Effect of proteases on the functional properties of pea protein isolates

Effect of proteases on the functional properties of pea protein isolates | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of proteases on the functional properties of pea protein isolates Ashwitha Amin, Ourania Gouseti, Gernot J. Abel, Poul Erik Jensen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8923282/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Plant proteins are promising candidates for the green transition in foods, but they have low functionality. Partial hydrolysis is gaining interest as a way to modify the functionality of plant proteins. The degree of hydrolysis (DH) is the “golden standard” to characterize partial hydrolysis. In this work, we hydrolyzed pea protein isolate (PPI) with three enzymes (Novozym, Alcalase, and Neutrase) at a range of enzyme concentrations, and we characterized the hydrolysates for DH and a range of food relevant functionalities including fermentation induced gel formation. The results indicate that the DH was insufficient to correlate with functional properties. For example, hydrolysis increased viscosity in all systems, with more extensive hydrolysis resulting in a greater increase in viscosity for individual enzymes. However, at low DH (≈ 0.5–1%), Alcalase hydrolysates exhibited ~ 400% higher viscosity than those from Novozym, while Neutrase hydrolysates showed the lowest viscosity despite having the highest DH. Similarly, hydrolysis increased the foaming ability of PPI, but the sample with the highest DH (hydrolyzed with Neutrase) showed a medium effect, and at a similar DH, Alcalase hydrolysis increased foamability to a larger extent. These findings indicate that DH alone was insufficient to explain variations in PPI functionality, and that enzyme specificity, leading to distinct hydrolysate compositions, played a key role. Moreover, hydrolysates formed harder gels during fermentation (4–171 g) only when transglutaminase was added. partial hydrolysis fermentation functional properties protein gelation protein-crosslinking Transglutaminase Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Pea ( Pisum sativum ) is one of the most promising legumes for the development of plant-based food products due to its high protein content (20−25%), availability, and cost effectiveness [ 1 , 2 ]. Pea proteins are rich in essential amino acids and are low in allergens compared to other commonly used plant protein sources like soybean and wheat [ 3 , 4 ]. Therefore, many food production and processing industries are increasingly attempting to utilize pea proteins to develop plant-based foods and beverages, including alternatives to meat and dairy products. However, the utilization of pea proteins in food applications faces challenges due to factors like their size, degree of aggregation, poor emulsification, foaming, and gelation properties [ 4 – 9 ]. Change in structure, interaction of proteins with other components of the seed matrix, and reorganization of the native state of proteins during extraction are among the factors reported to contribute to their poor functional properties [ 10 ]. To overcome these limitations, modification of pea proteins is often required. Chemical, physical, and enzymatic modification are common techniques used to improve structural and physicochemical properties of food proteins [ 11 ]. Among these, enzymatic modifications using proteases have gained increasing attention over the past two decades due to the mild processing conditions required, minimal by-product formation, high specificity, and ease of process control [ 12 , 13 ]. Proteases catalyze the hydrolysis of proteins into smaller peptides; and the process may simultaneously cause a certain degree of protein unfolding and exposure of previously buried hydrophobic, hydrophilic, or ionizable groups [ 14 ]. These modifications can affect techno-functional properties of plant proteins. In contrast to most animal-derived proteins, seed storage proteins are more difficult to hydrolyze because of their highly aggregated structure. The effect of proteases is typically measured by the degree of hydrolysis (DH), referring to the percentage of peptide bonds hydrolyzed under investigated conditions [ 14 ]. Many studies have demonstrated improved solubility, emulsion stability, and foaming properties of plant proteins upon partial protein hydrolysis [ 13 , 15 ]. However, selecting the appropriate enzymes and determining the optimal DH are critical factors in achieving the desired functional property enhancements [ 14 , 16 ]. Proteases with different specificities may result in diverse effects on protein modification, as they may release peptides with varying amino acid compositions, sizes, and conformations [ 17 , 18 ]. In addition, controlling the DH is crucial, as too little hydrolysis may not significantly alter the functional properties, while extensive hydrolysis may produce small peptides and free amino acids with reduced functionality and compromised texture and flavor properties [ 14 ]. Therefore, understanding the relationships among the type of protease, DH, and functional properties is crucial for developing novel food structures with desired quality attributes. Protein gelation is an important functional property that significantly impacts the structure, texture, rheology, and creaminess of food products, influencing both sensory perception, downstream processing and storage parameters. Various methods are employed to induce protein gelation, with heating and acidification using glucono-𝛿-lactone (GDL) being the most common approaches [ 19 – 21 ]. Acidification using GDL is a clean label gelation method with various advantages such as rapid and controlled acidification, but it does not improve flavor and nutritional properties [ 22 ]. Alternatively, fermentation using lactic acid bacteria has been explored to induce protein gelation [ 1 , 23 – 25 ]. Fermentation also provides additional benefits such as improvement of flavor properties due to formation of flavor-eliciting amino acids, peptides, and volatile compounds [ 26 , 27 ]. Furthermore, enzymatic protein cross-linking through transglutaminase (TG) has also been investigated as TG offers a selective protein gelation by facilitating isopeptide bond formations between glutamine and lysine residues, resulting in intermolecular or intramolecular protein cross-linking, which ultimately strengthen gel structures [ 23 , 25 , 28 ]. The use of TG in food applications is generally recognized as safe (GRAS). Although a few studies have independently investigated fermentation and TG-induced cross-linking of plant proteins, the combined impact of fermentation and TG-induced crosslinking of hydrolysates remains less explored [ 1 , 25 ]. The aim of the present study is to understand the impact of enzymatic partial hydrolysis combined with subsequent fermentation and TG-induced crosslinking on the functional properties of a pea protein isolate. Three different proteases with different specificities (Novozym, Neutrase and Alcalase) were used and their effects on the acquired functional properties were compared. Novozym is a serine endopeptidase of microbial origin, and exhibits specificity towards hydrolysis of peptide bonds involving aromatic amino acids such as phenylalanine, tryptophan, and tyrosine [ 29 ]. Neutrase, a protease of bacterial origin, has a broad specificity, typically preferring to cleave peptide bonds adjacent to hydrophobic amino acids such as leucine and phenylalanine [ 29 ]. Alcalase is an endopeptidase with broad specificity and has been reported to hydrolyze peptide bonds with aromatic, acidic, basic and aliphatic amino acid residues [ 9 ]. From these enzymes, Novozym is the least explored for food applications [ 30 – 32 ]. These enzymes are commercially available, and providing comparative data on their effects in modulating the functional properties of seed storage proteins will help in selecting the most appropriate enzyme for large-scale applications. In this study, therefore, hydrolysates were comprehensively characterized for various functional properties essential in the development of plant-based foods and beverages, including particle and molecular size distribution, viscosity, suspension and emulsion stability, foaming properties, zeta potential, isoelectric point, and surface tension. Furthermore, the effectiveness of hydrolysates in forming protein gels was investigated using a combination of TG-induced crosslinking and fermentation. Materials and methods Materials Pea protein isolate (PPI) NUTRALYS® S85F (isolated from Pisum sativum ) was donated by Roquette, Lestrem, France. Novozym® 11028 (EC 3.4.21.1; serine endoprotease from Bacillus licheniformis ; 75000 PROT/g; reported optimal conditions: pH 6–7, temperature 50–75°C), Alcalase® 2.4 L FG (EC 3.4.21.62; alkaline serine endoprotease from Bacillus licheniformis ; 2.4 AU-A/g; reported optimal conditions: pH 7–9, temperature 30–65°C), Neutrase® 0.8 L (EC 3.4.24.28; metallo endoprotease from Bacillus amyloliquefaciens ; 0.8 AU-N/g; reported optimal conditions: pH 7, temperature 40–50°C), and transglutaminase (TG) (EC 2.3.2.13, from Bacillus licheniformis ) were donated by Novonesis, Lyngby, Denmark and referred to as Novozym, Alcalase, Neutrase, and TG respectively. The fermentation starter culture Vega™ Harmony (a blend containing Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus paracasei, Lactobacillus acidophilus, and Bifidobacterium ) was obtained from Novonesis, Hørsholm, Denmark. Rapeseed oil was purchased from a local supermarket and was used without any modifications. Picrylsulfonic acid solution (1 molL − 1 in H 2 O, TNBS), glucose (99%), sucrose (99%), L-alanine (99.5%), and Brilliant Blue G were purchased from Sigma Aldrich, Copenhagen, Denmark. NuPAGE™ MES SDS Running Buffer 20X, SeeBlue™ Plus2 Pre-stained protein standard, and NuPAGE™ LDS Sample Buffer 4X were purchased from Invitrogen, Waltham, USA. The other chemicals used in this study were of analytical grade. Milli-Q water (Merck, Germany) was used throughout the study. Partial protein hydrolysis Pea protein isolate suspension (40 mL) with a protein concentration of 8% w/v was prepared using Milli-Q water in a 50 mL falcon tube (the pH of the protein suspension was determined to be 7.8 ± 0.0) and the suspension was hydrated for 1 h with vertical mixing at room temperature. The hydrated sample was preheated at 90°C for 10 min in a shaking water bath (LSB Aqua Pro, Grant, UK) and immediately cooled down by placing the sample tube in ice cold water for 5 min. Thereafter, the sample was heated to 50°C in a hot air oven and proteases were added in different concentrations in independent experiments (n = 3): Novozym was added at four different concentrations (0.01, 0.05, 0.15, and 0.5% v/w of protein,) while Alcalase and Neutrase were added at two concentrations (0.05 and 0.15% v/w of protein, n = 2). The sample mixtures were then incubated for 30 min in a hot air oven with vertical mixing to achieve partial protein hydrolysis. Following hydrolysis, the enzymes were inactivated by heating the samples at 90 °C for 10 min in a water bath (inactivation time was based on preliminary experiments). After cooling to room temperature, the hydrolysates were used for subsequent functional property analyses. A control sample was generated by following all the steps as described above except addition of enzymes. Degree of hydrolysis (DH) The DH of protein samples was determined using the trinitrobenzenesulfonic acid (TNBS) total amine assay following a protocol described in the literature with some modifications [ 33 ]. Briefly, a stock solution of L-alanine (0.2 mg/mL) was prepared in borate buffer (0.05 molL − 1 , pH 10) and diluted to 2−200 µg/mL in the buffer to prepare calibration standards. Sample hydrolysates were diluted appropriately (10−100 fold) in the borate buffer. A 0.1% TNBS solution was freshly prepared in Milli-Q water and kept in dark. In the assay, 100 µL each of blank (0.05 molL − 1 borate buffer, pH 10), calibration standards and sample hydrolysates were transferred to a 96-well microplate. Subsequently, 100 µL of 0.1% TNBS solution was added to each well and the absorbance was measured continuously at 37 °C for 10 min at 450 nm using a