Structural evolution of pea-derived albumins during pH and heat treatment studied with light and X-ray scattering.

Pea albumins are found in the side stream during the isolation of pea proteins. They are soluble at acidic pH and have functional properties which differ from their globulin counterparts. In this study, we have investigated the aggregation and structural changes occurring to pea albumins under different environmental conditions, using a combination of size-exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALS) and small-angle X-ray scattering (SAXS). Albumins were extracted from a dry fractionated pea protein concentrate by precipitating the globulin fraction at acidic pH. The albumins were then studied at different pH (3, 4, 4.5, 7, 7.5, and 8) values. The effect of heating at 90 °C for 1, 3, and 5 min on their structural changes was investigated using SAXS. In addition, size exclusion of the albumins showed 4 distinct populations, depending on pH and heating conditions, with two large aggregates peaks (∼250 kDa): a dimer peak (∼24 kDa) containing predominantly pea albumin 2 (PA2), and a monomer peak of a molar mass of about 12 kDa (PA1). X-ray scattering intensities as a function of q were modeled as polydisperse spheres, and their aggregation was followed as a function of heating time. Albumins was most stable at pH 3, showing no aggregation during heat treatment. While albumins at pH 7.5 and 8 showed aggregation after heating, solutions at pH 4, 4.5, and 7 already contained aggregates even before heating. This work provides new knowledge on the overall structural development of albumins under different environmental conditions, improving our ability to employ these as future ingredients in foods.

Electrostatic spray drying: A new alternative for drying of complex coacervates.

Complex coacervation can be used for controlled delivery of bioactive compounds (i.e., flaxseed oil and quercetin). This study investigated the co-encapsulation of flaxseed oil and quercetin by complex coacervation using soluble pea protein (SPP) and gum arabic (GA) as shell materials, followed by innovative electrostatic spray drying (ES). The dried system was analyzed through encapsulation efficiency (EE) and yield (EY), morphological and physicochemical properties, and stability for 60 days. Small droplet size emulsions were produced by GA (in the first step of complex coacervation) due to its greater emulsifying activity than SPP. Oil EY and EE, moisture, and water activity in dried compositions ranged from 75.7 to 75.6, 76.0-73.4 %, 3.4-4.1 %, and 0.1-0.2, respectively. Spherical microcapsules were created with small and aggregated particle size but stable for 60 days. An amount of 8 % of quercetin remained in the dried coacervates after 60 days, with low hydroperoxide production. In summary, when GA is used as the emulsifier and SPP as the second biopolymer in the coacervation process, suitable coacervates for food applications are obtained, with ES being a novel alternative to obtain coacervates in powder, with improved stability for encapsulated compounds. As a result, this study helps provide a new delivery system option and sheds light on how the characteristics of biopolymers and the drying process affect coacervate formation.

sRAGE-binding and antimicrobial bioactivities of soy and pea protein after heating and in vitro infant digestion.

During infant formula production, proteins are always heated, potentially affecting their digestibility and the bioactivities of resulting peptides. Although plant proteins are a promising dairy alternative for infant formula, they remain understudied, necessitating further investigations. Therefore, this research aimed to fill this gap by assessing the impact of different heating modes on soy protein (SP) and pea protein (PP), focusing on glycation levels, peptide formation during in vitro infant digestion, and immune protection potential (sRAGE-binding and antimicrobial activities) of the resulting peptides. Consequently, dry heating led to increased glycation and glycated peptide production, particularly with higher glycation in PP than SP. Moreover, PP exhibited an overall stronger sRAGE-binding capacity than SP, regardless of heating and digestion conditions. Regarding antimicrobial activity, both SP and PP-derived peptides displayed reduced effectiveness against Enterobacter cloacae after dry heating. Additionally, Staphylococcus epidermidis was differently inhibited, where PP-derived peptides showed inherent inhibition. The primary determinant of sRAGE-binding and antimicrobial potential in digestion-derived peptides was the protein source. Subsequent bioinformatics analysis predicted 519 and 133 potential antimicrobial peptides in SP and PP, respectively. This study emphasises the importance of protein source for infant formula to ensure infant health.

Microencapsulation of flaxseed oil in pea protein-gum arabic complex coacervates delays lipid digestion in liquid yoghurt.

