Impact of gamma irradiation on the structural, functional and physicochemical properties of pea protein isolate

Abstract

BACKGROUND

The properties of pea protein isolate (PPI), comprising a sustainable plant‐derived protein for which the full functional potential in complex food matrices remains unrealized as a result of inherent limitations in food applications, are investigated in this work under gamma (γ) irradiation (0–10 kGy). The study used non‐thermal physical treatment, namely γ irradiation, using ionizing radiation from a ⁶⁰Co source, which causes controlled structural rearrangements in macromolecules.

RESULTS

Fourier transform infrared spectroscopy deconvolution revealed irradiation‐induced changes: partial unfolding of secondary structures (α‐helix/β‐sheet), whereas scanning electron microscopy showed fragmented morphology (4–8 kGy) and aggregated clusters. Functional properties peaked at 8 kGy with solubility, emulsifying activity and water/oil‐holding capacity, whereas 10 kGy enhanced emulsion stability but reduced solubility because of disulfide cross‐linking.

CONCLUSION

This study establishes 8 kGy as the critical threshold for balancing structural flexibility and functionality of PPI, providing a non‐thermal strategy for tailoring plant proteins in next‐generation foods. Physicochemical studies linked these changes to disulphide bond breaking, free sulfhydryl dynamics and fluorophore exposure. These results highlight γ irradiation as a flexible approach to modify PPI activity, hence balancing structural changes with particular dietary applications. © 2026 Society of Chemical Industry.

INTRODUCTION

The worldwide transition to plant‐based proteins has established pea protein isolate (PPI) as a prominent contender for sustainable food innovation, as a result of its nutritional adequacy, hypoallergenic properties and ecological advantages. However, the adoption of PPI in textured foods and emulsions is limited by inherent functional constraints: low solubility, weak emulsification and subpar water/oil‐binding capacities.^{1, 2} These limitations stem from the rigid tertiary structure of PPI stabilized by disulfide bonds and hydrophobic interactions. Although chemical modification exists, non‐thermal physical methods such as γ irradiation offer ‘clean‐label’ advantages by avoiding residual solvents.^{1, 2, 3} Physical modification methods such as extrusion, high‐pressure processing and γ irradiation have evolved as clean replacements for chemical treatments that address these concerns. ¹

Food irradiation finds an appealing approach in ionizing radiation as a result of γ rays from radionuclides ⁶⁰Co or ¹³⁷Cs or high‐energy electrons generated by machine sources. ⁴ Molecules (proteins, polysaccharides) form solids through a hierarchy of structures: from native protein complexes formed during extraction/storage (e.g. vicilin hexamers) to complex supramolecular structures. Gamma irradiation disrupts molecular organization, allowing targeted modulation of functional properties in food systems. This non‐thermal technique effectively modifies plant‐derived macromolecules via controlled energy deposition, which drives structural rearrangements. Research on finger millet and pea fiber suggests that γ irradiation changes crystalline structures, reduces particle size and enhances porosity, therefore improving functional properties such as water retention and adsorption capacity.^{5, 6} The irradiation of pea fiber at 2 kGy considerably increased oil‐holding capacity (8.12 g g^–1) and swelling characteristics (19.75 mL g^–1), at the same time as modifying cellulose and lignin content, indicating its potential for customized functionality. ⁶ Similar structural changes in polysaccharides and starches have been associated with improved digestibility and retention of bioactive compounds, demonstrating the adaptability of this technique.^{1, 7} Despite these advances, little research has been carried out on how γ irradiation alters PPI. Recent studies on plant proteins such as soy isolates and rapeseed showed that irradiation can alter surface hydrophobicity, solubility and emulsion stability by breaking down disulphide linkages and reorganizing secondary structures.^{1, 3} For example, extruded PPI matrices treated with β‐mercaptoethanol exhibited reduced hardness and water‐holding capacity, emphasizing the critical role of molecular interactions in texture modulation. However, there are no systematic studies on how γ radiation specifically affects the structural hierarchy of PPI, from the primary arrangement of amino acids to tertiary conformations.^{1, 2, 6}

In particular, no previous studies have combined multiple analytical techniques across a full dose series to probe the response of PPI to γ irradiation. Most prior work used a single technique or a narrow dose range, and so the relationship between molecular changes and functionality remains unclear for PPI. We addressed this gap by irradiating PPI at a series of doses (0, 2, 4, 6, 8 and 10 kGy) and applying multiple characterization methods. We used sodium‐dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) to detect changes in polypeptide profiles, Fourier transform infrared spectroscopy (FTIR) spectroscopy to monitor secondary structure, scanning electron microscopy (SEM) to visualize microstructure and intrinsic fluorescence to assess tertiary conformation. We then correlated these structural data with key functional measurements: protein solubility, surface hydrophobicity and emulsion activity/stability. This multi‐technique, dose‐dependent approach is novel for PPI. By contrast to soy or rapeseed proteins, PPI has a distinct amino acid composition that may influence its cross‐linking behavior under irradiation. By combining quantitative structural analyses with functional assays, our study could provide a comprehensive view of how increasing γ irradiation doses affect PPI, adding new depth beyond the partial insights from earlier soy, rice and canola studies.

