The impact of compound addition sequence on functional, antioxidant, and thermal properties of dielectric barrier discharge plasma-produced ternary complexes

Abstract

The present study aimed to form a ternary complex using carboxymethyl cellulose (CMC), soy protein isolate (SPI), and curcumin (CUR) through dielectric barrier discharge (DBD) plasma technology and to investigate the effects of this treatment on the functional, antioxidative, and thermal properties of the ternary complex. The antioxidant activity was similar in the mixtures before and after plasma treatment, except for the SPI-CUR and CUR (SPI-CMC) mixtures. Plasma treatment increased carbonyl content in protein-containing mixtures, with the exception of CMC (SPI-CUR). Surface hydrophobicity increased by 233 % and 364 % in SPI-CMC and CMC (SPI-CUR) post-plasma, respectively. The complexes exhibited approximately 250–430 % higher solubility and 10–40 % higher emulsifying activity than SPI, except for SPI-CUR. TGA results indicated higher thermal resistance after the formation of the complex. The SPI (CMC-CUR) complex displayed superior antioxidant activity, foaming capacity, foam stability, emulsifying activity, emulsion stability, and thermal resistance than other ternary complexes.

Highlights

• •The sequence of compound addition influenced the properties of complexes.
• •The SPI-CUR mixture had the highest antioxidant activity which reduced post plasma.
• •The thermal resistance improved after formation of ternary complex by plasma.
• •The SPI (CMC-CUR) complex solubility was 254 % higher than SPI.
• •The emulsion stability of SPI (CMC-CUR) complex was 224 % higher than SPI.

1. Introduction

Soy protein is an abundant protein with high nutritional value that is inexpensive and easy to access. Similar to most proteins, soy protein has functional properties like emulsification, gelation, and foaming (Wan, Wang, Wang, Yuan, & Yang, 2014). However, the globular nature of its subunits, especially glycinin, limits its functional properties and practical applications (Han et al., 2023). Numerous attempts have been made to modify soy protein to enhance its functional properties such as solubility, surface activity and foaming properties.

Recent research has shown that modifying proteins with polyphenols can improve their functional properties. Polyphenols exhibit increased biological activity and stability when bound to proteins (J. Li & Chen, 2022). Curcumin is a particularly appealing polyphenol compound due to its antioxidant, anti-inflammatory, and anti-proliferative functions (Racz et al., 2022). Moreover, CUR has a high reactivity with proteins, allowing it to interact with active sites in protein molecules to form complexes that alter the functional and structural properties of the protein. Han et al. (2023) observed increased emulsifying activity and antioxidant activity in SPI after conjugation with epigallocatechin gallate, epigallocatechin, and epicatechin. S. Chen, Zhang, and Tang (2016) reported a decrease in surface hydrophobicity of SPI after forming complexes with CUR. The interaction between proteins and polyphenols involves non-covalent interactions such as hydrophobic interactions, hydrogen bonding, and van der Waals forces. Additionally, during the oxidation of polyphenols, the resulting quinones can covalently react with nucleophilic groups in proteins (Yildirim-Elikoglu & Erdem, 2018).

Although the protein-polyphenol complex can enhance interfacial activity and protein flexibility, this binary complex is not stable under harsh environmental conditions. Additionally, in the presence of a excessive polyphenols, proteins may aggregate, leading to a decrease in the stability of the protein at the interfacial layer (W. Chen et al., 2019). Polysaccharides can be incorporated into the protein-polyphenol binary complex to enhance its functional properties. Due to their hydrophilic nature, polysaccharides can increase the solubility and water absorption of the complex. They can also increase the viscosity of the continuous phase, providing steric hindrance and electrostatic repulsion, which effectively enhances emulsion and foam stability (Huang, Luo, Ning, Ye, & Liu, 2024). Several protein-polysaccharide-polyphenol ternary complexes have been successfully designed, such as lactoferrin-oat β-glucan-curcumin (Yang et al., 2020), whey protein concentrate-high methoxyl pectin-phenolic acid (Zhang, Li, Yang, Wang, & Zhang, 2022), and CMC (or pectin)-SPI-epigallocatechin-3-gallate (Zhao et al., 2020).

The ternary complex is primarily formed using the Maillard reaction along with acidic or alkaline treatment. Some limitations of this method for producing conjugates include the prolonged time and high temperature required for the Maillard reaction, the production of carcinogenic byproducts, the alteration of flavor and color, as well as a reduction in nutritional value (Sharafodin, Soltanizadeh, & Barahimi, 2023). Non-thermal plasma is a promising method recently considered for complex formation. In this method, electrical energy is applied to carrier gases to create ions and reactive radicals such as H⁺, H₃O⁺, O⁺, OH⁻, N₂⁺, N₂, O₂, O₃, H₂O₂, O^•, H^•, OH^•, and NO^• (Yu et al., 2021). The most common source of plasma production is based on the dielectric barrier discharge (DBD) method. This innovative technology has great potential in improving the formation of bonds between different compounds due to the diverse reactive species generated at low temperatures. Previous research has shown that complex formation using cold plasma can be effective. Ji et al. (2020) were able to form a peanut protein-dextran complex with a binding degree of 21.62 % after 1.5 min of treatment. Yu et al. (2021) formed a covalent complex between high-denatured peanut protein isolate and sesbania gum. The results showed that cold plasma treatment facilitates the grafting reaction. C. Liu et al. (2024) used cold plasma to form a covalent complex between ovalbumin and gallic acid. The results indicated that the antioxidant activity and emulsifying properties of ovalbumin were enhanced after conjugation with gallic acid.

Our previous research indicated that the order of compound addition during the production of a ternary complex using DBD plasma affects the glycation degree and structural properties of the produced ternary complex. Therefore, the aim of this study was to evaluate the effect of the order of conjugation of SPI, CMC, and CUR using the innovative DBD plasma technique on the interfacial, functional, antioxidative, and thermal properties of binary and ternary mixtures and complexes produced during this treatment.

2. Materials and methods

2.1. Materials

CUR with a purity of 95 % was obtained from Osareh Tabiat Company (Iran). SPI with 90 % protein was bought from Tianji Company (China). Corn oil was procured from Golshahd Company (Iran). Ethyl acetate, sodium hydroxide, bovine serum albumin (BSA), ethanol, urea, sodium tetraborate, methanol, β-Mercaptoethanol, sodium dodecyl sulfate (SDS), and CMC (DS = 0.7–0.9, MW ≈ 250,000 g/mol) were obtained from Merk company (Germany). Gallic acid, Folin-Ciocalteu reagent, 8-anilinonaphthalene-1-sulfonate (ANS), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were procured from Sigma-Aldrich Company, USA. Hydrochloric acid, 2,4-Dinitrophenuylhydrazine (DNPH) and guanidine hydrochloride were obtained from Samchun Company (Korea).

2.2. Preparation of binary mixtures and complexes

The binary complexes and mixtures studied in this research involved the various combinations of SPI with CMC and CUR. To prepare the SPI-CMC mixture, each biopolymer was dissolved in distilled water at a concentration of 3 % (w/v) and left to hydrate at 4 °C for 24 h. The two dispersions were then mixed in a 1:1 ratio and stirred for 3 h before being freeze-dried (Dena vacuum, Iran) at −18 °C. The SPI-CUR and CMC-CUR mixtures followed a similar procedure but with a 30:1 ratio and the addition of ethanol to aid in the dispersion of CUR. The resulting dried mixtures were stored at −18 °C until needed and exposed to plasma treatment (Kavoshyaran Co., Iran) at 18 kV for 10 min to form binary complexes.

2.3. Preparation of ternary mixtures and complexes

Three ternary mixtures were prepared, including SPI (CMC-CUR), CUR (SPI-CMC) and CMC (SPI-CUR). For the CUR (SPI-CMC) mixture, SPI-CMC complex was first dissolved in distilled water (3 % w/w) and combined with CUR solution at the 30:1 ration after 24 h of hydration at 4 °C. The mixtures were then frozen at −18 °C, freeze-dried, ground, and treated with DBD plasma at 18 kV for 10 min. Other ternary mixtures were prepared following the same procedure with a 30:30:1 ratio between SPI, CMC, and CUR.