Flaxseed oil coacervates were produced by complex coacervation using soluble pea protein and gum arabic as shell materials, followed by either spray or electrostatic spray drying and their incorporation to yoghurt. Three yoghurt formulations were prepared: yoghurt with spray-dried microcapsules (Y-SD); with electrospray-dried microcapsules (Y-ES); with the encapsulation ingredients added in free form (Y). The standardised semi-dynamicin vitrodigestion method (INFOGEST) was employed to study the food digestion. The structure was analysed by confocal laser scanning microscopy and particle size distribution. Protein and lipid digestion were monitored by cumulated protein/free NH2 release and cumulated free fatty acids release, respectively. Stable microcapsules were observed during gastric digestion, but there was no significant difference in protein release/hydrolysis among samples until 55 min of gastric digestion. Formulation Y showed less protein release after 74 min (40.46 %) due to the free SPP being available and positively charged at pH 2-4, resulting in interactions with other constituents of the yoghurt, which delayed its release/hydrolysis. The total release of protein and free NH2 by the end of intestinal digestions ranged between 46.56-61.15 % and 0.83-1.57 µmol/g protein, respectively. A higher release of free fatty acids from formulation Y occurred at the end of intestinal digestion, implying that coacervates promoted the delayed release of encapsulated oil. In summary, incorporating protein-polysaccharides-based coacervates in yoghurt enabled the delay of the digestion of encapsulated lipids but accelerated the digestion of protein, suggesting a promising approach for various food applications.

Influence of emulsifier on lipid oxidation in spray-dried microencapsulated O/W emulsions.

Lipid oxidation limits the shelf-life of dried microencapsulated oils (DMOs), such as infant formula. However, it is poorly understood how lipid oxidation is affected by different types of emulsifiers. To improve our understanding, we prepared DMOs with different emulsifiers (whey protein isolate (WPI), pea protein isolate (PPI), and non-proteinaceous CITREM) and studied lipid oxidation in both the free and encapsulated fat. Only a small difference in oxidation rate was observed between these fat fractions for all formulations. We ascribed this to a non-discrete distribution of the fractions and the subsequent low fractionation selectivity as shown by Raman microscopy. The DMO with PPI showed hardly any oxidation during a 7-week incubation at 40 °C, whereas the DMOs with WPI and CITREM both reached significantly higher contents of oxidation products (lipid hydroperoxides, aldehydes, and epoxides). The enhanced stability of DMO-PPI could not be ascribed to the presence of phytic acid. In conclusion, we demonstrate the potential of using PPI to produce oxidatively stable DMOs.

Plant and animal protein mixed systems as wall material for microencapsulation of Manuka essential Oil: Characterization and in vitro release kinetics.

Combination of plant and animal protein diet is becoming a valuable source of nutrition in the modern diet due to the synergistic functional properties inherent in these protein complexes. Moreover, the synergy between animal and plant proteins can contribute to the high stability and improved solubility of the encapsulated bioactive ingredients (e.g., essential oils). Therefore, the study was designed to evaluate the plant (pea protein (PP) and lupine protein (LP)) and animal protein (whey protein, WP) mixed systems as a wall material for microencapsulation of manuka essential oil, as an example of bioactive compound. Moreover, physicochemical properties and in vitro release profile of encapsulated manuka essential oil were studied. Manuka essential oil microcapsules exhibited low moisture content (5.3-7.1 %) and low water activity (0.33-0.37) with a solubility of 53.7-68.1 %. Change in wall material ratio significantly affected the color of microcapsules, while microcapsules prepared with 1:1 protein/oil ratio demonstrated a high encapsulation efficiency (90.4 % and 89.4 %) for protein mixed systems (PP + WP and LP + WP), respectively. Microcapsules further showed low values for lipid oxidation with a high oxidative stability and antioxidant activity (62.1-87.0 %). The zero order and Korsmeyer-Peppas models clearly explained the release mechanism of encapsulated oil, which was dependent on the type and concentration of the protein mixed used. The findings demonstrated that the protein mixed systems successfully encapsulated the manuka essential oil with controlled release and high oxidative stability, indicating the suitability of the protein mixed systems as a carrier in encapsulation and application potential in development of encapsulated functional foods.

In vitro protein digestibility of RuBisCO-enriched wheat dough: a comparative study with pea and gluten proteins.