MATERIALS AND METHODS

Materials

PPI with 80% protein on a w/w basis was obtained from Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China). All additional chemicals and reagents used in the studies came from regional companies and were of analytical or higher grade.

Gamma irradiation of PPI

Samples of PPI were packed in polyethylene bags for γ irradiation treatment. The irradiation test was conducted at Guangzhou Huada Biological Technology Co., Ltd (Guangzhou, China). The dose of γ radiation is control (0), 2, 4, 6, 8 and 10 kGy, respectively, were performed with a ⁶⁰Co source irradiator. The dose rate is 0.8 kGy h^–1, and the total activity of the radioactive source is approximately 1.14 million curies. The treated samples were stored for two weeks at 4 °C for further examination.

Structural properties

SDS‐PAGE

SDS‐PAGE of PPI was performed using a modified technique of Zhuang et al. ⁸ Briefly, 30 μL of sample solution and 10 μL of loading buffer (pre‐thawed) were collected and then poured into a 0.5‐mL tube and placed in a heater for 20 min at 100 °C, after which 10 μL of the prepared samples were applied to the gel (BeyoGel™ Plus Precast PAGE Gel; Beyotime, Shanghai, China) (Hepes, 4–20%). Molecular weight standards (Beijing Solarbio Science & Technology Co., Ltd, Beijing, China) were in the range 11–245 kDa. Electrophoresis was performed at a voltage of 100 V for 40 min using a compact vertical electrophoresis device DYC‐Mini4 (Nanjing Yucheng Experimental Equipment Co., Ltd, Nanjing, China). After electrophoresis, it was treated with an electrophoretic staining solution for 20 min and solution was discolored for 1 day.

Particle size and zeta potential

Particle size and zeta potential were measured using a ZEN3600 nano laser particle sizer (Malvern Instruments Ltd, Malvern, UK). Protein solutions from various irradiation treatments were diluted to 1 mg mL^–1 with a phosphate buffered saline (PBS) (pH 7.4) and the average particle size and zeta potential of the solutions was measured at 25 °C. Three repeats were performed for each radiation dose.

FTIR

The FTIR spectrum of PPI were obtained using an FTIR spectrometer (Vertex 70; Bruker Ltd, Ettlingen, Germany). Pre‐dried spectrally pure potassium bromide (100 mg) and lyophilized samples (1 mg) were weighed and ground in an agate mortar. All of the ground powder was placed into the tablet die and pressed into a powder tablet press (NL‐15; TianJinNuoLeiXinDaTechnology Co., Ltd, Tianjin, China) under a pressure of 10 MPa. For detection purposes, the pressed light‐transmitting tablet was placed in an FTIR spectrometer. The peak spectra and data of each sample were acquired in the range 4000–400 cm⁻¹, and each sample was scanned three times.

Microstructure of proteins

The protein's microstructure was analyzed using the approach previously described by Zhu et al. ⁹ The PPI was dehydrated using a supercritical dryer SCD‐350 M [Xi’an Jia (Beijing) Biotechnology Co., Ltd, Beijing, China]. The desiccated samples were affixed to circular aluminium specimen stakes and thereafter transported to a QI50TS sputtering trans coater (Quorum Corporation, Edinburgh, UK) for the purpose of applying a layer of gold via spraying, which lasted for a duration of 60 s. Afterwards, the protein's microstructures were examined using a field emission scanning electron microscope (Nano SEM‐450; FEI Corporation, Hillsboro, OR, USA). The magnification was 5000×, the accelerating voltage was 5 kV and the accompanying graphs were recorded using SEM software (xT Microscope Control software (FEI Company, Hillsboro, OR, USA)).

Functional properties

Emulsifying activity and emulsifying stability

The emulsifying activity and emulsifying stability were assessed using the methodology outlined by Zhu et al. ¹⁰ Initially, a protein solution with a concentration of 10 mg mL^–1 was prepared using PBS buffer. Subsequently, 8 mL of the protein solution was combined with 2 mL of avocado oil in a 50‐mL centrifuge tube. The mixture was then homogenized at a speed of 10 000 × g for 1 min using an XHF‐DY high‐speed homogenizer (Ningbo Xinzhi Bio‐Technology Co., Ltd, Ningbo City, China). Immediately after the homogenization process, a 50‐μL sample of the emulsion was extracted from the bottom. Then, 5 mL of a solution that contained 0.1% SDS was added and thoroughly mixed. The absorbance of the emulsion was then measured at a wavelength of 500 nm, both at the start (0 min) and after 10 min. The blank control consisted of a solution containing 0.1% SDS. The formulas used to determine emulsifying activity index (EAI) and emulsification stability index (ESI) were:

$$\mathrm{EAI}\left({\mathrm{m}}^2{\mathrm{g}}^{-1}\right)=\left(2\times 2.203\times A_0\times N\right)/\left(C\times B\times 10000\right)$$ (1)

$$\mathrm{ESI}\left(\%\right)=\left(A_{10}\times 100\right)/A_0$$ (2)

where N is the number of dilutions, B is the volume fraction of oil in the formed emulsion, C is the initial concentration of protein (g mL^–1) and A ₀ and A ₁₀ are refer to the absorbance of the emulsion at 0 and 10 min, respectively. Each sample was measured three times in parallel.

