Enhancing the techno-functional properties of Quinoa protein isolate through cold plasma treatment: a comprehensive study on pH effects

Abstract

This study explores the enhancement of functionality in quinoa protein isolate (QPI) through Vacuum-cold plasma (VCP) treatment across varying pH levels (2.0–10.0). Quinoa, a gluten-free pseudocereal, is recognized for its high-quality protein content (12–23%) and favorable amino acid profile. However, its further application in food products is limited by challenges related to solubility and functional properties. Our findings revealed that VCP treatment significantly improves the solubility, dispersibility, and techno-functional properties of QPI. Specifically, solubility increased from about 4% (untreated) to 72% at alkaline pH, with dispersibility rising from 25% to 54%. Techno-functional properties exhibited enhancement, including water holding capacity (2.8–5.9%) and oil holding capacity (2.3–3.2%) at pH 10.0. Emulsifying activity index (EAI) increased to 9.24 m²/g while emulsion stability index (ESI) reached 71.6 min at acidic conditions (pH 2.0). Furthermore, the foaming capacity (FC) improved from 43.75% to 78.54% and foaming stability (FS) ranged from 8.45% to 89.65%. Additionally, particle size analysis demonstrated that VCP reduced aggregate sizes, resulting in greater surface area and hydrophilicity, thus enhancing WHC and OHC. Zeta potential indicated increased electrostatic repulsion following plasma treatment, correlating with improved emulsion stability. FTIR analysis confirmed structural alterations in QPI, highlighting increased hydrogen bonding contributing to enhanced functional properties. Overall, this research substantiates the potential of VCP as a non-thermal processing method to improve the quality and functionality of plant-based proteins, making quinoa protein a viable ingredient for various food applications catering to growing dietary preferences. Future studies should focus on optimizing VCP conditions to maximize these benefits while maintaining protein integrity.

Introduction

Plant proteins have attracted considerable interest in the food industry due to their various advantages over animal proteins. They provide convenience, are cost-effective, and cater to diverse dietary needs, including those of vegans and individuals with cultural or religious dietary restrictions^1,2. However, the applications of plant proteins in food products are frequently hindered by challenges such as low solubility and inadequate functional properties³. Among these plant proteins, pseudocereal proteins are recognized as an excellent source of high-quality protein, making them especially valuable as bifunctional ingredients⁴.

Quinoa seed (Chenopodium quinoa), in particular, is gaining attraction as a high-quality protein source, owing to possessing essential amino acids such as lysine, methionine, cysteine, and threonine⁵. It is a gluten-free pseudocereal that provides not only high-quality protein but also dietary fibers, vitamins, minerals, and bioactive compounds⁶. With a protein content ranging from 12% to 23%, quinoa surpasses some cereals like corn, rice, rice bran, and oats^7,8, although it remains lower than that found in wheat germ⁹. Quinoa protein is composed of 11 S globulin (37%) which contain six pairs of basic subunits (22–23 kDa) and acidic subunits (32–39 kDa) that combine to form hexamers through non-covalent interactions, and 2 S albumin (35%) which consists of smaller subunits (3–4 kDa) and larger subunits (8–9 kDa) that are connected by disulfide bonds, forming a heterodimer structure¹⁰. The amino acid composition of quinoa protein is similar to that of complete proteins, as acknowledged by the Food and Agriculture Organization (FAO)^11,12. Nevertheless, due to its botanical origin, quinoa protein has saponins which are anti-nutritional and prone to contamination and thus cannot be readily utilized in food products. Therefore, various chemical, physical, and non-thermal treatments are under consideration, including High Hydrostatic Pressure (HHP), sonication, Pulsed Electric Field (PEF), irradiation, ozone, and Vacuum-cold plasma (VCP).

VCP represents the fourth state of matter and including a balanced mix of photons, electrons, and excited neutral atoms, remaining electrically neutral despite its chemical activity¹³. VCP is a non-thermal processing method broadly employed in the food sector for microbial decontamination, while decreasing the loss of food nutrient in comparison with thermal treatments. Additionally, the chemical modification of proteins can enhance their techno-functional properties through alterations in functional groups, which can lead to changes in surface characteristics via graft polymerization or depolymerization¹⁴. The extent of changes brought about by VCP treatment is closely related to the intensity of the VCP.

Enhancing the functionalities of plant proteins — such as their gelling, foaming, and emulsifying abilities — through non-thermal methods is a promising avenue. VCP has the advantage of minimizing impacts on food quality, nutrient retention, color integrity, and texture, compared to other physical treatments¹⁵. Recent studies on cold plasma treatment, particularly dielectric barrier discharge cold plasma, have revealed significant enhancements in the structural and functional properties of plant proteins, such as pea protein and soy protein^16,17. It has been found that plasma-treated proteins exhibit improved functional properties, making them more versatile for various food applications, including food packaging and healthier formulations such as noodles¹⁶. In this context, several reviews have examined the effects of cold plasma on the structure of plant proteins and enzymes^18–20. Despite numerous studies exploring the physicochemical effects of cold plasma on proteins^21–24, research focused on the functional and interfacial properties of proteins remains limited^25,26. Moreover, there have no studies specifically investigating the techno-functional properties of quinoa protein at different pH levels after VCP treatment.