2.4. Plasma treatment

The plasma treatment was conducted on SPI, CMC, and CUR individually, followed by exposure of all complexed binary and ternary compounds to DBD plasma. Each compound (2 g) was placed in a 9 cm diameter petri dish with a 2 mm wall thickness, then placed in a Pyrex container serving as a dielectric barrier (0.34 cm thickness). The upper electrode was brought into contact with the petri lid, resulting in a 1 cm distance between electrodes. A 10-min exposure to atmospheric pressure cold plasma of the DBD type was carried out, applying an 18 kV voltage with constant power (50 W) and frequency (50 kHz) settings. Interfacial, functional and antioxidative characteristics of the samples were subsequently analyzed.

2.5. Antioxidative properties

2.5.1. Phenolic content

The phenolic content of the samples was determined using the Folin-Ciocalteu method (Shi et al., 2022). Samples with a protein concentration of 1 mg/ml were prepared in 10 mM potassium phosphate buffer (pH = 7). To measure the phenolic content in CUR, 0.16 mg of pure CUR was dissolved in 10 ml ethanol to achieve the equivalent CUR concentration in the complexes. Subsequently, 0.5 ml of the sample was mixed with 2.5 ml of Folin-Ciocalteu reagent (10 % in distilled water). After 5 min, 2 ml of sodium carbonate (0.075 g/ml) was added, and the solution was kept in the dark for 2 h. The absorbance was then measured using a spectrophotometer at 760 nm (Hanon, China). The phenolic content of the samples was determined based on the standard curve of gallic acid at concentrations ranging from 10 to 200 ppm, and was expressed as grams of gallic acid per 100 g.

2.5.2. Antioxidant activity

The antioxidant activity of the samples was assessed using the DPPH method. Initially, 0.2 g of each sample was incubated with 5 ml of methanol for 3 h in a shaker incubator (IKA, Germany). The mixture was then centrifuged at 10,000g for 10 min, and 100 μl of the supernatant was combined with 1.5 ml of 0.1 mM DPPH solution. Methanol was utilized as a blank, while a control comprised of 100 μl of methanol and 1.5 ml of 0.1 mM DPPH solution. The absorbance of both the control and samples was measured at 517 nm, and the percentage of antioxidant activity was calculated using Eq. 1 (Khudhayer Oglah & Fakri Mustafa, 2020):

$$\text{Antioxidant activity}\left(\%\right)=\left(\left(\text{sample absorbance}-\text{control absorbance}\right)/\text{control absorbance}\right)\times 100$$ (1)

2.5.3. Carbonyl content

Samples containing 0.2 % (w/v) SPI were prepared for the experiment. Subsequently, 400 μl of each sample was added to two separate centrifugal tubes. One tube had 1 ml of 2 N hydrochloric acid solution added, while the other was mixed with 1 ml of 2 % DNPH solution prepared in 2 N HCl. The tubes were then shaken for 1 h at room temperature and centrifuged at 8000g for 10 min (Hermel, Germany) to separate the suspensions. To eliminate excess DNPH, the supernatant was removed, and the remaining precipitates were treated with 1 ml of ethanol: ethyl acetate solution (1,1) and centrifuged at 8000g for 10 min. This process was repeated three times. The sediments from the centrifugal tubes were dissolved in 2 ml of 6 M guanidine chloride solution in phosphate buffer (20 mM, pH = 6.5) and then centrifuged at 8000g for 10 min. The absorbance of the DNPH-reacted samples was read at 370 nm, while the other tube's absorbance was measured at 280 nm to determine protein concentration using the standard curve of BSA in 6 M guanidine hydrochloride. The carbonyl content was calculated using the protein absorption coefficient (22 mM⁻¹ cm⁻¹) and reported as nmol DNPH per mg of protein (Levine et al., 1990).

2.6. Solubility

The solubility of the samples was measured using a weighing method. A 0.2 % solution (in 10 mM potassium phosphate buffer) was prepared from all samples. The samples were hydrated for 24 h, then centrifuged for 10 min at 4 °C. The supernatant was discarded, and the sediment was dried at 50 °C for 6 h. The centrifugal tube containing the dried sample was then weighed, and the solubility value was calculated using the Eq. 2.

$$\text{Solubility}\left(\%\right)=\left({\mathrm{W}}_1-{\mathrm{W}}_2\right)/\mathrm{M}\times 100$$ (2)

where W₁ is the weight of centrifugal tube containing sample, W₂ is the weight of centrifugal tube and sample after drying, and M indicates the initial sample weight.

2.7. Superficial properties

2.7.1. Surface hydrophobicity

To assess surface hydrophobicity, the solutions were prepared using complexes and mixtures containing 0.5 mg/ml protein in 10 mM potassium phosphate buffer (pH = 8) with 4 M urea. Subsequently, each sample was diluted to protein concentrations of 0.4, 0.3, 0.2, and 0.1 mg/ml using 10 mM potassium phosphate buffer (pH = 8). A 4 ml of each dilution was then mixed with 20 μl of ANS solution (8 mM). After incubating for 20 min in the dark, the fluorescent intensity was measured using a fluorimeter (Shimadzu, Japan) with excitation and emission wavelengths of 390 nm and 470 nm, respectively. Surface hydrophobicity (H0) was determined by plotting fluorescent intensity against protein concentration for each sample (Ji et al., 2020).

2.7.2. Interfacial tension

The interfacial tension between corn oil and distilled water, with 1 % (w/v) of the samples, was measured before and after plasma treatment using the du Nouy ring method and a tensiometer (Kruss, Germany) for 1200 s. Samples were prepared by making a 1 % (w/v) solution containing 1 g of protein and/or polysaccharide, hydrating it, and then introducing it into the instrument vessel followed by the addition of oil. A ring was placed into the liquid, and the force needed to detach the ring from the liquid surface was recorded (Lee, Chan, Ravindra, & Khan, 2012).

2.7.3. Emulsifying activity and emulsion stability

The emulsifying activity of the samples was assessed following the turbidity method of Pearce and Kinsella (1978). Initially, a suspension of 0.5 % (w/v) was prepared from the samples and hydrated for 45 min on a stirrer. The suspension was then homogenized with 10 ml corn oil at 13000 rpm for 5 min using a homogenizer (IKA, Germany). Immediately after emulsion formation, 10 μl of the prepared emulsion was mixed with 5 ml of 0.1 % SDS, and the absorbance of the mixture was measured at 500 nm. The emulsifying activity was evaluated using the initial absorption.

Emulsion stability was tested by keeping the emulsions at room temperature for 2 h, and after mixing with SDS, the absorbance was measured at 500 nm. Emulsion stability was calculated using the Eq. 3:

$$\%\mathrm{ESI}=\left(\mathrm{A}/{\mathrm{A}}_0\right)\times 100$$ (3)

where ESI indicates emulsion stability, A₀ and A are absorption at the moment of emulsion formation, and absorption after 2 h, respectively.

2.7.4. Foaming capacity and foam stability

The foaming capacity and foam stability were measured according to the method of Onodenalore and Shahidi (1996). For this purpose, the sample solution containing 0.5 % protein was prepared from all samples. After complete hydration, the solution was stirred for 5 min with a high speed mixer (Moulinex, France) and the volume of the resulting foam was read immediately. The foaming capacity was calculated using Eq. 4:

$$\text{Foaming capacity}\left(\%\right)=\left({\mathrm{V}}_{\text{t0}}/\mathrm{V}\right)\times 100$$ (4)

where Vt₀ and V depicted the volume of foam immediately after preparation (ml) and V is the initial volume of solution (ml).

After 60 min, the volume of the remaining foam was read and the stability of the foam was measured using the following equation:

$$\text{Foam stability}=100-\left(\left(\left({\mathrm{V}}_{\text{t0}}-{\mathrm{V}}_{\mathrm{t}60}\right)/{\mathrm{V}}_{\text{t0}}\right)\times 100\right)$$ (5)

2.8. Thermal properties

A simultaneous thermal analyzer (449 F3, Netzsch Co., Germany) was applied to evaluate the thermal properties and the weight loss of samples in the presence of nitrogen. About 5 mg samples was weighted in the aluminum pan and the temperature was increased from 25 to 550 °C at the rate of 5 °C/min. An empty pan was used as the reference. The measurement was performed after calibrating the instrument using indium (Tm,onset = 156.6 °C, ΔH = 28.45 J/g) (Bruni et al., 2020).

2.9. Statistical analysis

In this study, all complexes and plasma treated compounds were prepared trice and used for further experiments. Statistical analysis was performed using a completely random design in the form of a factorial test. Data analysis was done at the 95 % level using Statistix 8 software.