Foaming capacity (FC) and foam stability (FS)

The method described by Xie et al., ¹¹ Stone et al. ¹² and Wang et al., ¹³ used to determine the foaming properties and FS of the protein. A protein solution was produced by dissolving protein in PBS buffer at a concentration of 10 mg mL^–1. After that, 10 mL of the solution was mixed using XHF‐DY high‐speed homogenizer (Ningbo Xinzhi Bio‐Technology Co., Ltd ) at 11 000 × g for 1 min. After homogenization, the level of foam generated within the time frame of 0 to 30 min was measured and recorded. The calculation formula was:

$$\mathrm{FC}\left(\%\right)=\left(V_0–10\right)/10\times 100$$ (3)

$$\mathrm{FS}\left(\%\right)=V_{30}/V_0\times 100$$ (4)

where V ₀ is the foaming volume after homogenization of 0 and V ₃₀ is the foaming volume after homogenization for 30 min.

Water‐holding capacity (WHC) and oil‐holding capacity (OHC)

WHC and OHC of PPI was assessed using the methodology developed by Stone et al. ¹² The suspension of 0.5 g of protein powder in 5 g of water or oil within a 50‐mL centrifuge tube allowed determination of the WHC and OHC. After 10 s every 5 min for 30 min overall, the dispersions were vortexed and then centrifuged at 3000 × g for 15 min. The leftover particle was weighed after carefully decant the supernatant. Calculating the weight obtained by the protein samples against the original weight of the samples allowed determination of the water/oil retention capacity. Measurement of grams of water or oil per gram of sample allowed determination of the water and oil retention capabilities.

Physicochemical properties

Protein solubility

First, the irradiated PPI was diluted at different doses to 10 mg mL^–1 using PBS (pH 7.4). A test tube which contained 1 mL of the sample solution was centrifuged at 11 000 × g for 20 min at 4 °C. ¹⁴ We used a spectrophotometer (N6000; Shanghai Yoke Instrument Co., Ltd, Shanghai, China) with an ultraviolet and visible spectrum to measure the optical density at 540 nm. The Coomassie Brilliant Blue method was used to determine the protein content in the supernatant. The ratio of the total protein concentration in the suspension to the fraction of protein in the liquid filler before serving for centrifugation was expressed as protein solubility.

Surface hydrophobicity

The quantification of protein surface hydrophobicity was conducted by measuring the level of bromophenol blue binding (BPB). ¹⁵ A protein solution with a concentration of 5 mg mL^–1 was prepared using a PBS buffer. Then, 5 mL of the protein solution was thoroughly mixed with 1 mL of BPB. The reaction was carried at a temperature of 25 °C for 10 min. The mixture was centrifuged at a speed of 2000 × g for 15 min at a temperature of 4 °C. The resulting liquid was then diluted 10 times, and the absorption was measured using a UV scanning spectrophotometer N6000 (Shanghai Yoke Instrumentation Co., Ltd) at a wavelength of 595 nm. The BPB (H₀) ratio was calculated using:

$$\text{Ratio}\mathrm{BPB}\left(\mathrm{\mu g}\right)=\left(A_0–A\right)/A_0\times 500\mathrm{\mu g}$$ (5)

where A ₀ is the absorbance of the BPB solution without protein and A is the absorbance of BPB solution incubated with the protein sample.

Free sulfhydryl and disulfide bond content

Ellman's method was used to determine the free sulfhydryl (F‐SH) and total sulfhydryl (T‐SH) content of the sample, with some modifications. ¹⁶ Initially, proteins exposed to various doses of irradiation were diluted to a concentration of 1 mg mL^–1 using phosphate buffer. A Tris‐Gly buffer solution was prepared with a concentration of 0.086 mol L^–1 Tris, 0.09 mol L^–1 Gly and 0.04 mol L^–1 EDTA (i.e. ethylenediaminetetraacetic acid) was prepared. The pH of the buffer was then adjusted to 8.0 using a 2 mol L^–1 HCl solution. The preparation of Ellman's reagent involved the use a Tris‐Gly buffer containing free sulfhydryl groups to obtain a solution of 5,5′‐dithiobis‐(2‐nitrobenzoic acid) (DTNB) with a concentration of 4 mg mL^–1. The composition of the Tris‐Gly buffer for the measurement of total sulfhydryl groups were as follows: the solution consisted of Tris at a concentration of 0.086 mol L^–1, Gly at a concentration of 0.09 mol L^–1, EDTA at a concentration of 0.04 mol L^–1 and Urea at a concentration of 8 mol L^–1. The Ellman's reagent, used to measure the total sulfhydryl groups, was obtained from the Tris‐Gly buffer and had a concentration of 4 mg mL^–1 of DTNB. Next, 5 mL of Tris‐glycine buffer and 25 μL of Ellman's reagent were added to 0.75 mL of the protein solution and thoroughly mixed. A blank control was prepared using a mixture of Tris‐Gly buffer and Ellman reagent. The sample was immersed in a water bath maintained at a temperature of 25 °C for 1 h and kept away from any light source. It was then centrifuged at 11 000 × g for 10 min at 4 °C. The liquid part was collected, and the intensity of light absorption was determined at a wavelength of 412 nm using an N6000 UV‐visible spectrophotometer manufactured (Shanghai Yoke Instrument Co., Ltd). The calculation formula was:

$$\text{Free}-\mathrm{SH}/\text{total}-\mathrm{SH}\left(\text{μmol}{\mathrm{g}}^{-1}\right)=73.35\times A_{412}\times \text{dilution ratio}/\text{protein concentration}$$ (6)

$$\text{Disulfide bond content}\left(\text{μmol}{\mathrm{g}}^{-1}\right)=\left(\text{total}-\mathrm{SH}–\text{free}-\mathrm{SH}\right)/2$$ (7)

where A ₄₁₂ is the absorbance at 412 nm.