Growing demand for sustainable, plant-based protein sources has stimulated interest in new ingredients for food enrichment. This study investigates the nutritional and digestive implications of enriching wheat dough with RuBisCO, in comparison to pea protein-enriched and gluten-enriched doughs. The protein quality and digestibility of these enriched doughs were analysed through dough characterization, in vitro digestion experiments and biochemical analysis of digesta. Our findings indicate that an enrichment at 10% of RuBisCO or pea proteins improves the chemical score and the in vitro PDCAAS (IV-PDCAAS) score of wheat dough as compared to the control dough. Digestibility assays suggest that RuBisCO introduction modifies the protein hydrolysis kinetics: the nitrogen release is lower during gastric digestion but larger during intestinal digestion than other samples. The analysis of the protein composition of the soluble and insoluble parts of digesta, using size-exclusion chromatography, reveals that the protein network in RuBisCO-enriched dough is more resistant to gastric hydrolysis than the ones of other doughs. Indeed, non-covalently bound peptides and disulfide-bound protein aggregates partly composed of RuBisCO subunits remain insoluble at the end of the gastric phase. The digestion of these protein structures is then mostly performed during the intestinal phase. These results are also discussed in relation to the digestive enzymatic cleavage sites, the presence of potential enzyme inhibitors, the protein aggregation state and the secondary structures of the protein network in each dough type.

Enhancing the functionality of pea proteins by conjugation with propylene glycol alginate via transacylation reaction assisted with ultrasonication.

Pea proteins lack the desirable functional characteristics for food and beverage applications. In this study, transacylation reaction assisted with ultrasonication was used to glycate pea proteins with propylene glycol alginate to enhance their functional properties. The reaction was carried out at pH 11.0 for different pea protein isolate: propylene glycol alginate mass ratios and time durations in a sonic bath at 40 °C. Glycation was confirmed in gel electrophoresis, and ultrasonication enhanced the glycation, with optimal degrees of glycation observed at 45 min reaction time and mass ratios of 2:1 (37.73%) and 1:1 (35.96%). The transacylation reaction increased random coil content of pea proteins by 28% and enhanced their solubility by 2.02 times at pH 7.0, water holding capacity by >50% at pH 7.0, foaming properties, emulsifying properties, and heat stability. This study offers a novel approach that can enhance functionality and applicability of pea proteins.

Acid-induced pea protein gels pretreated with media milling: Gelling properties and the formation mechanism.

This study explored the effect of stirred media mill (SMM) processing on the acid-induced gelling properties of pea protein. Results showed that SMM treatment enhanced the gel strength from 75.06 g to 183.89 g and increased the water holding capacity from 46.64 % to 73.50 %. The minimum gelation concentration achieved for SMM-treated pea protein was 4 %, significantly lower than that of heat-pretreated pea protein (9 %). SMM decreased protein aggregate size from 104 µm to 180 nm. Microscopy analysis revealed that the small aggregates facilitated the formation of uniform gel networks with tight connections. Linear rheology indicated that small protein aggregates resulted in slower gelation rates with a higher G' for the formed gels. The SMM-pretreated protein gel showed strain hardening, shear thinning behaviors, and satisfactory stability to withstand large-amplitude oscillatory shear. Overall, SMM emerges as a promising technology for producing protein gel products with strong mechanical attributes and customizable rheological properties.

In vitro gastrointestinal simulated digestion of three plant proteins: determination of digestion rate, free amino acids and peptide contents.

Cassava protein (CP), barley protein (BP) and yellow pea protein (YPP) are important nutrient and integral constituent of staple in pet foods. It is known that the digestion of proteins directly influences their absorption and utilisation. In the present work, we performed in vitro simulated gastrointestinal digestion of three plant proteins as a staple for dog and cat food. The digestion rate of CP, BP and YPP in dog food was 56.33 ± 0.90%, 48.53 ± 0.91%, and 66.96 ± 0.37%, respectively, whereas the digestion rate of CP, BP, and YPP in cat food was 66.25 ± 0.72%, 43.42 ± 0.83%, and 58.05 ± 0.85%, respectively. Using SDS-polyacrylamide gel electrophoresis to determine the molecular weight (MW) of each protein and the products of their digestion, it was revealed that MW of digestion samples decreased, and MW during the small intestine phase was lower than that during the gastric phase. Peptide sequences of digested products were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and it was found that the total number of peptides in the small intestine digestion samples was higher than that in the gastric phase samples. The MW of peptides obtained from CP was within the range of 1000-1500 Da, while MW of peptides derived from BP and YPP was within the range of 400-2000 Da. In addition, free amino acids were mainly produced in the small intestine phase. Furthermore, the percentage of essential amino acids in the small intestine phase (63 ~ 82%) was higher than that in the gastric phase (37 ~ 63%). Taken together, these findings contribute to the current understanding of the utilisation of plant proteins in dog and cat foods and provide important insights into the selection and application of plant proteins as a staple in dog and cat foods.