Therefore, this study aims to improve the functionality of quinoa protein through the application of vacuum-cold plasma treatment at various pH levels. Acknowledging the need to enhance the functional characteristics of plant-based proteins, this research recognizes VCP as an innovative technology capable of modifying functional groups and techno-functional properties. The investigation will focus on how VCP treatment influences the techno-functional attributes of quinoa protein isolate in relation to different pH environments.

Materials and methods

Materials

Quinoa seeds were provided from a local company (Tejarat Sabz Ilia Co. Mashhad, Iran). Sunflower oil was obtained from national market. All other chemicals used in this study, such as urea disodium hydrogen phosphate, and monosodium di-hydrogen phosphate (purity ˃ 99.55%), were of analytical quality and were purchased from Merck Chemical Co. (Darmstadt, Germany). Purified water was obtained from a Milli-Q Ultrapure water purification system (Millipore Corp., Bedford, MA, USA).

Preparation of Quinoa protein isolate

Quinoa protein isolate (QPI) was prepared according to our previous works with some modifications^3,8. In summary, quinoa seeds were first ground and then sieved through a 50-mesh screen. To prevent lipid oxidation, the resulting quinoa flour was defatted twice using commercial hexane at a 1:4 (w/v) ratio while stirring at 400 rpm in a laboratory stirrer (Alfa, Model HS860, Iran) for one hour. The mixture was then centrifuged (Universal 320R, Andreas Hettich GmbH & Co. KG, Germany) at 4,000 × g for 10 min at room temperature (25 °C). The defatted quinoa was air-dried overnight under a fume hood, subsequently ground using a Moulinex mill (Model Depose 00022, France), passed through an 80-mesh sieve (U.S. Standard sieve), and finally packaged in polyethylene bags for storage at 5 °C until needed for experimentation. Then, defatted quinoa powder were dispersed in distilled water at a ratio of 1:4, the pH was adjusted to 10.0 using 1.0 N NaOH, and thoroughly mixed for 60 min. The insoluble materials were removed by centrifugation at 4,000 × g for 15 min. Supernatant pH was adjusted to 4.5 using 1.0 N HCl and the precipitated protein was collected by centrifugation. The precipitated protein was washed and dispersed with deionized water, freeze-dried (Zirbus VaCo 5, Laboratory Freeze Dryer, Germany), and stored at −5 °C.

Vacuum-cold plasma treatments

The quinoa powder underwent treatment with cold plasma using a vacuum-cold plasma system (Femto Science Co., Ltd., Hwaseong, Republic of Korea) operating at a power supply of 80 W and a frequency of 50 MHz. The low-pressure vacuum plasma was adjusted to 0.66 mbar (0.5 Torr). The quinoa samples were contained in an open glass vessel which was rotated continuously throughout the plasma treatment to ensure uniform exposure of the powder to the plasma species. The total duration of the vacuum cold plasma (VCP) treatment was 15 min, divided into three 5-minute cycles with 2-minute rest intervals to mitigate significant temperature rises that could adversely affect the protein structure. Exceeding treatment duration of 20 min may lead to excessive oxidation, which could result in undesirable structural alterations in the protein, including the potential degradation of essential amino acids^18,25. Both VCP-treated quinoa protein and untreated samples were subsequently analyzed to assess their physicochemical, techno-functional, structural, and surface properties.

Physicochemical properties

Yield of QPI

Chemical properties of quinoa protein including moisture (925.10), ash (923.03), fiber (920.86), crude fat (920.39), and crude protein (920.87) were determined according to the Association of Official Analytical Chemists (AOAC) procedure²⁷. The carbohydrate content was also calculated by subtracting the other amounts from 100. Quinoa protein content was determined by Kjeldahl method according to the AOAC procedure with slight changes⁸. The Kjeldahl digestion and distilling system (B-322, BÜCHI, Co., Switzerland) was used to digest the protein and determine nitrogen content of the protein samples. Then, the protein content was calculated by multiplying the nitrogen content by the conversion factor of 6.25. Protein yield was measured according to the Eq. 1:

1

Bulk density

Bulk density (ρ_b), tapped/true density (ρ_t), and compressibility index of the quinoa protein was determined according to the European Pharmacopoeia. A total of 25 g of quinoa protein powder was placed into a 100 mL graduated measuring cylinder without any packing. The cylinder was then tapped gently 100 times against a laboratory bench. Both the bulk density (ρb) and tap density (ρt) were recorded in g/mL. The compressibility index, which indicates the tendency of the powder to compress, was calculated using Eq. 2 as follows:

2

Where V₀ and V_f are the unsettled apparent volume and final tapped volume, respectively.

