prepared by a washing process, substantially as described by Walia and Chen (2020). Briefly, pea protein powder is mixed with distilled water to form a suspension, preferably at a 1:7 ratio (w/v). The pH of the suspension may be adjusted as desired, for example to pH 9 with 1 M NaOH. After stirring, the solid fraction may be collected, such as by centrifuging or filtration. The solid fraction may then be dispersed in distilled water and adjusted to pH 7 by adding 1 M NaOH. Next, the mixture was dialyzed (3.5 kDa MWCO dialysis tubing) against distilled water for three days and then dried or lyophilized to obtain the PPC powder. The protein content of the powder may preferably be between about 70-95% (w:w).
Some embodiments of the present invention are based on the use of atmospheric cold plasma (ACP) as a treatment to improve pea protein gelling properties. As used herein, a “plasma” is a fully or partially ionized gas containing neutrals, ions, free radicals and electrons, which can be produced by a variety of electrical discharges.
ACP is well-known to those skilled in the art, and is conventionally used as an alternative to traditional thermal processing for microbial inactivation to extend the shelf life of different food products such as fruits, vegetables, meats, eggs, and cereals (Miao, Nyaisaba, Koddy, Chen, Hatab, & Deng, 2020). ACP technology has the advantages of shorter treatment time and no thermal damage to food physical properties (e.g., color, texture), flavor and nutritive value (e.g., vitamins and flavonoids) due to its lower temperature (<60° C.) compared to traditional thermal processing (Deng, Ruan, Mok, Huang, Lin, & Chen, 2007; Mandal, Singh, & Singh, 2018).
At atmospheric pressure, when air is used, an ACP process generates reactive oxygen species (ROS) such as O3, O, O−, O2−, O2−2, H2O2 or —OH, and reactive nitrogen species (RNS) such as NO, NO2 or N2. These reactive species are known to trigger lipid peroxidation in microbial cell membranes, and oxidize proteins and DNA, leading to microbial inactivation (Feizollahi, Misra, & Roopesh, 2020). The active species generated by ACP can also break covalent bonds and initiate some chemical reactions (Kim, Lee, & Min, 2014). ACP treatment can expose active sites on the protein surface, thus increasing the affinity between protein and water molecules. The enhanced surface polarity of the protein molecules by ACP treatment is associated with the improved solubility (Dong, Gao, Xu, & Chen, 2017; Ji, et al., 2018; Ji, et al., 2019). In addition, ACP technology could affect the formation of disulfide bonds in proteins.
ACP may be applied with suitable plasma systems, which are generally commercially available. For example, a dielectric barrier discharge plasma system (PG 100-D, Advanced Plasma Solutions, Malvern, USA) is representative of an exemplary suitable system.
In some embodiments, a pea protein suspension (such as 12 wt. %) may be prepared by dispersing and stirring protein powder in water, preferably ensuring thorough hydration. The protein suspension is then placed between a high-voltage electrode and a ground electrode of a plasma system. The distance from the sample surface to the high-voltage electrode may between about 1 mm to about 5 mm, preferably between about 2 mm and 4 mm. The frequency may be between about 1000 Hz and 5000 Hz, preferably between about 2000 to about 3500 Hz, such as about 3000 Hz. The duty cycle may be varied, such as 70%. The voltage output may be 2-10 kV, 0-20 kV, or 0-30 kV, the current output may be 0.0 to 1.0 A. The pulse width may be 10 μs. The length of treatment may vary between seconds and minutes, for example between about 2 to about 20 min, preferably between about 5 to about 15 min, such as 10 min. The process may be repeated, for example five times. The temperature of ACP-treated sample may slightly increase as a result of such treatment, for example 1-3° C., however, the process is preferably non-thermal in that the suspension is not heated.
The ACP-treated PPC may optionally be processed to remove air bubbles, and heated at gelation temperature between about 70° and about 100° C., preferably above 70° C., followed by cooling, to strengthen the gel network. At 70° C., the protein does not significantly unfold and the undenatured protein may form particulate aggregates to build up a coarse and particulate network together with the initially formed fibrillar aggregates by ACP treatment, leading to self-standing but relatively weak gels (the compressive strength of 0.53 kPa). At about 80° C. and above, ACP treated pea protein unfolds to develop a strong fibrillar network mainly stabilized by hydrogen bonding together with other interactions. This resulted in a gel with a compressive strength of 2.70 kPa, a water holding capacity of 85%, and good viscoelasticity. At 90° C., a stronger gel with a more homogenous porous structure was formed because the more fully unfolded protein exposed more hydrophobic groups and active sites. In this way, protein chains were crosslinked to the fibrillar aggregates formed by ACP treatment through more interactions. In preferred embodiments, the gel strength reaches 6.27 kPa, water holding capacity is 88%, and with good viscoelasticity.
Without restriction to a theory, it is believed that the gelling mechanism of atmospheric cold plasma (ACP) treated pulse protein is based on the protein tertiary structure partially unfolding, exposing internal hydrophobic side chains and active sites. The active groups on the surface of protein molecules (such as —SH groups) may react with some free radicals generated by ACP to form covalent bonds (e.g., —S—S— bonds). At the same time, the ACP treated pea protein concentrate (PPC) suspension contained a significant amount of negative charge, which prevents protein random aggregation, thus the protein grows linearly based on hydrophobic patches, to form fibrillar aggregates.
Protein conformational changes are sensitive to pH variations due to electrostatic repulsion. As used herein, “pH-shift” or “pH-shifting” means to treat the protein with acidic or alkaline conditions sufficient to substantially disrupt the tertiary structure of the protein. Thus, in some embodiments, pulse proteins may be treated by exposure to alkaline pH (greater than pH 9.0, preferably about pH 10 to about 12) or acidic pH (lower than pH 5, preferably about pH 1.5 to about 3.5), followed by adjusting the pH back to neutral. Heating the pH-shifted proteins induces gel formation. The shift to an alkaline or acidic pH induces an intermediate conformational state during protein unfolding, which has a similar secondary structural content as the native state, but with a disrupted tertiary structure.
When pulse protein is exposed to more alkaline conditions, for example pH 12, more extensive protein unfolding and disruption of disulfide bonds occurred to expose hydrophobic side chains. Pulse protein also loses some side-chain interactions to create free active sites, thus becoming more flexible. The increase of ionic interactions and hydrogen bonds between charged proteins and water contributes to improved protein solubility.
The pH-shifting process preferably comprises treatment at a highly alkaline pH, e.g. greater than pH 10, such as pH 12, because the higher pH significantly improved pea protein solubility and emulsifying properties when compared to other samples modified by pH-shifting at pH 2, 4, and 10. It is believed the protein underwent more unfolding under pH 12 for modification of functional properties.
Without restriction to a theory, it is believed that pulse protein molecules which exist as mostly hexamers and trimers, dissociate to individual subunits upon alkali or acid treatment. The subunits partially unfold to facilitate protein aggregation to form initial protein crosslinking. When pH is adjusted back to neutral, certain refolding is expected due to neutralization of the negative charges. The refolding of the protein upon neutralization was negatively correlated to the degree of unfolding when exposed to alkaline pH, so the extent of unfolding in protein coagulum treated for an extended period (48 h holding) was expected to be the highest. Polymer-like gels develop upon heating because partial protein unfolding, and crosslinked aggregates were developed prior to heating. Further unfolding and protein aggregations both took place during heating, where extensively unfolded protein developed at a lower heating temperature, which might not quickly induce aggregations so that polymer-like gels with uniform network structure develop. Heating native protein molecules without pH-shifting pre-treatment results in unfolding at a higher temperature followed by quick aggregation, thus are more likely to form gels with particulate microstructures.
The uniform polymer-like network structure which results contributes to strong and elastic gels when compared to particulate gels obtained from native untreated protein directly. In addition, such a polymer-like network created more active sites to promote protein molecular interactions, resulting in gels enforced by improved hydrogen bonds and hydrophobic interactions to withstand large deformations. Moreover, the fine-stranded polymer-like gels exhibited evenly distributed network with small pore size, giving high water holding capacity and minimum water loss.