Intrinsic fluorescence analysis

The protein obtained as a result of various irradiation treatments was diluted with a PBS to 1 mg mL^–1, and then the protein content in the solution was measured at room temperature using an RF‐6000 fluorescent spectrophotometer (Shimadzu Manufacturing Co., Ltd, Kyoto, Japan). The excitation wavelength was 283 nm, the slit width was 10 nm and the radiation wavelength range was 300–400 nm. Each sample was measured six times in parallel.

Statistical analysis

All the studies were conducted three times, and the findings were presented as the mean ± SD. The data was subjected to a one‐way analysis of variance. SPSS, version 27.0 (IBM Corp., Armonk, NY, USA) was used for all statistical analyses. Duncan's multiple range test was then used to identify any significant differences between the means of the samples. P < 0.05 was considered statistically significant. Data visualizations were performed using Origin 2022 ( OriginLab Corp., Northampton, MA, USA ).

RESULTS AND DISCUSSION

Structural properties

SDS‐PAGE

The results of the analysis of the PPI after γ irradiation are shown in Fig. 1. Because bands of more than 100 kDa were not detected, it is likely that the larger quaternary structures of legumin, vicilin, and albumins were compromised during extraction.^{17, 18} All irradiated PPI had protein bands with molecular weights of at least 97 kDa, which are probably lipoxygenases. Moll et al. ¹⁸ and Schumacher et al. ¹⁹ also discovered such protein fields with molecular weights smaller than 100 kDa. Globulins (legumin, vicilin and convicilin) are presented in all irradiated samples. A dim band around 11 kDa (minor albumin fraction) was observed in all samples, indicating that albumins were a minor component. Only a very slight decrease in the intensity of bands below 20 kDa was observed at 6, 8 and 10 kGy, indicating minor fragmentation or partial degradation of low molecular weight polypeptides at higher doses. It is important to note that the formation of high molecular weight cross‐linked particles was not detected under reducing conditions.

Figure 1.

SDS‐PAGE of pea protein isolate solution treated with different irradiation doses.

Particle size and zeta potential

The particle size measurements of γ‐irradiated PPI are illustrated in the Fig. 2. There was a slight decrease in particle size at 2 kGy, followed by a gradual increase with radiation dose escalation culminating in 10 kGy. At 6 and 8 kGy, the absolute value of the zeta potential decreased, indicating a decrease in electrostatic repulsion. This effect, in turn, stimulated aggregation. Gamma irradiation caused structural changes in proteins, which then led to aggregation and an increase in particle size. At irradiation intensities of 6 and 8 kGy, partial denaturation of the protein occurred, which contributed to aggregation and, consequently, an increase in particle size. Protein aggregation occurred due to the formation of new covalent bonds, such as disulfide bridges, or hydrophobic interactions between molecules exposed to radiation. Excessive γ irradiation caused to the formation of cross‐links in proteins, electrostatic and hydrophobic interactions, as well as the formation of disulfide bonds. As a result, the molecular weight increased, and the particle sizes became larger. ²⁰ The zeta potential, which describes the surface charge of a particle, is an important factor in its stability in solution. The use of γ irradiation potentially enhanced the electrostatic repulsion and potential, thereby contributing to the stability of the solution. ²¹ The observed decrease in zeta potential at 6 and 8 kGy was most likely reflected by changes in the surface charge of protein aggregates. The aggregation of larger entities diminished surface charge density, which, in turn, resulted in a lower absolute zeta potential. A reduction in zeta potential contributed to the particle aggregation and an increase in their size, which indicated a decrease in electrostatic repulsion between the particles. The observed increase in particle size and decrease in zeta potential of proteins exposed to γ radiation suggested protein aggregation and a change in surface charge. The observed changes were consistent with the formation of protein aggregates. FTIR and sulfhydryl content analysis showed that these aggregates were formed by disulfide bonds and hydrophobic interactions. Xu and Liu ²² funded a similar effect: that the γ irradiation destroys charged groups in rice proteins, decreasing their polarity and potential value.

Figure 2.

The particle size and zeta potential of pea protein isolate solution treated with different irradiation doses. Each value represents the mean ± SD, n = 3. Different superscript letters indicate statistically significant differences among the means (P < 0.05).

FTIR

Figure 3 shows the FTIR spectra of the PPI caused by γ irradiation (0–10 kGy). According to the Fig. 3(A), all samples of PPI showed similar curves, while retaining characteristic stripes. In the work of Cheng et al. ⁶ where samples of pea fiber after γ irradiation showed a similar result. At a peak of 3298–3542 cm⁻¹ (O‐H/N‐H), a decrease in intensity was observed with an increase in the radiation dose. Absorption peaks 2931–2940 cm⁻¹ (C—H) showed a decrease in intensity at doses above 4 kGy.