Nitrogen solubility, dispersibility and wettability

Nitrogen solubility of the proteins at 1% (w/v) was determined by the method of Bradford in a pH range of 2.0 to 10.0²⁸. The pH adjustment of the protein dispersions was performed using either 0.1 N HCl or NaOH. The suspensions were stirred over a magnetic stirrer for 1 h at room temperature (25 °C). Then, the suspensions were centrifuged at 3,000 × g for 15 min. The protein analysis was performed on supernatant and bovine serum albumin was used as a standard curve. Nitrogen solubility (NS) was expressed as % of nitrogen content of the sample according to the Eq. 3:

3

The Sharma procedure was used to measure the dispersibility of quinoa protein at various pH levels¹². 3 g of QPI was dispersed in distilled water in a 50 mL measuring cylinder, and then the pH was adjusted using dilute HCl and NaOH solutions. After adding distilled water to reach a volume of 30 mL, the mixture was vigorously stirred and left to settle for 2 h. The dispersibility was then calculated using the Eq. 4:

4

Wettability was assessed using the Sharma method¹². Two grams of powder were added to a beaker containing 80 mL of distilled water, and the interaction of the powder with the water surface was observed immediately. After 30 min, the mixture was stirred on a magnetic stirrer to create a vortex reaching the bottom of the beaker for 1 min. Wettability was then rated as excellent, good, fair, or poor based on the dispersion behavior and time.

Surface characteristics

Zeta potential and aggregate size

The aggregate size of quinoa proteins at a concentration of 0.05% (w/v) was measured using dynamic light scattering (DLS) with a nanoparticle zetasizer (Microtrac MRB, Nanotrac wave II). The light scattering angle was set at 173° and conducted at 25 °C, with refractive indices of 1.456 for the protein and 1.33 for the dispersion medium. The pH of the samples was adjusted from 2.0 to 10.0 using 0.1 N HCl or NaOH, and measurements were taken in triplicate²⁸. Zeta potential (mV) was also measured using the same equipment, calculated based on the electrophoretic mobility of the protein solutions using the Smoluchowski equation.

Surface hydrophobicity

Surface hydrophobicity (H₀) of the QPI was determined using the fluoresence 1-anilino-8-naphthalenesulfonate (ANS) binding method²⁹. The 0.003% quinoa protein solutions were prepared in milli Q water, and pH was adjusted to desired pH value (2–10) with 0.5 and 0.1 N HCl or NaOH. Fluorescence intensity (FI) was measured through adding 20 µl of ANS (8 mM) to 4 mL of the protein solution using a fluorescence spectrophotometer (Cary Eclipse, Model FP 6200; Jasco Inc, Maryland, USA) at excitation and emission wavelengths of 390 and 470 nm, respectively. S₀ was also determined by centrifugation of protein solutions at 10,000 × g for 15 min and adjusting pH to 7.0 at different protein concentrations from 0.0015 to 0.015%. The coefficient of linear regression analysis of the FI vs. protein concentration (%) was used as an index of the protein surface hydrophobicity (H₀).

Techno-functional properties

Water and oil holding capacity

WHC and OHC of the protein samples were assessed using a modified method from He et al.³⁰. A 0.5 g sample was dispersed in 10 mL of 0.01 M phosphate buffer at pH 2.0–10.0. After mixing for 1 min and standing for 30 min, the sample was centrifuged at 3,000 × g for 15 min at 25 °C. The supernatant was removed, and excess water was drained for 15 min; the water retained per gram of sample was then calculated. For OHC, 0.5 g of the protein was mixed with 10 mL of sunflower oil for 30 min and centrifuged at 3,000 × g for 20 min. After decanting the supernatant, the remaining pellets were weighed to calculate the oil retained per gram of sample.

Emulsion activity and stability index

The emulsifying properties of quinoa protein were evaluated across pH levels (2–10)⁸. Quinoa protein dispersions (1% w/v) were prepared with deionized water and adjusted to pH 2.0–10.0 using 0.1 N HCl or NaOH. A mixture of 20 mL soybean oil and 36 mL of 1% (w/v) quinoa protein dispersion was homogenized at 12,700 rpm for 1 min. An aliquot (100 µL) was taken from the bottom of the container at 0 and 10 min post-homogenization and mixed with 10 mL of 0.1% sodium dodecyl sulfate solution. The absorbance of the emulsions was measured at 500 nm using a spectrophotometer. The absorbance readings at 0 and 10 min represented the emulsion activity index (EAI) and emulsion stability index (ESI), respectively.

5

where A₀ and A₁₀ are the absorbance at 0 min and after 10 min, respectively⁸.

Foaming capacity and stability

Foaming capacity (FC) and foam stability (FS) were assessed by the Ghorbani et al.³. The SPI solutions were prepared at (1%, w/v), and the pH was adjusted from 2.0 to 10.0 using either 0.1 NHCl or NaOH. The solutions were agitated in graduated plastic tubes at high speed 12,700 rpm with homogenizer for 1 min. Foam capacity was reported as Eq. 6:

6

A similar procedure was used to determine the foam stability, but the samples were allowed to stand for 20 min at room temperature and the residual foam volume was measured. The following formula (Eq. 7) was used to calculate FS:

7

Structure behavior

FTIR

FTIR analysis was conducted to determine the functional groups and interactions within the QPI protein samples. The samples were mixed with KBr, followed by pressing into pellets in which a pressure of 100 kg/cm² was applied for roughly two minutes to create KBr pellets (Perkin-Elmer, Beaconsfield, Bucks., UK). The secondary structure of the protein was assessed using a FTIR spectrophotometer (Agilent Cary 630 FTIR, USA), with measurements taken across a wavelength range of 650 to 4000 cm^–1 at resolution of 4 cm^–1 and at ambient temperature²⁸.