Improving the Three-Dimensional Printability of Potato Starch Loaded onto Food Ink.

This study focuses on improving the 3D printability of pea protein with the help of food inks designed for jet-type 3D printers. Initially, the food ink base was formulated using nanocellulose-alginate with a gradient of native potato starch and its 3D printability was evaluated. The 3D-printed structures using only candidates for the food ink base formulated with or without potato starch exhibited dimensional accuracy exceeding 95% on both the X and Y axes. However, the accuracy of stacking on the Z-axis was significantly affected by the ink composition. Food ink with 1% potato starch closely matched the CAD design, with an accuracy of approximately 99% on the Z-axis. Potato starch enhanced the stacking of 3D-printed structures by improving the electrostatic repulsion, viscoelasticity, and thixotropic behavior of the food ink base. The 3D printability of pea protein was evaluated using the selected food ink base, showing a 46% improvement in dimensional accuracy on the Z-axis compared to the control group printed with a food ink base lacking potato starch. These findings suggest that starch can serve as an additive support for high-resolution 3D jet-type printing of food ink material.

Enhanced functional properties of pea protein isolate microgel particles modified with sodium alginate: Mixtures and conjugates.

Protein microgels are emerging as versatile soft particles due to their desirable interfacial activities and functional properties. In this study, pea protein isolate microgel particles (PPIMP) were prepared by heat treatment and transglutaminase crosslinking, and PPIMP were non-covalently and covalently modified with sodium alginate (SA). The effects of polymer ratio and pH on the formation of PPIMP-SA mixtures and conjugates were investigated. The optimal ratio of PPIMP and SA was found to be 20:1, with the optimal pH being 7 and 10, respectively. PPIMP-SA conjugates were prepared by Maillard reaction. It was found that ultrasound (195 W, 40 min) enhanced the degree of glycation of PPIMP, with a highest value of 37.21 ± 0.71 %. SDS-PAGE, browning intensity and FTIR data also confirmed the formation of PPIMP-SA conjugates. Compared with PPIMP and PPIMP-SA mixtures, PPIMP-SA conjugates exhibited better thermal stability, antioxidant, emulsifying and foaming properties, which opens up opportunities for protein microgel in various food applications.

Chemical acylation of pea protein isolate hydrolysate with fatty acid N-hydroxysuccinimide esters: Effect on structure and functional properties.

Applications of pea protein in the food industry have been greatly restricted by its poor functional properties. In order to solve this problem, a novel technique combining enzymatic hydrolysis and fatty acid acylation has been applied in this work to construct a pea protein-fatty acid covalent complex that aims to improve its functional properties. The processed pea protein with increased water solubility tends to decrease the chance of self-aggregation. Additionally, emulsifying and antioxidant properties have also been found after this process. On top of that, the modified pea protein has been characterized by Fourier transform infrared and circular dichroism spectroscopy. These results demonstrate that these properties were mainly caused by the acylation of the amino group from hydrolyzed pea protein and the carboxyl group from the fatty acid. The enzymatic hydrolysis/fatty acid acylation research provides insights into manufacturing high-quality functional lipoproteins from inexpensive pea protein for the food industry.

Synthesis of taste active γ-glutamyl peptides with pea protein hydrolysate and their taste mechanism via in silico study.