Microplate Spectrophotometer (Biotek Instruments, EPOCH 2). The measurements were taken in technical duplicates for each biological replicate. Mean velocity was calculated using Gen5 software (version 3.11, BioTek, Winooski, USA) and used as the response in the calculations. The concentration of total amines was calculated using standard calibration curve. The degree of hydrolysis was calculated using the following Eq. 1: \(\:DH\:\left(\%\right)=\frac{\left(Cs-Cc\right)}{Cmax}\:⨉\:100\:\) Eq. 1 where Cc = Concentration of total amines in control sample Cs = Concentration of total amines in partially hydrolyzed sample Cmax = Concentration of total amines in completely hydrolyzed pea protein isolate. The completely hydrolyzed pea protein isolates were prepared using total acid hydrolysis (n = 3) following the procedure described by Poojary et al. (2020) with some modifications [ 34 ]. Briefly, 10–15 mg of pea protein isolate were placed in a microwave vial and then 3 mL of 6 molL − 1 HCl were added. A magnetic bead was placed into the vial and then the vial was purged with nitrogen before immediately sealing it with an aluminum crimp fitted with a Teflon septa. The sealed vial was kept in a microwave synthesizer (Biotage® Initiator+, Sweden) and heated at 150 °C for 1 min followed by 165 °C for 10 min to achieve complete protein hydrolysis. Thereafter, the hydrolysate was neutralized by mixing it with equal volume of 6 molL − 1 NaOH. The hydrolysate was diluted before the analysis as described above. Particle size distribution Particle size distributions were measured based on laser diffraction using a Mastersizer (3000, Malvern, UK) following the manufacturer’s instruction. In a typical experiment, the sample was diluted in a degassed water tank until the obscuration level reached between 10 and 20%. All the samples were measured based on the refractive index (RI) of 1.52 for pea protein [ 1 ]. The measurements were taken in technical duplicates for each biological replicate. Molecular weight distribution Sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the soluble protein samples was performed under non-reducing conditions according to the method described in Jansson et al. (2017) with some modifications [ 35 ]. All samples (4 mL each) were centrifuged at 8000 g for 20 min at 25 °C, and the resulting supernatants were used for subsequent analysis. The supernatant (50 µL) was added with 950 µL of SDS buffer (5% SDS in 0.1 molL − 1 Tris buffer, pH 8). A sample mixture was then prepared by combining 65 µL of the diluted supernatant, 25 µL of 4X LDS sample buffer, and 10 µL of Milli-Q water. The mixture was then heated under agitation (350 rpm) for 10 min at 80°C in a thermoblock (Provocell™ Microplate Incubator, Esco, Singapore). The running buffer (1X) was freshly prepared by mixing 50 mL of 20X running buffer and 950 mL of cold Milli-Q water. A 2% of brilliant blue solution was prepared in Milli-Q water. An equilibration buffer was prepared by dissolving 20 mL of concentrated phosphoric acid, 150 g of ammonium sulphate, and 180 mL ethanol in 1000 mL Milli-Q water. The protein standard (marker) was used directly without any sample preparations. A gel (NuPAGE™ 4−12% BT mini gels, Invitrogen) was placed into the electrophoresis system tank (Mini Gel Tank, Life Technologies, USA) and the tank chambers were filled with 1X running buffer. Protein standard (3 µL) and sample mixtures (5 µL each) were loaded into the gel. Thereafter, the gel ran at 200 V for 35 min. Following electrophoresis, the gel was placed in a container containing 100 mL of equilibration buffer and 1 mL of brilliant blue and allowed to stain overnight on a rocking table in the fume hood. Subsequently, the gel was destained by using Milli-Q water and scanned using a ChemiDoc MP Imaging System (BIO-RAD, California, USA). Zeta potential and isoelectric point determination The zeta potential of samples was determined using a Zetasizer NANO ZSP (Malvern, UK) without adjusting initial pH of the samples. The hydrolysates were diluted in Milli-Q water to reach the final protein concentration of 1% and the zeta potential was measured at the refractive index of 1.52. To determine the isoelectric point (pI), the zeta potential of the diluted samples was measured across pH 7.8−3.0, and the pH was controlled by dropwise addition of 0.5 or 1 molL − 1 HCl. The pH corresponding to a zeta potential of 0 mV was considered as the isoelectric point (pI). The measurements were taken in technical duplicates for each biological replicate. Surface tension A bubble pressure tensiometer (SITA pro line t15) connected with an automated liquid handler (Hamilton Microlab® STAR) was used to measure the surface tension. Samples were diluted in deionized water at a 1:1 ratio, and 1.8 mL of the diluted samples were added into the wells of a 24-well multiwell tissue culture plate. The automated liquid handler connected to the tensiometer immersed the capillary tube into the sample and the surface tension was measured at the bubble lifetime of 100 ms at room temperature (23 °C). The measurements were taken in technical duplicates for each biological replicate. Viscosity The shear viscosity of the samples was measured using a rotational rheometer (Kinexus pro+, Malvern, UK) with a C25 (DIN) cup/bob geometry. Each sample (17 mL) was placed in a cup and then the bob was lowered and brought to a temperature of 22 °C. Shear viscosity was determined by increasing the shear rate logarithmically (1−100 s − 1 ), with 10 measurements points per decade based on the method described in Fang et al. (2021) with minor modifications [ 36 ]. The shear rates of 1−100 s − 1 were selected based on the assumption that they are relevant for oral food processing [ 37 ]. The measurements were taken in technical duplicates for each biological replicate. Shear viscosity versus shear rate plots were constructed, and curves were fitted to Eq. 2 (power law equation) to determine the flow behavior index (n) and the flow consistency index (K). Foaming capacity and stability The foaming capacity and stability of protein hydrolysates were determined using an automated liquid hander (Hamilton Microlab® STAR, Reno, NV, USA). The hydrolysates were diluted in Milli-Q water at a 1:1 ratio, and 1.8 mL of each diluted sample was added into the wells of a micronic tube plate. Foam generation was facilitated at room temperature (23 °C) by the conductive tips of the automated liquid handler, which pipetted 300 µL of sample from each well and dispersed it into the same well at high speed from the height of 60 mm. This process was repeated 15 times to generate the foam. The sample height was automatically measured before and after foaming using the conductive tips. The foam height was calculated by subtracting the sample height before foaming from the sample height after foaming. The foam height immediately after the foam creation was considered as foam capacity. To determine the foam stability, the drop in foam height was measured at each 5 min for total 55 min. The measurements were taken in technical duplicates for each biological replicate. Suspension stability The accelerated physical stability of the samples was determined using Lumisizer® (LUM GmbH, Germany). The instrument measures the transmittance (%) across the sample during centrifugation to determine physical stability. Each sample was diluted in deionized water at a 1:1 ratio and loaded (400 µL) into rectangular sample cells. The sample cells were then placed horizontally within the in-built centrifuge of the Lumisizer®. The analysis was conducted under the following parameters: centrifugation speed of 2300 g, temperature set at 22 °C, wavelength of 865 nm, total analysis duration of 60 min, transmission profile recording time of 30 s, and a light factor of 1.0. The instability index, ranging from 0 to 1, was calculated using SEPView software (LUM GmbH, Germany). A value of 0 indicates very stable suspension, while a value 1 indicates complete instability (which is characterized by complete phase separation and 100% transmittance). Emulsion stability Emulsions (6.8 mL) were prepared by homogenizing the hydrolysates with rapeseed oil using an Ultraturrax homogenizer (T25, IKA, Germany) at 13,500 rpm for 1 min in 15 mL falcon tubes. The resulting emulsions had protein and oil contents of 7% and 12.5%, respectively. The stability of the emulsion was determined using the Lumisizer® following the same procedure as described in the section above. Gelation For gelation, protein hydrolysates were prepared according to the procedure described in the section “Partial protein hydrolysis” above, but in higher volumes (500 mL) in two biological replicates (n = 2). The gelation experiments included two steps, including emulsion preparation and fermentation of emulsions, which is described below. Emulsion preparation The emulsions were prepared by mixing hydrolysates (6.4% protein w/w), rapeseed oil (10.5% w/w), sucrose (1% w/w), and glucose (1% w/w) using T 25 Digital Ultra-turrax® (IKA, Germany) at 13500 rpm for 2 min. The total weight of the final emulsion was 571 g. The emulsion was further homogenized using a high-pressure homogenizer (PandaPLUS 2000, GEA, Italy) at two stages (150 and 50 bars) in one pass. The homogenized emulsion was pasteurized at 90 °C for 10 min in a water bath and then cooled until the sample reached a temperature of 43 °C by placing it on a cold water for subsequent gelation step. Fermentation of emulsions TG (3% v/w of protein) was added to the pasteurized emulsions and the emulsions were then inoculated with Vega™ Harmony (0.02% inoculum). The inoculated emulsions were transferred into the sterile sealed cap containers and fermented at 43 °C for 20 h in a dry oven. Another set of gels was prepared using the same method but without adding TG. Initial pH (immediately after the inoculation) and final pH (after 20 h of incubation) of the gels were recorded using a pH meter (Testo 205, Buhl & Bønsøe, Smørum, Denmark). Gels were further characterised as detailed below. Gel texture analysis Protein gels were subjected to a penetration test using a Texture Analyser (TA.XTplusC, Stable Micro Systems, UK) to measure gel hardness as described in Masiá et al. (2022) [ 1 ]. The hardness was measured at 20°C in the same containers where samples were fermented. The height and diameter of the container was 70 and 55 mm, respectively, and the height of the samples used for gelation was ~ 30 mm. Once gelation was completed, the gels were penetrated to 6 mm depth with a delrin cylindrical probe (P/10, 10 mm diameter, 45 mm long) at a 1 mm/s speed, return speed of 10 mm/s, and trigger force of 1 g using a 5 kg load cell. The software Exponent 32 (Stable Micro Systems, UK) was used to process the results and the hardness (g) was obtained as the maximum value detected during sample penetration. The measurements were taken in technical triplicates for each biological replicate. Gel rheological measurements Storage modulus (G’) was measured based on the method described in Masiá et al. without any modifications [ 1 ]. The gels were sliced to 1 mm thickness. The linear viscoelastic region of the gel slices was determined by performing a preliminary amplitude sweep test from 0.01% to 100% strain at 1 Hz for each sample at 20 °C using a rotational rheometer (Kinexus pro+, Malvern, UK). A strain of 0.1%, which was within the linear viscoelastic region of the materials, was then used to perform frequency sweep tests from 0.01 to 10 Hz for all samples at 20 °C. The measurements were taken with a parallel plate-to-plate geometry with flat surfaces (65 mm diameter lower plate and 20 mm diameter top plate) and a 1 mm gap. For the technical duplicates, two different slices of each biological replicate were used. Moisture content of gels Gels (1 g each) were dried on an aluminum sample plate in an oven at 105 °C for 4 h, and their weights were recorded after cooling them to room temperature in a desiccator. The drying process was repeated (1 h for each cycle) until constant weights were obtained. The measurements were taken in technical duplicates for each biological replicate. The moisture content was calculated using the following Eq. 3: Statistical analysis The results are expressed as mean ± one standard deviation (SD). The data were processed using GraphPad (Prism 10, version 10.1.2) and OriginPro 2020 (version 9.7) software. One-way analysis of variance (ANOVA) was performed with Duncan test using IBM SPSS software (version 29) to determine the statistically significant differences between the means. Differences were considered significant when p < 0.05. Results and discussion In the present study, partial hydrolysis of pea protein isolate was carried out using Alcalase, Neutrase, and the less studied Novozym, to modify its functional properties. As proteins are known to change their structure and properties as a function of pH and temperature, all hydrolyses were carried out at identical pH (7) and temperature (50°C) conditions, to eliminate protein modifications due to factors other than the enzymatic hydrolysis. The selected conditions fall within the optimal working range for all investigated enzymes. In all experiments, the DH was determined and used as a measure of the level of hydrolysis in the discussions. The least explored enzyme in the literature, Novozym, was studied at four different concentrations (0.01, 0.05, 0.15, and 0.5% based on the supplied enzyme preparation), while two concentrations (0.05 and 0.15%) were selected for the other two enzymes. In the following sections, DH is presented first, followed by characterisation of the hydrolysates (particle size distribution, molecular weight of soluble proteins, ζ-potential, surface tension), their functionality (foamability, viscosity, storage stability) and an application-relevant property (gelation of an emulsion, relevant to cheese analogues). Degree of hydrolysis All tested proteases resulted in partial hydrolysis of the pea proteins, with DH values ranging from 0.4–5.4% (Fig. 1 ). The DH increased significantly with higher enzyme concentrations for Novozym (DH of 0.4–5.4% at dosing from 0.01–0.5%) and Neutrase (DH of 1.8 and 3.4% at dosing of 0.05 and 0.15%, respectively). For Alcalase, under investigated conditions, the DH was low (DH ≈ 0.5%), and similar to that of Novozym-treated PPI at 0.01% enzyme concentration. When the three enzymes were compared at the same dosage (0.05% and 0.15%), the DH order was: Neutrase > Novozym > Alcalase. This could be attributed to variations in enzyme activity and specificity of the different enzymes towards the PPI and its aggregates as a substrate at the tested conditions. Interestingly, higher DH has been previously reported for PPI treated with Alcalase, compared to Neutrase, at enzyme/substrate concentration of 0.5% (both enzymes sourced from the same supplier as in the present work). DH is key in modulating protein functionality of hydrolysates. High DH can lead to generation of bioactive peptides (< 10 KDa). However, partially hydrolyzed proteins with low to moderate DH (< 10%) is generally regarded beneficial for food applications requiring emulsification, foaming, or gelation, largely due to the potential to control the size and behaviour (e.g., surface properties) of the resulting peptides [ 38 – 40 ]. This is therefore discussed in the present work. Characterization of hydrolysates: molecular and particle sizes Particle size distribution Particles with sizes up to ≈ 250µm in diameter were determined in all investigated systems, supporting the existence of protein aggregates in the commercial PPI samples (Fig. 2 a), as previously reported [ 41 ]. Excluding the sample treated with Novozym at the highest enzyme concentration (i.e., at 0.5%), all others showed generally bimodal size distributions, with one peak maximum at ≈ 0.1 µm, and one at ≈ 10µm. The surface moment mean diameters (D 32 ) is a measure of the volume/surface area of the particles. For the samples with bimodal distribution, D 32 values were unchanged at 80 nm (see Table 1 ). This may support a hydrolysis mechanism where the enzymes react largely in the outer layer of the particles, without causing substantial alternations in their sizes (e.g., without diffusing into the particle and breaking it into smaller fragments). It should be noted that treatment with Alcalase at 0.15% enzyme concentration reduced D 32 to 70 nm, which was a significant reduction if statistical analyses excluded the mono-dispersed system (data not shown). This may imply formation of small, fragmented protein particles or aggregated peptides due to increased inter-peptide interactions, as previously hidden hydrophobic regions become exposed (also previously reported) [ 13 ]. This will be discussed further in the following sections. The volume moment mean diameter (D 43 ) is often influenced by the large aggregates in the sample. The somehow decreasing D 43 of particles after hydrolysis with increasing enzyme concentration may therefore indicate erosion of the large aggregates, which become somehow smaller as the enzymes remove peptides from the surface. This is further supported by the D10, D50, and D90 values, where the enzymatic treatment caused no changes to the “fine” end of the size distribution (D10), minimal changes to the “median” (D50), and small but more pronounced changes in the “coarse” end of the size distribution curve (D90), where hydrolysis reduced the size of the large particles. It is interesting to note that despite its low DH (< 1%), Alcalase caused a more prominent reduction in D43 and D90, compared to the other two enzymes in all investigated conditions. Reduction in mean particle size of soy protein isolate has been previously reported on hydrolysis with Alcalase at DH of 2.5%, albeit for substantially smaller particles (of the order of 0.2 µm), and of peanut protein on hydrolysis with papain [ 42 , 43 ]. The PPI sample treated with Novozym at 0.5% enzyme/substrate ratio showed a unimodal distribution with maximum at ≈ 10µm and no particles < 1µm. This may suggest that the increased Novozym concentration extensively hydrolyzed the small particles to sizes below the detection limit of the used method ( < ≈ 0.01µm). Table 1 Mean diameters (D [3;2] and D [4;3]) and particle size distributions (D10, D50, D90) for control and protease treated samples. Here, D [3;2] refers to the area mean diameter, D [4;3] refers to the volume mean diameter, and D10, D50, D90 indicate the particle diameters corresponding to 10%, 50%, and 90% of the cumulative volume, respectively. Significant differences between values within the same column are denoted by different letters (P < 0.05). Sample Particle size (µm) D [3;2] D [4;3] D (10) D (50) D (90) Control 0.08 ± 0.00 a 9.68 ± 2.82 a 0.03 ± 0.00 a 0.17 ± 0.02 a 18.85 ± 2.06 a Novo_0.01% 0.08 ± 0.00 a 7.18 ± 1.53 a 0.03 ± 0.00 a 0.17 ± 0.02 a 16.60 ± 1.40 a Novo_0.05% 0.08 ± 0.00 a 6.78 ± 0.86 a 0.03 ± 0.00 a 0.17 ± 0.02 a 16.17 ± 0.45 a Novo_0.15% 0.08 ± 0.00 a 6.28 ± 2.03 a 0.03 ± 0.00 a 0.15 ± 0.02 a 15.28 ± 2.21 a Novo_0.5% 8.94 ± 0.84 b 22.07 ± 3.88 b 4.22 ± 0.44 b 11.80 ± 1.56 b 46.05 ± 13.81 b Alca_0.05% 0.08 ± 0.00 a 6.12 ± 0.77 a 0.03 ± 0.00 a 0.16 ± 0.00 a 15.50 ± 0.71 a Alca_0.15% 0.07 ± 0.00 a 6.27 ± 0.35 a 0.03 ± 0.00 a 0.14 ± 0.01 a 14.58 ± 0.25 a Neut_0.05% 0.08 ± 0.00 a 7.32 ± 0.73 a 0.03 ± 0.00 a 0.17 ± 0.00 a 16.63 ± 1.03 a Neut_0.15% 0.08 ± 0.00 a 7.56 ± 1.59 a 0.03 ± 0.00 a 0.15 ± 0.00 a 15.33 ± 0.11 a Molecular weight distribution SDS-PAGE was carried out to visualise peptide distribution upon partial protein hydrolysis. The undigested control PPI exhibited complex protein profiles with poor resolution and had several low and high molecular weight (MW) proteins in the range of 14–200 kDa (Fig. 2 b). Among them, distinct protein bands at 70, 68, 62, 49, 38, and 20 kDa were observed, which may correspond to subunits of convicilin, albumin, legumin, vicilin, legumin acidic subunit, and legumin basic subunit, respectively, as per previous literature [ 4 , 44 ]. It was evident in the SDS-PAGE gels that even at the lowest DH (0.4%), protease treatment led to the loss of the high MW bands and the formation of new peptide bands, particularly in the low MW range (3−30 kDa). Increasing enzyme concentration increased the number and intensity of the new peptide bands for a single enzyme. Interestingly, treatment with Alcalase at 0.05% and 0.15% showed insignificant differences in DH (Fig. 1 ), but changes in MW size distributions were (qualitatively) evident, with the higher enzyme concentration resulting in increased hydrolysis of large peptides, suggesting the potency of this non-specific enzyme to reduce MW of proteins. At the most extensively hydrolyzed sample (i.e., the sample treated with Novozym at 0.5%, DH 5.4%), the high molecular weight proteins almost completely disappeared, and the band intensity of peptides at molecular weights 3–30 kDa also reduced, suggesting the possible formation of smaller peptides and possibly amino acids that cannot be retained on the gels (< 3 kDa). SDS-PAGE further confirmed the different specificities of the three proteases. For example, the band at ≈ 72 kDa (likely convicilin fraction), persisted at all investigated Neutrease treatments despite the relatively high DH. Besides Neutrase, this band has previously shown resistance to other well-known proteases, such as Papain and Flavorozyme [ 13 , 18 , 45 ]. It was found susceptible to Alcalase at 0.15% concentration, as also previously reported, and it was further observed to hydrolyze with the less studied Novozym, albeit only at concentrations ≥ 0.15% (DH ≥ 1.2) [ 13 , 18 ]. The band at ≈ 62 kDa persisted in all hydrolysates, suggesting the resistance under investigated conditions, albeit band intensity weakened when PPI was hydrolyzed with Novozym at 5.4% DH. This band could be a dissociated subunit of 11S hexameric legumin, and it has previously shown resistance to Flavourzyme and Nautrase, and susceptibility to Papain and Alcalase (at higher DH) [ 4 , 13 , 18 ]. Two other interesting bands are the one at ≈ 50 kDa and the one at ≈ 26 kDa. The former has been previously associated with vicilin, certain fractions of which have shown allergenic potential [ 13 , 18 ]. Alcalase and Neutrase were unable to hydrolyze this band, which has been previously reported [ 13 , 18 ]. Interestingly, the less studied Novozym appears more potent in breaking down this fraction, indicating potential for reducing pea allergenicity, but further studies are required. Similarly, the band at 26 kDa (likely albumin fraction) showed resistance to Alcalase and Neutrase, and besides these two enzymes, it has previously shown resistance to other 8 enzymes [ 13 , 18 ]. Interestingly, its intensity decreased in the Novozym-treated PPI, although it did not disappear completely. Surface properties Particle surface properties: ζ-potential and isoelectric point Zeta potential is an indicator of the average surface charge, and it is therefore key in determining particle-particle and particle-solvent interactions, which subsequently affect food material properties such as rheology, foaming, and stability [ 46 ]. Figure 3 a confirms that all investigated samples carried a net-negative charge, which was expected as the pH of the measurement (≈ 7) was above the isoelectric point of PPI (≈ 4.5). The control sample showed the highest absolute ζ-potential value (-30 mV), and protease treatment caused a small but statistically significant reduction (down to -24 mV), with the level of reduction overall following the order of the DH. Similar ζ-potential values have been previously reported for protein isolates from chickpea, faba bean, lupin, pea, soy, quinoa [ 47 – 50 ]. In the literature, absolute values of ζ-potential have been reported to moderately reduce upon partial hydrolysis (DH ≤ 5%) of quinoa protein isolate with Alcalase, to remain constant for PPI treated with Alcalase (DH 5%), and to slightly increase for lentil protein isolate treated with trypsin (DH 5%) and PPI treated with trypsin (DH 2%) and papain (DH 9–12%) [ 50 – 53 ]. It should be noted that in all works, the changes were moderate, in agreement with the results of the present study. The less studied enzyme Novozym (at DH 4.6%), and also Alcalase (at DH 6.1%), have been used in one publication to hydrolyze lentil protein, and showed insignificant changes in ζ-potential, but concentrate rather than isolate was used [ 54 ]. In the same study, hydrolysis shifted the pI to lower values, from ≈ 4.7 in the control to ≈ 4 in the