Morphological properties

The surface morphology of quinoa protein isolate (QPI) was examined using scanning electron microscopy (SEM, JSM-700 LF JEOL, Japan) with an acceleration voltage of 20 kV. To improve electrical conductivity and achieve clearer images, a 10-nm gold-palladium alloy coating (Quorum SC7620, UK) was applied to the sample surfaces prior to imaging. SEM images were taken at a magnification of 5000 x.

Statistical analysis

Statistical analysis was performed on experiments conducted in triplicates, reported as means ± standard deviations (SD) by one-way analysis of variance (ANOVA). Regression analysis using Excel version 2010 was employed, and the Duncan’s test was used to assess significant differences (P < 0.05) between the means for each treatment, utilizing SPSS version 26. P values < 0.05 is considered as statistically significant difference.

Results and discussions

Physicochemical properties of Quinoa protein isolate

Bulk density (ρ_b), true density (ρ_t), and compressibility of the quinoa protein powders are evaluated as some of physical properties. Bulk density plays a vital role in powdered ingredients, influencing both the flowability and solubility of the product, as well as having a significant impact on packaging. Additionally, a higher bulk density can raise the risk of oxidation due to permitting more air to occupy the spaces between granules. The values of the ρ_b, ρ_t, and compressibility index of the QPI were 0.68 ± 0.01, 0.74 ± 0.01, and 20.18 ± 1.07, respectively. These values are in line with similar protein isolates obtained from sesame and rice bran proteins^3,8,12. While the values of the ρ_b and ρ_t are close together, the interparticulate interactions are less and the powder has more flowability. The compressibility index as a measure of the powder ability to settle was 20.18 which confirmed the flowability of the quinoa protein.

The chemical composition of quinoa protein isolate including moisture, protein, fat, minerals, and carbohydrate were 5.47 ± 0.53, 82.45 ± 0.67, 0.21 ± 0.11, 3.24 ± 0.17, and 12.63 ± 0.23, respectively. Similar results in the composition of quinoa protein have been previously reported, with finding the protein content of QPI to be in the range of 77 to 83.5%³¹. The high protein content of quinoa has confirmed its nutritional properties which have been similarly reported^32,33. According to these works, the highest extraction yield of protein has been achieved at the isoelectric point of quinoa protein (pH = 4.5). Therefore, the pH 4.5 was used for extraction of QPI in this study and the yield was 83.52% which is lower than that of the previous work (88.74%)³². Furthermore, the protein content of QPI was less than sesame protein isolate in previous works^3,34. It has been understood that alkaline extraction of QPI induced more yield, it led to higher solubility which affect the functionality of the protein³⁵.

Protein solubility of QPI

Protein solubility is a crucial factor influencing protein functionality and serves as an essential indicator in both food and pharmaceutical applications. It significantly impacts food characteristics, including color, texture, foaming ability, emulsification, and sensory properties³⁶. Therefore, we examined the protein solubility (PS) of QPI during alkaline extraction and after cold plasma treatment across a pH range of 2.0 to 10.0 (Fig. 1a). The solubility of the proteins exhibited a classic U-shaped curve for both treated and untreated samples when exposed to atmospheric cold plasma. The lowest solubility, approximately 4%, occurred for untreated proteins at the isoelectric point (pI), which is around pH 4.5. In contrast, following VCP treatment of quinoa protein, solubility increased to 8.5%. The low solubility at the protein’s pI can be attributed to its net charge and zwitterionic state. Indeed, nitrogen solubility is affected by multiple factors, including concentration, pH (ionic strength), zeta potential, conformation, and temperature, all of which influence the presence of charged amino acids. In opposition, protein solubility increased steadily to 24% at neutral pH (7.0) and exceeded 72% at alkaline pH levels (9–10). Although there was an increase in solubility at acidic pH values (23% at pH 2.0), it was lower than the solubility observed at alkaline pH. The solubility characteristics of QPI at both acidic and alkaline pH can be attributed to the presence of 11 S quinoa globulins and albumins, with albumins demonstrating higher solubility than globulins at pH 10. Previous research has been shown a strong correlation between protein solubility and the emulsifying and foaming properties³⁷. Our findings align with earlier studies by Abugoch (2008) and Elsohaimy (2015)^31,32, which indicated the highest solubility of QPI at alkaline pH values.

Fig. 1.

pH profiles of solubility of the quinoa protein isolate (a), and dispersibility (b) for control, alkaline extraction (blank symbol) and VCP treated (filled symbols) as a function of pH (2.0–10).

Comparatively, the solubility profiles of VCP-treated proteins differed from untreated samples, revealing higher solubility in the range from pH 5.0 to 10.0 (Fig. 1a). This observation can be linked to the increased protein aggregation induced by plasma treatment, which enhances quinoa protein solubility. The enhancement of electrostatic repulsion induced by VCP treatment is believed to be a key factor contributing to the increase in protein solubility. The degradation of proteins caused by VCP processing results in smaller particle sizes and an increased specific surface area, leading to a greater availability of active sites and, therefore, improved solubility^26,38. Therefore, it can be concluded VCP like as thermal acid hydrolysis can increase quinoa protein’s solubility³⁹.