Figure 3.

Results of (A) Protein secondary structure (%) and (B) FTIR spectra of pea protein isolate treated with different irradiation doses.

Figure 3(B) shows the secondary structural changes in the PPI caused by γ irradiation (0–10 kGy). The regions of amide I (1600–1700 cm⁻¹) and amide II (1500–1600 cm⁻¹), which were crucial for the analysis of the secondary structure of the protein, revealed modifications caused by irradiation. Changes in intensity of the β‐sheets during irradiation indicated a change in hydrogen bonds and aggregation. Random spirals became more noticeable at higher doses (8–10 kGy), which indicated denaturation caused by radiation. α‐Helix associated with spiral conformations gradually decreased at ≥ 4 kGy, which reflected the unwinding of the spiral because of the rupture of hydrogen bonds. A decrease in β‐turns at higher doses indicated a loss of structural rigidity. At 0–2 kGy, minimal structural destruction occurred and the predominance of β‐sheets persisted. At 4–6 kGy, the content of α‐helices decreased with a simultaneous increase in the number of arbitrary turns, which indicated a partial unfolding. At 8–10 kGy, the peaks of amide‐I and the β‐sheet shifted to the red side, indicating the formation of cross‐links or aggregation. A decrease in the content of α‐helices and β‐turns correlated with increased protein flexibility, which potentially increased its solubility. The aggregation of β‐sheets at high doses may explain the improved stability of emulsification as a result of the formation of a film on the interface. Gamma irradiation destroyed hydrogen bonds and cleaved disulfide bonds, destabilizing the native conformation. The transition from ordered structures (α‐helix, β‐sheets) to disordered structures (random turns) was consistent with reports of radiation effects on plant proteins, where controlled energy release adapted the functionality to food products. Zhang et al. ²³ reported that the secondary structure of rice proteins was redistributed after electron beam irradiation, with decreases in α‐helices and increases in β‐sheets, β‐turns and random coils. Wang et al. ²⁴ found that electron beam irradiation improves the functional qualities of wheat germ protein hydrolysates.

Microstructure of proteins

The attached SEM results (Fig. 4) showed the morphological properties of PPI exposed to γ irradiation. PPI (Fig. 4(A)–(C)) contained irregularly shaped protein aggregates with a rough and textured surface. These particles combined to form a porous network. The granular appearance (Fig. 4(A)) indicated the presence of tightly packed protein subunits held together by hydrophobic interactions and disulfide bonds. In Fig. 4(B), small depressions are indicated, which are most likely caused by γ irradiation, which destroys tertiary protein structures and reduces particle adhesion. Figure 4(C) shows representative areas of partial fragmentation caused by radiation‐induced cleavage of polypeptide chains or cross‐link modifications. An increase in depressions (Fig. 4(D)) indicated depolymerization caused by irradiation and rearrangement of protein matrices. Analogous surface fragmentation and an increase in specific area resulting from electron irradiation of rice proteins was shown in the study by Zhang et al. ²³ The markings in Fig. 4(E) indicate smoother areas that contrast with the rougher morphology of the untreated PPI, which was caused by local structural rearrangement or surface smoothing as a result of denaturation and reorientation of protein chains after irradiation. According to Wang et al., ²⁵ soy protein irradiation increased the Maillard reaction with maltose, which led to the formation of more hydrophilic surfaces. This knowledge helps to better understand the development of smooth zones resulting from contact to polar groups. In Fig. 4(F), stitching occurred in the recesses, indicated a partial unfolding of the spiral or β‐sheet structure, potentially improving functional aspects such as water binding, in line with the pea fiber results of Cheng et al. ⁶ These results were confirmed by the results of FTIR findings: low‐dose γ‐rays begin to disrupt and partially unfold PPI (surface has become irregular), whereas exposure to high doses led to the formation of larger clusters. Zhang et al. ²³ found that electron irradiation of rice proteins produced a rise in β‐sheet and random tangling content, hence lowering the α‐helices. This showed how radiation resulted in a redistribution of the secondary structure, which finally increased both convenience and flexibility. After γ irradiation, the protein surface showed increased pore formation and greater fragmentation of protein particles, as well as increased pore depth. Gamma irradiation adversely affected the surface architecture of protein molecules, resulting in increased roughness and pronounced indentation of pores.^{26, 27} Zhao et al. ²⁸ found that electron beam irradiation treatment causes a considerable decrease in the surface quality of walnut protein powder. This led to the powder becoming less compact, more porous and uneven, with smaller particle fragments. Furthermore, these particles coalesced and adhered to each other at higher doses.

Figure 4.

Scanning electron microscopy changes in the microstructure of pea protein isolate treated with different doses. (A–F) Corresponding to γ irradiation doses of 0, 2, 4, 6, 8 and 10 kGy, respectively, all at 5000× magnification.

