Protein Isolate Research Articles

Heterologous Expression of Secreted Proteins from Phytophthora in Pichia pastoris Followed by Protein Purification by Immobilized Metal Ion Affinity (IMAC).

This chapter describes the protocol for heterologous expression of Phytophthora proteins in the yeast Pichia pastoris. Two methods to prepare the constructs for expression are described, using two different strains of P. pastoris, as well as methods for protein expression and purification by immobilized metal ion affinity (IMAC). The combined protocol was used to successfully produce a Phytophthora pluvialis small, secreted cell death elicitor protein and a Phytophthora agathidicida glycoside hydrolase 12, PaXEG1, also a cell death-inducing protein.

Recombinant Protein Production and Purification of Rift Valley Fever Virus Nucleoprotein from Escherichia coli Expression Systems.

The recombinant expression and purification of viral proteins are a key component in the study of the immune response of viruses, as well as the creation of diagnostic techniques for the detection of viruses. For structurally simple proteins, one commonly used technique is the production of recombinant proteins in bacterial expression systems, which enable the large-scale synthesis and purification of recombinant viral proteins. In this technique, the cDNA encoding for a viral protein is cloned into a bacterial expression vector (with an appropriate purification tag), produced in a modified bacterial culture, and optimized for maximum protein production in a minimal amount of time. In this chapter, a protocol for the production of Rift Valley fever virus nucleoprotein is described. This protein has been previously shown to highly antigenic (and thus, is used in diagnostic tests), and has also been shown to be a potent inducer of the T-cell response following Rift Valley fever virus infection. The protocol outlined in this chapter describes the cloning of the cDNA of RVFV nucleoprotein into a bacterial expression vector, which also contains a fusion protein for optimal protein expression and solubilization, as well as a poly-histidine tag for efficient purification This chapter also describes the steps required for bacterial transformation, culture, lysis, purification and dialysis of RVFV nucleoprotein, resulting in a recombinant protein preparation, which can be upscaled to produce milligram quantities of protein product, which in turn can be used for downstream immunological and diagnostic applications.

Simplifying Recombinant Protein Production: Combining Golden Gate Cloning with a Standardized Protein Purification Scheme.

Recombinant protein production is pivotal in molecular biology, enabling profound insights into cellular processes through biophysical, biochemical, and structural analyses of the purified samples. The demand for substantial biomolecule quantities often presents challenges, particularly for eukaryotic proteins. Escherichia coli expression systems have evolved to address these issues, offering advanced features such as solubility tags, posttranslational modification capabilities, and modular plasmid libraries. Nevertheless, existing tools are often complex, which limits their accessibility and necessitate streamlined systems for rapid screening under standardized conditions. Based on the Golden Gate cloning method, we have developed a simple "one-pot" approach for the generation of expression constructs using strategically chosen protein purification tags like hexahistidine, SUMO, MBP, GST, and GB1 to enhance solubility and expression. The system allows visual candidate screening through mScarlet fluorescence and solubility tags are removable via TEV protease cleavage. We provide a comprehensive protocol encompassing oligonucleotide design, cloning, expression, His-tag affinity chromatography, and size-exclusion chromatography. This method, therefore, streamlines prokaryotic and eukaryotic protein production, rendering it accessible to standard molecular biology laboratories with basic protein biochemical equipment.

BRISC-Mediated PPM1B-K63 Deubiquitination and Subsequent TGF-β Pathway Activation Promote High-Fat/High-Sucrose Diet-Induced Arterial Stiffness.

Metabolic syndrome heightens cardiovascular disease risk primarily through increased arterial stiffness. We previously demonstrated the involvement of YAP (Yes-associated protein) in high-fat/high-sucrose diet (HFHSD)-induced arterial stiffness via modulation of PPM1B (protein phosphatase Mg2+/Mn2+-dependent 1B)-lysine63 (K63) deubiquitination. In this study, we aimed to elucidate the role and mechanisms underlying PPM1B deubiquitination in HFHSD-induced arterial stiffness. Enzymes governing PPM1B deubiquitination were identified through siRNA screening and mass spectrometry. Glutathione S-transferase pull-down, coimmunoprecipitation, protein purification, and immunofluorescence were used to explore the mechanism underlying PPM1B deubiquitination. Doppler ultrasound was used to evaluate HFHSD-induced arterial stiffness in mice, and telemetry was used to record pulsatile (systolic and diastolic) blood pressure. Smooth muscle cell-specific PPM1B overexpression attenuated HFHSD-induced arterial stiffness in mice in a PPM1B-K326-K63-linked polyubiquitination-dependent manner. Mechanistically, ABRO1 (Abraxas brother 1; a core BRCC36 [BRCA1/BRCA2-containing complex subunit 36] isopeptidase complex component) directly bound YAP and underwent liquid-liquid phase separation with YAP and PPM1B in a YAP-dependent manner, which in turn promoted PPM1B deubiquitination. Furthermore, smooth muscle cell-specific Abro1-knockout mice and Brcc3-knockout mice showed attenuated HFHSD-induced arterial stiffness and activation of transforming growth factor-β-Smad signaling. We elucidated the PPM1B deubiquitination mechanisms and highlighted a potential therapeutic target for metabolic syndrome-related arterial stiffness.