3. Results and discussion

3.1. Antioxidative properties

3.1.1. Phenolic content

The chemical stability of ternary complexes is influenced by the presence of polyphenols, which is attributable to their antioxidant properties as noted by F. Liu, Ma, Gao, and McClements (2017). Table 1 shows an increase in the phenolic content of CUR following plasma treatment. This may be attributed to the breakdown of larger polyphenols into smaller compounds, leading to an increase in phenolics (Pogorzelska-Nowicka et al., 2021).

Table 1.

Phenolic content (g gallic acid/100 g), antioxidant activity (%) and carbonyl content (nmol/mg protein) of SPI (soy protein isolate), CMC (carboxymethyl cellulose), CUR (curcumin) and their binary and ternary mixtures before and after plasma treattment.

Sample	Phenolic content (gr gallic acid/100 g)	Antioxidant activity (%)	Carbonyl content (nmol/mg protein)
SPI before plasma	2.17 ± 0.20C	2.92 ± 0.03J	6.00 ± 0.09E
SPI after plasma	1.67 ± 0.14D	3.37 ± 0.07J	9.94 ± 0.07 A
CMC before plasma	–	–	–
CMC after plasma	–	–	–
CUR before plasma	19.24 ± 0.31B	87.79 ± 0.37AB	–
CUR after plasma	20.50 ± 0.59 A	85.11 ± 0.60CD	–
SPI-CMC before plasma	1.00 ± 0.20E	2.89 ± 0.25J	6.02 ± 0.01E
SPI-CMC after plasma	1.17 ± 0.11E	2.93 ± 0.16J	6.45 ± 0.07C
SPI-CUR before plasma	2.09 ± 0.21C	89.11 ± 0.12 A	5.91 ± 0.05E
SPI-CUR after plasma	1.96 ± 0.03CD	86.38 ± 1.31BC	6.85 ± 0.07B
CMC-CUR before plasma	0.57 ± 0.07FG	83.70 ± 0.63DE	–
CMC-CUR after plasma	0.50 ± 0.07G	82.16 ± 0.38E	–
CMC (SPI-CUR) before plasma	0.94 ± 0.04EF	50.94 ± 0.81I	5.25 ± 0.07F
CMC (SPI-CUR) after plasma	1.05 ± 0.02E	50.18 ± 0.13I	5.36 ± 0.06F
SPI (CMC-CUR) before plasma	1.25 ± 0.01E	77.69 ± 1.91F	5.97 ± 0.03E
SPI (CMC-CUR) after plasma	1.21 ± 0.07E	76.31 ± 0.11F	6.36 ± 0.06CD
CUR (SPI-CMC) before plasma	0.91 ± 0.05EF	69.92 ± 1.39G	5.95 ± 0.07E
CUR (SPI-CMC) after plasma	0.91 ± 0.04EF	60.51 ± 2.75H	6.25 ± 0.07D

Different letters in each column indicate significant differences between samples (p < 0.05).

Soybeans are rich in phenolic compounds, with phenolic acids and flavonoids, particularly isoflavones, being the most abundant (Dueñas et al., 2012). Some of these compounds can be retained in SPI. Table 1 indicates a decrease in the phenolic content of SPI after plasma treatment. The energetic particles from cold plasma can lead to the degradation of phenolic compounds of SPI.

The combination of SPI and CMC resulted in a reduction in SPI phenolic compounds (Table 1). This decline may be due to the interaction between SPI and CMC to form hydrogen and covalent bonds. Interestingly, plasma treatment did not alter the phenolic content of SPI-CMC. Despite the decrease in phenolic content of SPI following plasma treatment, the coating of SPI by CMC and the bond formation between them appear to have protected the phenolic compounds from the reactive species in plasma.

After the SPI and CUR were mixed, the phenolic content of CUR significantly decreased, likely due to the formation of CUR-SPI bonds during freeze-drying (Table 1). This could be due to the hydrogen bond formed between the hydroxyl groups of phenolic compounds with the sulfhydryl and free amine groups of SPI (Tamsen, Soltanizadeh, & Fathi, 2025). The phenolic content before and after plasma treatment did not show any significant differences. The Folin reagent is capable of detecting reducing compounds by reacting with them to produce a blue color, which can then be measured spectrophotometrically (Pérez, Dominguez-López, & Lamuela-Raventós, 2023). It was observed that the reducing compounds of CUR increased after plasma treatment, while no change was noted in the reducing compounds of the SPI-CUR mixture before and after plasma treatment. The presence of a covalent bond between CUR and SPI may explain why no significant difference was observed after plasma treatment compared to the untreated sample. The SPI-CUR complex exhibited the highest phenolic content among the various binary complexes due to the simultaneous presence of phenolic compounds in both SPI and CUR.

The addition of CMC to CUR led to a significant reduction in the phenolic content of CUR. As a result, the CMC-CUR complex exhibited the lowest phenolic content among the binary and ternary complexes (Table 1). It appears that the coating of CUR by CMC and the formation of a bond between the two compounds inhibited the reduction of the Folin reagent by CUR, leading to the lowest observed amount of phenolic content among the mixtures and complexes. Research conducted by Sarika, James, Kumar, Raj, and Kumary (2015) indicated that when an Arabic gum-CUR complex is formed, CUR is situated in the inner core while Arabic gum is located in the outer shell of the mixture. Plasma treatment did not affect the phenolic content of the CMC-CUR mixture (Table 1), likely due to the protective coating of CUR by CMC, thus shielding CUR from the energetic plasma particles.

In all ternary mixtures and complexes, the phenolic content of SPI and CUR decreased, indicating bond formation between CUR, SPI, and CMC. The addition of CMC to SPI and CUR reduced the phenolic content of the SPI-CUR complex. This decrease in phenolic content is associated with higher encapsulation efficiency and bond formation of polyphenol components with other compounds (Gali, Bedjou, Ferrari, & Donsì, 2022).

3.1.2. Antioxidant activity

Table 1 shows the percentage of radicals scavenged by CUR, SPI, and their complexes. SPI exhibited minimal antioxidant activity, and cold plasma treatment did not alter its level. Previous research suggested that SPI has antioxidant activity due to the presence of small amounts of flavonoids and cinnamic acids (Pratt & Birac, 1979).

CUR, which is derived from turmeric, is a phenolic antioxidant, and its effectiveness as an antioxidant is mainly due to the phenolic hydroxyl group, which neutralizes radicals by donating its H atom (Tapal & Tiku, 2012). Plasma treatment led to a decrease in the antioxidant activity of CUR. This could be due to the interaction of plasma-induced reactive species on antioxidant compounds and their degradation (Hemmati et al., 2021).

Mixing SPI with CMC had no effect on the antioxidant activity of SPI, and no changes were observed after the plasma treatment. It appears that despite the bonding between SPI and CMC during plasma treatment, the antioxidant activity of SPI flavonoids remained unaffected.

The combination of SPI and CUR exhibited significantly higher antioxidant activity compared to SPI alone, with no significant difference observed relative to pure CUR. This increase in activity may be attributed to the presence of antioxidant compounds in both CUR and SPI. However, when cold plasma was applied to form the SPI-CUR conjugate, the antioxidant activity decreased. The degradation of phenolic compounds during plasma treatment could account for this reduction. Additionally, sulfhydryl groups play a crucial role in scavenging free radicals, and increasing the surface sulfhydryl content may be a mechanism for enhancing antioxidant activity in complexes (Z. Jiang et al., 2023). Since plasma treatment led to a reduction in the free sulfhydryl content of the SPI-CUR mixture, this reduction may be responsible for the decrease in antioxidant activity of SPI-CUR after plasma treatment. However, the SPI-CUR complex exhibited the highest level of antioxidant activity among binary and ternary complexes.

The antioxidant activity of SPI was not affected by mixing it with CMC, and no changes were observed after plasma treatment (Table 1). Despite the formation of bonds between SPI and CMC during plasma treatment, SPI flavonoids remained intact and their antioxidant activity was preserved.

Comparatively, the CMC-CUR mixture displayed lower antioxidant activity than CMC-SPI (Table 1), possibly due to the absence of phenolic compounds in CMC. Plasma treatment did not impact the antioxidant activity of CUR-CMC. It appears that CMC acted as a protective layer for CUR by encapsulating its particles, thereby maintaining the antioxidant property of CUR and preserving its phenolic compounds during the plasma process.

The mixture of CMC (SPI-CUR) exhibited the lowest antioxidant activity compared to the other ternary mixtures and complexes, with no observed changes after plasma treatment (Table 1). It appears that the coating of CUR and SPI with CMC acted as a barrier, hindering their antioxidant activity.