Functional properties

Emulsifying activity and emulsifying stability

Figure 5(A)) illustrates the EAI and the ESI of PPI treated with γ irradiation at doses in the range 0–10 kGy. At control (0 kGy, 7.4 ± 0.005 m² g^–1), the initial EAI level was low, which corresponded to the limited surface activity of untreated PPI as a result of its rigid tertiary structure and poor solubility. At 2–8 kGy, EAI progressively raised, peaking at 8 kGy (10.34 ± 0.005 m² g^–1), indicative of structural alterations induced by irradiation, including partial unfolding of the α‐helix/β‐sheet conformations and enhanced surface hydrophobicity. These modifications improved protein adsorption at the oil–water interface. At 10 kGy, the EAI decreased slightly and this indicated possible excessive aggregation or excessive denaturation, which reduced the flexibility of the interface. At control (0 kGy, 109.5 ± 0.14%), the initial ESI was moderate, limited by weak interfacial film formation. At 2–8 kGy, the ESI increased sharply with an increasing radiation dose, reaching at 8 kGy (124.7 ± 0.06%). This surge correlated with the cross‐linking of the β‐sheet aggregates under the action of radiation, which strengthened the emulsion structure and slowed down phase separation. Cieśla et al. ²⁹ found that γ‐irradiated calcium caseinate‐whey protein isolate films have increased β‐sheet content and improved mechanical properties due to cross‐linking. Liu et al. ³⁰ found that whey protein isolate solutions treated with γ irradiation increased viscosity and formed bigger molecular weight aggregates, indicating better protein–protein interactions. These structural modifications contributed to the reinforcement of the emulsion network, thereby slowing down phase separation and improving emulsion stability. Partial denaturation improved surface adsorption (EAI) at optimal levels (4–8 kGy), whereas higher dosages (10 kGy) promoted cross‐linking, which improved emulsion adhesion. The negative connection between EAI and ESI at 8 kGy represented a compromise between surface activity and structural rigidity. The γ irradiation disrupted the orderly arrangement of proteins, leading structural disorder. This allowed the identification of internal hydrophilic groups and hydrophobic side chains, which improved interaction with oil and water phases. Although hydrophobic segments stabilized oil droplets by steric interactions, hydrophilic areas bound water molecules, thereby enhancing the emulsification activity and stability.^{13, 31} However, excessive irradiation led to the production of intramolecular disulphide bonds and protein cross‐linking, which reduced the hydrophobicity of the surface. These changes diminished emulsification ability and prevent protein adsorption at water–oil interfaces.^{32, 33} Studies on agricultural crops have shown different effects of γ irradiation depending on genetic elements, protein composition and irradiation intensity. Emulsion capacity dropped in African oil bean and cowpea seeds,^{34, 35} but improved in soybean and sunflower seeds.^{36, 37}

Figure 5.

Functional properties of pea protein isolate solution treated with different irradiation doses. (A) Emulsifying activity and emulsifying stability, (B) foam capacity and foam stability, and (C) water holding capacity and oil holding capacity. Each value represents the mean ± SD, n = 3. Different superscript letters indicate statistically significant differences among the means (P < 0.05).

FC and FS

The FC and FS of γ‐irradiated PPI are presented in Fig. 5(B). Gamma irradiation could modify the structure and functional properties of proteins, including PPI. Specifically, during irradiation, the tertiary and quaternary structures of protein molecules underwent changes, as new chemical bonds are established and existing connections are disrupted. As a result, these structural changes could affect the behavior of proteins on surfaces, which then affected their ability to foam and stability. The FC increased depending on the dose and ranged from 26.03 ± 2.3% (0 kGy) to 78.3 ± 1.5% (10 kGy), which suggested that irradiation promotes rapid air absorption. The observed improvement was probably the result of the partial unfolding of proteins. This process increased the surface hydrophobicity, which helped the proteins move to the air–water interface. When proteins were exposed to low levels of radiation, they could partially unfold. This unfolding could increase the hydrophobicity of the proteins surface, which could improve its foaming ability. However, higher radiation doses caused more extensive unfolding and aggregation of protein, which may have reduced their functional properties. FS depended on how easily air passes through its structure and on the strength of the protein layer. These parameters, in turn, were influenced by the hydrophobicity of the surface, the secondary and tertiary structures of protein molecules, and their flexibility. ³⁸ FS demonstrated a progressive decrease with escalating doses, commencing at 79.1 ± 1.4% at 0 kGy and ultimately reaching 61.3 ± 4.6% at 8 kGy, followed by a slight increase to 67.2 ± 5.4% at 10 kGy. This gradual stability reduction suggested that irradiation promoted aggregation and cross‐linking, although these phenomena were beneficial for emulsion stability, they reduced the elasticity and adhesion of the surface film, which was critical for long‐term retention of foam. The results indicated a trade‐off between how the foam formed and how long it persisted when exposed to γ radiation. Hassan et al. ³⁹ found that exposure to high level of γ radiation on low‐fat sesame seeds significantly reduced the ability of their flour to form foam. This decrease was evident when comparing the treated samples with both untreated controls samples and samples that received lower doses of radiation. Meanwhile, Wang et al. ²⁵ discovered that irradiating glycosylated soy proteins with 7.5 kGy enhanced their FC.