hydrolysates. Similar, but more moderate, trend was seen in the present study, where the control sample exhibited a pI of 4.7, and hydrolysis led to a slight decrease in pI values, ranging from 4.7 to 4.4 (Fig. 3 b). More substantial decrease in pI (from ≈ 4.3 to ≈ 2) has been reported upon hydrolysis of rice protein isolate with Flavourzyme (DH 2%), but the authors did not comment on possible reasons [ 55 ]. High absolute values of ζ-potential indicate increased electrostatic repulsion between particles, which can enhance stability or lower the tendency for aggregation/flocculation [ 46 ]. The absolute value of 30 or 40mV has been suggested as a threshold for (moderate) particle stability, with values < 30 mV related to systems with more compromised stability [ 56 , 57 ]. In the present work, ζ-potential values are close to the threshold, indicating surfaces that are overall repulsive and can support some level of electrostatic stability, particularly for the control sample, but electrostatic particle-particle interactions cannot be ruled out. It appears that hydrolysis reduced the surface charge, likely due to the overall reduction in ionizable groups. Surface tension In food systems, where proteins exist in air-water interfaces, surface tension significantly influences adsorption, aggregation, and interaction properties of proteins, which collectively impact the stability and functionality of the food product [ 30 , 58 , 59 ]. The control sample had a surface tension of 72 mN/m (Fig. 3 c). This was at first surprising, as it equals the equilibrium surface tension of pure water, without any proteins suspended [ 60 ]. The discrepancy can be attributed to kinetic limitations, as at such fast measurements as those used in the present study (≤ 100 ms), the reported dynamic surface tension of pure water is 90mN/m, and therefore a 20% reduction in the presence of the PPI can be ascribed [ 61 ]. Partial protein hydrolysis had a small but significant influence on the surface tension of samples, which overall followed the same trend as the DH. At DH 1%, Novozym reduced surface tension down to 63 mN/m at 0.5% concentration, and Neutrase resulted in surface tension of 67 mN/m for both investigated enzyme concentrations. Similar small yet significant reduction in surface tension on hydrolysis with Neutrease and other proteases has been reported for milk protein isolate [ 62 ]. An earlier study has also shown that the surface tension of soy protein isolate was notably decreased after hydrolysis with different proteases including Papain, Pancreatin, and Alcalase [ 30 ]. Interestingly, the authors measured dynamic surface tension, indicating that while for some hydrolysates reduction was evident immediately, the effect of Alcalase was limited at the beginning (as reported in the present study), and reached ≈ 7% after 5 minutes, supporting the kinetic role of protein hydrolysis. Due to the fast nature of the measurements, one possible factor contributing to the observed reduction in surface tension on enzymatic hydrolysis could be the decrease in MW of the peptides, as small peptides can have a quick effect by diffusing faster to the surface [ 63 ]. Hydrolysis may further increase solubility of the PPI, which can enhance surface tension reduction. Another reason may be related to the observed reduction in aggregation size of the large particles (see Fig. 2 a), which may have increased their mobility and conformational flexibility, as also previously reported [ 64 ]. In addition, the possible exposure of the inner hydrophobic regions induced by the enzymatic hydrolysis may further increase affinity to the air/water interface and result in associated reduction in surface tension, as previously reported [ 59 , 63 ]. In one study, surface hydrophobicity of PPI increased after Alcalase treatment with DH of 2%, followed by a modest reduction at DH > 6% [ 13 ]. In the same study, Neutrase appeared to moderately reduce hydrophobicity at DH of 2%. However, in another study, Alcalase treatment appeared to reduce surface hydrophobicity by 10-fold even at DH < 1% [ 65 ]. This indicates an important variation of how hydrolysis with the same enzyme can impact protein properties, which could be at least partially attributed to variability in the substrate. Suspension viscosity and foamability Suspension Viscosity Viscosity is key in determining the texture, consistency, and mouth feel of foods. The apparent viscosity curves of the control and hydrolyzed PPI are shown in Fig. 4 a. As the shape of the flow curves confirmed a power-law relationship between viscosity and shear rate for the investigated shear regime, the power-law model was used to analyse the obtained data, and the relevant power-law indices and consistencies are shown in Table 2 . The control sample exhibited a constant shear viscosity of 19 cP (i.e., about 20 times the viscosity of water), with Newtonian behaviour and a power law index close to 1 (n = 0.9) [ 66 ]. Hydrolysis overall increased the apparent viscosity, but interestingly, the effect did not overall follow the order of DH. For example, Alcalase caused > 10-fold increase in power-law consistency, and induced shear-thinning (n = 0.7) at DH as low as 0.5%, while Neutrase had limited effect on viscosity at DH as high as 3.4%. The effect of Novozym was in-between that of Alcalase and Neutrase, being moderate at DH ≤ 1.3% and stronger at increasing DH. The highest consistency (≈ 1300 cP s n−1 ) and lowest viscosity index (0.4) were observed for the PPI treated with 0.5% of Novozym. An interesting observation relates to the two Alcalase treatments, which resulted in similar and low DH (both at ≈ 0.5%), but viscosity increased by more than 10-fold when dose increased from 0.05 to 0.15%. Similar differences were observed for the MW of the hydrolysates (section “Molecular weight distribution”) for the two Alcalase-treated samples, indicating high sensitivity to Alcalase hydrolysis, with minimal increase in DH causing substantial changes in the hydrolysates’ properties. Protein hydrolysis can increase or decrease apparent viscosity, depending on the substrate properties, enzyme, and conditions. For example, the observations made in the present study align with previous work where an increase in the viscosity of lentil proteins was observed after Alcalase and Flavorzyme treatment with DH of 6% and 5%, respectively [ 54 ]. However, in another study, apparent viscosity of soy protein isolate was decreased by bromelain at DH 2%, but the initial substrate had different properties compared to that used in the present work (e.g., consistency of 7500 cP s n−1 , viscosity index of 0.35). At higher DH (≈ 10%), hydrolysis with Alcalase and Neutrase have been shown to reduce viscosity of PPI suspensions, ascribed to the extensive hydrolysis [ 67 ]. For dairy proteins, Doucet et al. observed that whey protein hydrolysates produced using Alcalase treatment exhibited higher viscosity at DH greater than 15% [ 68 ]. The viscosity of protein suspensions vary depending on factors such as the total number of molecules/particles, their size and shape, and their interactions [ 69 – 71 ]. Schematically, hydrolysis of protein-based particles can remove small peptides from the outer layer (e.g., by a mechanism of erosion) or larger entities (e.g., in the form of small fragments). The obtained viscosity data may result from the different patterns of hydrolyses from the three enzymes. Due to its relatively aggressive, non-specific nature, Alcalase may have penetrated the surface of the parent large PPI particles to partially fragment their outer layer to produce small daughter particles even at low DH [ 72 ]. It may also have resulted in the formation of small particulate aggregates through increased hydrophobic interactions, as previously discussed. Increased inter-particle interactions due to the increased surface area, potentially supported by exposure of hydrophobic regions previously hidden within the particle, as Alcalase has previously shown to increase surface hydrophobicity at DH < 6%, may have caused the observed shear viscosity increase [ 13 ]. It is known that for the same volume, particle size reduction increases viscosity [ 69 ]. Shear may further weaken these interactions and orient the particles to the flow, further causing the observed shear-thinning [ 69 , 70 ]. On the other hand, the milder Neutrase may have largely hydrolyzed peptides from the outer surface of the large particles at DH up to 3.4%, in an erosion-type mechanism and without fragmenting particles or causing extensive aggregation [ 72 ]. The resulting small peptides and amino acids will have a reduced effect on friction during shearing, and hence viscosity, further possibly supported by a reduction in surface hydrophobicity, as previously reported for this enzyme [ 13 ]. Novozym appears to have an in-between result that possibly produces both small particles and peptides, depending on the DH. Changes in molecular size and surface charge distribution also affect the hydration layers surrounding these molecules, substantially changing their effective hydrodynamic size, which can further influence viscosity [ 69 – 71 ]. It should be noted that the K values measured for the samples treated with Novozym (0.15 and 0.5%) and Alcalase (0.15%) had a large standard deviation. One possible explanation could be related to the poor suspension stability of these samples, indicating fast particle sedimentation, which may affect homogeneity of the sampling (discussed in section “Suspension stability”). Despite the large variations, the samples showed significantly different K values. Table 2 power-law viscosity indices and consistencies of the PPI control and hydrolyzed samples. Sample n K (cP s n−1 ) Control 0.9 ± 0.0 a 19 ± 3 a Novo_0.01% 0.9 ± 0.0 a 11 ± 1 a Novo_0.05% 0.9 ± 0.0 a 13 ± 0 a Novo_0.15% 0.5 ± 0.1 d 706 ± 360 c Novo_0.5% 0.4 ± 0.1 e 1296 ± 579 d sAlca_0.05% 0.9 ± 0.0 a 12 ± 1 a Alca_0.15% 0.7 ± 0.1 c 246 ± 63 b Neut_0.05% 0.9 ± 0.0 a 18 ± 0 a Neut_0.15% 0.8 ± 0.0 b 64 ± 3 a Foaming capacity and foam stability of PPI and hydrolysates The foaming of protein solutions is primarily governed by the adsorption kinetics and interfacial properties of proteins at the air-water interface [ 30 , 73 – 76 ]. When protein solutions are agitated, proteins migrate to the air-water interface due to their amphiphilic nature, wherein, their hydrophobic and hydrophilic regions interact with air and water, respectively. At the air-water interface, protein rearrangements and protein-protein interactions facilitate the formation of a viscoelastic film, preventing air bubble coalescence, and ultimately resulting in the formation of stable foam [ 73 ]. In this study, the control sample showed the lowest foaming capacity with foam height of 8.0 mm when it was measured immediately after the foam generation (Fig. 4 b). Protease treatment increased the foaming capacity with increasing enzyme concentrations, irrespective of type of enzyme. At low DH (≈ 0.5%, i.e., samples treated with Alcalase at 0.05 and 0.15%, and with Novozym at 0.01%) small improvement in foaming properties was observed. As DH increased to ≈ 1.5% with Novozym or Neutrase (at 0.05% dose), the effect also increased and higher foam heights were recorded (11 mm), which were similar for both enzyme treatments. However, as DH increased further, differentiation between enzymes was observed, with Novozym having lower DH (2.8%) but more pronounced effect on foam height (13.6 mm), compared to Neutrase (DH 3.4%, foam height 12.6 mm). This will be discussed below. It should be noted that further hydrolysis with Novozym (to DH 5.4%) had no apparent effect on foaming properties. A similar observation has been previously reported, where PPI treated with Alcalase, Esperase, and Papain showed foaming capacity that was enhanced at DH in the range of 4−7% and reduced at DH 5–10% [ 18 ]. PPI treated with Trypsin has also shown increased foaming capacity, which the authors attributed to the presence of small, mobile peptides [ 15 ]. Foaming capacity of the samples partially correlated with the decrease in surface tension (Fig. 3 c), as also previously reported [ 58 ]. At low DH, surface tension reduction was limited, and improvement in foaming properties was also limited. At increasing DH, the