Identification and comparison of N-glycome profiles from common dietary protein sources

The N-glycomes of bovine whey, egg white, pea, and soy protein isolates are described here. Glycoproteins from four protein isolates were analyzed by HILIC high performance liquid chromatography and quadrupole time-of-flight tandem mass spectrometry (HILIC-FLD-QTOF-MS/MS). A total of 33 N-glycans were identified from bovine whey and egg white, and 10 structures from soy and pea glycoproteins. The type of N-glycans per glycoprotein source were attributable to differences in biosynthetic glycosylation pathways. Animal glycoprotein sources favored a combination of complex and hybrid glycan configurations, while the plant proteins were dominated by oligomannosidic N-glycans. Bovine whey glycoprotein isolate contained the most diverse N-glycans by monosaccharide composition as well as structure, while plant sources such as pea and soy glycoprotein isolates contained an overlap of oligomannosidic N-glycans. The results suggest N-glycan structure and composition is dependent on the host organism which are driven by the differences in N-glycan biosynthetic pathways.

Silkworm cocoon-like nanocoatings for single-cell encapsulation of probiotics: Construction, mechanism and characterization

Single-cell encapsulation technology is an advanced method to protect probiotics from adverse environmental conditions by forming coating on the surface of individual probiotics. In this study, Lactiplantibacillus plantarum (L. plantarum) was used as model probiotic. Four polysaccharides, namely carboxymethyl chitosan (CMCS), chitooligosaccharide (COS), amylopectin (AP) and pectin (PE), as well as four proteins, namely gelatin (GE), whey protein isolate (WPI), ovalbumin (OVA) and soy protein isolate (SPI), were selected as the initial coating materials. The coated probiotics were prepared by single-cell encapsulation under pH 3.0–6.0 for the first time. The feasibility, mechanism and effect of these coatings were then explored. Results indicated that the positively charged materials were conducive to the formation of coating, and the optimal pH was 4.0. Silkworm cocoon-like coatings with a thickness of 100–150 nm on the surface of L. plantarum were observed. Subsequently, fourier transform infrared spectroscopy (FTIR) analysis showed that electrostatic interaction, hydrogen bonding and hydrophobic interaction were the main driving forces between coatings and L. plantarum. In addition, COS coating fostered the growth and reproduction of L. plantarum, while CMCS and OVA inhibited it. Finally, COS, WPI and GE coatings as barriers all improved the survival rate of L. plantarum under pasteurization, freeze-thaw cycle and storage conditions. Among them, COS as a marine oligosaccharide had the best protective effect on L. plantarum due to its excellent properties and the dense coating it formed. The results may provide more support for further development of nanocoated probiotics.

Ultrasonication modifies the structural, thermal and functional properties of pumpkin seed protein isolate (PSPI).

Protein isolates from pumpkin seeds were prepared and then treated with high-intensity ultrasound (HIUS) using a probe-based method. The impact of ultrasonication on the physicochemical, molecular, and thermal properties of these isolates were analyzed and compared to untreated controls. Results showed significant improvements (p≤0.05) in color (L*, a*, b* values), solubility, emulsification capacity, and stability, as well as a reduction in molecular weight, indicating enhanced functionality of the pumpkin seed protein isolates (PSPIs) after HIUS treatment. However, HIUS treatment decreased the denaturation temperature (Td), denaturation enthalpy (ΔH), thermal stability, and particle size of the isolates. With treatment durations ranging from 5 to 20min, Td dropped from 67.31°C to 56.38°C, and ΔH declined from 45.78 to 35.43J/g, likely due to structural and conformational modifications from ultrasonic-induced molecular bond disruptions. The greatest reduction in particle size, from 117.46μm to 85.26μm, was observed after 20min of ultrasonication. X-ray diffraction (XRD) analysis showed two distinct diffraction peaks at 2θ=10° and 2θ=20°, indicating altered crystallite sizes post-ultrasound treatment. Ultrasonication induced structural and conformational changes in the pumpkin seed protein isolates, as confirmed by SDS-PAGE and weight loss analyses. Alterations in the SDS-PAGE profile and reduced weight loss were associated with improved solubility and enhanced thermal and functional properties in the treated pumpkin seed protein isolates. This emphasizes the potential of PSPI to increase their value-added potential through ultrasonication.