The highest antioxidant activity among the ternary complexes was achieved by mixing SPI with CMC-CUR conjugate, with no significant difference noted after plasma treatment (Table 1). This increase in activity may be attributed to the positioning of SPI on the CMC-CUR conjugate, preventing CMC from forming a barrier layer that would block the access of these antioxidant compounds to donate hydrogen atoms.

After plasma treatment, the antioxidant activity of the CUR (CMC-SPI) mixture decreased (Table 1). CUR particles were observed on the surface and within the gaps of the SPI-CMC complex (Data not shown). In this scenario, CUR was more exposed to the active species of plasma, leading to a decrease in its antioxidant activity. Direct exposure of CUR to plasma causes it to act as a pro-oxidant rather than an antioxidant, as it can exhibit both properties (Ahsan, Parveen, Khan, & Hadi, 1999).

3.1.3. Carbonyl content

The formation of free carbonyl groups (C=O) in amino acids serves as a primary stable chemical indicator to identify protein oxidation (Mehr & Koocheki, 2020). It is important to evaluate protein oxidation and its stability during oxidative processes, such as plasma treatment. According to Table 1, the carbonyl content of SPI increased after plasma treatment and reaching the highest amount among all samples. The increase in protein oxidation leads to a rise in free carbonyl groups due to the production of strong oxidizing compounds, like ozone, during DBD plasma treatment. When reactive oxygen species (ROS) oxidize amino acid side chains, carbonyl groups are produced, especially on lysine, arginine, threonine, and proline (Weng et al., 2017).

Plasma treatment increased the carbonyl content of SPI in the SPI-CMC mixture, as shown in Table 1. This increase is a result of protein oxidation during the plasma treatment process. The oxidation level in the SPI-CMC mixture is significantly lower compared to SPI alone, suggesting that coating SPI with CMC can mitigate its oxidation. Gaurav et al. (2023) conducted a study on various polysaccharides, including CMC, to investigate their impact on protein oxidation in fried fish. Their findings revealed that coating fish with CMC could effectively prevent the increase in carbonyl content and reduce the rate of protein oxidation during frying.

On the other hand, the carbonyl content of SPI remained unchanged in the SPI-CUR mixture (Table 1), indicating the protective effect of CUR against SPI oxidation. Quan and Benjakul (2019) studied the formation of a complex between duck egg albumen hydrolysate and epigallocatechin gallate. They found that increasing the amount of epigallocatechin gallate in the complex resulted in a decrease in carbonyl production during oxidation. This decrease was attributed to the formation of C—N and C—S covalent crosslinks between the components of the complex, as well as the ability of epigallocatechin gallate to prevent protein oxidation. After plasma treatment, the carbonyl content of the SPI-CUR mixture increased, showing the highest carbonyl content among binary and ternary complexes (Table 1). The change in the protein structure, which might exclude hydrophilic amino acids such as lysine, can decrease carbonyl formation. This increase in carbonyl content can be attributed to the presence of free radicals and oxidizing compounds in the cold plasma, which oxidize alcoholic CO groups and transform them into carbonyl groups. During plasma treatment, SPI is exposed to plasma active species, resulting in increased oxidation and carbonyl content. Furthermore, CUR reacts with surrounding molecules such as protein during plasma treatment, converting to a pro-oxidant compound and contributing to oxidation (Medina & Trigos, 2022).

Mixing CMC with the SPI-CUR complex reduced SPI carbonyl content, as shown in Table 1. The coating of SPI by CMC prevented DNPH reagent from accessing carbonyl groups. After plasma treatment, the carbonyl content remained unchanged, being the lowest among the complexes, possibly due to the protective effect of CMC against protein oxidation. Additionally, CUR acts as an antioxidant and can inhibit carbonyl formation.

The SPI (CMC-CUR) mixture exhibited a similar carbonyl content to SPI (Table 1). Since this treatment process does not induce protein oxidation, no change in carbonyl content was expected. However, after plasma treatment, the carbonyl content increased due to the exposure of SPI on the surface, leading to increased protein oxidation by energetic plasma particles. The presence of phenolic compounds in this complex helped lower the carbonyl content of SPI compared to plasma-treated SPI.

In the case of the CUR (SPI-CMC) mixture, the carbonyl content was initially the same as SPI, but increased after plasma treatment (Table 1). Interaction with plasma active species transformed CUR on the surface of this complex into a pro-oxidant, intensifying oxidation.

The sequence in which compounds were added when forming the ternary complex played a significant role in determining carbonyl levels. Placing CMC on the surface reduced the protein oxidation intensity, highlighting the protective impact of this compound.

3.2. Solubility

Solubility is a crucial characteristic of any compound that influences its properties. According to Table 2, the solubility of SPI is lower than that of CMC but higher than that of CUR, and plasma treatment did not affect its solubility.

Table 2.

Surface hydrophobicity, Solubility (%), Emulsifying activity and Emulsion stability (%) of SPI (soy protein isolate), CMC (carboxymethyl cellulose), CUR (curcumin) and their binary and ternary mixtures before and after plasma treatment.

Sample	Surface hydrophobicity	Solubility (%)	Emulsifying activity (a.u.)	Emulsion stability (%)
SPI before plasma	73.77 ± 1.42C	24.17 ± 1.41H	0.43 ± 0.01H	41.36 ± 0.42H
SPI after plasma	98.48 ± 0.92 A	21.42 ± 1.30HI	0.42 ± 0.01H	57.31 ± 1.61F
CMC before plasma	–	95.00 ± 2.12 A	0.51 ± 0.01F	66.89 ± 0.76E
CMC after plasma	–	90.25 ± 0.35BC	0.54 ± 0.00DE	88.07 ± 5.34C
CUR before plasma	–	10.83 ± 1.18J	–	–
CUR after plasma	–	12.42 ± 1.53J	–	–
SPI-CMC before plasma	42.75 ± 1.05F	57.95 ± 3.83G	0.57 ± 0.01BC	75.28 ± 1.49D
SPI-CMC after plasma	98.05 ± 2.40 A	64.57 ± 3.51EF	0.50 ± 0.00F	72.86 ± 1.30D
SPI-CUR before plasma	34.35 ± 2.60G	19.57 ± 1.50I	0.35 ± 0.00J	52.27 ± 0.97G
SPI-CUR after plasma	21.80 ± 1.94H	20.78 ± 1.71HI	0.35 ± 0.00J	52.13 ± 2.20G
CMC-CUR before plasma	–	89.51 ± 1.37C	0.54 ± 0.01D	88.49 ± 1.11BC
CMC-CUR after plasma	–	93.34 ± 0.01AB	0.59 ± 0.00 A	98.47 ± 0.26 A
CMC (SPI-CUR) before plasma	22.47 ± 1.15H	64.95 ± 0.26E	0.38 ± 0.01I	86.29 ± 3.78C
CMC (SPI-CUR) after plasma	81.72 ± 2.70B	65.95 ± 1.32DE	0.46 ± 0.01G	84.89 ± 0.52C
SPI (CMC-CUR) before plasma	69.11 ± 1.60D	68.96 ± 0.45D	0.55 ± 0.01CD	86.58 ± 1.40C
SPI (CMC-CUR) after plasma	51.54 ± 2.42E	61.06 ± 0.17FG	0.58 ± 0.02AB	92.68 ± 3.29B
CUR (SPI-CMC) before plasma	20.98 ± 1.74H	67.37 ± 1.46DE	0.52 ± 0.01EF	73.01 ± 2.32D
CUR (SPI-CMC) after plasma	21.75 ± 1.40H	67.89 ± 0.96DE	0.54 ± 0.02D	74.49 ± 1.08D

Different letters in each column indicate significant differences between samples (p < 0.05).

After plasma treatment (Table 2), the solubility of CMC decreased. Oxidation by reactive plasma species can cause CMC molecules to bind together, forming aggregates with lower solubility (Ji et al., 2020). Additionally, the hydrophilicity of hydrocolloids may decrease during plasma treatment due to the etching process. During etching, the hydroxyl groups are dissociated from CMC, leading to reduced solubility of CMC (Amirabadi, Milani, & Sohbatzadeh, 2020).

There was no significant difference in the solubility of CUR before and after plasma treatment, as it has the lowest solubility among the samples due to its hydrophobic nature. As previously stated, CUR has very low water solubility (<10 μg/ml).