Consequently, the solubility of QPI is dependent on pH and relates to the hydrophilic-hydrophobic balance of proteins as well as their interaction with the solvent³⁵. The presence of negatively charged regions at alkaline pH, resulting from the ionization of carboxyl groups and the deprotonation of amine groups, leads to enhanced protein-solvent interactions and an increase in quinoa protein solubility. This pattern of reduced solubility at acidic pH and elevated solubility at alkaline pH has been observed in various plant proteins, including rice bran, peas, chickpeas, soybeans, and sesame proteins^8,40–42.

The hydration of food powders is closely linked to their dispersibility and hydration properties. The dispersibility of QPI treated and untreated with VCP as a function pH of 2.0 to 10.0 is illustrated in Fig. 1b. As shown, the dispersibility of QPI follows a trend similar to that of its solubility, with the lowest dispersibility observed at pH 4.5. The small particle size of the QPI powder, which results in a large surface area relative to its mass, contributes to lower dispersibility and wettability during rehydration. Consequently, this reduced hydration enhances the rehydration rate of QPI, which is considered a beneficial technological characteristic. Conversely, above the protein’s isoelectric point (pI), increased dispersibility is associated with a decreased likelihood of agglomeration. The presence of fine particles adversely affects the dispersibility by reducing wettability during the rehydration process. Similar behaviors have been noted for sesame protein isolate^12,40. The more dispersibility for VCP treated QPI may be related to the particle sizes and an increased specific surface area, leading to a greater availability of active sites and, therefore, improved dispersibility as stated for the solubility of protein.

Zeta potential and particle size

Zeta-potential (ζ) represents the charge strength of the functional groups of proteins at the surface and is considered as a measure of electrostatic repulsion. It can principally influence on the solubility, gelation, emulsifying, foaming, surface activity, stability and interactions with other biopolymers⁴³. Electric charge interactions significantly affect structure, stability, rheological behavior, texture, color, shelf life and flavor of food systems. ζ-potential and particle size of quinoa protein treated and untreated with VCP at pH range of 2.0 to 10.0 is given in Fig. 2. At the pI of the QPI (4.5), the net charge of the protein is zero, although by increasing the pH from 2.0 to 10.0, it was gradually decreased from positive values to negative ones, which can be attributed to the gradual protonation and deprotonation of carboxyl groups and amine groups, respectively^44,45. The negative value of ζ-potential of QPI can be related to more contribution of aspartic and glutamic acids. The same behavior of ζ-potential have been reported for plant proteins such as soybean, chicken pea, lentil, and sesame, where, ζ-potential was positive at pH less than pI and gradually declined to 0 adjusting pH to the pI (pH = 4.5), and then exhibited a negative trend with further increasing pH value^45,46. The ζ-potential of QPI was − 16.32 ± 1.67 mV at pH 10.0, which is less than that of the ζ-potential of QPI at the same pH. They have found that protein extraction at pH 10 is more effective and other pH values have not shown any difference³³.

Fig. 2.

Effect of pH and cold plasma treatment on zeta-potential (a) and particle size (b) of quinoa protein isolate. Intact QPI is filled symbol and VCP treated is blank symbols.

In comparison, by applying VCP, the ζ-potential of QPI was decreased to more negative values which may affect on the structure and stability of the protein. These significant changes on the ζ-potential of treated QPI are supposed to be induced by the proteins unfolding and more exposure of the hydrophilic groups at the surface. Aggregation induced by active species of VCP treatment is supposed to hide the negatively charged amino acids and consequently decrease the absolute zeta potential as demonstrated⁴⁷. Increased hydrophobicity and decreased zeta potential observed at Fig. 2a is attributed to VCP-induced conformational changes, which has been observed for QPI²⁶.

Particle size has a great effect on solubility, functionality and stability of the protein depending on thermodynamic dimension. The particle size of QPI for both treated and untreated samples with VCP as a function of across pH 2.0–10.0 is illustrated in Fig. 2b. However, the mean particle size D_[4,3] of QPI decreased at acidic and alkaline pH, the highest value was obtained at pH around 4.5, which is in the pI range and zero net charge that makes larger aggregates through strong hydrophobic interactions. It has been found that particle size of QPI increased to highest value at high thermal treatment, which could be due to the denaturation of protein and aggregate formation³⁰. A similar finding was also found for high hydrostatic pressure treated quinoa protein, which the aggregation and re-association of protein structures can be explained the slightly increased solubility and reduced particle size of QPI, particularly at alkaline pH^48–50. Furthermore, it has been understood that VCP decreased the particle size and improve the emulsification properties of QPI in an intensity dependent manner²⁶.