small additional reduction in surface tension correlated with increasing foaming capacity. However, it appears that surface tension alone cannot explain the differences between Novozym and Neutrase at the highest investigated DH. The hydrolysis patterns due to different specificities may be linked with the observed results. In the previous section, it was schematically hypothesised that Alcalase may remove small particles from the protein aggregates, Neutrase may produce peptides without greatly affecting the mother aggregates, and Novozym may result in a mixture of small particles and peptides as DH increases. It appears that the presence of small peptides (evident in Fig. 2 b) that can rapidly cover the air/water surface, and small, possibly more flexible compared to large, particles that can mechanically enhance the viscoelastic film surrounding the bubbles may be beneficial for foam development. This is further supported by the somehow enhanced foam stability in the Novozym-treated, compared to the Neutrase-treated samples (15% vs 20% reduced foam height after 55 minutes storage), as it is known that small, flexible particles can sterically enhance foam stability through a Pickering-type mechanism [ 77 ]. Long-term stability Suspension stability Accelerated stability was further measured to evaluate the behaviour of the samples during long-term storage. This was performed by subjecting the samples to centrifugal force of 2300g for 1h and evaluating phase separation in the tube. In a scale from 0 (very stable) to 1 (completely phase separated), all samples had instability indices > 0.6 (Fig. 5 a), indicating high tendency to phase separate, with clarification at the top and sedimentation at the bottom of the tube (Fig. S1 ). Interestingly, ranking of suspension stability did not follow the same order as the DH, but was rather enzyme specific. No effect was seen on treating the PPI with Novozym at DH ≤ 1.4% (instability index 0.68), but the system became increasingly less stable at DH 2.8 and 5.4% (instability index up to 0.83). For Alcalase, treatment with 0.05% dose did not affect suspension stability, but increasing enzyme concentration to 0.15% increased phase separation (instability index 0.8), even though no significant differences in DH were recorded (Fig. 1 ). By contrast, treatment with Neutrase benefited suspension stability, and a small but significantly different reduction in the instability index (down to 0.6) was observed. Obtained results could be correlated with the hydrolysis patterns discussed in the section “Degree of hydrolysis”. Hypothesizing formation of particles by Alcalase and considering the previously reported possible increase in surface hydrophobicity of the produced hydrolysates, increased instability may be due to the increasing insoluble material and inter-particle interactions [ 13 ]. Novozym shows similar effect at higher DH levels, possibly after hydrolysing the “easily accessible” targets to peptides, further producing also small particles. On the other hand, Neutrase may have largely produced peptides, without forming insoluble aggregates, thus enhancing stability by a mechanism that may include increased solubility. It should be noted that in the present study, reduction in ζ-potential (Fig. 3 a) showed limited correlation with suspension stability, suggesting that the acquired absolute values (all > 25) were sufficient to ensure sufficient repulsions. Emulsion stability The emulsifying properties of proteins play a crucial role in determining their suitability for various products containing both oil and water phases, such as plant-based milk, ingredients utilized in the production of meat analogues, and plant-based cheese. In this study, emulsions were prepared using pea protein hydrolysates and rapeseed oil, and emulsion stability was evaluated by Lumisizer® under accelerated centrifugation. Creaming was observed at the top of the control sample with time of centrifugation (Fig. S2), indicating a level of instability, and instability index of 0.22 (Fig. 5 b). Creaming was also observed in all hydrolysates as the sample was centrifuged, with instability indices either similar to that of the control or higher (up to 0.28), except for the sample treated with Alcalase at 0.15% which had an instability index of 0.15. Once again, it was observed that while Alcalase treatment at the two investigated enzyme concentrations resulted in similar DH, the effect on the functional properties was dissimilar. Observed results correlate well with the increased hydrophobicity previously reported for PPI treated with Alcalase at low DH, which can enhance affinity to the oil/water interface by exposing previously buried hydrophobic regions [ 13 , 39 , 78 ]. In addition, related the hypothesis discussed in the section “suspension viscosity”, formation of small, fragmented particles by Alcalase activity may increase flexibility of the produced protein aggregates, further enhancing the emulsifying properties of these hydrolysates. On the other hand, peptides, particularly small peptides, may form less stable emulsions by ways including the limited ability to bind strongly to the oil/water interface due to fewer interaction sites, or potentially increasing aggregation due to exposed hydrophobic regions that are not well attached to the interface [ 39 ]. These factors may result in larger oil droplets, with higher tendency to cream. It should be noted that at 8% protein content in the aqueous phase, exposed oil surfaces are not expected, and saturated coverage of oil droplets is anticipated due to the excess proteinic substances present. The existing literature also demonstrates a varied effects on emulsion stability based on the types of proteins and proteases employed. For example, Liu et al. has shown that hydrolysing a fava bean protein isolate with Alcalase up to DH of 4% did not significantly affect the emulsion stability [ 79 ]. However, hydrolysing it further up to DH of 9 and 15% resulted in increasing instability when stored for 7 days [ 79 ]. Shuai et al. have demonstrated improved emulsion stability of pea protein isolate when it hydrolyzed with Flavourzyme, Neutrase, Alcalase, and Trypsin (DH = 2−6%) [ 13 ]. On contrary, in another study, a reduction in emulsion stability was observed after hydrolysing the lentil protein isolate with trypsin with DH ranging from 4−20% [ 52 ]. Gelation As emulsions showed better long-term stability compared to the suspensions (see section above), and fermentation was carried out for 20 h, gels were prepared using emulsified PPI and hydrolysate samples. Gels are formed when a stable three-dimensional protein network with characteristics of a soft solid is produced [ 80 ]. In this study, proteins and hydrolysates were first fermented, and the resulting pH reduction enhanced protein-protein attractive interactions by decreasing electrostatic repulsions as the pI of the proteins was approached. TG was further used to study the effect of crosslinking on gel mechanical properties. pH monitoring during fermentation (Table S1 ) confirmed reduction from 6.8–7.4 to 4.1–4.3, close to the pI (as determined in Fig. 3 b). The moisture content in all the gels was measured, and it ranged between 75 and 82% (Table S1 ). Gel texture Without addition of TG, all produced gels were soft, with hardness ≤ 60 g. The hardest one (58 g) was that prepared with the control sample. Partial protein hydrolysis overall decreased the gel hardness, reaching values in the range of 4–41 g, with increasing enzyme concentrations, irrespective of enzyme type, but the differences were generally considered small due to the low values of hardness (Fig. 6 a). Incorporating TG during fermentation significantly increased the hardness by 2-4-fold, depending on the treatment. Final gel hardness reached values of 52−171 g in the gels prepared with control and hydrolyzed samples, wherein the control sample yielded the highest hardness (171 g). As also observed in previous sections for other properties, ranking of hardness did not follow the order of DH. It was observed that, among the three investigated enzymes, Alcalase was the most effective, and Neutrase the least effective in reducing gel hardness. For example, at a DH 0.76%, gel hardness was reduced by ≈ 47% when hydrolysis was performed with Alcalase, whereas for significantly similar DH, hydrolysis with Novozym resulted in only a 16% reduction (Fig. 6 b). Besides, statistically similar gel hardness reductions were observed when the protein was hydrolyzed with Novozym at a DH of 0.44% and with Neutrase at a DH of 1.8% (Fig. 6 a and b). Interestingly, increasing Alcalase concentration to 0.15% had marginal effect on DH (which was ≈ 0.5%, see Fig. 1 ) but a significant effect on reducing gel hardness. In general, protease treatment may enhance or reduce the gelation ability depending on the products and extent of hydrolysis. For example, it can enhance gel formation by increasing the hydrophobic and electrostatic interactions (non-covalent interactions) and disulphide bond formation (covalent interaction) as it exposes buried hydrophobic groups and cysteine residues upon protein unfolding, as reported for partially hydrolyzed soy protein isolate [ 9 , 27 , 80 ]. Conversely, a reduction in molecular size during hydrolysis and the formation of polypeptides with a poor ability to interact with each other can decrease the ability of proteins to form firm gels [ 81 , 82 ]. Covalent cross-linking can further greatly enhance the stability of the gel structures [ 83 ]. In this study, the large protein particles present in the samples (Section “Particle size distribution”) imply that the produced gel is of particulate rather than strictly polymer nature. Fragmentation to smaller particles by Alcalase, discussed as a possible mechanism in previous sections, may result in weakening of the particle-based network partially due to steric effects, and the observed reduced gel hardness (Fig. 7 ). On the other hand, formation of small peptides through Neutrase action may explain the reduced effect of this enzyme on gel hardness at DH 1.7%, as limited steric effects are expected (Fig. 7 ). At similar DH (1.4%), the effect of Novozym on reducing gel hardness was somehow more pronounced, possibly related to the previously hypothesised formation of mixtures of peptides and small fragments (Fig. 7 ). As DH increases, disruption of the gel network further increases. In addition, interactions between the possible fragmented particles, the peptides, and the protein aggregate network may further affect gel hardness, for example by a mechanism similar to inactive filler, or repulsive forces. Although the overall gel hardness was lower when hydrolysates were used for gel preparation, it was interesting to note that the extent of increase in gel hardness by TG was notably higher in the gels prepared with hydrolyzed proteins (Table S2). Specifically, the gels prepared with the control sample exhibited a twofold increase in hardness in the presence of TG, while the increase ranged between 2.5 and 4 folds when protein hydrolysates were used. Among the three tested proteases at a concentration of 0.15%, Novozym-treated samples showed the highest increase in hardness (4-fold) compared to Neutrase (3.2-fold) and Alcalase (2.7-fold) treated samples. This observation suggests that Novozym treatment might have resulted in the exposure of more lysine and glutamine residues, thus causing increased protein-protein crosslinking in the presence of TG. However, further increasing the Novozym concentration to 0.5% did not result in an increase in gel hardness in the presence of TG. This indicates that although there were more exposed lysine and glutamine residues, the formation of smaller peptides, due to excessive hydrolysis (as shown in Sections “Degree of hydrolysis” and Molecular size distribution”), resulted in the formation of a weak gel network. These observations further highlight that a balance between the exposure of lysine or glutamine residues and peptide size is crucial to achieve optimal gel hardness. Gel rheology The rheological characterization provides valuable insights into the viscoelastic behaviour of gels. The G’ of the gels prepared by fermentation of control samples was 7408 Pa, which increased to 8548 