The hydrophilic nature of CMC can increase the solubility of the SPI-CMC mixture, which is further enhanced by plasma treatment (Table 2). The attachment of polysaccharide to protein can improve solubility, while steric hindrance from CMC can prevent SPI precipitation and boost solubility. Structural changes in SPI during plasma treatment, such as protein unfolding and increased flexibility from disordering the structure, play a role in enhancing solubility (Segat, Misra, Cullen, & Innocente, 2015a). After the formation of the peanut protein-sesbania gum conjugate using cold plasma, Yu et al. (2021) reported a two-fold increase in solubility. They attributed the increase in protein solubility to five factors: i) The conjugation of the protein-polysaccharide through the etching effect of reactive plasma species; ii) The oxidation of the protein, which increases polar groups such as C-O-H and C O on the protein surface; iii) The higher steric and electrostatic repulsion which prevents protein-protein aggregation and precipitation; iv) The increase of hydrogen and electrostatic binding between the conjugate and water which causes the formation of a weak hydration layer and thereby increases the protein-polysaccharide conjugate solubility; v) The enhancement of hydrophilic side chains by polysaccharide.

The solubility of the SPI-CUR mixture was lower than SPI and higher than CUR (Table 2). It seems the addition of the hydrophobic CUR to SPI reduced the solubility of the mixture compared to SPI. With the formation of bonds between SPI and CUR during freeze-drying, the formation of protein aggregates might be enhanced, which can reduce the solubility of SPI. The exposure of SPI hydrophilic groups on the surface of the SPI-CUR mixture increased the solubility compared to pure CUR. The plasma treatment did not have any significant effect on SPI-CUR solubility. The SPI-CUR mixture and conjugate indicated the lowest solubility among all binary and ternary complexes.

The mixture of CMC-CUR had lower solubility compared to CMC (Table 2) which originates from the hydrophobic nature of CUR. Plasma treatment increased the solubility of the CMC-CUR complex making it the most soluble sample overall and did not have a significant difference with CMC. The binding of CUR and CMC during plasma treatment could increase the solubility of hydrophobic CUR. Previous studies confirmed the improved solubility of hydrogel-stabilized CUR compared to pure CUR (Stachowiak, Mlynarczyk, & Dlugaszewska, 2024).

In general, all ternary complexes and mixtures demonstrated higher solubility compared to SPI and CUR, yet lower solubility than CMC. Mixing CMC with SPI-CUR conjugate increased the solubility of the conjugate due to CMC's hydrophilic nature, as shown in Table 2. As previously noted, the steric repulsion generated by CMC can prevent the precipitation of SPI and CUR particles. The encapsulation of the SPI-CUR conjugate by CMC can ultimately boost the solubility of the conjugate. The solubility of the CMC (SPI-CUR) mixture remained consistent both before and after plasma treatment, displaying no significant difference.

The SPI (CMC-CUR) conjugate exhibited lower solubility in comparison to the (CMC-CUR) conjugate due to the presence of SPI on the surface of the mixture and its affinity for binding to one another, consequently decreasing solubility. Moreover, the steric hindrance effect of CMC decreased in this scenario. After plasma treatment, the solubility of the SPI (CMC-CUR) mixture decreased, likely attributed to the formation of disulfide bonds between SPI molecules during plasma treatment, leading to increased aggregation and sedimentation of the conjugate.

Mixing CUR with CMC-SPI conjugate had no effect on the solubility of CMC-SPI conjugate. Similarly, the solubility of CUR (SPI-CMC) before and after plasma treatment did not exhibit any significant difference. Overall, the addition of CUR to SPI, CMC, and SPI-CMC conjugate did not significantly alter the solubility levels, possibly due to the relatively low amount of CUR compared to other compounds as highlighted in Table 2.

3.3. Interfacial properties

3.3.1. Surface hydrophobicity

Based on Table 2, the surface hydrophobicity of SPI increased following plasma treatment. It appears that the unfolding of the protein structure and the exposure of hydrophobic groups on the surface were caused by the plasma treatment. In a study by Segat et al. (2015a) on whey protein isolate, an increase in surface hydrophobicity due to slight structural changes and protein unfolding was reported, enabling greater access of hydrophobic groups to ANS.

Mixing CMC with SPI resulted in a reduction in the surface hydrophobicity of SPI (Table 2) possibly due to the presence of hydroxyl groups in CMC. The interaction between protein and polysaccharide during freeze drying can enhance the hydrophilicity of the protein piece while decreasing surface hydrophobicity. Non-thermal plasma treatment, on the other hand, increased the surface hydrophobicity of SPI-CMC, showing no significant difference compared to plasma-treated SPI and displaying the highest value among all samples (Table 2). This suggests that plasma treatment increased the hydrophobicity of the complex by influencing CMC and altering its hydrophilicity. Previous studies have shown that plasma treatment can intensify surface roughness through the impact of active and energetic species, along with an etching process, which in turn increases the surface hydrophobicity of the polysaccharide (Amirabadi et al., 2020). Moreover, the binding of SPI to CMC during plasma treatment may induce changes in protein structure leading to increased surface hydrophobicity. It is possible that the unfolding of SPI structure during binding to CMC exposes its hydrophobic groups, further enhancing surface hydrophobicity (Segat, Misra, Cullen, & Innocente, 2015b).

The mixture of SPI-CUR exhibited lower surface hydrophobicity compared to SPI, and its amount decreased post plasma treatment (Table 2). The involvement of the hydrophobic region of SPI in interaction with CUR may account for the decrease in surface hydrophobicity. Djuardi, Yuliana, Ogawa, Akazawa, and Suhartono (2020) examined the characteristics of SPI and tea extract polyphenols complex, revealing that an increase in polyphenol concentration led to decreased surface hydrophobicity due to the presence of oxidized polyphenol groups on the protein surface. Additionally, polyphenol aromatic rings may hide hydrophobic amino acids like tryptophan through hydrophobic bonds.

The addition of CMC to SPI-CUR conjugate significantly lowered surface hydrophobicity (Table 2). The reduction may be attributed to the hydrophilic nature of CMC and the coating of SPI-CUR conjugate. The CMC (SPI-CUR) conjugate demonstrated higher surface hydrophobicity in comparison to its mixture. The exposure of CMC to DBD plasma may have resulted in the removal of OH groups from its surface, decreasing its hydrophilicity. Amirabadi et al. (2020) observed a shift in the hydrophilic nature of Arabic gum to hydrophobic after cold plasma treatment.

The mixture of SPI (CMC-CUR) resulted in a decrease in the surface hydrophobicity of SPI, as shown in Table 2. This decrease may be attributed to the steric repulsion that occurs. The conjugates of CUR-CMC can prevent ANS from reaching the hydrophobic regions of the proteins. Moreover, plasma treatment along with forming of the SPI (CMC-CUR) complex contributed to reducing the surface hydrophobicity of the sample. Plasma treatment involves exposing substances to highly reactive species like oxygen-containing radicals, which induce chemical alterations on the material's surface. These alterations involve the disruption of hydrophobic bonds between protein molecules (Xu, Chen, Keidar, Leng, & Materials, 2019), thereby decreasing surface hydrophobicity. Furthermore, the binding of SPI to CUR-CMC adds hydrophilic groups to the complex's surface, enhancing surface hydrophilicity. Binding SPI to CMC and CUR through plasma treatment can induce structural modifications that result in reduced surface hydrophobicity in the protein.

Mixing CUR with SPI-CMC conjugate notably lowered the surface hydrophobicity of SPI to a level comparable to that of the CMC (SPI-CUR) mixture, and this level remained unchanged after plasma treatment. Once again, CMC functioned effectively in protecting SPI from exposure to ANS.

The results demonstrated that the presence of CMC or CUR on the complex's surface could reduce surface hydrophobicity, and the effects of plasma treatment varied based on the nature of the surface compound, resulting in increased, decreased, or unchanged surface hydrophobicity.

3.3.2. Interfacial tension

Interfacial tension refers to the attractive force between molecules at the interface of two fluids. An effective emulsifier should rapidly adsorb at the interface and form stable layers that can shield droplets by generating strong hydrophobic, steric repulsion, or electrostatic forces to prevent their coalescence. The surface behavior of emulsifiers is influenced by the number of hydrophilic and hydrophobic groups they contain (Y.-H. Jiang, Cheng, & Sun, 2020).