In some embodiments, the invention may comprise a method of forming a pulse protein gel, comprising the step of treating pulse protein with ACP and pH-shifting, simultaneously or in either order. Satisfactory pulse protein gels may be formed at reduced temperatures (e.g., as low as 70° C.) that are lower than the denaturation temperature, even with relatively short heating times (e.g., 10-20 min), and shorter exposure to the combined ACP and pH-shifting treatment (e.g. 10 min) than with either treatment individually.
The same factors and features described herein for each treatment individually applies when the treatments are combined. In some embodiments, the combined treatment may permit significantly shorter treatment time when the treatments are applied, either consecutively or simultaneously. The resultant gels from the combined treatment show strength comparable to soy protein gels.
Specific embodiments of the present invention are described with reference to the following Examples. These Examples are provided for the purpose of illustration only.
Pea protein powder (53.1% protein, 33.7% carbohydrate, 2.0% fat, 5.3% ash, and 5.9% moisture) was provided by AGT Foods Research & Innovation Centre (Saskatoon, SK, Canada). Sodium dodecyl sulfate (SDS), 2-mercaptoethanol (2-ME), 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), 1-anilinonaphthalene-8-sulfonic acid (ANS), 2,4,6-trinitrobenzene sulfonic acid (TNBS) (5% (w/v) in H2O), glycine, L-lysine, Trizma® base and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma-Aldrich (St. Louis, MO, USA), urea and other chemicals (analytical grade) were purchased from Fisher Scientific (Fair Lawn, NJ, USA).
Preparation of Pea protein Concentrate (PPC)
PPC was prepared as described by Walia and Chen (2020) with minor modifications. Briefly, pea protein powder was mixed with distilled water at a 1:7 ratio (w/v) and the pH of the suspension was adjusted to 9 using 1 M NaOH. After stirring for 1 h at room temperature, the mixture was centrifuged at 8000×g for 15 min (Acanti® J-E centrifuge, Beckman Coulter, USA). The supernatant was collected and adjusted to pH 4.5 using 1 M HCl. Then the suspension was centrifuged at 8000×g for 15 min and the precipitate was collected. The pellet was dispersed in distilled water and adjusted to pH 7 by adding 1 M NaOH. Next, the mixture was dialyzed (3.5 kDa MWCO dialysis tubing) against distilled water for three days and then lyophilized to obtain the PPC powder. The protein content was 87.2% measured by a Leco nitrogen analyzer (Leco®, USA) using a nitrogen-to-protein conversion factor of 5.96 (Fujihara, Kasuga, & Aoyagi, 2001).
ACP with a dielectric barrier discharge plasma system (PG 100-D™, Advanced plasma solutions, Malvern, USA) was used to treat the PPC sample. Pea protein suspension (12 wt. %) was prepared by dispersing PPC (1.2 g) in distilled water (8.8 g) with magnetic stirring (700 rpm) at room temperature (22-23° C.) for 2 h and then stirred overnight at 4° C. in refrigerator to ensure thorough hydration. In the next day, the PPC suspension was taken out from the fridge (4° C.) to return to room temperature before further experiments. Approximate 2 mL of 12 wt. % PPC suspension in a 4-cm diameter and 0.5-cm height plastic container (METER®, Pullman, WA, USA) was placed between a high-voltage electrode and a ground electrode. The distance from the sample surface to the high-voltage electrode was maintained at 2 mm, the frequency was 3500 Hz and the duty cycle was 70%. The voltage output was 0-30 kV, the current output was 0-1 A, and the pulse width was 10 μs. The sample was treated for 2 min, followed by well stirring. This process was repeated five times, so the total treatment time was 10 min. The temperature of ACP-treated sample increased from 21.3° C. to 23.5° C.
Approximately 5 mL of ACP-treated PPC (12 wt. %, pH 6.0) was transferred into a 10 mL (BPA-free) of cryovial (Simport™ polypropylene T310-10A) at room temperature. All tubes were centrifuged at 3000 ×g for 1 min (Centrifuge 5810 R, Eppendorf, Germany) to remove air bubbles, and then heated at 70, 80, and 90° C. for 30 min followed by cooling in an ice-water bath for 10 min, and then stored in a fridge (4° C.) for 24 h prior to analysis. The gels were also prepared from untreated PPC suspension (pH 6.9) at the same concentration and temperature for comparison. Each sample was replicated three times.
The impact of various ACP treatments on pea protein properties, and suitable ranges and optimized conditions have been determined (3500 Hz, 10 μs, 10 min, 0-30 kV, and 0-1 A) which can improve the gelling capacity of pea protein. As shown in
After ACP treatment, PPC was found to form gels even at 70° C., which was significantly lower than the denaturation temperature (legumin of around 92° C.) of pea protein (Liang, et al., 2013).
Mechanical properties of gel samples were measured using an Instron 5967 universal testing instrument (Instron Corp., MA, USA) equipped with a 50 N load cell (Yang, Wang, Vasanthan, & Chen, 2014). The gel samples had a height of 8 mm and a diameter of 14 mm. The sample was compressed to 30% of its original height with a rate of 1 mm/min at room temperature (22-23° C.). Compressive strength was calculated as the maximum compressive stress in the stress-strain curve. Young's modulus was determined by the slope of the linear section (the compressive strain of 0-10%) in the stress-strain plots.
The gel formed at 70° C. showed a compressive strength of 0.53 kPa, and this value increased to 2.70 kPa and 6.27 kPa when the temperature was raised to 80° C. and 90° C., respectively. The Young's modulus of the gel formed at 90° C. was 5.84 kPa, followed by gel heated at 80° C. (4.21 kPa) and 70° C. (1.43 kPa). It was noted that the gels formed at 80° C. from 12 wt. % ACP treated PPC showed even higher mechanical strength than those of native pea protein gels (15 wt. %) heated at 95° C. for 30 min (1.03 kPa) (Munialo, van der Linden, & de Jongh, 2014).
Young's modulus of the gel formed by ACP-treated PPC (4.21 kPa) was higher than that of gel formed from soy protein isolate at the same conditions (80° C. for 30 min) was higher than that of the gel formed by soy protein isolate (15 wt. %, pH 7, 10 mM CaCl2) at 80° C. for 30 min (1.84 kPa) (Brito-Oliveira, Bispo, Moraes, Campanella, & Pinho, 2018).
The storage modulus (G′) and loss modulus (G″) indicate the elastic and viscous properties of gels. As shown in
As ACP-treated PPC gels showed solid-like behavior based on rheological results, the
morphology of PPC with or without ACP treatment was observed by TEM. The PPC in its native state was shown in
The microstructures of PPC gels prepared at 70, 80, and 90° C. are shown in SEM images (
Rheological measurements were performed on a TA Discovery HR-3 rheometer (TA Instruments, DE, USA) using a parallel plate geometry with a diameter of 40 mm. Unless described otherwise, all measurements were conducted at a gap of 1 mm by applying a constant strain of 1%. Frequency sweeps were done to determine the storage modulus G′ and loss modulus G″ at oscillation frequency from 0.1 to 100 rad/s. To study the changes in viscoelastic properties as a function of temperature, the PPC suspensions with or without APC treatment were subjected to a temperature ramp at a rate of 2° C./min from 25 to 90° C., held at 90° C. for 30 min, and then cooled down to 25° C., and held at 4° C. for 30 min. Silicone oil was applied to cover the surface of samples to prevent evaporation during the tests. There was no sample weight loss before and after the tests and the effect of silicone oil on the measurements was regarded as negligible. The temperature of the plate was controlled with a Peltier system. All rheological measurements were performed within a predetermined linear viscoelastic region at the strain value at 1%.