Pa when the control samples were treated with TG during fermentation (refer to Fig. 8 ). Generally, gels prepared with protein hydrolysates exhibited a lower G’ (ranging from 36 to 6650 Pa) than the control samples, regardless of the type of protease used. Furthermore, the addition of TG during fermentation increased the G’ of gels prepared with hydrolysates, although the values did not surpass those of the control samples treated with TG. In the case of gels prepared in the presence of TG using hydrolysates obtained from Novozym treatment, a decrease in G’ was observed with increasing enzyme concentration. Conversely, enzyme concentration did not have any significant impact on the G’ of gels prepared with hydrolysates obtained from Neutrase and Alcalase-treatment in the presence of TG. The reduction in molecular sizes and changes in other intermolecular interactions induced by hydrolysis can make the proteins less effective in forming extensive networks within the gel matrix, which may impact the elastic behaviour of the gels. Hydrolysis can also result in different extents of solubility of the proteins, which may also affect the elastic properties of the gels. A previous study showed that higher soluble proteins result in higher G’ in fermented pea protein gels than insoluble proteins [ 84 ]. Insoluble proteins can act as inactive fillers and can weaken the emerging gel matrix [ 84 ]. Conclusions The present study highlights the importance of protease specificity, complementary to the DH, on determining the functionality of pea-protein hydrolysates. For example, at similar DH, Novozym treatment led to an increase in suspension viscosity, whereas Neutrase treatment had no significant effect on viscosity compared to the control. For all investigated systems, absolute value of zeta potential, pI, and surface tension were slightly reduced after enzymatic hydrolysis, while the effect of proteolysis on functional properties, including suspension viscosity, foaming capacity, and gel hardness, was largely enzyme specific. Based on the SDS-PAGE profiling, particle size analysis, and DH characterisation of partial hydrolysates, the study suggests that the size and properties of the released peptides play a key role in controlling functional properties of the material. Therefore, selecting the appropriate protease is essential for achieving desired functional properties. A possible mechanism of hydrolysis is suggested where the three enzymes result in small, fragmented particles or aggregates, peptides, or their mixture. Acquired results support this hypothesis, albeit full verification requires further work. From a product development perspective, hydrolysates with high viscosity (e.g., from Novozym treatment) could be utilized in thickened sauces, soups, or yogurt preparations, while hydrolysates with minimal viscosity change (e.g., from Neutrase treatment) may be ideal for beverages. Further research involving characterization and sequencing of the resulting peptides using mass spectrometry is needed to establish clearer structure–function relationships. Future research is also needed to design protein hydrolysate-based systems with predictable behavior for food applications. In addition, more studies are required to better understand protein functionality in large-scale production systems. Abbreviations Novo Novozym® Alca Alcalase® Neut Neutrase® DH Degree of hydrolysis TG Transglutaminase GDL Glucono-𝛿-lactone SDS-PAGE Sodium dodecyl-sulphate polyacrylamide gel electrophoresis Declarations CRediT author statement Ashwitha Amin: Conceptualization, Methodology, Formal Analysis, Investigation, Validation, Visualization, Writing - Original Draft, Writing - Editing. Ourania Gouseti: Conceptualization, Methodology, Validation, Investigation, Writing-review & editing, Supervision. Gernot J. Abel: Methodology, Resources Poul Erik Jensen: Conceptualization, Methodology, Validation, Investigation, Resources, Writing-review & editing, Supervision. Funding The Milk Levy Fund (PLANTCURD: Funktionelle planteproteiner som ostemasse/ PLANTCURD: Functional Plant Proteins for Cheese Curd.) Acknowledgments The authors would like to acknowledge Signe Munk Rydtoft, Line Friis Bakmann Christensen, and Søren Lillevang (Arla Foods Innovation Centre, Aarhus, Denmark) and Hanna Maria Lilbæk (Novonesis, Lyngby, Denmark) for scientific discussions and suggestions.The authors would like to thank Natascha Krog Bager (Novonesis, Lyngby, Denmark) for technical help in physical stability analyses.The authors would like to thank Roquette, Lestrem, France and Novonesis, Lyngby, Denmark for donating pea protein isolate and enzymes, respectively. Data availability All data used in this study are presented in the illustrated figures and Supplementary Information, and the raw data will be made available upon request to the corresponding author. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8923282","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":604522856,"identity":"30ee90cd-479b-4416-9654-4ce1b6eafd30","order_by":0,"name":"Ashwitha Amin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYFACHgaGBIYDDPzMB0DsBKBIGjFaEg4wSLYlkKKFAajF4BixWvj7zx6TePjjjrzxMeaDD94wpCU2sKcl4NUicSMvTSIh4ZnhtmNsyYZzGHISG3ieHcBvzQ0esxsJCYcZt93vMZPmYahIbJBIb8CrQ/78GbAW+81tPERqMTiQA9aSuIENrAXoMIk0/A4zvJFj/iMh7XDyDLBfDNKM23ieJeDVInf+jLHhD5vDtv1toBCrSJbtZ08zwKsF3Z0MDGykqB8Fo2AUjIJRgB0AACUHSn32K275AAAAAElFTkSuQmCC","orcid":"","institution":"University of Copenhagen","correspondingAuthor":true,"prefix":"","firstName":"Ashwitha","middleName":"","lastName":"Amin","suffix":""},{"id":604522857,"identity":"95d6e072-4740-430f-aec4-939764e7a3cb","order_by":1,"name":"Ourania Gouseti","email":"","orcid":"","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Ourania","middleName":"","lastName":"Gouseti","suffix":""},{"id":604522858,"identity":"00148201-ad84-40e5-aba5-8c065fe95ca4","order_by":2,"name":"Gernot J. Abel","email":"","orcid":"","institution":"Novonesis A/S, Enzyme Research","correspondingAuthor":false,"prefix":"","firstName":"Gernot","middleName":"J.","lastName":"Abel","suffix":""},{"id":604522859,"identity":"6f995fa5-b4b1-48ba-a06e-031175f62e03","order_by":3,"name":"Poul Erik Jensen","email":"","orcid":"","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Poul","middleName":"Erik","lastName":"Jensen","suffix":""}],"badges":[],"createdAt":"2026-02-20 07:23:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8923282/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8923282/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104781038,"identity":"eaec47da-933f-4126-ab4d-97683ad86498","added_by":"auto","created_at":"2026-03-17 07:54:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":51817,"visible":true,"origin":"","legend":"\u003cp\u003eDegree of hydrolysis resulted in the investigated treatments\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8923282/v1/662ac39d572589a6068779e4.png"},{"id":104496052,"identity":"412e322e-8261-4e33-8c53-ef1a53eff660","added_by":"auto","created_at":"2026-03-12 12:46:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":282356,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Particle size distribution in the control and investigated hydrolysates; (b) molecular weight distribution of soluble proteins and peptides determined with SDS-PAGE\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8923282/v1/8f6ec499e1ebe70b9bd8456a.png"},{"id":104781427,"identity":"9db9dc13-d7a4-4180-b28a-6869090c6515","added_by":"auto","created_at":"2026-03-17 07:55:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":70790,"visible":true,"origin":"","legend":"\u003cp\u003e(a) zeta-potential; (b) evaluated isoelectric point; and (c) surface tension of the PPI control and hydrolysates\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8923282/v1/7d901d31fae6b892ab9441d4.png"},{"id":104780712,"identity":"5634136e-769b-4005-87c6-61f330772f93","added_by":"auto","created_at":"2026-03-17 07:53:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":133216,"visible":true,"origin":"","legend":"\u003cp\u003e(a) suspension viscosity; and (b) foaming properties of the PPI control and hydrolysates\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8923282/v1/ecea4163fa0699e71f173c4d.png"},{"id":104780597,"identity":"71b971a3-ad8a-4ed2-88ed-015ec26b51c6","added_by":"auto","created_at":"2026-03-17 07:53:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":111790,"visible":true,"origin":"","legend":"\u003cp\u003eInstability indices of (a) suspensions; and (b) emulsions prepared from the PPI control and its investigated hydrolysates\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8923282/v1/0c1632d9efe41de6c0274541.png"},{"id":104781226,"identity":"f923d648-9c95-4aea-88a6-44b31ea63d3c","added_by":"auto","created_at":"2026-03-17 07:55:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":83244,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Hardness of gels prepared with the PPI control and hydrolysed; and (b) Relationship between gel hardness and DH of samples prepared with Alcalase, Novozym, and Neutrase. Gels were prepared with the addition of transglutaminase (TG). This plot highlights the effect of protease choice on gel hardness at comparable DH levels\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8923282/v1/98e8eb2825a83a88874569e4.png"},{"id":104781208,"identity":"99240fce-26e3-4501-ae16-2ac4120082e9","added_by":"auto","created_at":"2026-03-17 07:55:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":134873,"visible":true,"origin":"","legend":"\u003cp\u003eA suggested mechanism for the PPI hydrolysis under investigated conditions discussed in the present work\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8923282/v1/c25b0f5339a43679fd90a16e.png"},{"id":104780880,"identity":"b0b549bb-9fc0-42f1-a4d0-47698b09872c","added_by":"auto","created_at":"2026-03-17 07:54:12","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":88425,"visible":true,"origin":"","legend":"\u003cp\u003eStorage modulus measured at 1 Hz for gels prepared from the control and hydrolysed samples\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8923282/v1/d8d399802a3fc08cb4f2e224.png"},{"id":104808692,"identity":"54cf05d0-4ff2-4918-94d5-abe25f7cf125","added_by":"auto","created_at":"2026-03-17 12:39:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1968216,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8923282/v1/12604b3f-1197-4481-99c7-33032e5ed6c0.pdf"},{"id":104781272,"identity":"98c93da2-d0f6-427d-864a-2076f385fc75","added_by":"auto","created_at":"2026-03-17 07:55:16","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1396773,"visible":true,"origin":"","legend":"","description":"","filename":"Appendix1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8923282/v1/46fc15bce0ef94cfbabf749c.docx"}],"financialInterests":"Competing interest reported. Gernot J. Abel is employed at Novonesis A/S, Denmark.","formattedTitle":"Effect of proteases on the functional properties of pea protein isolates","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePea (\u003cem\u003ePisum sativum\u003c/em\u003e) is one of the most promising legumes for the development of plant-based food products due to its high protein content (20\u0026minus;25%), availability, and cost effectiveness [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Pea proteins are rich in essential amino acids and are low in allergens compared to other commonly used plant protein sources like soybean and wheat [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, many food production and processing industries are increasingly attempting to utilize pea proteins to develop plant-based foods and beverages, including alternatives to meat and dairy products. However, the utilization of pea proteins in food applications faces challenges due to factors like their size, degree of aggregation, poor emulsification, foaming, and gelation properties [\u003cspan additionalcitationids=\"CR5 CR6 CR7 CR8\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Change in structure, interaction of proteins with other components of the seed matrix, and reorganization of the native state of proteins during extraction are among the factors reported to contribute to their poor functional properties [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. To overcome these limitations, modification of pea proteins is often required.