Intrinsic fluorescence and surface hydrophobicity

Structural evaluation of proteins was conducted, specifically focusing on the alterations in the fluorescence emission spectra of tryptophan (Trp) residues in quinoa protein isolate (QPI) across varying pH levels, as well as the impact of treatments using vacuum-cold plasma (VCP). The fluorescence intensity (FI) and maximum emission wavelength (λ_max) of QPI at different pH levels are illustrated in Figs. 3a, b. It has been proposed that Trp is considered to be enveloped in a nonpolar environment if λ_max < 335 nm and recognized as being in a polar environment if λ_max > 335 nm⁵¹. The λ_max of Trp that is buried within protein structures generally measures less than 335 nm, whereas solvent-exposed Trp displays λ_max values exceeding 335 nm³³. The findings indicated that pH significantly affects intrinsic fluorescence, as evidenced by a red shift in λ_max as the pH was elevated from 4.0 to values between 5.0 and 9.0. A red shift was also observed when the protein moved beyond its isoelectric point (pI) under both acidic and alkaline conditions, which may be attributed to the transition of Trp residues from a hydrophobic environment to a more polar and hydrophilic one, generally resulting in a detrimental effect on Trp fluorescence intensity⁴². Furthermore, the maximum fluorescence intensity was recorded under both acidic (pH = 2.0) and alkaline (pH = 10) conditions, while the minimum intensity occurred near the pI of the protein at pH = 4.0. Similar results have been documented for plant protein isolates, which exhibit maximum fluorescence intensity under alkaline conditions as compared to pH values near the pI^46,52. This phenomenon may be linked to the formation of aggregates at pH 5.0, which sequester most intrinsic Trp residues, despite the electrostatic repulsion that promotes their migration to polar surfaces in an alkaline environment. The enhanced interface characteristics of QPI appear to derive from a greater exposure of hydrophobic groups.

Fig. 3.

Effect of pH (a) and vacuum-cold plasma treatment (b) on fluorescence intensity of quinoa protein isolate.

In comparison to untreated protein samples, QPI that underwent VCP treatment displayed a pronounced red shift and a significant reduction in fluorescence intensity when subjected to extreme pH values. This decrease was greater than that observed for untreated QPI (Fig. 3b). Comparable findings have been reported concerning thermal treatment, microwave heating, steaming, boiling, and baking of QPI^30,37.

Surface hydrophobicity (H₀) serves as a valuable metric for assessing the denaturation and aggregation of proteins and is commonly determined by the initial slope of FI versus concentration plots. It was determined that surface hydrophobicity is pH-dependent, with increased pH correlating with decreased H₀. Thus, the highest and lowest H₀ values were recorded at pH 2.0 and pH 10, respectively. The elevated H₀ at lower pH is likely due to the greater exposure of hydrophobic residues resulting from protein dissolution⁵². The H₀ for QPI was found to be 38, surpassing that of rice bran protein (33, 37)⁸ but lower than that of bovine serum albumin (BSA) (86.4)³⁷. There exists a correlation between surface hydrophobicity and the functional properties of proteins, with higher H₀ values typically associated with enhanced protein functionality. Studies have indicated that the H₀ values for animal proteins are generally more than plant proteins, however, H₀ values of quinoa protein were found to be greater than those of casein and ovalbumin. Overall, surface hydrophobicity plays a critical role in influencing emulsifying properties, with the activity of emulsions being primarily affected by H₀. Conversely, the lower surface hydrophobicity of quinoa protein limits protein-lipid interactions, thereby reducing emulsifying capabilities³⁷. Nevertheless, treatments involving heat and enzymatic methods can enhance H₀ and improve the emulsifying activity of quinoa protein by exposing hydrophobic amino acids that are typically buried within the protein structures. Research has shown that both pH and heating time affect the H₀ of quinoa globulins, with heating improving H₀ due to protein denaturation⁵³. Similar to thermal processing, QPI’s H₀ under VCP treatment increased to 43, which may be attributed to modifications in the tertiary structure of the protein. This transformation is believed to involve a shift in the secondary structure of QPI from ordered forms (α-helix and β-turn) to disordered structures (β-sheet and random coil), as reported in related studies²⁶. Comparable outcomes have also been recorded concerning treatments involving plasma and high hydrostatic pressure (HHP) in pea protein⁵⁴ and QPI^48–50. Considering these results, it is anticipated that the emulsifying properties of QPI will be enhanced as a result of VCP treatment³³.

Techno-functional properties

Water and oil holding capacity

Water holding capacity (WHC) and oil holding capacity (OHC) as functions of pH and VCP treatment are plotted in Fig. 4a. The QPI had a similar WHC (2.8–5.6%) to that of soy protein isolate (4.3%) and wheat protein (3.67%)^31,55. The same trend was also found for OHC (2.3–3.2%) compared with wheat protein (1.58%) and SPI (2.10%)³². In the view point of pH, there was a direct relationship between protein solubility, WHC and OHC. The highest WHC and OHC were observed at pH values beyond Ip of protein, however the lowest values of WHC and OHC were found at Ip of QPI which clearly confirmed its relation with protein solubility. The high solubility of QPI at pH = 10 (78%) closely correlate with its high WHC (5.89%) and OHC (3.2%). Thus, the WHC and OHC were pH dependent, revealing that changing in the polar groups causing those interactions with the water and oil molecules. Since QPI is gluten-free and have higher water and oil holding capacity than that of wheat protein, it is expected to have improved texture in different food applications^56,57.

Fig. 4.

Effect of pH and cold plasma treatment on techno-functional properties of quinoa protein isolate: WHC and OHC (A), ESI and EAI (B), and foam capacity and foam stability (C). Different lowercase letters show the significant differences between samples (p < 0.05).