Molecular interactions involved in the gel formation were studied by a rheological measurement in presence of dissociation reagents (Nieto-Nieto, Wang, Ozimek, & Chen, 2015; Yang, Wang, Vasanthan, & Chen, 2014). The gel samples were cut into approximately a height of 1.0 cm and a diameter of 1.4 cm. The resulting gel disks were immersed into 0.2 M 2-mercaptoethanol (2-ME), 2 M urea, or 1% (w/v) sodium dodecyl sulfate (SDS) solution for 48 h at room temperature. The gel disks immersed in the distilled water were used as control samples. A frequency sweep analysis was conducted on the soaked gels to measure G′ and G″.
Dynamic rheological properties of the PPC suspensions with or without ACP treatment were studied when subject to a temperature ramp to simulate the gelation process (
The gels formed at 90° C. were immersed into dissociation reagents including urea, sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (2-ME) in order to disrupt hydrogen bonds, hydrophobic interactions and disulfide bonds, respectively. As shown in
Water holding capacity of a gel is an important property for its application in food production because a lack of water has a negative impact on the texture and quality of a foodstuff.
Water holding capacity (WHC) was determined according to a method described by Nieto-Nieto et al. (2016) with slight modification. PPC gel samples (1.0 g) were placed into a Vivaspin 20 centrifugal filter unit (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and centrifuged at 4000×g for 20 min. The weight of the gel was recorded before or after centrifugation. The WHC of gels was calculated according to equation (1):
Where M1 represents the mass of total water in the gel before centrifugation (g), M2 represents the mass of water released in the gel by centrifugation (g).
The morphology of protein samples before or after ACP treatment in suspension was observed using a transmission electron microscope (TEM, Morgagni 268, Philips-FEI, Hillsboro, USA) at an accelerating voltage of 80 kV. A drop of the diluted PPC suspensions (0.03 wt. %) was transferred onto a carbon film-covered 400 mesh copper grid. Subsequently, a drop of 4% uranyl acetate (staining solution) was added for negative staining for 45 s, then the excess solution was removed with filter paper.
A Zeiss EVO M10 scanning electron microscope (SEM) (Carl Zeiss AG, Oberkochen, Germany) was used to examine the microstructure of the PPC gels. The gel samples were rapidly frozen in liquid nitrogen, and then freeze-dried. The cross-sections of the gel samples were sputter-coated with gold under vacuum for 90 s and then was observed to record the microstructure at 20 kV.
The surface charge (zeta potential) of PPC suspensions with or without ACP treatment were determined by laser Doppler velocimetry using a Zetasizer Nano-ZS (Malvern Instruments Ltd., UK). The protein refractive index (RI) was set at 1.45 and dispersion medium RI was 1.33. The samples were properly diluted before measurement.
The contents of free —SH groups including total and exposed —SH were measured using the method described by Dong et al. (2017) with modification. PPC suspensions (12 wt. %) with or without ACP treatment were diluted five times with Tris-glycine buffer (pH 8.0). Then 1.0 mL of the diluted sample suspension was mixed with 2.0 mL of Tris-glycine buffer (pH 8.0) and 0.02 mL of Ellman's reagent (DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid), 4.0 mg/mL) in 5.0 mL of microcentrifuge tubes. After the mixture was incubated at 25° C. for 5 min, the exposed free —SH group content was determined at 412 nm using a SpectraMax M3 microplate reader (Molecular Devices, USA). The total —SH group content was evaluated as well. First, 0.5 mL of diluted samples was mixed with 0.05 mL of 2-mercaptoethanol and 2.5 mL of 10 mol/mL urea in Tris-glycine buffer (pH 8.0) in 15 mL centrifuge tubes. After incubating at 25° C. for 1 h, 2.5 mL of 12% trichloroacetic acid (TCA) was added and the mixture was further incubated for 1 h at 25° C. Subsequently, the tubes were centrifuged at 3000×g for 15 min and the precipitate was suspended in 2.0 mL of 12% TCA. The mixture was centrifuged again to remove the 2-mercaptoethanol. Finally, the precipitate was dissolved in 3.0 mL of 8.0 mol/mL urea in Tris-glycine buffer (pH 8.0) and then 0.05 mL Ellman's reagent was added. After 5 min, the absorbance was read at 412 nm using a SpectraMax M3 microplate reader (Molecular Devices, USA). The content of the free —SH group was calculated according to equation (2).
Where A is the absorbance at 412 nm; D is the dilution multiple; C is the sample concentration (mg/mL).
The content of free —NH2 groups was evaluated by the trinitrobenzene sulphonate (TNBS) assay (Habeeb, 1966) with slight modification. The PPC suspensions with or without ACP treatment were diluted with reaction buffer (0.1 M sodium bicarbonate, pH 8.5). And 0.01% (w/v) TNBS solution was prepared using reaction buffer as the diluent. Then, 0.5 mL of the sample was mixed with 0.25 mL of the 0.01% TNBS solution. After incubating at 37° C. for 2 h, 125 μL of 1 N HCl was added to the mixture to terminate the reaction. Then the absorbance of the mixture was recorded at 335 nm using a SpectraMax M3 microplate reader (Molecular Devices, USA). L-lysine solution (2.5, 5, 10, 20, and 40 μg/mL) was used as a standard instead of a sample to repeat the above experimental steps and record absorbance. The content of free —NH2 groups was calculated by the standard curve.
The surface hydrophobicity (H0) of samples was evaluated using the fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (ANS) (Kato & Nakai, 1980) according to Chalamaiah et al. (2017) with slight modification. Serial dilutions (protein concentration of 0.001 to 0.5 mg mL−1) of the PPC suspensions with or without ACP treatment were prepared by diluting with 0.1 M phosphate buffer (pH 7.4). Then, 20 μL of 8 mM ANS solution was mixed with 4 mL diluted sample. The fluorescent intensity was measured at an excitation wavelength of 390 nm and an emission wavelength of 460 nm using a SpectraMax M3 microplate reader (Molecular Devices, USA) after the mixture was kept in the darkroom for 15 min. The initial slope of the corrected fluorescence intensity (the fluorescence intensity of protein suspension with ANS subtracted protein suspension without ANS) versus protein concentration plot was calculated by linear regression analysis and used as an index of surface hydrophobicity.
Table 1 summarizes the changes in the surface charge of untreated and ACP-treated PPC suspensions. Since the isoelectric point of pea protein (about pH 4.5) was lower than the pH value of the native PPC suspension (pH 6.9) and the ACP-treated PPC suspension (pH 6.0), both samples were negatively charged as reflected in their zeta potential values. The decrease in pH by ACP treatment might be due to the formation of some acidic substances, such as acidic H3O+ ions formed from the reaction between water molecules and H2O2 generated by ACP, and HNO3 and HNO2 generated from NO through NO2 (Ji, et al., 2018). Because the inherent minerals of pea protein caused relatively low salt levels in suspension, the protein molecules aggregated in an ordered ‘string-of-beads’ via the weakened electrostatic repulsion after ACP treatment and possible attractive hydrophobic interactions between protein molecules (Bryant & McClements, 1998).
On the one hand, protein unfolding can expose free —SH groups that are originally hidden inside the protein globular structure, on the other hand, —SH groups are easily oxidized by reactive oxygen species (ROS) generated by ACP, such as ·OH (Zhang, et al., 2020) and H2O2 (Sullivan & Sebranek, 2012). Thus, the changes of the total and exposed —SH groups, as well as the free —NH2 groups of PPC before and after ACP treatment were measured and the results are displayed in Table 2. ACP treatment reduced the total —SH groups from 10.22 to 8.28 μmol/g protein. However, the content of exposed free —SH groups was slightly increased after ACP treatment. These results suggest protein unfolding to expose more —SH groups, although the generated free —SH groups reacted with reactive species to form —S—S— bonds at the same time. Meanwhile, the free —NH2 groups of PPC had no significant changes after ACP treating, which means no significant protein hydrolysis occurred.