A Novel Tpp-like Protein from Bacillus thuringiensis Strain GXUN31-2 with Nematicidal Activity against Meloidogyne enterolobii

The plant root-knot nematode Meloidogyne spp. is an endoparasite with worldwide distribution that is detrimental to the growth of a wide range of plants. The insecticidal crystal proteins produced by Bacillus thuringiensis are widely used as a biological insecticide to control Lepidopteran, Hemiptera, and Coleopteran pests. However, there are few reports of toxicity against plant-parasitic nematodes. In this study, we report the cloning and characterization of a novel Tpp-like protein from B. thuringiensis strain GXUN31-2 that exhibits significant nematicidal activity against the plant root-knot nematode Meloidogyne enterolobii. The full-length CG_5628 gene was 951 bp and encoded a 34.4 kDa protein consisting of 316 amino acid residues. The gene was ligated into pET30a(+) and expressed in Escherichia coli BL21(DE3). Following purification of the Tpp-like protein, bioassays against the second-stage juvenile (J2) of M. enterolobii revealed lethal concentration (LC50) values of 144.3 μg/ml (95% Confidence interval: 133.5–155.7μg/ml). Analysis of the conserved domain revealed the presence of a Ricin B lectin domain with residues located at positions 8–155. Our results indicate the potential of this protein as a new and effective nematicide.

Keratinocyte-derived extracellular vesicles in painful diabetic neuropathy.

Painful diabetic neuropathy (PDN) is a challenging complication of diabetes with patients experiencing a painful and burning sensation in their extremities. Existing treatments provide limited relief without addressing the underlying mechanisms of the disease. PDN involves the gradual degeneration of nerve fibers in the skin. Keratinocytes, the most abundant epidermal cell type, are closely positioned to cutaneous nerve terminals, suggesting the possibility of bi-directional communication. Extracellular vesicles are lipid-bilayer encapsulated nanovesicles released from many cell types that mediate cell to cell communication. The role of keratinocyte-derived extracellular vesicles (KDEVs) in influencing signaling between the skin and cutaneous nerve terminals and their contribution to the genesis of PDN has not been explored. In this study, we characterized KDEVs in a well-established high-fat diet mouse model of PDN using primary adult mouse keratinocyte cultures. We obtained highly enriched KDEVs through size-exclusion chromatography and then analyzed their molecular cargo using proteomic analysis and small RNA sequencing. We found significant differences in the protein and microRNA content of high-fat diet KDEVs compared to KDEVs obtained from control mice on a regular diet, including pathways involved in axon guidance and synaptic transmission. Additionally, using an in vivo conditional extracellular vesicle reporter mouse model, we demonstrated that epidermal-originating GFP-tagged KDEVs are retrogradely trafficked into the dorsal root ganglion (DRG) neuron cell bodies. This study presents the first comprehensive isolation and molecular characterization of the KDEV protein and microRNA cargo in RD and HFD mice. Our findings suggest a potential novel communication pathway between keratinocytes and DRG neurons in the skin, which could have implications for PDN.

Dialysis and lyophilization of the mesenchymal stromal cell secretome for wound healing.