Pea (Pisum sativum L.) protein hydrolysate (PPH) has a bitter taste, which has limited its use in food industry. γ-Glutamylation is used to debitter PPH. Results showed that the bitterness of PPH was decreased significantly due to the formation of γ-glutamyl peptides, including 16 γ-[Glu](n=1/2)-amino acids (AAs) and 8 newly discovered γ-glutamyl tripeptides (γ-Glu-Asn-Phe, γ-Glu-Leu-Val, γ-Glu-Leu-Tyr, γ-Glu-Gly-Leu, γ-Glu-Gly-Phe, γ-Glu-Gly-Tyr, γ-Glu-Val-Val, and γ-Glu-Gln-Tyr). Their total production concentrations were 27.25 µmol/L and 77.76 µmol/L, respectively. The γ-Glu-AA-AAs presented an umami-enhancing, salty-enhancing, and kokumi taste when their concentration reached 1.67 ± 0.20 âˆ¼ 2.07 ± 0.20, 1.65 ± 0.25 âˆ¼ 2.29 ± 0.45 and 0.68 ± 0.19 âˆ¼ 1.03 ± 0.22 mmol/L, respectively. The γ-Glu-AA-AAs exhibited a kokumi taste by entering the Venus flytrap (VFT) of the calcium-sensing receptor and interacting with Ser147, Ala168, and Ser170. γ-Glu-AA-AAs can enhance the umaminess of Monosodium Glutamate (MSG) as they can enter the binding pocket of the taste receptor type 1 subunit 3 (T1R3)-MSG complex.

Functionality of pea protein isolate solutions is affected by reconstitution conditions.

Pea protein isolate (PPI), a high-concentration protein ingredient derived from peas, is increasingly utilized in food applications, including beverages, meat or dairy alternatives, and baked goods. The protein extraction process typically used to manufacture PPI renders the protein highly denatured, which can have a negative impact on its functionality. Therefore, it is critical to understand how to prepare and utilize PPI to maximize its functionality. The current study evaluates the effect of select reconstitution conditions on the structure and functionality of PPI, across a range of protein concentrations (4%-10%) relevant to a variety of food applications. Temperature during reconstitution with water and hydration time impacted both protein hydration and its functionality. Increasing reconstitution temperature from 20 to 60°C and increasing hydration time from 10 to 40 min decreased PPI particle size in solution and increased PPI solubility. Viscosity of PPI solutions also increased with mild heating and longer hydration time, whereas their flow behavior was highly dependent on protein concentration. Experimental data demonstrates that reconstitution conditions have a significant impact on PPI functionality. These findings can help food formulators develop high-quality food products that utilize PPI as a functional ingredient. PRACTICAL APPLICATION: Protein in commercially available pea protein isolates (PPIs) is usually highly denatured, and thus, it is important to find ways to maximize its functionality in practical applications. The findings of this study inform food scientists how to leverage PPI at various protein concentrations with optimal reconstitution conditions to develop high-quality products. Generally, mild heating and longer hydration times improve PPI functional performance.

Correlationship between self-assembly behavior and emulsion stabilization of pea protein-high methoxyl pectin complexes treated with ultrasound at pH 2.0.

This study investigated the effects of ultrasound on the self-assembly behavior of pea protein (PP)-high methoxyl pectin (HMP) complexes at pH 2.0 through transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and intrinsic fluorescence analysis. The emulsion stabilization mechanism of PP-HMP treated with ultrasound (PP-HMP-US) was also elucidated. The results indicated that ultrasound increased the emulsifying activity index (EAI) and emulsifying stability index (ESI) of PP-HMP. Moreover, PP-HMP-US-based emulsions formed small, dispersed oil drops, which were stable during storage. PP-HMP- and PP-HMP-US-based emulsions did not demonstrate any creaming. The TEM results revealed that ultrasound can regulate the self-assembly behavior of PP and HMP to form spherical particles with a core-shell structure. This structure possessed low turbidity, a small particle size, and high absolute zeta potential values. The FTIR and intrinsic fluorescence spectra demonstrated that ultrasound increased the α-helix and ß-sheet contents and exposed the tryptophan groups to more hydrophilic environments. Ultrasound also promoted the PP-HMP self-assembly through electrostatic interaction and improved its oil-water interfacial behavior, as indicated by the EAI and ESI values of PP-HMP-US-based emulsions. The current results provide a reference for the development of an innovative emulsifier prepared by ultrasound-treated protein-pectin complexes at low pH.

Non-thermal ultrasonic contact drying of pea protein isolate suspensions: Effects on physicochemical and functional properties.