FTIR spectra of the PPC in suspensions and gels were recorded using a Nicolet 6700 Fourier transform infrared spectrophotometer (Thermo Fisher Scientific Inc., MA, USA). All data were collected in the spectral range from 4000 to 800 cm−1 during 128 scans at a spectral resolution of 4 cm−1. D2O was used as a solvent for the PPC suspensions and gels preparation. The samples were placed between two CaF2 windows separated by a 25 μm polyethylene terephthalate film spacer for test. The spectrophotometer was continuously purged with dry air from a lab gas generator (Parker Hannifin Corp., USA). Protein secondary structures (α-helices, β-sheets, β-turns, and random coils) were determined by analyzing the amide I band region (1700-1600 cm−1) according to the Fourier self-deconvolution (FSD) algorithm using Omnic 8.1 software (Thermo Fisher Scientific, MA, USA) at a bandwidth of 24 cm−1 and an enhancement factor of 2.5.
The fluorescence spectra of the PPC suspensions with or without ACP treatment were measured by a SpectraMax M3 microplate reader (Molecular Devices, USA). The samples were properly diluted before measurement. The excitation wavelength was 295 nm. The emission spectra at 300-400 nm were recorded at 10 nm/s scanning speed. The emission and excitation slits were set at 3 nm.
The tertiary structure of proteins can be evaluated by studying intrinsic fluorescence spectra from tryptophan, tyrosine, and phenylalanine residues.
The protein surface hydrophobicity (H0) was also determined to assess protein denaturation. The H0 value of PPC suspension increased from 19800 to 23000 after ACP treatment (
The changes in protein secondary structure were studied by FTIR through the analysis of the amide I band (1600-1700 cm−1) (
Heating of the native PPC suspension at 90° C. for 30 min only partially unfolded pea protein due to its high denaturation temperature. Heating of ACP treated PPC at 70° C. also did not lead to significant protein denaturation, but interestingly a self-standing gel was able to be formed. When heating ACP-treated PPC at 80 and 90° C., obvious protein denaturation was observed as reflected by the disappearance of the major protein secondary structural components, accompanied by significant increase of the absorptions at 1691 and 1608 cm−1, suggesting pea protein aggregation after unfolding. The band at 1619 cm−1 corresponded to intermolecular β-sheets caused by aggregation via hydrogen bonds (Clark, Saunderson, & Suggett, 1981), while the one at 1691 cm−1 indicated antiparallel β-sheets (Byler & Susi, 1986). This result indicates that ACP treatment enabled pea protein denaturation at reduced temperature of (80-90° C.) to facilitate protein interaction development for gel formation.
All experiments in this section were performed in three independent batches and the data were reported as mean±standard deviations. Statistical evaluation was analyzed using a paired T-test or One-way analysis of variance followed by post-hoc test (Fisher's Least Significant Difference (LSD)). All of the analyses were conducted using SPSS 21.0 software (IBM Corp., USA) with a probability of p<0.05 considered to be significant.
Pea protein concentrates (PPC 55% protein) were provided by AGT Foods and Ingredients (Saskatchewan, Canada). Reagents and chemicals used in the experiments were purchased from Sigma-Aldrich (MO, USA) and Fisher Scientific (Ontario, Canada).
PPC were dissolved in distilled water with the solid to solvent ratio of 1:6 to make a PPC suspension. Sodium hydroxide (NaOH, 6 mol/L) aqueous solution were added until pH 9 to further solubilize pea protein, followed by one hour of stir at room temperature (22° C.). Afterwards, the suspension was centrifuged at 8000 rpm for 15 min using high performance centrifuge (Acanti® J-E centrifuge, Beckman Coulter, USA) to obtain the supernatant, followed by acidic precipitation to pH 4.5 using 6 mol/L HCl solution and centrifugation with the same condition as above to obtain the sediment. The sediment was washed with distilled water and adjusted the pH back to 7.0 by addition of 6 mol/L NaOH solution, stirring well to form a solution. The protein solution was dialyzed using regenerated cellulose tubing (Spectra/Por® 3, 54 mm flat width, molecular weight cut-off: 3.5 kDa, Spectrum Chemical Mfg. Corp., Gardena, USA) against distilled water for 96 h. The dialyzed pea protein solution was freeze dried using Labconco Freezone 6L Console Freeze Dryer System with Stoppering Tray Dryer for 7 days to obtain the pea protein isolate (PPI). The protein content was 88.94% determined by Leco nitrogen analyzer (Leco, USA) using nitrogen-protein conversion factor of 5.96 (Fujihara, Kasuga, & Aoyagi, 2001).
The PPI suspensions were prepared in phosphate buffer saline (1×PBS, pH 7.4) solvent at room temperature. 0.02% (w/v) sodium azide was added as antimicrobial agent. PPI solutions were adjusted to pH 12 using 1 mol/L NaOH solution and were held for different periods of time. The PPI solution were then titrated back to pH 7 with 1 mol/L HCl solution at room temperature while stirring to maintain a homogeneous suspension. The suspensions after pH-shifting treatment were heated at 92° C. for 1 or 2 h to form gels. Afterwards, the heated samples were cooled to room temperature and held at 4° C. overnight before analysis. The effect of protein concentration (17%, 21%), holding time at alkaline pH (1, 24 and 48 h) and heating time (1 and 2 h) on the pea protein gel properties was studied. The detailed sample preparation condition is described in Table 3, and the samples were labeled as 17% HO1-HE2, 17% HO24-HE1, 17% HO24-HE2, 17% HO48-HE2, and 21% HO48-HE2 (“HO” as holding time, “HE” as heating time, “%” as pea protein concentration). A heating temperature at 92° C. was chosen which was reported to be the pea protein denaturation temperature in the presence of NaCl (Shand et al., 2007).
The gel samples with about 7 mm in length and 8.5 mm in diameter were analyzed by a universal testing machine (Instron® 5967, Instron Corp., MA, USA) with a 50 N load cell. The gel samples were compressed twice to 50% strain at room temperature with the crosshead speed of 1 mm/min. Textural parameters including hardness, springiness, and cohesiveness were computed by software (Blue Hill 2). Hardness is related to compressive stress and calculated as peak force (N) during the first compression cycle. Springiness indicates the ability of the gel to spring back after the deformation during the first compression cycle, which is measured by distance 2/distance 1 in the force-time curve (Ferreira, Calvinho, Cabrita, Schacht, & Gil, 2006). Cohesiveness indicates how well the gel perform against a second deformation compared to its resistance to the first deformation, which is represented by the ratio Area 2/Area 1 in the force-time curve (Ferreira et al., 2006).
Protein concentration significantly impacts the gel hardness value, which improved from 104.78 N to 155.27 N when the concentration was increased from 17 to 21%, while springiness and cohesiveness were not significantly affected. Protein concentration is believed to be a major factor affecting gel strength due to the fact that more unfolded protein molecules are available in close proximity for molecule interactions and gel network building.
The effects of heating time did not show significant difference in gel mechanical properties. It is worth noting that the springiness of 21% HO48-HE2 gel reached a mean of 0.82, which is comparable to the value of 0.86 from 10% gelatin gels (Marfil, Anhê, & Telis, 2012). Gelatin gels are known to have excellent elasticity due to the high solubility of gelatin molecules and the unique gel network structure created by rigid triple helix entangled by flexible cross-links, which are able to support large deformations.
SEM images of the 3D protein coagulum treated by pH-shifting method with different holding and heating period and the resultant gels are shown in
The corresponding resultant gels in
holding treatment on the establishment of pea protein gel structure. It is noticeable that polymer-like porous protein networks were established in all resultant gel samples. The gel with 1 h holding has thick connective walls and large pore size of more than 7 μm (
Globular proteins normally form particulate or filamentous gel by heating, depending on the environmental pH (Lefèvre, & Subirade, 2000). At a pH near a protein isoelectric point, heating cannot split the globular subunits, leading to the formation of spherical particles and a particulate gel structure. At a pH that deviates from the isoelectric point, a strong repulsive force causes the dissociation of subunits into monomers and induces extensive protein unfolding. The association of these unfolded protein chains results in flexible linear strands and filamentous gel or fine-stranded structure. The uniform polymer-like gel network microstructure created by methods of the present invention indicate a high level of pea protein unfolding. pH-shifting treatment at pH 12 (or acidic pH) can effectively interrupt disulfide bonds, hydrophobic interactions, and hydrogen bonds that establish the protein secondary and tertiary structures. This made the protein chain flexible and created more active sites to promote molecular interactions including hydrogen bonds and hydrophobic interactions when the protein was neutralized and heated, leading to a well-established gel network with better mechanical properties performance.