To some extent, VCP treatment improved the WHC and OHC. Like as some non-thermal processing, irradiation and plasma, is found to denature proteins or change their conformation to influence their contact with oil and/or water. VCP treatment likely induces crosslink formation or conformational changes, affecting the techno-functional properties of proteins^26,58. The increase observed may be induced by the more exposure of QPI hydrophilic groups induced by active cites through VCP treatment^26,59. For OHC, it may be interpreted that the hydrophilic groups hidden inside the protein structure is supposed to be more available by VCP treatment which is induced by proteins unfolding and conformational changes. This functional property of QPI can assist to improve mouthfeel and flavor retention of various food products such as meat and bakery systems⁵².

Emulsifying activity and stability indices

Emulsifying properties mainly depend on the ability of proteins to lower the surface tension, and prevent the coalescence of droplets as well as increase the surface hydrophobicity, rather than overall protein hydrophobicity³⁵. Proteins are known as the frequently used emulsifiers in food industry with potentially strong absorbance at oil–water interface through their amphiphilic nature⁴². The emulsifying activity index (EAI) and emulsion stability index (ESI) of quinoa protein as a function of pH and VCP treatment are shown in Fig. 4b. The results showed that the pH was effective in EAI and ESI of quinoa protein and the lowest EAI (0.15 ± 0.01) and ESI (4.56 ± 0.01 min) were observed at pH 5.0. Although, EAI and ESI of quinoa protein were increased at upper and lower pH level of pI of protein and the highest EAI and ESI were found at pH 10.0 and 2.0, respectively. Similar findings for EAI and ESI have been reported in which QPI showed higher EAI and ESI than that of millet, wheat and soy protein³². Therefore, low EAI (0.15–9.24 m²/g) but good ESI (5.70–71.6 min) was observed for QPI.

While EAI represents the proteins ability to be oriented at the oil and water interface, the ESI value is an indicator of its potential to influence the stability of emulsions⁶⁰. By applying VCP on the QPI samples, it was found a negligible change in EAI and ESI. This change in EAI and ESI values may be related to the protein conformations changes induced by VCP treatment. Combined effect of pH and VCP treatment on emulsion activity of QPI found that at alkaline pH, the highest ESI (32.56) and ESI (124 min) were seen, which may be attributed to the increased Coulombic repulsions between neighboring droplets, coupled with the high hydration of the charged protein molecules that finally decrease the interface energy and combination of emulsion droplet. Indeed, the protein solubility directly affected on the emulsion activity of protein, which both reduced at pI when the ζ-potential tends to zero (see Sect. 3.3 and 3.4). As a result, Coulombic forces induce the molecules to be more stable and Brownian motion promotes droplet suspension than emulsion formation⁶¹. Compared to BSA as a good emulsifier, although it can be interpreted that QPI had low EAI, owning to lower surface hydrophobicity, it has good ESI. As a result, QPI treated by VCP due to its good emulsifying properties, can be utilized in bakery products for celiac person.

Foaming capacity and stability

Foaming capacity and foaming stability are critical factors in the functional properties of QPI. There is a direct relationship between foaming of QPI and its protein hydrophobicity and interfacial properties⁶². FC and FS of QPI as affected by pH and VCP treatment are depicted in Fig. 4c. FC of untreated QPI samples was ranged from 43.75 ± 2.35% to 78.54 ± 2.36%. It was also found the lowest FC and FS at pH = 5.0 near to the pI of QPI; and the greatest FS was seen at alkaline pH (9.0), which may be related to QPI’s hydrophobicity. However, the FC was gradually increased away from the pI, the most changes in FC occurred at alkaline conditions. Similar results have been also reported for other plant proteins^40,45. FS was ranged from 8.45 ± 1.12 to 89.65 ± 1.67% after 60 min. The high foaming stability of QPI is comparable to SPI and lower than egg albumin and wheat⁴⁰. Considering an egg albumin, as a reference, FC was ranged from 43% to 78%⁶¹. So, it can be stated that QPI had an ability to make foam less than egg albumin but showed foam stability similar to it. The obtained results evidence the high ability of quinoa protein to make foam with high stability that is raising its potential for using in food processing.

High foam ability of protein (43.75%) may be associated with highly globular proteins resist to denaturation which is related to the high globular fraction of quinoa protein. Although, plant proteins have been exhibited more FS than dairy proteins due to the higher molecular weight of globulins in plant proteins, which forms adsorption film with high elasticity⁴⁶. Therefore, similar trends have been reported in FS and FC of RBP isolate⁸, and sesame protein³. Indeed, by increasing net charge of proteins, the hydrophobic interactions are weakened and proteins become more flexible, leading to foam formation. High foamability of QPI can be attributed to good solubility, which enables it to dissolve in aqueous phase and quickly developing a cohesive layer at the interface layer, which induce the low surface tension. In contrast, due to the high intermolecular cohesiveness and elasticity enables the QPI to produce stable foams^45,46.

Considering the independent variables assessed at this study, it has been observed that using VCP, improved the FC and FS of QPI. On the basis of solubility parameter, it seems that soluble fraction of proteins play a detrimental role in FC determination which is in accordance with previous works^30,61. Increased surface hydrophobicity is supposed to form denser foams and consequently increased foaming stability⁵². Considering the importance of foaming characteristics in functionality determination of proteins, VCP treatment can be considered as an improving treatment.