As shown in Fig. 1-A, plasma-treated CMC exhibits the highest interfacial tension among all samples. CMC, being a highly hydrophilic compound, lacks amphiphilic properties and therefore lacks interfacial activity. Consequently, it is unable to effectively reduce interfacial tension. Within the aqueous phase of the solution, CMC increases the viscosity of the continuous phase, ultimately lowering interfacial tension (Barbosa, Ushikubo, de Figueiredo Furtado, & Cunha, 2019). The rate of interfacial tension reduction decreases for CMC following plasma treatment (Fig. 1-A). Plasma treatment can result in the creation of cross-links in the polysaccharide structure. Initially, plasma active species break O—H and C-OH bonds, then form C-O-C glycosidic bonds between the two polysaccharide chains (Wongsagonsup et al., 2014). These cross-links may cause CMC to clump together, reducing its ability to prevent oil droplets from coalescing after emulsion formation. Solubility test results also indicate a decrease in CMC solubility after plasma treatment (Table 2), suggesting the formation of cross-links in the polysaccharide.

Fig. 1.

Interfacial tension of (A) binary and (B) ternary mixture of SPI (soy protein isolate), CUR (curcumin) and CMC (carboxymethyl cellulose) before and after DBD plasma treatment.

Unlike CMC, SPI exhibits lower interfacial tension attributed to its surface activity and presence of hydrophobic groups. Notably, cold plasma treatment does not alter this characteristic of SPI (Fig. 1-A).

After the addition of CMC to SPI, the interfacial tension rapidly decreased (Fig. 2-A). This could be attributed to the formation of bonds between SPI and CMC during freeze-drying. In this scenario, the protein's surface activity and CMC's steric hindrance work together to reduce interfacial tension. Following plasma treatment, not only did the interfacial tension decrease, but the rate of reduction also significantly increased (Fig. 1-A). The formation of the SPI and CMC conjugate during plasma treatment results in the protein being placed at the interface and the polysaccharide in the aqueous phase, ultimately increasing the thickness of the interface and reducing the interfacial tension (Barbosa et al., 2019). Additionally, there is a direct correlation between surface hydrophobicity and the rate of interfacial tension reduction (Mehr & Koocheki, 2020). Hence, the rate of interfacial reduction in the CMC-SPI mixture may attributed to its surface hydrophobicity.

Fig. 2.

DSC curve of (A) CUR (curcumin), CMC (carboxymethyl cellulose), SPI (soy protein isolate), and their (B) binary and (C) ternary mixtures before and after DBD plasma treatment.

Mixing SPI with CUR decreased interfacial tension, and after plasma treatment, the interfacial tension reached its lowest value among the binary complexes (Fig. 1-A). The protein-polyphenol complex can increase hydrophobicity and, by increasing the thickness of the interfacial layer, prevent droplet coalescence. This phenomenon was attributed to increased steric repulsion and hydrophobicity, leading to a thicker interfacial layer. When evaluating the egg white-catechin complex interfacial tension, Gu et al. (2017) found that the interfacial tension of egg white protein alone was higher than that of the catechin-egg white protein mixture, and even higher than the catechin-egg white protein conjugate. They found that the presence of polyphenols increased the surface activity of proteins, particularly when covalently attached. The researchers suggested that catechin's covalent binding to the protein surface may have enhanced their surface hydrophobicity or altered their structure, enabling them to more effectively reduce interfacial tension at the interface. Conversely, the surface hydrophobicity of the SPI-CUR mixture and complex decreased compared to SPI (Table 2). Therefore, it appears that the increase in surface hydrophobicity is not necessarily the root cause of the decrease in interfacial tension. The spherical soy protein rarely resides at the interface of water and oil. Upon unfolding of the protein structure through mixing and the formation of a complex with CUR, its capability of adsorption at the interface increased, resulting in a reduction of interfacial tension.

Fig. 1-A illustrates that the inclusion of CMC with CUR diminished the interfacial tension of CMC and accelerated the rate of tension reduction. Conversely, plasma treatment did not impact this property. The hydrophobic characteristic of CUR, combined with the hydrophilic nature of CMC, contributed to an increase in adsorption potential at the water-oil interface, thus preventing droplet coalescence. Additionally, the steric repulsion caused by CMC could lead to a decrease in interfacial tension.

The findings demonstrated that ternary mixtures and conjugates exhibited a quicker reduction in interfacial tension compared to individual SPI and CMC (Fig. 2-B).

The reduction rate of interfacial tension in CMC (SPI-CUR) mixture was higher than SPI and CMC (Fig. 1-B). CMC can cover SPI-CUR conjugate. It seems that steric repulsion had an effective role in increasing the rate of reduction of interfacial tension. Also, plasma treatment did not have any influence on this property.

The mixture of SPI (CUR-CMC) showed a faster rate of reduction in interfacial tension compared to SPI and CMC alone (Fig. 1-B). The amphiphilic properties of CUR-CMC conjugate were enhanced after the addition of SPI, which increased the rate of adsorption on the droplet surface. Plasma exposure slightly increased the interfacial tension. It appears that the formation of aggregates by crosslinking between ternary complex compounds prevented easy placement at the water-oil interface. Additionally, results from surface hydrophobicity tests (Table 2) showed that plasma treatment decreased the surface hydrophobicity of the complex. A decrease in surface hydrophobicity can lower the adsorption potential at the water-oil interface, ultimately leading to an increase in interfacial tension.

The mixture of CUR (SPI-CMC) decreased interfacial tension more rapidly than other ternary complexes (Fig. 1-B). The presence of CUR on the mixture's surface may have heightened hydrophobicity and thus the adsorption potential at the water-oil interface. Plasma treatment slightly increased interfacial tension. The binding of CUR to SPI and CMC during plasma exposure could alter the complex's structure, resulting in an increase in interfacial tension.

3.3.3. Emulsifying capacity and emulsion stability

Proteins are amphiphilic macromolecules with a moderate emulsifying capacity. The formation of a conjugate between proteins and certain compounds, such as polysaccharides and polyphenols can enhance this property (Djuardi et al., 2020). During emulsion formation, the hydrophobic groups of proteins adsorb at the oil-water interface to form a cohesive viscoelastic layer, while polysaccharides, with hydrophilic groups, contribute to colloidal stability by increasing thickness and forming a gel in the aqueous phase (steric repulsion) (Kato, 2002).

According to Table 2, the emulsifying capacity of SPI did not differ significantly before and after plasma treatment, but its emulsion stability increased. It appears that plasma treatment has induced structural modifications in the protein, thereby enhancing the stability of the resulting emulsion. The free sulfhydryl group analysis conducted in the first part of this study also revealed that the protein structure underwent unfolding during plasma treatment (Tamsen et al., 2025). This structural disruption, along with partial denaturation, likely contributed to improved prevention of oil droplet coalescence. The emulsifying capacity of CMC surpasses that of SPI, highlighting the role of steric repulsion in emulsion formation. Additionally, SPI exhibited lower solubility compared to CMC (Table 2), which potentially affected its emulsifying properties. After plasma treatment, both the emulsifying capacity and stability of CMC increased, suggesting that plasma treatment effectively prevents oil droplets from coalescing by altering the structure of CMC and enhancing steric repulsion. CMC contributes to steric repulsion by forming a gel-like structure (Kato, 2002).

The combination of SPI and CMC enhances the emulsification properties of both components. The results of interfacial tension (Fig. 1-A) showed that mixing SPI with CMC accelerated the reduction of interfacial tension, allowing this mixture to quickly cover the surface of oil droplets. The presence of both CMC and SPI enhances steric repulsion and surface hydrophobicity, preventing oil droplets from coalescencing and thereby improving emulsifying properties. However, after plasma treatment, the emulsifying capacity of SPI-CMC decreased (Table 2). The formation of disulfide bonds and the aggregation of SPI may contribute to this decline in emulsifying capacity. Protein aggregation decreases the ability of the complex to be positioned at the water-oil boundary, thus reducing the emulsifying properties.

The SPI-CUR mixture and complex exhibited the lowest emulsifying capacity and stability (Table 2). It appears that the emulsifying ability decreased as the solubility of SPI was reduced by mixing with CUR. Reduction in solubility inhibits protein accessibility to the oil-water interface (Segat et al., 2015a), as reflected in the solubility results (Table 2). Additionally, the decrease in surface hydrophobicity of SPI-CUR in comparison to SPI, along with the imbalance between hydrophilic and hydrophobic groups, diminishes the ability of the complex to prevent oil droplet coalescence.

The inclusion of CUR in CMC enhanced its emulsification capability (Table 2). The CMC-CUR mixture exhibited lower interfacial tension and a higher rate of interfacial tension reduction compared to CMC alone, suggesting its ability to impede oil droplet coalescence through steric repulsion. Following plasma treatment and creation of the CMC-CUR complex, emulsion capacity and stability improved due to increased solubility (Table 2).