The gel water holding capacity was determined following the method from Kocher and Foegeding (1993) with modifications. Approximately 0.5 gram of the gel sample was loaded in ultrafiltration tube from Vivaspin Turbo 15, 3,000 MWCO (Sartorius Stedlm Lab Ltd., Stonehouse, UK) and centrifuged at 4000 rpm for 10 min at room temperature. The separated water was discarded and the weights of the tube together with the sample gel before and after centrifugation were recorded as W1 and W2. The weight of ultrafiltration tube was recorded as W. WHC was calculated as equation (1):
Approximately 0.5 gram of gel samples were soaked into 30 mL of water for up to 24 h at room temperature. Swelling ratio of pea protein gels was determined at different time point by equation (2):
As shown in
The pea protein gels exhibited considerably higher or comparable WHC value to the protein gels with good water holding properties, for example, the gel prepared from egg white at neutral pH had a WHC value of 90.03% (Khemakhem, Attia, & Ayadi, 2019); soy protein gels were reported to show a WHC range of 60-95% based on different preparation methods (Maltais, Remondetto, Gonzalez, & Subirade, 2005; Zhou et al., 2019). Such high WHC observed in this work was likely attributed to the uniform and dense crosslinked gel network structure achieved from the pea proteins after pH-shifting treatment. It is suggested that a gel with evenly distributed network structure tends to have better WHC as water is more tightly trapped in the dense structure (Munialo et al., 2014). Higher WHC was also found in finer-stranded network compared to thick-stranded network for the gel prepared from whey protein isolates (He, Azuma, & Yang, 2010).
The formed gels were tested on a DHR-3 rheometer (TA Instruments, DE, USA) to evaluate their viscoelastic properties. Samples were loaded on 40 mm parallel plate geometry with a gap of 1 mm. The value of the strain amplitude was set as 1%. The frequency sweep measurements were performed at 25° C. at an angular frequency (w) from 0.1 to 100 rad/s. Lines of storage modulus (G′) and loss modulus (G″) in response to frequency were plotted.
In order to monitor the change of rheological properties during the formation of pea protein gels, the protein suspensions after pH shifting back to pH 7 were tested during temperature ramps at a rate of 5° C./min from 25 to 92° C. and then cooled to 25° C. at a rate of 5° C./min. Parallel plate geometry (40 mm) with a gap of 1 mm was used to measure viscoelastic parameters including storage modulus G′ and loss modulus G″. Approximately 1.5 mL of each suspension sample was loaded for the dynamic temperatures sweep measurements and the angular frequency (w) was set to be 1 Hz.
The rheological behavior of the resultant gels is shown in
Temperature dependence of the gel rheological properties was shown in
Sharp increase in both G′ and G″ occurred at different temperature for samples treated by different holding time, with 1 h and 24 h holding sample at approximately 65° C. (
It is noticeable that the G′ value barely went up during the cooling procedure in all treatments, which suggests that heating process played much more important roles in establishing the formation of protein crosslinks than the cooling process did.
Protein suspensions after pH-shifting treatment and the formed gels were frozen in liquid nitrogen, and then freeze-dried. Cross-sectional view of the freeze-dried 3D pea protein coagulum and gels were sputter-coated with gold and the morphology was observed using a Philips XL-30 scanning electron microscope (SEM) at an acceleration voltage of 20 kV.
Pea protein was analyzed by FTIR (Fourier Transform Infrared Spectroscopy) at different steps in both dried and wet forms during the gel preparation to understand the protein conformational change. Pea protein isolate powder and the freeze dried pea protein gel samples were analyzed in KBr plate with KBr to sample ratio as 1:100 in weight and the spectra was recorded by Nicolet 6700 spectrophotometer (Thermal Fisher Scientific Inc., Pittsburgh, PA, USA) along the wavenumber from 400 to 4000 cm−1 with 128 scans at a 4 cm−1 resolution. Wet samples including 3D coagulum at pH 12 and the resultant gel samples were prepared with D2O, 1M DC1, and 1M NaOD. The wet samples were analyzed between CaF2 windows separated by a 25 mm polyethylene terephthalate film spacer with the spectra recorded at the same condition as previously described. Fourier self-deconvolution for amide I region was analyzed using Omnic 8.1 software at a bandwidth of 24 cm−1 and an enhancement factor of 2.5.
FTIR spectra results are displayed in
In order to understand the molecular interactions involved in the formation of pea protein gels, the gel samples prepared with different holding and heating time were soaked into water (controls), 2M urea, 0.02M 2-mercaptoethanol (2-Me), and 1 wt % sodium dodecyl sulfate (SDS) solution as dissociation reagents for 48 h to disrupt hydrogen bond, disulfide bond, and hydrophobic interactions, respectively. Frequency sweep analysis were performed on the soaked gel samples over a range of 0.1-100 rad/s at 25° C. with 1% strain. Storage modulus (G′) in response to frequency were plotted and shown in
The gels that immersed in 2-Me became too rigid to be tested, which might due to the replacement of water in the gel by 2-Me solvent. This also suggests that disulfide bonds might not be involved in the formation of the pea protein gel. This is probably owing to the low cysteine content in pea proteins. A very low amount of cysteine content as 0.87% was reported for pea protein in the study from Pownall, Udenigwe, and Aluko (2010). In the gel with 48 h holding time during pH-shifting treatment (
All experiments in this section were performed in three independent batches and the data were reported as mean±standard deviations. Analysis of variance (ANOVA) was performed to analyze the effects of holding time, heating time, and protein concentration on mechanical properties of gels, followed by Tukey's test to compare multiple means. A probability of p<0.05 was considered to be statistically significant.
Pea protein flours were provided by AGT Foods Research & Innovation Centre (Saskatoon, SK, Canada), which contained protein (53.1%), carbohydrate (33.7%), fat (2.0%), ash (5.3%), and moisture (5.9%) (S. Zhang et al., 2020). Broad-range SDS-PAGE molecular weight standards, SDS-PAGE 4-15% gels (Mini-PROTEAN® TGX™), sample buffer, and running buffer were purchased from Bio-Rad Laboratories Inc. (Hercules, CA, USA). Ethylenediaminetetraacetic acid (EDTA), Trizma® base, sodium dodecyl sulfate (SDS), 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), 2,4,6-trinitrobenzene sulfonic acid (TNBS) (5% (w/v) in H2O), 2-mercaptoethanol (2-ME), 1-anilinonaphthalene-8-sulfonic acid (ANS), L-lysine, and Bradford Reagent were purchased from Sigma-Aldrich (St. Louis, MO, USA). Urea, bovine serum albumin (BSA), and other chemicals (analytical grade) were purchased from Fisher Scientific (Ottawa, ON, Canada).