WHC and OHC

All irradiation samples, as the Fig. 5(C) shown, had more WHC than their control. At an irradiation dosage of 8 kGy (4.34 ± 0.19 g/g), WHC increased most significantly, suggesting that γ irradiation clearly influences WHC. Furthermore, the value showed the OHC of irradiation and control proteins. Reaching a high at 8 kGy (2.28 ± 0.01 g g^–1), the OHC levels of irradiation samples raised above those of the non‐modified sample. The partial unfolding of proteins in response to irradiation, thereby exposing more non‐polar residues inside the molecules, could be responsible for this rise in OHC. This structural change improved the contact between the protein and the oil, therefore improving the absorption of the oil. Structural changes, such as an increase in β‐sheet content, which occurred at high concentrations, led to the proteins acquiring a more dense and rigid structure. As a result of the reduced interaction with water and oil, this diminished the water‐holding and oil‐holding capacities. ⁴⁰ Additionally, the structures of fiber networks were damaged by strong energy, which affected their ability to retain water. The fiber's capacity to retain water has further diminished because of severe radiation damage to its structure. ⁴¹

Physicochemical properties

Protein solubility

Protein solubility enhanced with an increase in the irradiation dose to 8 kGy and reached 47.5 ± 0.1%, which significantly exceeded the control (38.4 ± 0.3%). At 10 kGy, solubility decreased slightly to 45.2 ± 0.4%, but it was still higher than the control measurement (Fig. 6(A)). This dual‐phase reaction was consistent with the structural changes observed via FTIR and sulfhydryl analysis: intermediate doses (4–8 kGy) caused partial unfolding, which exposed hydrophilic groups and enhanced solubility, whereas excessive cross‐linking at 10 kGy promoted aggregation and partially reduced solubility. The enhanced solubility observed at 8 kGy was the result of the destruction of stable three‐dimensional configurations, which made it possible to identify the polar regions of the molecule. This was evidenced by the increased hydrophobicity of the surface and changes in the secondary structure. The slight decrease observed at 10 kGy was a result of the formation of insoluble aggregates, a process caused by disulfide cross‐linking and hydrophobic interactions, which subsequently diminished the accessibility of polar groups for hydration. This aggregation led to the formation of large protein complexes, which have reduced solubility in aqueous environments. A significant level of denaturation revealed hydrophobic residues and amplified hydrophobic protein–protein interactions. ⁴² This led to the hydrophobic intermolecular interactions stabilizing aggregated protein complexes. ⁴³ Consequently, a consistent increase in irradiation dose reduced solubility due to the formation of insoluble aggregates even after reaching maximum solubility at 8 kGy. Therefore, the balance between the exposure to hydrophobic regions, which enhanced solubility, and protein aggregation at higher dosages, which reduced solubility, determines changes in protein solubility under γ irradiation. These results confirmed that γ irradiation at 8 kGy optimally balances structural flexibility and solubility.

Figure 6.

Physicochemical characterization of pea protein isolate solution treated by different irradiation doses. (A) Protein solubility (%), (B) surface hydrophobicity (μg), (C) sulfhydryl content (μmol g^–1) and (D) intrinsic fluorescence spectroscopy. Each value represents the mean ± SD, n = 3. Different superscript letters indicate statistically significant differences among the means (P < 0.05).

Surface hydrophobicity

Figure 6(B) shows the surface hydrophobicity of γ‐irradiated PPI at different doses. Gamma irradiation increased the hydrophobicity of all treated proteins compared to the non‐irradiated sample, indicating that γ‐rays can significantly disrupt the stable conformation of proteins. The hydrophobicity values increased with the dose up to 8 kGy (217.1 ± 7.5 μg), followed by a slight decrease at 10 kGy. Elevated hydrophobicity values suggested that γ irradiation can induce protein degeneration. When proteins underwent degeneration, their compact structure unfolded, exposing hydrophobic groups and increasing the relative amount of hydrophobic residues on the protein surface. Gamma irradiation affected proteins by altering their structure and, consequently, their functional properties. At irradiation doses up to 8 kGy, partial denaturation of the protein occurred, which led to the exposure of hydrophobic regions previously hidden inside the molecule. This increase in surface hydrophobicity enhanced functional properties such as FC and FS. However, at higher irradiation doses, such as 10 kGy, more extensive protein denaturation and aggregation occurred. These processes reduced the accessibility of hydrophobic regions on the protein surface because aggregated structures protected these areas. As a result, a slight decrease in hydrophobicity was observed at 10 kGy compared to 8 kGy. Therefore, the changes in protein hydrophobicity caused by γ irradiation were the result of a balance between the exposure of hydrophobic regions at moderate doses and their concealment because of aggregation at higher irradiation doses. The increasing surface hydrophobicity was consistent with our FTIR and SEM findings of structural loosening. Similarly, Wang et al. ²⁴ reported that the hydrophobicity of wheat germ protein hydrolysates' surface reached its lowest point at 10 kGy following electron beam irradiation. Zhang et al. ²³ found that the hydrophobicity of rice protein surfaces was enhanced following exposure to electron beam irradiation. The improvement in hydrophobicity was dose‐dependent, with the largest improvement observed at a dose of 30 kGy. Furthermore, Malik and Saini ³⁷ found that γ irradiation enhanced the surface hydrophobicity of sunflower protein isolates. Furthermore, Wang et al. ¹³ reported an augmentation in the hydrophobic nature of the surface of pea protein hydrolysates following irradiation. Cheng et al. ⁶ revealed that γ irradiation alters the structure of pea fiber, leading to an enhancement in its porosity and specific surface area. As a result, the functional attributes of the fiber, including oil and water retention were improved. Li et al. ⁴⁴ found that γ irradiation of watermelon seed protein led to an increase in surface hydrophobicity because of the disclosure of the protein structure and exposure of internal hydrophobic residues.