The clinical translation of mesenchymal stromal cell secretome (MSC-S) has been challenging owing to a lack of appropriate methods in downstream processing. Dialysis is an age-old method of protein purification by the exchange of small molecules through a semi-permeable membrane. In this study, we investigated the potential of three forms of umbilical cord-derived MSC secretome (UC-MSC-S)-native (S), dialyzed (DS), and lyophilized (LDS)-for wound healing applications. We dialyzed the UC-MSC-S using Slide-A-Lyzer G3 Dialysis Cassettes (20K MWCO) and then lyophilized it to obtain secretome powder. The DS fraction exhibited an 86.01-fold decrease compared with S, whereas LDS showed a 613.71-fold increase in the total protein concentration. Growth factor analysis revealed a significant decrease in the levels of interleukin-6 (IL-6; 54.44-fold), angiopoietin-1 (79.56-fold), angiopoietin-2 (51.76-fold), IL-8 (54.4-fold), platelet endothelial cell adhesion molecule-1 (PECAM-1; 63.25-fold), phosphatidylinositol glycan anchor biosynthesis class F (PIGF; 40.42-fold), vascular endothelial growth factor (VEGF; 39.64-fold), and tumor necrosis factor alpha (TNF-α; 24.62-fold) after dialysis as analyzed by the LEGEND plex multi-analyte flow assay kit on a FACS analyzer. Post-lyophilization, the levels of IL-6 (392.21-fold), angiopoietin-1 (823.04-fold), angiopoietin-2 (397.69-fold), IL-8 (584.83-fold), PECAM-1 (341.28-fold), PIGF (342.85-fold), VEGF (2209.42-fold), and TNF-α (194.4-fold) were enriched in LDS. The highest wound closure (64.07%) and a significant increase in angiogenesis were seen in DLS at the concentration of 1 µg/µL of protein by wound scratch and in ovo yolk sac membrane assay, respectively. Dialysis followed by lyophilization is a simple and cost-effective method to fractionate and enrich the bioactive components of MSC-S without compromising the bioactivity for tailor-made applications.

Preparation and characterization of lutein functional salt by microemulsion stabilized by whey protein isolate and maltodextrin: Exploration of a novel dietary nutritional supplement

Excessive salt intake has become one of the leading causes of various non-communicable diseases such as hypertension and cardiovascular diseases. Researchers have been looking for alternative ways to reduce sodium intake without compromising the sensory properties of food. One promising approach is to develop hollow salts, which can provide the same salty taste while significantly reducing sodium consumption. Hollow salts were developed using microemulsion stabilized by whey protein isolate (WPI) and maltodextrin (MD) via spray drying in this study. The Maillard reaction between WPI and MD was confirmed through grafting and browning degree measurements. Optical microscopy revealed that the WPI-MD emulsion had more uniform droplet sizes, producing hollow salt particles with a size about 5 to 15 μm. Scanning electron microscopy images confirmed the successful formation of the hollow structure, while energy dispersive spectroscopy results showed that the surface of the salt microspheres was mainly composed of sodium and chlorine elements. The use of MD improved the stability and prevented agglomeration of hollow salt. Lutein functional salt was successfully prepared with a high encapsulation efficiency about 77.57 %, excellent antioxidant activity, and stability. In conclusion, this study demonstrated that stable lutein functional salt can be prepared using WPI and MD spray drying methods.

Impact of various physical treatments on physicochemical and microstructural characteristics of vegetable oil-based whipped cream stabilised by faba bean protein isolate

Impact of various physical treatments on physicochemical and microstructural characteristics of vegetable oil-based whipped cream stabilised by faba bean protein isolate

Cold atmospheric plasma-induced alterations in the multiscale structural and functional properties of guar gum.

The use of guar gum in the food industry faces challenges owing to its large molecular weight and high viscosity. Cold atmospheric plasma (CAP) is a novel technique that utilizes the reactive species produced by high voltage discharge to modify food ingredients. In this study, guar gum was treated with CAP at different powers and duration, and its rheological properties, molecular structure, thermal stability, emulsifying activity, and stability were evaluated. CAP reduced the molecular weight of guar gum and altered its M/G ratio, leading to a decrease in the apparent viscosity of guar gum and an enhancement in its thermal stability and emulsifying function for soy protein isolate emulsions. Molecular structure analysis revealed the CAP treatment did not destroy the basic structure of guar gum, but caused alterations in the linkages between its glycosidic bonds and/or carbohydrate units. Scanning electron microscopy showed guar gum changed from a dense surface structure to a porous and loose structure after CAP treatment. Therefore, CAP effectively modifies guar gum, enhancing its potential in food and other industries.