Atmospheric Cold Plasma (ACP) and pH-Shifting Pre-Treatment
Pea protein concentrate (PPC) was extracted from the pea protein flour by the method described previously (J. Yang, Liu, Zeng, & Chen, 2018) with slightly modification. Briefly, the pea protein flour was dispersed into water at a flour-to-water ratio of 1:7. The mixture was adjusted to pH 9 and stirred for 1 h. After centrifuged at 8000 g for 15 min, the supernatant was adjusted to pH 4.5, followed by centrifugation to precipitate the proteins. Finally, the protein precipitate was neutralized and dialysis to remove salt. After freeze-drying, the PPC was stored in 4° C. before analysis. The obtained PPC has a protein content of 85.6% as determined by a nitrogen-to-protein conversion factor of 5.96 (Fujihara, Kasuga, & Aoyagi, 2001) using a Leco nitrogen analyzer (Leco®, USA). PPC suspension in distilled water (12 wt. %, pH 7.0) was stirred at 350 rpm overnight at 4° C. After raising to room temperature (22-23° C.), the PPC suspension was adjusted to pH 12 with 1 M NaOH. 2.5 g of PPC suspension (12 wt. %) was transferred to a plastic container (4 cm in diameter and 0.5 cm in height, METER®, Pullman, WA, USA). Then the plastic container was placed between a high-voltage electrode and a ground electrode for treatment by a dielectric barrier discharge plasma system (PG 100-D, Advanced plasma solutions, Malvern, USA). The processing conditions were set up as follows: frequency of 3500 Hz, the duty cycle of 70%, the pulse width of 10 μs, the distance from the sample surface to the high-voltage electrode of 2 mm, the current output of 0-1 A, the voltage output of 0-30 kV, and a total treatment time of 10 min (2 min/time, 5 times). The temperature of ACP-treated samples increased from around 21.0° C. to 24.0° C. The system pH decreased from 12 to around 11.5. Immediately after the ACP treatment, the pH was adjusted back to 7.0 with 1 M HCl. Finally, the treated dispersions were spray-dried to obtain the modified pea protein powder labeled as PPCtreated. The control samples were protein powder treated with pH-shifting alone labeled as PPCpH-shifting, and protein powder treated with ACP alone labeled as PPCACP, as well as protein sample without any treatments labeled as PPCuntreated.
Key parameters of ACP including voltage output (0-15 and 0-30 kV), frequency (2000 and 3500 Hz), and treatment time (2-15 min) were tested. From there, we identified 0-30 kV of voltage output, 3500 Hz of frequency, and over 10 min of treatment time were preferred as allowing formation of stronger pea protein gels. Longer exposure time was not preferred for economic reasons. Thus, the following conditions (3500 Hz, 10 μs, 10 min, 0-30 kV, and 0-1 A) were applied.
Transmission electron microscopy (TEM) was used to analyze the morphology of the modified PPC powders. A drop of PPCuntreated, PPCpH-shifting, PPCACP, and PPCtreated suspensions of 0.05 wt. % was deposited on carbon film-covered 400 mesh copper grids and allowed to settle for one minute, respectively. Excess was removed with filter paper. Next, the samples were negatively stained with 4% uranyl acetate for several seconds, and then the excess was wicked away. After sufficient drying, grids were examined at 80 kV on a transmission electron microscope (TEM, Morgagni 268, Philips-FEI, Hillsboro, USA).
Morphology of the PPCuntreated, PPCpH-shifting, PPCACP, and PPCtreated suspensions are presented in
Protein conformation before and after treatment was characterized by Fourier transform infrared spectroscopy (FTIR) and intrinsic fluorescence spectroscopy. FTIR spectra of samples were recorded by a Nicolet 6700 Fourier transform infrared spectrophotometer (Thermo Fisher Scientific Inc., MA, USA). All untreated and treated PPC powder samples were dissolved in D2O to prepare suspensions and gels, which were placed between two CaF2 windows separated by a 25 μm polyethylene terephthalate film spacer for measurement. The sample was tested from 4000 to 1111 cm−1 under 128 scans at 4 cm−1 resolutions. The background was the spectrum of air tested at the same condition and subtracted automatically. Fourier self-deconvolution (FSD) on amide I band (1700 -1600 cm−1) in each spectrum with bandwidth at 24 cm−1 and enhancement factor at 2.5 was analyzed with Omnic 8.1 software (Thermo Fisher Scientific, MA, USA) to evaluate the secondary structures (T. Lefèvre & Subirade, 1999). The intrinsic fluorescence spectra of protein suspensions were determined using a SpectraMax M3 microplate reader (Molecular Devices, USA). The samples were diluted with distilled water to 2.5 mg/mL before testing. Emission spectra between 300 to 400 nm were recorded at an excitation wavelength of 295 nm. The slit width was set to 3 nm for both excitation and emission, and the scanning speed was 10 nm/s.
The protein tertiary structural changes can be reflected by the intrinsic fluorescence spectrum of tryptophan because it is sensitive to the polarity of microenvironments. All samples were diluted to 2.5 mg/mL with distilled water before testing.
The absorption bands of deconvoluted FTIR spectra of the amide I band (1600-1700 cm−1) can be assigned as follows: 1608 cm−1 (vibration of amino acid residues), 1629 cm−1(β-sheet), 1643 cm−1 (unordered structures), 1658 cm−1 (α-helix), 1691 cm−1 (β-sheet). When the sample was only treated by pH-shifting, the PPC was exposed to pH 12 for 10 min, then adjusted back to pH 7 followed by spray-drying. When the sample was treated only by ACP, the protein concentrate was treated by ACP at neutral pH for 10 min followed by spray-drying. The PPCpH-shifting and PPCACP showed similar absorptions in FTIR when compared to PPCuntreated. While for the pea protein concentrates by combined ACP and pH-shifting treatment, two new peaks appeared at 1619 and 1681 cm−1 in the spectrum of PPCtreated. (Ellepola, Choi, & Ma, 2005) The band at 1619 cm−1 is assigned to intermolecular β-sheets via hydrogen bonds resulting from aggregation, while the band of 1681 cm−1 indicates that the β-sheets are antiparallel. Considering the α-helix at 1658 cm−1 and β-sheet at 1629 cm−1 were still obvious after the treatment, the result suggests that the combined ACP and pH-shifting treatment triggered partial unfolding of pea protein and aggregation.
Zeta potential—The zeta potential measurement of PPCuntreated, PPCpH-shifting, PPCACP, and PPCtreated suspensions (1 mg/mL) at neutral pH was carried out on a Zetasizer Nano-ZS (Malvern Instruments Ltd., UK) at 25° C. The particle (pea protein) and dispersant (distilled water) refractive index (RI) was set to 1.45 and 1.33, respectively, and the absorption index was 0.001 (R. Wang, Tian, & Chen, 2011).
The zeta-potential values are summarized in Table 4. After pH-shifting or combined ACP and pH-shifting treatment, the pH of the protein suspensions was adjusted back immediately, thus the four samples were measured at the neutral pH. PPCuntreated, PPCpH-shifting, PPCACP, and PPCtreated were all negatively charged due to the deprotonation of the —COO− group at neutral pH. Protein colloids with a high absolute value of the zeta potential (˜30 mV) are electrically stabilized (Klost, Brzeski, & Drusch, 2020). The absolute values of PPCuntreated (−33.80 mV), PPCpH-shifting (−31.73 mV), PPCACP (-31.03 mV), and PPCtreated (−31.50 mV) samples were above 30 mV, suggesting that pea protein with or without treatments were not susceptible to coagulate or flocculate.
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed under non-reducing (NR) and reducing (R) conditions (Chihi, Mession, Sok, & Saurel, 2016). Protein samples (5 mg/mL) were mixed with the 2×sample buffer (Bio-Rad Laboratories Inc.) with (R) or without (NR) 5% 2-mercaptoethanol (2-ME). Then the mixtures were boiled for 5 min and cooled to room temperature. All the sample mixtures were centrifuged at 10,000×g for 5 min before electrophoresis. 12 μL of supernatants and 5 μL of Broad-range SDS-PAGE molecular weight standards (Bio-Rad Laboratories, Hercules, CA) were loaded onto SDS-PAGE 4-15% gels (Mini-PROTEAN® TGX™, Bio-Rad Laboratories Inc.), respectively. After the running of protein lanes at a constant voltage of 80 V, the gel was stained for 20 min using 0.1% (w/v) Coomassie brilliant blue-R250 in methanol/ acetic acid/water (2:9:9, v/v/v) and destained in methanol/ acetic acid/ water (1:1:8, v/v/v) overnight.