Free sulfhydryl and disulfide bond content

The changes in the free sulfhydryl and disulphide bonds of PPI after γ irradiation are shown in Fig. 6(C). Gamma irradiation modified the composition of sulfhydryl groups (‐SH) and disulfide bonds (‐S‐S‐), affecting the structure and functions of PPI. The SH/S‐S exchange process under the influence of radiation doses from 0 to 10 kGy proceeded in two phases. At low doses (0–4 kGy), γ irradiation predominantly cleaved disulfide bonds by radiolysis, which resulted to an increase in the concentration of free sulfhydryl. At intermediate radiation doses, from 4 to 8 kGy, some oxidation occurred, and disulfide bonds were reformed in a controlled manner. This created new cross‐links between the molecules at the same time as maintaining some structural flexibility. At 8 kGy, a balance was achieved between unfolding, which exposed functional groups, and cross‐linking, which enhanced the stability of the interfacial films. Conversely, at 10 kGy, excessive oxidation resulted to the formation of inflexible, crosslinked structures. This equilibrium, in turn, diminished both the solubility and the flexibility of the interface. Consequently, this equilibrium explained the observed functional maximum of approximately 8 kGy. The disulfide bonds underwent minimal changes at low dosages but decreased markedly at elevated doses because of cleavage and oxidation, which led to protein denaturation and aggregation. Consequently, γ irradiation caused structural changes in PPI, showing a moderate effect at low doses and more significant denaturation at elevated doses. Zang et al. ²³ found that, as a result of the ‐SH/S‐S exchange process during protein unfolding, rice protein pretreatment with electron beam irradiation reduced SH_T and increased S—S. Lv et al. ⁴⁵ found that myofibrillary proteins of Tegillarca granosa meat contained less ‐SH with increased electron beam irradiation.

Intrinsic fluorescence analysis

This study assessed the intrinsic fluorescence spectra of γ‐irradiated proteins were evaluated, with the results being illustrated in Fig. 6(D). Stable protein molecules induced the denaturation of normal proteins, thereby exposing tryptophan residues to polar solvents. Elevated fluorescence intensity, conversely, indicated that γ irradiation enhanced the exposure of tryptophan residues. Gamma irradiation caused structural changes in proteins, which affect their fluorescence properties. A partial protein denaturation occurred when the irradiation dose reached 8 kGy, with hydrophobic regions and amino acid residues, such as tryptophan, that were previously hidden becoming revealed. This exposure enhanced the accessibility of tryptophan residues for excitation and emission, therefore increasing the quantum yield of fluorescence. However, a subsequent increase in the irradiation dosage triggered protein aggregation and the formation of new chemical bonds, changing the microenvironment of fluorophores and, as a result, reducing fluorescence intensity. Accordingly, an increase in the fluorescence intensity by 8 kGy corresponded to the ideal level of structural changes, at which fluorophores were most accessible and effective in radiation. Consequently, the increase in fluorescence intensity at 8 kGy irradiation occurred as a result of exposure to hydrophobic areas and amino acid residues, which increased the quantum yield of fluorescence. At higher irradiation doses, structural changes led to a decrease in fluorescence intensity due to aggregation and changes in the microenvironment of the fluorophore. Li et al. ⁴⁴ reported that γ irradiation promoted the formation of tryptophan from watermelon seed protein in conjunction with other amino acids with side chains, which resulted to the aggregation of protein molecules. This led to a decrease in the fluorescence intensity.

CONCLUSIONS

In conclusion, γ irradiation significantly changed the structural hierarchy of PPI, which enabled an accurate change of its functional and physicochemical properties. At moderate dosages (4–8 kGy) irradiation‐induced unfolding of rigid tertiary structures enhanced surface hydrophobicity, solubility and interfacial activity, thereby improving emulsifying capacity (8 kGy), FS and water/oil holding capacity. Higher doses (8–10 kGy) increased the cross‐linking and β‐sheet aggregation, which improved emulsion stability but decreased flexibility because of excessive denaturation. The ratio between the emulsifying activity and the emulsifying stability emphasized the need to optimize the dosages: 8 kGy was the optimal threshold for achieving a balance of solubility, emulsification and foaming, whereas 10 kGy increased the emulsion stability. This comprehensive approach confirmed some general trends from other legumes, but also highlighted that PPI behaves differently because of compositional differences. The combined data confirmed our revised interpretation: functional changes in irradiated PPI were not random or speculative, but directly resulted from the dose‐dependent balance between deployment and aggregation observed in the data. The structural–functional correlations confirmed by the FTIR and SEM methods allowed γ irradiation to be considered as a scalable, non‐thermal technique for improving PPI in next‐generation plant‐based meals. This work fills crucial gaps in plant protein engineering by providing practical information for the food sector to use irradiation as a tool for functional optimization.

FUNDING INFORMATION

National Key Research and Development ‘Fourteenth Five‐Year Plan’ project number: 2021YFD1600705‐04. Key R&D projects number in Shaanxi Province: 2023‐YBNY‐160. Shaanxi Province Technology Innovation Guidance Special Project number: 2022QFY12‐07.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.