Electrostatic spraying encapsulation of probiotic-loaded W/O/W emulsion in sodium alginate microspheres to enhance probiotic survival stability

Water-oil-water (W/O/W) double emulsions have been widely studied and applied in probiotic encapsulation. However, challenges remain in enhancing emulsion stability, protecting encapsulated probiotics from adverse environmental conditions, and improving their viability. This study aimed to optimize the functional components of each phase of the W/O/W emulsion to address these issues. First, the prebiotic fructooligosaccharide, which promotes bacterial growth, was incorporated into the inner water phase. The oil phase (O) was composed of sunflower oil, polyglyceryl polyricinoleate, and different proportions of cocoa butter to investigate the critical role of cocoa butter in maintaining emulsion stability. The effect of varying ratios of whey protein isolate and gum arabic complexes in the outermost water phase on emulsion stability was also systematically investigated. Finally, combined with electrostatic spraying technology, sodium alginate was used as the encapsulating wall material for the probiotic-encapsulated emulsion, and the stability of the system during in vitro gastrointestinal digestion was evaluated. This study utilized electrostatic spray technology to create a protective “armor” around the emulsion encapsulating probiotics. The combination of emulsion encapsulation and electrostatic spray encapsulation significantly improved the survival stability of probiotics, providing a method for maintaining high viability in complex food media.

Preparation and characterization of microcapsules and tablets for probiotic encapsulation via whey protein isolate-nanochitin complex coacervation

This research delved into the feasibility of utilizing three nanochitin—chitin nanocrystal (CNC), chitin nanofiber (CNF), and chitin nanosphere (CNS) in complexation with whey protein isolate (WPI) to fabricate complex coacervation and create microcapsules for probiotic encapsulation. The results showed that CNC, CNF, and CNS exhibited notable differences in morphologies, dimensions, and properties due to the respective synthesis methodologies. Nevertheless, all of them maintained a positive charge and were capable of assembling into microcapsules with WPI via electrostatic interactions at optimal pHs. The inclusion of Lactobacillus casei (L. casei) during the complex coacervation phase engendered a shell-like formation around the bacterium within the microcapsule, which enhanced probiotic viability and increased colony-forming unit count. Additionally, these probiotic-loading microcapsules were also processed into tablets, displaying robust structural integrity, augmented protective capabilities, and a distinctive sustained-release profile compared to the microcapsules alone. In summary, this study pioneered the employment of nanochitin formulations in complex coacervation to encapsulate L. casei, spearheading an innovative approach to the creation of a compressed probiotic supplement and contributing to the advancement in the design and fabrication of encapsulation vehicles for active ingredients.

Effect of Fortification with High-Milk-Protein Preparations on Yogurt Quality.

Protein-enriched yogurts have become increasingly popular among consumers seeking to boost their daily protein intake. The incorporation of milk proteins and protein preparations in yogurt production not only enhances nutritional value but also improves texture, viscosity, and overall sensory properties-key factors that influence consumer acceptance. The main objective of this study was to evaluate the influence of casein and whey protein preparations on the physicochemical properties, viability of lactic acid bacteria, and sensory attributes of yogurts. Yogurts were enriched with 2% (w/w) protein preparations, including micellar casein preparation (CN85), whey protein isolate (WPI), whey protein concentrate (WPC60), and protein preparations obtained from skim milk by membrane filtration: micellar casein concentrate (CN75) and serum protein concentrate (SPC). The yogurts were produced using the thermostatic method, and their chemical composition, rheological properties, syneresis, firmness, lactic acid bacteria population, and sensory attributes were evaluated. The effects of high-protein preparations derived from skim milk through laboratory-scale membrane filtration processes (SPC, CN75) were compared with those of commercially available protein preparations (SMP, CN85, WPI, and WPC). Obtained results demonstrated that the membrane filtration-derived preparations (SPC and CN75) exhibited advantageous physicochemical properties and supported robust viability of yogurt and probiotic bacteria. However, their sensory quality was marginally inferior compared to the commercial preparations (SMP, CN85, WPI, and WPC). These findings indicate the potential applicability of membrane filtration-derived protein preparations in yogurt production while underscoring the necessity for further investigation to enhance and optimize their sensory characteristics.

A mechanistic insight into whey protein isolate (WPI) fibrillation driven by divalent cations.

Protein fibrillation complex mechanisms led to an emerging trend in research for years. The mechanisms behind whey protein isolate (WPI) fibrillation driven by divalent cations remained still a matter of speculation. All cations (Ca2+, Fe2+, Mg2+, and Zn2+) enhanced the microenvironment polarity through π-π stacking, and the amide I and II shifts confirmed the fibrillation. The Fe2+ followed the nucleation-growth mechanism attested by spider-like microstructure, maximum fibril length (654.96nm) and β-sheets. The hydrophobic forces were mainly involved while disulfide bonds had no key role. The Ca2+ and Mg2+ followed the nucleated conformational conversion and electrostatic shielding which induced mature multilayer fibrils. The Zn2+ induced worm-like fibrils probably showing the low conversion rate regarding the high binding affinity toward WPI which stabilized the structure, so simple nucleated polymerization conducted the fibrillation. More ionic strength improved the exposure of hydrophobic amino acids in the surface, accelerating the growth phase by increased number of nuclei.