The SDS-Page patterns of the PPCuntreated, PPCpH-shifting, PPCACP, and PPCtreated were obtained under non-reducing (NR, line 1-4) and reducing conditions (R, line 5-8) with the former conducted in the presence of SDS, and the latter with both SDS and 2-ME (
Surface hydrophobicity (H0) was measured by using the 1-anilinonaphthalene-8-sulfonic acid (ANS) as a fluorescent probe, according to the method of Haskard (Haskard & Li-Chan, 1998) with some modification. PPCuntreated, PPCpH-shifting, PPCACP and PPCtreated suspensions (0.005-1.25 mg/mL) were prepared in 0.1 M phosphate buffer (pH 7.4). 20 μL of 8 mM ANS solution was blended with 4 mL of each dilution, followed by keeping in dark for 15 min. The fluorescent intensity was analyzed at 460 nm (emission) with the excitation wavelength of 390 nm using a SpectraMax M3 microplate reader (Molecular Devices, USA) with a slit width of 3 nm. The initial slope of the corrected fluorescence intensity against the protein concentration plot was used as an index of surface hydrophobicity.
Both pH-shifting and ACP treatment alone slightly increased the protein surface hydrophobicity (H0) value from 1141 to 1249 and 1366, respectively. A greater level of H o increase (H0 value 1710) was observed when the pea protein was treated by combined ACP and pH-shifting (
Soluble protein content was quantified by the Bradford method (Bradford, 1976). A standard curve was established using bovine serum albumin (BSA) (0.1-1.6 mg/mL). The PPCuntreated, PPCpH-shifting and PPCtreated powders (0.5 g) were dispersed in 10 ml water, and then the suspensions were centrifuged at 10,000×g for 10 min at 4° C. 4 μL of the diluted sample (1 mg/mL) was mixed with 200 μL of Bradford Reagent in a 96-well clear polystyrene plate (Costar #9017, Corning, Inc., NY). Blank used distilled water instead of sample. After 20 min, the absorption value of the supernatant was measured at 595 nm using a SpectraMax M3 microplate reader (Molecular Devices, USA). The protein solubility was calculated by Equation (2):
Free —SH group levels were analyzed according to the method of Ji et al. (Ji et al., 2018). 0.5 mL of PPCuntreated, PPCpH-shifting, PPCACP, and PPCtreated suspensions (5 mg/mL) were mixed with 2.5 mL of Tris-Gly buffer with 8 M urea and 0.02 mL of Ellman's reagent (4 mg/mL). Blank used corresponding Tris-Gly buffer with 8 M urea. The mixture was then allowed to stand in dark for 1 h at 25° C., and the absorbance was recorded at 412 nm using a SpectraMax M3 microplate reader (Molecular Devices, USA). The —SH group contents were calculated by Equation (3).
Where A412 is the absorbance at 412 nm, D is the dilution factor, and C is the sample concentration (mg/mL).
The free —NH2 content was analyzed by the trinitrobenzene sulphonate (TNBS) method (Habeeb, 1966). 2 mL of the PPCuntreated, PPCpH-shifting, PPCACP, and PPCtreated solutions (0.2 mg/mL) in 0.1 M sodium bicarbonate was mixed with 1 mL of TNBS solution (0.01% (w/v) and the mixture was kept for 2 h at 37° C. After termination of the reaction by 0.5 mL of 1 M HCl, the absorbance of the mixture was recorded at 335 nm using a SpectraMax M3 microplate reader (Molecular Devices, USA). Serial dilutions of L-lysine (2.5, 5, 10, 20, and 40 μg/mL) instead of sample solutions were prepared to plot a standard curve.
The changes in —NH2 content of proteins can be considered as an indicator of the degree of hydrolysis. There was no significant difference in —NH2 content in the four samples (Table 1), indicating there was no obvious pea protein hydrolysis by pH-shifting or ACP treatment alone or their combination treatment. Meanwhile, the free —SH contents greatly decreased after combined ACP and pH-shifting treatment. This result is in accordance with the SDS-Page, suggesting that the disulfide bonds played an important role in pea protein aggregation and growth promoted by free radicals generated by ACP when the protein structure was partially unfolded. Similarly, ACP treatment decreased the free —SH groups in Grass pea protein isolate because of the high sensitivity of sulfur-containing amino acid side chains to oxidation and the conversion of —SH groups to disulfide bonds (Mehr & Koocheki, 2021). In contrary, an increase in the free —SH groups after pH 12-shifting for over 1 h was observed in ginkgo seed protein (Zhang et al., 2021), soy protein isolate (Jiang et al., 2009), and peanut protein isolate (Li et al., 2020) due to the exposure of internal —SH groups and breakage of disulfide bonds.
The viscosity of PPCuntreated, PPCpH-shifting, and PPCtreated suspensions (14 wt. %) was measured using a DHR-3 rheometer (TA Instruments, New Castle, DE, USA) with a 40 mm diameter and angle of 1.997° cone plate geometry. The sample (about 1.5 mL) was loaded onto the center of the parallel plate and the gap between plates was set to 1 mm. The sample was equilibrated for 5 min before testing to obtain a desirable temperature (25° C.). The flow measurement was performed over a shear rate range of 0.1-500 s−1, and the viscosity (11) curve was obtained from the data analysis software.
Protein powder rehydration ability is one of the most important physicochemical properties to enable good functional properties of protein ingredients for their wide applications. The PPCuntreated and PPCpH-shifting showed solubility of ˜70%. (
PPCuntreated, PPCpH-shifting, and PPCtreated powders were dispersed in distilled water with the final protein content of 14 wt. %. Afterward, the protein suspensions were incubated in a water bath at 70, 80, and 90° C. for 10 and 20 min, respectively. Since PPCuntreated and PPCpH-shifting only could not form gels or only form very weak gels, their protein content was increased to 18 wt. % and heated at 95° C. for 60 min to form self-standing gels for comparison. After gelation, the samples were immediately cooled in an ice-water bath for 10 min and stored in a fridge (4° C.) overnight before characterization. The prepared gel samples were labeled according to their preparation conditions, for example, T-70-10 and N-95-60 represent the gels prepared from PPCtreated at 70° C. for 10 min, and PPCuntreated at 95° C. for 60min, respectively.
Mechanical properties and TPA of the prepared gels were evaluated by an Instron 5967 universal testing instrument (Instron Corp., MA, USA) equipped with a 50 N load cell. Cylindrical geometries of each sample (14 mm diameter and 8mm height) were compressed to 70% strain with a deformation rate of 1 mm/min at room temperature. The stress-strain curve was obtained to calculate the compressive strength and compressive strain, which are the maximum stress and strain at break or at 70%. For TPA analysis, the gel samples were compressed twice to 40% strain with 1 mm/min speed. Textural parameters including hardness, cohesiveness, springiness, and chewiness were calculated according to the method of Ferreira et al. (Ferreira, Calvinho, Cabrita, Schacht, & Gil, 2006) using OriginPro 8.5 software (Origin Lab, USA). Hardness was obtained at the peak force during the first compression cycle. Cohesiveness is the ratio Area 2/Area 1 in the force-time curve. Springiness was represented by Distance 2/Distance 1 in the force-time curve. The chewiness was calculated as hardness multiplied by cohesiveness and springiness.
As shown in
As shown in
From the above results, gels were compressed to 40% of their height to study the textural profiles, and the gels prepared from PPCtreated and PPCuntreated were focused. The TPA test simulated oral mastication performed on the food matrix and the results are present in Table 2. Hardness is the resistance from solid material to other permanent deformation when compression is applied. Chewiness indicates the energy required to chew solid food into swallow-able food. Springiness reflects the ability to spring back to the original shape after removing the applied force. Cohesiveness signifies the internal binding force required for food formation. TPA values of the gels prepared from PPCtreated of 14 wt. % at 70° C. for 20 min or at 80-90° C. for 10 min were comparable or even higher than the gels from PPCuntreated (18 wt. %) heated at 95° C. for 60 min. For example, the PPCtreated gels heated at 80° C. for only 10 min and 70° C. for 20 min showed comparable harness and chewiness values to the PPCuntreated gels (60 min heating at 95° C.), while significantly higher cohesiveness and springiness values. High temperature and long heating time are required for pea protein (without treatment) to form gels because the pea legumin and vicilin proteins have high denaturation temperature (legumin: ˜92° C., vicilin: ˜83° C.) (Liang & Tang, 2013) due to their compact molecular structure. However, such intensive heating can be difficult to achieve by some industrial food processing. For example, the internal temperature of vegan burgers reaches only ˜75° C. during the cooking process (Services, 2017). The significantly reduced gelling temperature to 70-80° C. will allow the PPC-based gelling ingredient to be applied in many plant protein-based food products. The significantly reduced heating time from 60 min to 10-20 min also makes the gelling process more energy efficient.