Structural properties

Fourier transform infrared spectroscopy (FTIR) was used to investigate the conformational changes on the secondary structure of QPI treated and untreated by VCP and the peaks are provided in Fig. 5. FTIR region of the amide I band corresponds to the secondary structure in proteins as follows: 1610–1640 cm^{− 1} belongs to β-sheet, 1640–1650 cm^{− 1} belongs to random coil, 1650–1658 cm^{− 1} belongs to α-helix, and 1660–1670 cm^{− 1} belongs to β-turn⁶⁴. The deconvolution and curvefitting of the amide I region of QPI to obtain its second derivative spectrum was also analysed (Fig. 5b). The relative content of each secondary structure was obtained according to the second derivative spectrum of the amide I band of QPI.

Fig. 5.

FTIR analysis of quinoa protein isolates treated and untreated by VCP at pH = 7.0 (a), Deconvoluted spectra of QPI at the wavelength of amid I (b).

The relative content of α-helix, β-sheet, β-turn, and random coil in the secondary structure of QPI did not change after VCP treatment. The main peaks observed at 3296 cm^{− 1}, 3009 cm^{− 1} and 2885 cm^{− 1}, 1624 cm^{− 1} and 1530 cm^{− 1} correlated to the vibration of hydroxyle and N–H stretching, methyl group and amide groups, respectively⁶⁰. The enhancement observed in the band intensity at 3266 cm^{− 1} of VCP treated QPIs might be induced by more hydrogen bonding in the VCP treated protein chains⁶¹. Changes observed at peak intensities of 3009 and 2885 cm^{− 1} of VCP treated samples revealed the symmetric stretching of the methylene group and active species induced changes to a certain degree. Furthermore, VCP treatment did not alter the overall structure of proteins on the basis of its intensity which observed at the peak intensities of 1624 cm^{− 1} and 1530 cm^{− 1} corresponding to the amide groups of protein⁶⁰.

Microstructural properties

The morphological structure of QPI treated and untreated by VCP was illustrated in Fig. 6 (magnifications 5000). The QPI’s particles powder were irregular in shape and not uniform. The surface was rough, wrinkled, and the particles were more amorphous and agglomerated. It has been reported that the alkali extraction and isoelectric precipitation technique in quinoa protein isolates transformed the microstructure of the protein into a compact structure with a wrinkled surface³⁴. Untreated QPIs appear as large fragments, but VCP treatment reduces particle size, leading to a more uniform dispersion in solution⁶³. Crosslink formation induced by active species leads to formation of new protein fragments large in size which has lower solubility and distribution in the solution⁶⁴.

Fig. 6.

SEM images of QPI untreated (a), and treated by VCP (b) at magnification 5000 ×.

Conclusion

This study demonstrates that vacuum-cold plasma (VCP) treatment is a promising non-thermal processing method for enhancing the functionality of quinoa protein isolate (QPI). The findings indicate that VCP significantly improves key techno-functional properties, including solubility, dispersibility, water and oil holding capacities, emulsifying activity, and foaming properties. Specifically, VCP treatment resulted in a substantial increase in solubility from 4% to 72% at alkaline pH and boosted dispersibility from 25% to 54%. Additionally, the enhancement in water holding capacity (up to 5.9%) and oil holding capacity (up to 3.2%) suggests that QPI can effectively improve texture and moisture retention in various food applications.

The marked increases in emulsifying activity and stability, along with foaming capacity and stability, position QPI as an effective ingredient for emulsion-based and foamy products. Moreover, the reduction in aggregate size and alterations in surface characteristics indicate that VCP facilitates better protein-solvent interactions, enhancing the overall functionality of QPI. In conclusion, the application of VCP presents a valuable strategy for overcoming the limitations associated with quinoa protein in food formulations, catering to the growing demand for plant-based alternatives that meet dietary preferences such as noodle for celiac people. Furthermore, as plant-derived proteins are categorized as Generally Recognized As Safe (GRAS), their functional versatility and safety profile position them as promising candidates for innovative, sustainable food formulations for celiac people that align with health-conscious consumer demands. Future research should aim to optimize VCP treatment parameters to further refine the functional properties of quinoa protein, ensuring its broad applicability in the food industry while maintaining the nutritional integrity of this valuable plant-based protein source.

Author contributions

Conceptualization: L.Y, A.A. & E. M.; methodology: L.Y, A.A, E. M. & A.R.; formal analysis: L.Y., & A.R.; investigation: L.Y.; resources: A.A. and E.M.; data curation: L.Y, A.A. & E. M.; writing-original draft preparation: L.Y. & A. R.; writing-review and editing: L.Y., A.A. & A. R. All authors have read and agreed to the published version of the manuscript.

Funding

There is no funding source for this project.

Data availability

The data presented in this study are available on request from the corresponding author.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

The authors will adhere to the Ethical Responsibilities of Authors and COPE rules. On behalf of all co-authors, I believe the participants are giving informed consent to participate in this study.

Consent for publication

We, Leili Yousefi, Akram Arianfar, Elham Mahdian, and Ali Rafe give our consent for the submitted manuscript to be published in the journal of Scientific Reports.