Ultrasonic treatment of emulsion gels with different soy protein-hemp protein composite ratios: Changes in structural and physicochemical properties

To improve the emulsion gel system of single soybean isolate protein (SPI) and to broaden the application field of hemp protein isolate (HPI), ultrasonic treatment and HPI were introduced to improve the properties of SPI emulsion gel and to explore the mechanism. The results showed that the gel strength (218.6 g) and water-holding capacity (86.24 %) of the emulsion gels were improved under ultrasonic treatments when the ratio of SPI:HPI was >6:4, and the reticulation structure of the gels was enhanced. When the ratio of SPI:HPI was <6:4, the gel structure was loose and formless. Ultrasonic treatment has a significant effect on the emulsion gel with the ratio of SPI:HPI was >6:4. Appropriate ultrasonic treatment (400 W) changed the protein structure, improved the rheological properties of emulsion gels to form the protein-oil-coated network structure. However, excessive ultrasonic treatment (600 W) will destroy the conformation of the protein, reducing the stability of the structure. The effect of ultrasonic treatment on emulsion gels with the ratio of SPI:HPI was <6:4 is low, but improved the gel protein digestibility. This study provides a theoretical basis for the application of ultrasonic in composite protein emulsion gels systems and the development and application of HPI.

Effects of soy protein isolate hydrolysate on the structural and functional properties of yam starch during extrusion.

This study investigated the interaction between yam starch (YS) and soy protein isolate hydrolysate (SPH), and their effects on in vitro digestibility of starch through extrusion treatment. Results indicated that SPH with 6% hydrolysis degree had the lowest relative molecular mass. X-ray diffraction and Raman spectroscopy revealed an increase in the relative crystallinity of extruded yam starch (EYS) from 17.45% to 22.45% and a decrease in the half-peak width at 480cm-1. Its short-range ordering improved with increased SPH addition. Differential scanning calorimetry results demonstrated that SPH enhanced the thermal properties of EYS. Additionally, the solubility, peak viscosity, and setback viscosity of EYS decreased with an increase in the SPH content. Particle size analysis revealed that SPH addition decreased the particle size of EYS. Scanning electron microscopy and confocal laser scanning microscopy showed that the surface of EYS roughened and SPH molecules attached to the surface. In vitro digestion results indicated that SPH hindered the contact between EYS molecules and digestive enzymes and increased the resistant starch content of EYS from 8.31% to 23.39%. This study presents a new method for modification of starch using protein hydrolysates during extrusion.

Cold plasma reengineers peanut protein isolate: Unveiling changes in functionality, rheology, and structure.

This study investigates the effects of cold plasma (CP) treatment on peanut protein isolate (PPI), focusing on functionality, rheology, and structural modifications across various treatment times (0, 90, 180, 270, 360, and 450s) and voltages (120, 140, and 160kV). Key findings include a significant increase in solubility from 9.99mg/mL to 15.98mg/mL, as well as 161.07% enhanced water-holding capacity (WHC) and 448.45% oil-holding capacity (OHC). CP treatment also improved foaming capacity (FC) to 186.46% and increased emulsion capacity (EC) and emulsion stability (ES) by 185.90% at 160kV. Rheological analysis showed shear-thinning behaviour, with viscosity decreasing as the shear rate increased-higher voltages (140kV and 160kV) further reduced viscosity, indicating lower resistance to flow. Additionally, CP-treated PPI exhibited viscoelasticity, with increased storage and loss moduli at higher frequencies, indicating greater stiffness. Spectroscopic studies demonstrated shifts in the protein's secondary structure, altering the balance among alpha-helix, beta-sheet, and random coil components, which highlights CP's role in reengineering PPI. FTIR-ATR spectra revealed reductions in the 3200-3400cm-1 range, suggesting changes in protein backbone vibrations and hydrogen bonding. Particle size analysis showed significant increases, especially at higher voltages and longer treatment times, stabilizing after 270s. Zeta potential assays indicated a gradual decrease in negative surface charge, suggesting enhanced protein aggregation. Overall, CP treatment significantly improves the functional and rheological properties of PPI while inducing structural changes, making it more suitable for applications in the food and pharmaceutical industries.