Rheological behaviors of gels were studied by a TA Discovery HR-3 rheometer (TA Instruments, DE, USA) equipped with a parallel-plate geometry (diameter of 40 mm) and the gap was set at 1 mm. The gel sample was placed on the rheometer plate for the frequency sweep test. The storage modulus (G′) and loss modulus (G″) were recorded at oscillation frequency from 0.1 to 100 rad/s under a constant strain of 1%. For dynamic viscoelastic measurements, the temperature ramp test was conducted to study the relationship between viscoelastic modulus and temperature. Approximately 1.5 mL of PPCuntreated and PPCtreated suspensions (14 wt. %) were heated from 25 to respective 70, 80, and 90° C. at a rate of 2° C./min for 10 min, cooled down to 4° C. at 4° C./min and kept for 20 min under the frequency of 1 Hz and the strain value at 1%, which was within a predetermined linear viscoelastic region. Silicone oil and a solvent trap cover were used to prevent evaporation and keep a thermally stable environment during the measurements. G′ and G″ were obtained continually.
Rheological properties of PPC suspensions were studied with frequency sweep measurement in the limitation of the linear viscoelastic region (LVR) (
Water holding capacity (WHC) of the gels was measured by the method of Yang et al. (Yang, Wang, & Chen, 2017) with modifications. About 0.5 g gel sample was transferred into a Vivaspin 20 centrifugal filter unit (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), then centrifuged at 4,000×g for 30 min. The weight of the system of tubes and gel samples before and after centrifugation were recorded. WHC was calculated by Equation (3).
where W1 is the weight of total water in the gel before centrifugation (g), W2 is the weight of water released in the gel by centrifugation (g).
Water holding capacity (WHC) is the ability to interact with water in a gel and is an important quality attribute of food products. For example, the water holding capacity of the meat and meat analogue products is associated with juiciness and texture (Y. Jiang et al., 2021). Excellent water holding capacity was observed for both PPCuntreated and PPCtreated gels with the WHC value of 93.36-98.18% (
The microstructures of PPCuntreated and PPCtreated gels were observed by scanning electron microscopy (EVO M10 scanning electron microscope (SEM) (Carl Zeiss AG, Oberkochen, Germany) at an acceleration voltage of 20 kV and magnifications of 1500 and 5000. Each gel sample was quickly frozen in liquid nitrogen till it solidified well before freeze-drying. The cross-sections of the gel samples were fixed on a metal platform with conductive tape and sputter-coated with gold for 90 s in a vacuum coating unit before observation.
A uniform and dense gel network with neatly arranged pores was obtained from PPCtreated even at 70° C. for 20 min (
Molecular forces involved in the PPC gel formation were analyzed according to the method of Nieto-Nieto et al. (Nieto-Nieto, Wang, Ozimek, & Chen, 2015). Gel samples with cylindrical geometries (1.0 cm height and 1.4 cm diameter) were immersed into water (as a control), 0.6 M 2-mercaptoethanol (2-ME), 6 M urea, and 3% (w/v) sodium dodecyl sulfate (SDS) solution for 48 h at room temperature respectively. G′ and G″ of soaked gels were recorded with a frequency sweep analysis.
Gels were then immersed into different dissociation agents for 48 h including 6 M of urea, 3% of sodium dodecyl sulfate (SDS), and 0.6 M of 2-mercaptoethanol (2-ME) to break the hydrogen bonds, hydrophobic interactions, and disulfide bonds, respectively and the photographs are presented in FIGS. 22A1, B1, C1, and D1. The gels soaked into urea and SDS agents were destroyed, suggesting that both hydrogen bonds and hydrophobic interactions played important roles in the formation of gels from both PPCuntreated and PPCtreated. Since the gels soaked into water and 2-ME still maintained the original shape, a frequency sweep test was conducted to study the change of the gel rheological properties. As shown in FIGS. 7A2, B2, C2, and D2, the G′ of tested gels soaked into 2-ME solvent was higher than that of gels soaked in water, probably resulting in the replacement of water in the gel by 2-ME. This result suggests that although disulfide bonds were involved in pea protein aggregation by combined ACP treatment and pH-shifting method, they did not significantly contribute to gel network stabilization, possibly due to the fact that the cysteine content in pea protein is relatively low and the —SH groups were almost all engaged in the aggregate formation (Pownall, Udenigwe, & Aluko, 2010).
The FTIR spectra (
Similar FTIR profiles were observed for the gels from PPCtreated suspensions. The co-existence of the absorptions at 1619 and 1681cm−1 and the protein α-helix and β-sheet at 1658 cm−1 and 1629 cm−1 in the gel spectra suggests that the formed aggregates in PPCuntreated suspension did not undergo extensive unfolding by heating during the gel formation. Instead, the gel networks were built from the association of the aggregate. Since the aggregates were highly soluble, while possessing higher surface hydrophobicity, they could serve as active building blocks to gradually grow into a more ordered gel network with temperature increasing from 40° C. to 90° C. The gel structure was further enhanced during the cooling process.
Based on the above results, the PPCtreated gelation mechanism is proposed in
All the gel preparations were repeated three times. All results are expressed as the mean±standard deviation (SD) from three independent batches. Statistical analysis was examined by a One-way analysis of variance (ANOVA) followed by a post-hoc test (Fisher's Least Significant Difference (LSD)) using SPSS Statistics 21.0 (IBM Corp, USA) with significance defined at p<0.05.
The description of the present invention has been presented for purposes of illustration and description, but it is not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. Embodiments were chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated. To the extent that the following description is of a specific embodiment or a particular use of the invention, it is intended to be illustrative only, and not limiting of the claimed invention.
The corresponding structures, materials, acts, and equivalents of all means or steps plus function elements in the claims appended to this specification are intended to include any structure, material, or act for performing the function in combination with other claimed elements as specifically claimed.
References in the specification to “one embodiment”, “an embodiment”, etc., indicate that the embodiment described may include a particular aspect, feature, structure, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to combine, affect or connect such aspect, feature, structure, or characteristic with other embodiments, whether or not such connection or combination is explicitly described. In other words, any element or feature may be combined with any other element or feature in different embodiments, unless there is an obvious or inherent incompatibility between the two, or it is specifically excluded.
It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as “solely,” “only,” and the like, in connection with the recitation of claim elements or use of a “negative” limitation. The terms “preferably,” “preferred,” “prefer,” “optionally,” “may,” and similar terms are used to indicate that an item, condition or step being referred to is an optional (not required) feature of the invention.
The singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise. The term “and/or” means any one of the items, any combination of the items, or all of the items with which this term is associated.
As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. A recited range (e.g., weight percents or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.
As will also be understood by one skilled in the art, all ranges described herein, and all language such as “up to”, “at least”, “greater than”, “less than”, “more than”, “or more”, and the like, include the number(s) recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above.
References—the following references are provided to indicate the level of skill of one skilled in the art, and are incorporated herein by reference in their entirety. Any inconsistency between any reference and the disclosure herein may be considered to be alternative information.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2022/050223 | 2/16/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20240130395 A1 | Apr 2024 | US |
| Number | Date | Country | |
|---|---|---|---|
| 63149732 | Feb 2021 | US |