Blending CMC with SPI-CUR conjugate resulted in enhanced emulsifying capacity and stability compared to SPI-CUR mixture (Table 2), possibly due to CMC's thickening properties that prevent oil droplet coalescence. Post-plasma treatment and the formation of the CMC (SPI-CUR) complex, emulsification ability was heightened compared to the mixture (Table 2). The rise in surface hydrophobicity of the complex during cold plasma treatment (Table 2) can accelerate its localization at the water-oil interface, thereby boosting its emulsifying activity.

After the plasma treatment, the emulsifying capacity and stability of the CMC-CUR conjugate did not significantly change when SPI was added to the complex. However, the emulsification ability increased and reached the highest level among the ternary complexes after the formation of the SPI (CMC-CUR) ternary complex (Table 2). The enhancement of the emulsifying properties can be attributed to the structural changes through binding of SPI to CMC and CUR through plasma treatment. On the other hand, the presence of SPI on the surface of the complex increases the surface activity of the complex. Yang et al. (2020) studied the formation of a ternary complex of lactoferrin, CUR, and oat β-glucan by adding these compounds in three different orders. Their results revealed that the lactoferrin (CUR-oat β-glucan) complex exhibited the best emulsifying properties compared to CUR (lactoferrin-oat β-glucan), oat β-glucan (CUR-lactoferrin), and lactoferrin-oat β-glucan binary complex. This improvement was linked to the increase in viscosity and the creation of a gel-like structure in the emulsion, which prevented particle aggregation through reduction of the speed of particle movement. Changing the sequence of adding the components of the ternary complex led to the formation of different structures, causing variations in properties and functions.

The emulsifying properties of the SPI-CMC complex decreased when CUR was mixed with it (Table 2). Although the CUR (SPI-CMC) mixture showed higher solubility (Table 2) and lower interfacial tension (Fig. 1-B) compared to the SPI-CMC complex, the lower surface hydrophobicity may account for the lower emulsifying capacity relative to the SPI-CMC complex. Plasma treatment induced structural changes and the formation of a ternary complex after exposure, which could enhance the emulsifying capacity.

In summary, the formation of binary and ternary complexes leads to the creation of a more stable emulsion than CMC and SPI alone. Only the SPI-CUR complex did not alter the stability of the SPI emulsion.

3.3.4. Foaming capacity and foam stability

Foaming capacity is a key functional property of proteins, indicating the adsorption of soluble protein at the gas-liquid interface. The foam, being thermodynamically unstable due to the high free energy at the gas-liquid interface, ultimately leading to bubbles coalescence (Shen, Hong, & Li, 2022). The foaming behavior of proteins is influenced by their structure and composition (Geng et al., 2022).

Table 3 illustrates a notable difference in foaming capacity and foam stability of SPI before and after plasma treatment. Plasma exposure resulted in a decrease in the protein's foaming properties. This change may be attributed to alterations in the hydrophilic-hydrophobic balance, which affects these properties.

Table 3.

Foaming capacity (%) and foam stability (%) of SPI (soy protein isolate), CMC (carboxymethyl cellulose), CUR (curcumin) and their binary and ternary mixtures before and after plasma treatment.

Sample	Foaming (%)	Foam stability (%)
SPI before plasma	231.00 ± 1.41B	67.53 ± 0.20G
SPI after plasma	215.00 ± 0.04C	59.07 ± 0.66I
SPI-CMC before plasma	147.50 ± 0.71G	93.22 ± 0.03 A
SPI-CMC after plasma	150.00 ± 0.10F	93.66 ± 0.47 A
SPI-CUR before plasma	240.51 ± 0.71 A	65.28 ± 0.40H
SPI-CUR after plasma	216.50 ± 2.12C	66.98 ± 0.66G
CMC-CUR before plasma	–	–
CMC-CUR after plasma	–	–
CMC(SPI-CUR) before plasma	144.50 ± 0.71H	73.01 ± 0.85F
CMC(SPI-CUR) after plasma	150.00 ± 0.02F	81.00 ± 0.47D
SPI(CMC-CUR) before plasma	153.50 ± 0.71E	83.06 ± 0.08C
SPI(CMC-CUR) after plasma	165.50 ± 0.71D	89.73 ± 0.81B
CUR(SPI-CMC) before plasma	137.00 ± 1.41I	73.00 ± 0.75F
CUR(SPI-CMC) after plasma	144.50 ± 0.71H	75.43 ± 0.61E

Different letters in each column indicate significant differences between samples (p < 0.05).

When SPI was mixed with CMC, the foaming ability decreased, while foam stability increased (Table 3). The presence of CMC in the SPI-CMC mixture is believed to increase hydrophilic groups, such as hydroxyl, reducing protein surface hydrophobicity. As foaming capacity is directly related to surface hydrophobicity, its decrease aligns with the results of surface hydrophobicity measurements. The enhanced foam stability of the SPI-CMC mixture may be linked to CMC's thickening properties.

Following cold plasma treatment, the formation of the SPI-CMC complex increased foaming capacity without impacting foam stability. The enhanced foaming capacity post-plasma treatment may be attributed to reduced folding and increased molecular flexibility, improving foaming properties (R. Li et al., 2019). Surface hydrophobicity results (Table 2) also indicated increased surface hydrophobicity of SPI-CMC after plasma exposure. With heightened surface hydrophobicity, this complex could more efficiently position itself at the air-water interface, enhancing foaming capacity.

In the mixture of SPI-CUR, the foaming capacity is higher than SPI, while foam stability is lower (Table 3). CUR has the ability to make changes in the structure of SPI during freeze-drying, which increases its foaming capacity. Additionally, CUR has a hydrophobic nature, and when incorporated into SPI, it increases the foaming capacity due to its hydrophobic groups. You, Yang, Chen, Xiong, and Yang (2021) reported that the addition of epigallocatechin gallate to SPI increased the foaming capacity. They suggested that epigallocatechin gallate may partially unfold the SPI and increase the dispersibility of the protein in the solution. Furthermore, amino acids and polar groups, such as phenolic hydroxyl groups, may be drawn towards the liquid phase. Therefore, the SPI-CUR mixture can weaken the interactions with water molecules at the interface, reduce interfacial tension, adsorb regularly at the gas-liquid interface, and provide a stronger ability to form a cohesive surface film that entraps air and creates a stable foam. After plasma treatment and the formation of the SPI-CUR complex, the foaming capacity decreased, which can be due to the increase of CUR binding to SPI and the reduction of its free hydrophobic groups (Table 2).

The addition of CMC to the SPI-CUR complex reduced its foaming capacity but enhanced foam stability (Table 3). The coating of SPI by CMC may prevent it from unfolding and placement on the water-air interface and foam formation. Additionally, the addition of the hydrophilic groups of CMC to this conjugate reduces the hydrophobicity and the foaming activity of the ternary mixture. However, the enhancement of viscosity by CMC can create steric repulsion and prevent the incorporation of air bubbles. The foaming capacity and stability of the CMC (SPI-CUR) complex increased after plasma treatment. Surface hydrophobicity results also showed that plasma treatment increased surface hydrophobicity (Table 2). The increase in surface hydrophobicity leads to a better placement of this complex on the air-water boundary and improves foaming.

The SPI (CUR-CMC) mixture produced more foam than other ternary mixtures (Table 3). It appears that the foaming property increases by adding SPI as a foaming agent to this conjugate and placing it in the outer part of the ternary complex since CUR and CMC, as well as CUR-CMC conjugate, do not have the foam formation ability. Additionally, the amount of foam increased after plasma treatment. The SPI (CUR-CMC) ternary complex exhibited the highest foam stability. The positioning of the protein in the outer part of the ternary mixture and the complex formation could facilitate access to the air-water interface by the hydrophobic region of SPI.

When CUR was added as the last compound to the SPI-CMC complex, the foaming capacity decreased to the lowest amount among ternary complexes (Table 3). This indicated lower foam stability than the SPI-CMC complex. The positioning of CUR on the surface of the SPI-CMC complex reduced its surface hydrophobicity (Table 2), which negatively affected the foaming capacity and stability. However, due to the presence of CMC as a thickening agent in the mixture, the ternary mixture still exhibited better foam stability than SPI. Plasma treatment increased the foaming properties of the ternary conjugate, suggesting that the structural changes created by binding SPI to CUR and CMC increased the foaming ability.

The formation of the ternary complex led to a decrease in the foaming capacity of SPI. The presence of CMC with high hydrophilic properties appeared to decrease the surface hydrophobicity of SPI. Furthermore, the steric hindrance of CMC reduced the speed at which SPI was placed on the water-air interface.