\u003c/p\u003e \u003cp\u003eChemical, physical, and enzymatic modification are common techniques used to improve structural and physicochemical properties of food proteins [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Among these, enzymatic modifications using proteases have gained increasing attention over the past two decades due to the mild processing conditions required, minimal by-product formation, high specificity, and ease of process control [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Proteases catalyze the hydrolysis of proteins into smaller peptides; and the process may simultaneously cause a certain degree of protein unfolding and exposure of previously buried hydrophobic, hydrophilic, or ionizable groups [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These modifications can affect techno-functional properties of plant proteins. In contrast to most animal-derived proteins, seed storage proteins are more difficult to hydrolyze because of their highly aggregated structure. The effect of proteases is typically measured by the degree of hydrolysis (DH), referring to the percentage of peptide bonds hydrolyzed under investigated conditions [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Many studies have demonstrated improved solubility, emulsion stability, and foaming properties of plant proteins upon partial protein hydrolysis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, selecting the appropriate enzymes and determining the optimal DH are critical factors in achieving the desired functional property enhancements [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Proteases with different specificities may result in diverse effects on protein modification, as they may release peptides with varying amino acid compositions, sizes, and conformations [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In addition, controlling the DH is crucial, as too little hydrolysis may not significantly alter the functional properties, while extensive hydrolysis may produce small peptides and free amino acids with reduced functionality and compromised texture and flavor properties [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, understanding the relationships among the type of protease, DH, and functional properties is crucial for developing novel food structures with desired quality attributes.\u003c/p\u003e \u003cp\u003eProtein gelation is an important functional property that significantly impacts the structure, texture, rheology, and creaminess of food products, influencing both sensory perception, downstream processing and storage parameters. Various methods are employed to induce protein gelation, with heating and acidification using glucono-\u0026#120575;-lactone (GDL) being the most common approaches [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Acidification using GDL is a clean label gelation method with various advantages such as rapid and controlled acidification, but it does not improve flavor and nutritional properties [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Alternatively, fermentation using lactic acid bacteria has been explored to induce protein gelation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Fermentation also provides additional benefits such as improvement of flavor properties due to formation of flavor-eliciting amino acids, peptides, and volatile compounds [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Furthermore, enzymatic protein cross-linking through transglutaminase (TG) has also been investigated as TG offers a selective protein gelation by facilitating isopeptide bond formations between glutamine and lysine residues, resulting in intermolecular or intramolecular protein cross-linking, which ultimately strengthen gel structures [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The use of TG in food applications is generally recognized as safe (GRAS). Although a few studies have independently investigated fermentation and TG-induced cross-linking of plant proteins, the combined impact of fermentation and TG-induced crosslinking of hydrolysates remains less explored [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe aim of the present study is to understand the impact of enzymatic partial hydrolysis combined with subsequent fermentation and TG-induced crosslinking on the functional properties of a pea protein isolate. Three different proteases with different specificities (Novozym, Neutrase and Alcalase) were used and their effects on the acquired functional properties were compared. Novozym is a serine endopeptidase of microbial origin, and exhibits specificity towards hydrolysis of peptide bonds involving aromatic amino acids such as phenylalanine, tryptophan, and tyrosine [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Neutrase, a protease of bacterial origin, has a broad specificity, typically preferring to cleave peptide bonds adjacent to hydrophobic amino acids such as leucine and phenylalanine [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Alcalase is an endopeptidase with broad specificity and has been reported to hydrolyze peptide bonds with aromatic, acidic, basic and aliphatic amino acid residues [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. From these enzymes, Novozym is the least explored for food applications [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. These enzymes are commercially available, and providing comparative data on their effects in modulating the functional properties of seed storage proteins will help in selecting the most appropriate enzyme for large-scale applications. In this study, therefore, hydrolysates were comprehensively characterized for various functional properties essential in the development of plant-based foods and beverages, including particle and molecular size distribution, viscosity, suspension and emulsion stability, foaming properties, zeta potential, isoelectric point, and surface tension. Furthermore, the effectiveness of hydrolysates in forming protein gels was investigated using a combination of TG-induced crosslinking and fermentation.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eMaterials\u003c/h2\u003e\n \u003cp\u003ePea protein isolate (PPI) NUTRALYS\u0026reg; S85F (isolated from \u003cem\u003ePisum sativum\u003c/em\u003e) was donated by Roquette, Lestrem, France. Novozym\u0026reg; 11028 (EC 3.4.21.1; serine endoprotease from \u003cem\u003eBacillus licheniformis\u003c/em\u003e; 75000 PROT/g; reported optimal conditions: pH 6\u0026ndash;7, temperature 50\u0026ndash;75\u0026deg;C), Alcalase\u0026reg; 2.4 L FG (EC 3.4.21.62; alkaline serine endoprotease from \u003cem\u003eBacillus licheniformis\u003c/em\u003e; 2.4 AU-A/g; reported optimal conditions: pH 7\u0026ndash;9, temperature 30\u0026ndash;65\u0026deg;C), Neutrase\u0026reg; 0.8 L (EC 3.4.24.28; metallo endoprotease from \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e; 0.8 AU-N/g; reported optimal conditions: pH 7, temperature 40\u0026ndash;50\u0026deg;C), and transglutaminase (TG) (EC 2.3.2.13, from \u003cem\u003eBacillus licheniformis\u003c/em\u003e) were donated by Novonesis, Lyngby, Denmark and referred to as Novozym, Alcalase, Neutrase, and TG respectively. The fermentation starter culture Vega\u0026trade; Harmony (a blend containing \u003cem\u003eLactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus paracasei, Lactobacillus acidophilus, and Bifidobacterium\u003c/em\u003e) was obtained from Novonesis, H\u0026oslash;rsholm, Denmark. Rapeseed oil was purchased from a local supermarket and was used without any modifications. Picrylsulfonic acid solution (1 molL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in H\u003csub\u003e2\u003c/sub\u003eO, TNBS), glucose (99%), sucrose (99%), L-alanine (99.5%), and Brilliant Blue G were purchased from Sigma Aldrich, Copenhagen, Denmark. NuPAGE\u0026trade; MES SDS Running Buffer 20X, SeeBlue\u0026trade; Plus2 Pre-stained protein standard, and NuPAGE\u0026trade; LDS Sample Buffer 4X were purchased from Invitrogen, Waltham, USA. The other chemicals used in this study were of analytical grade. Milli-Q water (Merck, Germany) was used throughout the study.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003ePartial protein hydrolysis\u003c/h3\u003e\n\u003cp\u003ePea protein isolate suspension (40 mL) with a protein concentration of 8% w/v was prepared using Milli-Q water in a 50 mL falcon tube (the pH of the protein suspension was determined to be 7.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0) and the suspension was hydrated for 1 h with vertical mixing at room temperature. The hydrated sample was preheated at 90\u0026deg;C for 10 min in a shaking water bath (LSB Aqua Pro, Grant, UK) and immediately cooled down by placing the sample tube in ice cold water for 5 min. Thereafter, the sample was heated to 50\u0026deg;C in a hot air oven and proteases were added in different concentrations in independent experiments (n\u0026thinsp;=\u0026thinsp;3): Novozym was added at four different concentrations (0.01, 0.05, 0.15, and 0.5% v/w of protein,) while Alcalase and Neutrase were added at two concentrations (0.05 and 0.15% v/w of protein, n\u0026thinsp;=\u0026thinsp;2). The sample mixtures were then incubated for 30 min in a hot air oven with vertical mixing to achieve partial protein hydrolysis. Following hydrolysis, the enzymes were inactivated by heating the samples at 90 \u0026deg;C for 10 min in a water bath (inactivation time was based on preliminary experiments). After cooling to room temperature, the hydrolysates were used for subsequent functional property analyses. A control sample was generated by following all the steps as described above except addition of enzymes.\u003c/p\u003e\n\u003ch3\u003eDegree of hydrolysis (DH)\u003c/h3\u003e\n\u003cp\u003eThe DH of protein samples was determined using the trinitrobenzenesulfonic acid (TNBS) total amine assay following a protocol described in the literature with some modifications [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. Briefly, a stock solution of L-alanine (0.2 mg/mL) was prepared in borate buffer (0.05 molL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, pH 10) and diluted to 2\u0026minus;200 \u0026micro;g/mL in the buffer to prepare calibration standards. Sample hydrolysates were diluted appropriately (10\u0026minus;100 fold) in the borate buffer. A 0.1% TNBS solution was freshly prepared in Milli-Q water and kept in dark. In the assay, 100 \u0026micro;L each of blank (0.05 molL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e borate buffer, pH 10), calibration standards and sample hydrolysates were transferred to a 96-well microplate. Subsequently, 100 \u0026micro;L of 0.1% TNBS solution was added to each well and the absorbance was measured continuously at 37 \u0026deg;C for 10 min at 450 nm using a Microplate Spectrophotometer (Biotek Instruments, EPOCH 2). The measurements were taken in technical duplicates for each biological replicate. Mean velocity was calculated using Gen5 software (version 3.11, BioTek, Winooski, USA) and used as the response in the calculations. The concentration of total amines was calculated using standard calibration curve. The degree of hydrolysis was calculated using the following Eq. 1:\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u0026nbsp;\u003cspan class=\"mathinline\"\u003e\\(\\:DH\\:\\left(\\%\\right)=\\frac{\\left(Cs-Cc\\right)}{Cmax}\\:⨉\\:100\\:\\)\u003c/span\u003e\u0026nbsp;\u003c/span\u003e Eq. 1\u003c/p\u003e\n\u003cp\u003ewhere Cc\u0026thinsp;=\u0026thinsp;Concentration of total amines in control sample\u003c/p\u003e\n\u003cp\u003eCs\u0026thinsp;=\u0026thinsp;Concentration of total amines in partially hydrolyzed sample\u003c/p\u003e\n\u003cp\u003eCmax\u0026thinsp;=\u0026thinsp;Concentration of total amines in completely hydrolyzed pea protein isolate.