Pea protein isolate (PPI) is a popular plant-based ingredient, typically produced through alkaline-isoelectric precipitation and thermal drying. However, high temperatures and long drying times encountered in thermal drying can denature PPI and cause loss of functionality. This study investigated the use of an innovative ultrasonic dryer (US-D) at 30 °C for drying PPI suspensions, compared to conventional hot air drying (HA-D) at 60 °C. US-D led to an increase in the drying rate and correspondingly reduced the drying time by 55 %, when compared to HA-D. The average effective moisture diffusivity in the US-D process was 325 % higher than that in the HA-D process. The resulting PPI exhibited higher solubility, emulsification, and foaming properties than HA-D PPI, with a unique surface morphology and higher surface area. This study demonstrated that drying with acoustic energy is a promising approach for producing dried plant protein ingredients with improved functional properties, reduced processing time, and increased production efficiency.

Fabrication and Characterization of Chitosan-Pea Protein Isolate Nanoparticles.

Chitosan (CS) and pea protein isolate (PPI) were used as raw materials to prepare nanoparticles. The structures and functional properties of the nanoparticles with three ratios (1:1, 1:2 1:3, CS:PPI) were evaluated. The particle sizes of chitosan-pea protein isolate (CS-PPI) nanoparticles with the ratios of 1:1, 1:2, and 1:3 were 802.95 ± 71.94, 807.10 ± 86.22, and 767.75 ± 110.10 nm, respectively, and there were no significant differences. Through the analysis of turbidity, endogenous fluorescence spectroscopy and Fourier transform infrared spectroscopy, the interaction between CS and PPI was mainly caused by electrostatic mutual attraction and hydrogen bonding. In terms of interface properties, the contact angles of nanoparticles with the ratio of 1:1, 1:2, and 1:3 were 119.2°, 112.3°, and 107.0°, respectively. The emulsifying activity (EAI) of the nanoparticles was related to the proportion of protein. The nanoparticle with the ratio of 1:1 had the highest potential and the best thermal stability. From the observation of their morphology by transmission electron microscopy, it could be seen that the nanoparticles with a ratio of 1:3 were the closest to spherical. This study provides a theoretical basis for the design of CS-PPI nanoparticles and their applications in promoting emulsion stabilization and the delivery of active substances using emulsions.

Pea protein based nanocarriers for lipophilic polyphenols: Spectroscopic analysis, characterization, chemical stability, antioxidant and molecular docking.

The current research aims to construct and assess pea protein isolate (PPI) nanocarriers for lipophilic polyphenols of curcumin (CUR), quercetin (QUE) and resveratrol (RES), respectively. Fluorescence analysis demonstrated that the binding affinity declined in sequence of QUE > CUR > RES and about one polyphenol compound was bound to protein. Thermodynamic parameters revealed that hydrophobic interaction was mainly responsible for complexation between CUR/RES and PPI, while hydrogen bonding for QUE with PPI. All nanoparticles showed particle size of 154-159 nm. Three lipophilic polyphenols were successfully encapsulated into PPI, with loading capacity of RES > QUE > CUR. Complexation of three polyphenols did not change the secondary structure of PPI. Results of FTIR, DSC and XRD confirmed that polyphenols changed from crystalline to amorphous state after combination with PPI. SEM pictures exhibited regular spherical microstructure of nanocomplexes. PPI shielded polyphenols from sensitive environment of ultraviolet light and thermal treatment. ABTS and DPPH radical scavenging activity of polyphenols were considerably improved through complexation with PPI. Molecular docking studies showed binding energy with 11S legumin in sequence of QUE > RES > CUR, and stronger hydrogen bonds were built between QUE and the protein than the other two polyphenols. Data in the present work may provide helpful information for encapsulation of lipophilic polyphenols with pea protein and the potential application in food science, pharmaceutical and cosmetics industries in the future.

Structural, and functional properties of phosphorylated pea protein isolate by simplified co-spray drying process.

In this work, to improve the functionality of pea protein isolate (PPI), sodium hexametaphosphate (SHMP) was added during last step of protein extraction and co-spray dried. The influence of PPI to SHMP mixing ratios (95:5 and 90:10) and reaction pH conditions (pH 6, 7, 8, and 9) on reaction efficiency, structural and functional properties of phosphorylated PPI were evaluated. Results showed that both mixing ratios had a similar degree of phosphorylation, suggesting the high efficiency of a 95:5 mixing ratio. The mixing ratio affected powder yield and proximate composition whereas hydrophobicity and denaturation temperature were regulated by pH conditions. For functionality, both mixing ratios showed significantly increased solubility at pH 6. Moreover, an increase in foaming capacity was observed in all phosphorylated PPI. The result from the current study may work as a basis for PPI phosphorylation in the food industry using the simplified method.