3.4. Thermal analysis

Differential scanning calorimetry is a rapid method for measuring the thermal properties of samples. The SPI displays an endothermic peak at 68.4 °C, indicating the denaturation temperature of protein (Fig. 2-A). Furthermore, SPI exhibits an exothermic peak at 275.9 °C, which corresponding to the decomposition and structural alteration of the protein.

The DSC graph of CMC indicates an endothermic peak around 81 °C with an enthalpy of 162 J/g, showing the loss of water content between 45 and 160 °C (Akram, Taha, & Ghobashy, 2016). Additionally, two overlapping exothermic peaks are evident at 278.6 and 287.2 °C. The first exothermic peak is associated with the decomposition process, while the second peak is resulting from the breakdown of decomposed products like acetic acid and various volatile organic substances (Akram et al., 2016).

Fig. 2-A displays the characteristic peak of CUR at 172.1 °C with ΔH of 82.11 (J/g). CUR initially exists in a crystalline form but transitions to an amorphous form within the complex structure. The formation of the complex eliminates the observable peak (Wang, Lu, Ouyang, & Ling, 2020).

In all binary complexes, the denaturation temperature and its enthalpy are lower compared to untreated binary mixtures, indicating a reduction in intermolecular forces (Ji et al., 2020). Additionally, the exothermic nature of the coupling reaction leads to a decrease in enthalpy.

According to Fig. 2-B, the denaturation temperature of SPI decreased during mixing, followed by complex formation with CMC and CUR. This decrease may be attributed to the alteration in the natural structure of the protein as it forms a conjugate with these compounds. Ji et al. (2020) conducted a study where they formed a complex of peanut protein and sesbania gum using cold plasma. Their findings revealed that the denaturation temperature of the protein decreased from 80.6 °C to 76.9 °C and 74.6 °C after mixing with sesbania gum and undergoing cold plasma treatment, respectively. The researchers linked the decline in thermal stability of the products to the formation of a looser tertiary structure.

In ternary complexes, the denaturation and decomposition enthalpy also decreased (Fig. 2-C). The reduction of enthalpy indicates the bonding reaction between CMC, SPI, and CUR during plasma treatment. Also, the characteristic peak of CUR, CMC, and SPI is not seen in the mixtures and complexes, which can be due to the formation of bonds between the components in the freeze-drying stage and the intensification of bonds after applying cold plasma. Exothermic peaks in the temperature range above 200 °C are usually related to the decomposition of the structure due to high temperature. Wang et al. (2020) formed a complex using SPI, nanocrystals of cellulose, and CUR. Their results showed that the thermal stability of the SPI-CUR-cellulose complex was increased compared to SPI-CUR. They attributed their observations to the presence of cellulose in the ternary complex. They stated that cellulose increased the hydrophobic and electrostatic forces between the three compounds of CUR, cellulose, and SPI.

Thermogravimetric analysis (TGA) is used to evaluate the thermal stability of samples based on their weight changes during different temperatures under control conditions (Duhoranimana et al., 2018). According to Fig. 3-A, the initial weight loss in all samples is related to the loss of moisture. As the temperature increases, thermal decomposition and secondary weight loss occur. The weight loss of SPI occurred in two parts. The primary weight loss (40–110 °C) was related to moisture loss, and the second weight loss, occurring at about 200 °C, indicated thermal decomposition. The weight loss of SPI was 69 %. Changes in protein weight are due to the release of carbon monoxide, carbon dioxide, and ammonia and are related to the destruction of covalent bonds including C—N and C(O)-NH during the heating process (L. Chen et al., 2020). Qin et al.'s research (2019) showed that SPI has two degradation processes in the range of 30–130 and 270–500 °C, which were attributed to the evaporation of absorbed water and the thermal degradation of protein chains, respectively (Qin, Mo, Liao, He, & Sun, 2019).

Fig. 3.

TGA curve of (A) CUR (curcumin), CMC (carboxymethyl cellulose), SPI (soy protein isolate), and their (B) binary and (C) ternary mixtures before and after DBD plasma treatment.

The weight loss of CMC had three parts as shown in Fig. 3-A. The initial weight change, occurring at temperatures below 130 °C, was related to moisture loss. The secondary weight loss, starting at a temperature of about 250 °C and continuing up to 290 °C, can be attributed to the release of carbon dioxide from the polysaccharide. Due to the presence of COO- groups in the structure of CMC, decarboxylation occurs in this temperature range (L. Chen et al., 2020). The third weight loss of this compound occurred in the range of 290 to 480 °C, with a 62 % weight loss. Kumar et al. (2020) found that the major decomposition of CMC starts at 240 °C, due to side group destruction and chain scission, with decomposition after 335 °C attributed to pyrolysis reaction.

In CUR, primary decomposition occurred at 210 °C with a weight loss of 73 %, as shown in Fig. 3-A. The slope of weight loss was relatively gentle, consistent with previous research (El Kurdi & Patra, 2018). Varaprasad et al. (2019) attributed the weight loss of CUR at 190 to 210 °C to the dihydroxylation of OH groups due to the decomposition of CUR molecules.

Mixing SPI with CMC or CUR resulted in an increase in the thermal stability of SPI. The weight loss of these mixtures decreased after plasma treatment (Fig. 3-B). These findings indicate that plasma treatment enhanced the heat resistance of the mixture by creating strong bonds between SPI and CMC. Su, Huang, Yuan, Wang, and Li (2010) found that the thermal stability of SPI was improved through cross-linking with CMC.

In the CMC-CUR mixture, thermal stability reached approximately 240 °C. Additionally, the weight loss of this mixture was lower than that of pure CUR (Fig. 3-A, B). These observations suggest an enhancement in the thermal stability of CUR during its combination with CMC. Following plasma treatment, the weight loss increased from 62 % to 88 %, indicating potential damage and fragmentation of the CMC-CUR structure by high-energy plasma particles, subsequently reducing the thermal stability of the complex.

The main thermal decomposition of the CMC (SPI-CUR) mixture occurred at a temperature of approximately 230 °C (Fig. 3-C). After plasma treatment, the thermal resistance of the complex remained unchanged, however; the weight loss decreased. These results indicate that the thermal stability of the complex is enhanced by the formation of strong bonds between SPI, CMC, and CUR during plasma treatment.

The thermal decomposition of the SPI (CMC-CUR) mixture was observed at 210 °C (Fig. 3-C), which was the lowest among the ternary mixtures. This is likely due to the placement of SPI molecules on the surface of the mixture and its low thermal resistance. Following plasma treatment, the thermal stability increased to 230 °C, with the weight loss decreasing from 95 % to 79 %. These changes demonstrate the impact of cold plasma on enhancing the thermal stability of the complex, which also exhibited the lowest weight changes among the ternary complexes.

The main thermal decomposition of the CUR (SPI-CMC) mixture occurred at 230 °C (Fig. 3-C). While the thermal stability did not change after plasma treatment, there was a slight increase in weight loss. Since CUR is more exposed to plasma in this configuration and has a pro-oxidant effect, it can lead to increased oxidation, contributing to the weight changes observed during heating.

4. Conclusion

The results of the present study indicated that the addition sequence of the compounds can significantly influence the functional, antioxidant, and thermal properties of the plasma-produced ternary complexes of SPI, CMC, and CUR. Plasma treatment was found to alter the properties of the materials, leading to a reduction in surface hydrophobicity, foaming capacity, emulsifying activity, and solubility. Additionally, the antioxidant activity of the samples was adversely affected by plasma treatment, as indicated by an increase in carbonyl content, suggesting oxidation by plasma active species. Furthermore, denaturation and decomposition enthalpy decreased in all complexes after plasma treatment, implying bonding reactions among CMC, SPI, and CUR. Thermal resistance also improved following complex formation, with thermal decomposition occurring at higher temperatures. The SPI (CMC-CUR) complex exhibited superior functional properties, including enhanced foaming capacity, foam stability, emulsifying activity, and emulsion stability, as well as enhanced antioxidant activity and thermal stability. Consequently, this complex appears promising for applications such as the microencapsulation of bioactive compounds.

CRediT authorship contribution statement

Maryam Tamsen: Writing – original draft, Investigation, Formal analysis. Nafiseh Soltanizadeh: Writing – review & editing, Supervision, Project administration, Conceptualization. Milad Fathi: Writing – review & editing, Methodology.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the authors used Chat GPT in order to improve the language and readability of the manuscript. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.