\u003c/p\u003e\n\u003cp\u003eThe completely hydrolyzed pea protein isolates were prepared using total acid hydrolysis (n\u0026thinsp;=\u0026thinsp;3) following the procedure described by Poojary et al. (2020) with some modifications [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. Briefly, 10\u0026ndash;15 mg of pea protein isolate were placed in a microwave vial and then 3 mL of 6 molL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e HCl were added. A magnetic bead was placed into the vial and then the vial was purged with nitrogen before immediately sealing it with an aluminum crimp fitted with a Teflon septa. The sealed vial was kept in a microwave synthesizer (Biotage\u0026reg; Initiator+, Sweden) and heated at 150 \u0026deg;C for 1 min followed by 165 \u0026deg;C for 10 min to achieve complete protein hydrolysis. Thereafter, the hydrolysate was neutralized by mixing it with equal volume of 6 molL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaOH. The hydrolysate was diluted before the analysis as described above.\u003c/p\u003e\n\u003ch3\u003eParticle size distribution\u003c/h3\u003e\n\u003cp\u003eParticle size distributions were measured based on laser diffraction using a Mastersizer (3000, Malvern, UK) following the manufacturer\u0026rsquo;s instruction. In a typical experiment, the sample was diluted in a degassed water tank until the obscuration level reached between 10 and 20%. All the samples were measured based on the refractive index (RI) of 1.52 for pea protein [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]. The measurements were taken in technical duplicates for each biological replicate.\u003c/p\u003e\n\u003ch3\u003eMolecular weight distribution\u003c/h3\u003e\n\u003cp\u003eSodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the soluble protein samples was performed under non-reducing conditions according to the method described in Jansson et al. (2017) with some modifications [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. All samples (4 mL each) were centrifuged at 8000 g for 20 min at 25 \u0026deg;C, and the resulting supernatants were used for subsequent analysis. The supernatant (50 \u0026micro;L) was added with 950 \u0026micro;L of SDS buffer (5% SDS in 0.1 molL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Tris buffer, pH 8). A sample mixture was then prepared by combining 65 \u0026micro;L of the diluted supernatant, 25 \u0026micro;L of 4X LDS sample buffer, and 10 \u0026micro;L of Milli-Q water. The mixture was then heated under agitation (350 rpm) for 10 min at 80\u0026deg;C in a thermoblock (Provocell\u0026trade; Microplate Incubator, Esco, Singapore). The running buffer (1X) was freshly prepared by mixing 50 mL of 20X running buffer and 950 mL of cold Milli-Q water. A 2% of brilliant blue solution was prepared in Milli-Q water. An equilibration buffer was prepared by dissolving 20 mL of concentrated phosphoric acid, 150 g of ammonium sulphate, and 180 mL ethanol in 1000 mL Milli-Q water. The protein standard (marker) was used directly without any sample preparations. A gel (NuPAGE\u0026trade; 4\u0026minus;12% BT mini gels, Invitrogen) was placed into the electrophoresis system tank (Mini Gel Tank, Life Technologies, USA) and the tank chambers were filled with 1X running buffer. Protein standard (3 \u0026micro;L) and sample mixtures (5 \u0026micro;L each) were loaded into the gel. Thereafter, the gel ran at 200 V for 35 min. Following electrophoresis, the gel was placed in a container containing 100 mL of equilibration buffer and 1 mL of brilliant blue and allowed to stain overnight on a rocking table in the fume hood. Subsequently, the gel was destained by using Milli-Q water and scanned using a ChemiDoc MP Imaging System (BIO-RAD, California, USA).\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eZeta potential and isoelectric point determination\u003c/h2\u003e\n \u003cp\u003eThe zeta potential of samples was determined using a Zetasizer NANO ZSP (Malvern, UK) without adjusting initial pH of the samples. The hydrolysates were diluted in Milli-Q water to reach the final protein concentration of 1% and the zeta potential was measured at the refractive index of 1.52. To determine the isoelectric point (pI), the zeta potential of the diluted samples was measured across pH 7.8\u0026minus;3.0, and the pH was controlled by dropwise addition of 0.5 or 1 molL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e HCl. The pH corresponding to a zeta potential of 0 mV was considered as the isoelectric point (pI). The measurements were taken in technical duplicates for each biological replicate.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eSurface tension\u003c/h3\u003e\n\u003cp\u003eA bubble pressure tensiometer (SITA pro line t15) connected with an automated liquid handler (Hamilton Microlab\u0026reg; STAR) was used to measure the surface tension. Samples were diluted in deionized water at a 1:1 ratio, and 1.8 mL of the diluted samples were added into the wells of a 24-well multiwell tissue culture plate. The automated liquid handler connected to the tensiometer immersed the capillary tube into the sample and the surface tension was measured at the bubble lifetime of 100 ms at room temperature (23 \u0026deg;C). The measurements were taken in technical duplicates for each biological replicate.\u003c/p\u003e\n\u003ch3\u003eViscosity\u003c/h3\u003e\n\u003cp\u003eThe shear viscosity of the samples was measured using a rotational rheometer (Kinexus pro+, Malvern, UK) with a C25 (DIN) cup/bob geometry. Each sample (17 mL) was placed in a cup and then the bob was lowered and brought to a temperature of 22 \u0026deg;C. Shear viscosity was determined by increasing the shear rate logarithmically (1\u0026minus;100 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), with 10 measurements points per decade based on the method described in Fang et al. (2021) with minor modifications [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]. The shear rates of 1\u0026minus;100 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were selected based on the assumption that they are relevant for oral food processing [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. The measurements were taken in technical duplicates for each biological replicate. Shear viscosity versus shear rate plots were constructed, and curves were fitted to Eq.\u0026nbsp;2 (power law equation) to determine the flow behavior index (n) and the flow consistency index (K).\u003c/p\u003e\n\u003cp\u003e\u003cimg 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AhKQQCkRUEjUf7UsKQEJSEACEpCABCQgAQn8Q0Ah8Q8IXyQggbZIwDlJQAISkIAEJNBcBBQSzUXWdiUgAQlIQAISaDgBa0hAAiVDQCFRMkvlQCUgAQlIQAISkIAEJFA8BHIhUTwjciQSkIAEJCABCUhAAhKQQNETUEgU/RI5QAnURMB8CUhAAhKQgAQk0HoEFBKtx96eJSABCUig3Ag4XwlIQAJtiIBCog0tplORgAQkIAEJSEACEmhaArZWMwGFRM1sfCIBCUhAAhKQgAQkIAEJ1EBAIVEDGLNbm4D9S0ACEpCABCQgAQkUMwGFRDGvjmOTgAQkUEoEHKsEJCABCZQVAYVEWS23k5WABCQgAQlIQAL/I+A7CTSGQKsIib/++iu+/vrrePXVV+Onn35qzPjLtu5vv/0Wn376abz00kslyeDXX3+Nt956K0aPHl104x8zZky8+eab8csvvxTd2ByQBCQgAQlIQAISKBYCLS4k/vzzzyQgjjvuuOjRo0e88sorxcKiBcdRe1djx46Nu+++O84777w4Ma6gogAAEABJREFU++yz44EHHohvvvkmXnzxxVTxhx9+iHvvvTd22WWXWHnllVNeqf0ZNWpUbLfddnHkkUcW3dCvvPLK+Ne//hXvvvtu0Y3NAUlAAhKQgAQkIIFiIdDiQiLLsph++unj999/j++//75YOBTNONgFP+KII+LWW2+NTp06xZxzzhnPPPNM7LHHHnHiiSemcU488cQx++yzp13zlFGCf2adddY47bTTYv/99y+60ffu3TtOP/30mGOOOSqN7frrr6/02Q9lRsDpSkACEpCABCRQiUCLC4l27doFRuS8884bk0wySaXB+CHihhtuiBdeeCEZ2Jtttllg1B500EGx6qqrBicRMGrfvn0stthiMfXUU/OxJNNUU02VTqSWXHLJohv/3HPPHausskolvpwScUL0999/F914HZAEJCABCVRPwFwJSKB5CbS4kMink2VZ/tbXAgKDBw9OAmuaaaaJLPt/RpNOOmlytVlggQUqSmbZ/z+ryPBNsxH4448/4sYbb4zXXnut2fqwYQlIQAISkIAEJFBqBBolJPDTP/PMM4N0xRVXxI8//hhDhgxJn8kjffbZZymgeujQoSn/v//9byVGBF6/8cYbcdlll8Ull1wynl96HlPBc9q744474rvvvqto45133ombbropBR2PGDEixRTgCpQXIAYjr3vXXXfVGUA7bNiwNM6bb745jZuA5uuuuy7l3XLLLXXWz/ud0Ndu3bqlGJL//Oc/8cUXX1Q0M/nkk8dWW21V8bnwDQHrDz/8cJx77rmB+823335b+DidZDz44IPpOTEXnHhgHOeFcDG777770vOBAwcGnPI22IEnngFDGpYY04wNxqxN3gavr7/+euLEOpEeeughsoO2OGkhj/7JZG733HNP8L3gc56effbZ4Dty4YUXpnEQK5I/4/Xjjz9Opza0RbnnnnuO7EqJAPR8za+55pr48MMPKz1nvnfeeWecc8456Xt3//33x9tvv11Rhu8R688YyeQ7x2kEFwTA54ILLkj1GAOJz3Ch7CeffFLBAM7kmSQgAQlIQAISkEBbJNAoITHllFMmw7V///7pBqGJJpooOnToEIgKgqm5WQg3HPJ5jxFGbEQhSIxPjDQM4cMPPzx23333ZHjmZXIDN9+hP/DAA+OMM85I8RXc+oRhSh2MOYzDo48+OgUpY1wjAPDDn3baaYP+Dz300BgwYED8/PPPefOVXp9//vn46quv4vPPPw/6wSgnVgE3HETSYYcdFhi4lSoVfMBY32ijjaKudPLJJxfUqvx23333jS5duiRBtMMOO8Rtt91WMd6ll166cuFxnzDmKYPhi7G/5557JkN83KP0H7xh9NRTTyWffwxeeMGVuggK2F111VUx00wzxWSTTRbME1EHJwKOzz///LQul19+eSCmYIihnrtapY7G/cHVituOYMdaIH7GZacTFl75XsCSW5EuvfTS2G+//ZJ45Blp+PDhQXwI68V3a/DgwXHRRRfxKCWCzXfeeed04xcxIsy7T58+KRg9FRj3h/WBL6c4xOIwD5iwtuMep/8GDRoUw4YNS3EmrCvxEAgYHsLxhBNOSN8TRAF5c801Vyy44IK8jXnmmSf4DDdEBHNFcPD9pADfd9ygWAtEGHklkRykBCQgAQlIQAISaCCBRgmJFVdcMU455ZTUJcYVRtRqq62WAoOJhVhzzTWTsMA47dq1a+y1116x2267pfL5H04UMPYx+LiFiB1mTih4zu4zhiOBxptuumnss88+QcwARuzjjz8e3cbt3ueGHGIFg/Gxxx5LNwHRxkknnRQYlZtsskkcfPDBgSGOoZnvlNNHYSLugPbXW2+9wMgeNWpU7LjjjsFnDFwM01NPPbWwSqX33EKFQV5XwoCuVLHgA7EjGNAYruzYcwqx/fbbV9oxLygenOh07tw5BS5THmMekZCf2gwZMiTYYd97772TwIE1V69ioH/55ZdJYLGGCy20UGyxxRYB78UXXzwQdpwkdB23bjDBsGdHHt7wg2duPMc//2abbbZALK699tpJlMw333zpyRRTTJG+BwiYvn37xgwzzBCsCUHjqcA/f9j5X2qppdI4uDWJPjDYecytVawvt1Qxxs033zwOOeSQ5G6EOKIMpyQEbyPAttlmm+AVlogIjH4Mfoz7Rx99NPhubrzxxum7uvXWWyeOtLH++uunm7AQGHwmLbroojH//PPzNjbYYINgfjvttFP6nmVZlmJ+mDsF+I4QA7TtttsGHMkzSUACEmgOArYpAQlIoLUJtGvMALIsC4yshRdeODDOML4w6Pk888wzp5MJ2scYxA1q9dVXD56Tlyd279m5zsUGBjw71jzHCOzYsWO64QljeOTIkekmo48++ih4j1GIeKHsIosskm6DYjwYfRil7OzjmpTXxdhjjJwcUKdqwrBlfFmWpfgEgm7Z2c6yLI0bocFcGGNU8w/jnd9GqCvlO93VNJGy4IdLDzvljBlXIwxoXHE4WUmF/vlDwHrPnj0DDnBkzJwk4L5DEUQCBj2uOxjUnACxm//0008n1ynmi/EMP9qGLfOALYnnMEAYLr/88qkfYjXoJ8syuqiUGAcGOoxffvnl9IzxIBARnrRDYr2zrHJ9hAkCB0GEcFhiiSVSQDaNIF6YA2KCMZGHcENIITB5xbWL9YEVfWRZFvBDUPB9QFxSj5MUvlu4ajFnAtn5rvCMufI94H1tiTEg8GCBcESUUR6R9sgjj8Raa62VvkPkmSQgAQlIQAISkEBEtDkIjRIS0MD4Y5cWoYBLEIYUhjKGKbvhH3zwQbCzj4HJ7jZ16ptwq8EgvvbaayNPtM2uODvoGLo1tUVsxujRoyvqUR93J1yHGrNTjIjIhU7Vvt97771K/dFndQnRVbVu1c+49xxwwAGBixA78xjeuGbxuWrZ2j4jHHAJysdBrALCgh1/+qDdiy++OHA54qTn6quvDuZRW5u1PcOAR9jgAoSxTlnWn5MSjHo+15QwzBEhnCogGHCF2nXXXVNxxsQJCWNOGVX+IDxyocDpSf44y7J0MoA44vQry7LkUsX3CLcoXOHI5yQkr1PfV/pB7OHOhQscIgZ3K05cmH9927GcBCQgAQlIQAISKEUCjRYS7Ij36tUr7b7yw2mICXa1cWPC0Mf/np1fTihqMgJrA8cO8fHHH5/u9cdoyxPihb5rq0t/efnCV34IrbZ6tT1j53vs2LHVFsF1qrCfmt5jwFbbwLhMGI57qfgPVydceBATnDgQWF7xsJ5vlllmmfH4MTZOJoihICYAoYHAYPe+e/fu9Wy5+mJdunRJ19VykoLbEeuP+xXrUX2N/8/lNzM4dTnrrLOCUwGMfFzLEIBw5/vEqcr/l678N39OLiKS1zx1HHeqxfsZZ5yRl8D9jhiNfv36JRcuxArxMIjEVKABf2gLEUJ9BAksa1vfBjRtUQlIQAISkIAEJFDUBBotJJgd/vycQBA/QDAuBvUKK6wQxDBwsw674pwC4A5C+fomdrCJk2AXvbAOu9PESGAEF+YXvsclCp95XFoK83FtIhC2MK+Y3nPjUNXbfogxWGeddVJsAUZ1Q8aLWMCgx92qsB43FbErT1/cbLThhhsm4x/B19B1KmyX94g/YiA4rTrssMMCN6pcbPK8psSJCLv8uAXxntgYdviHDx8eiAzaQ2gU1kdcIK44BcDdis9VxQbCghie/AfmcEXidAwhQUwNrnAEWdd00lTYX9X3zBUh8sQTT8S///3vQIw1VohV7cPPEihHAs5ZAhKQgASKn0CTCAkMPAJy8RMndiH3hWcXHXcUDP9CVw92jzGIMfpwhcox4UtPHs8oQ6AxhiUuPf379w+MNWIHCJjGjYQTCVxWqE9d6vCehN8/QmTLLbeME088MTBGMaj322+/QGRQphgTcycgmt3tnA15Tz75ZDr14aSHcf/yyy8pIBxeeTl21HmPwOKVcgSJI04IDsaA5nYi4hBgiRFMHAMnLPRHPAmij1cEFydLBCjTF20SW0Cb9UmIS2IcYN6pU6fxmDM+1otXxk2bnFzgDkU/nEgQk4GoQUjxA3GIVeaAmGCc3NpFQD3jy7Is+OE+1haDHrc22sc1DnYE6yMm4EX8Bnk85zu03HLLpdgP+mQcfJcYE98xPpNw2cqyLLl94UKFyx75JE4kCN5GkHHzWJZlZJskIAEJSEAC5UTAuZYhgSYREnAjYJUdYW4H4jOJXWiMNG7YwWglj+BWfjuAkwsMQEQBJwTkcfKAEcc1owgSAllxjWKXl9MOBAo73PTDqQe3Ew0YMIBm029JXHnllek9f5Zddtk49thjg91hjM011lgjXS2KMcppCWWKMWE0s7uOqwwGPy5IuHbdfvvtyVCGM4Y++Rj6GOIID4xjgo1xJUK4wZC4AdxsiDnA5Yz3xC8gxnDJ4YYhXtmRpw3EGusAXwx14jEQHqwRu/q4P3GVbH24ERODaKMPBGFhnVGjRqXrbZkHImfQoEHpOl++I6wzrlyIAWIkuOmJteSkhPXkZIZxcpMWJxYIAUQC7fOdYKy4gCE+aYu2iQPhRjDiQCjHXHCbQmDy3eC0hluWGCtc+W5yOgF/GFOH7+Iss8wSlIM1MR/kk3CZgiO3OfF9I88kAQlIQAISkIAE2jqBJhMSGHoYXhjqOTQMOgJ5EQB5Hju7uJUcc8wx6TcSuKYT9xuuzMSAxIhj1x1jmjoE3+LmQjArxjRioW/fvoHB3blz5+DqTvIRGMQTUCdPxEJgUGMcU5+6GKYYlnmZ6l4J5KZPjPbqnjdnHqILA5hYBXb0cQ/D+Md4RgjQN0Y6Yggj+aabbgoMbXb9ue2JHX3mjGGLYY6hjdHMbjluQrBAcLDbz1rgMkQ7rB2nFuzqw5Kga1x2YMoasS4Y8og6xlCfhGFPwD3rW1ieUyYEAUKBWAWEDOMkHgLuiAS+Rxj/GPvMl/qcSjBO2uS3LbjSFbGSP6cMogLxyRWx8OC7d9RRR6Xff+B5lmXpSuAjjzwyllxyyYAxpzaMh9MPePMMIYe7F98z6uFuRR7ClVOu6aabjuyKhKBDMGWZpxEVUHwjAQlIQAISkECbJtBkQgJKuHhgEPI+T7i4YDjmn3EfIV4C16M8YQhjdOafec392TF22SHnRIF8rnfNhQDvycsTxnXeT/6K4UtdDEWM06rjy8sVvrIzjSGOa1Rhfku8x+jGMKbvlVZaKZgbRiw74vnYMWIROTwjMT+MZq7X5TMJ8ZFzxxhmbdZdd92gLH1gNDOfLMvSbyTAh3WgLH3TFuIQIUF7eaIc9eqTWGvGUbUsO/h5e7wyT+bMWjEG8kicZjGewvoITsbGOBACiKXC57zn+0IZ2kAodOjQgeyKRL1evXoltpRBPORs+VyY+C5QkRMIRAlrkYtc8kmcrPBDhnyvs0whARNT7QR8KgEJSEACEmgLBJpUSLQFIM5BAvUhgHsW7mCc8BC3gWjjpK0+dS0jAQlIQAIlR5SsoaYAABAASURBVMABS0AC1RBQSFQDpaYsAnl5VhiEy2dT+RHgx/HOO++8wM2OQHVc7PKTsvKj4YwlIAEJSEACEihHAsUtJIpsRbguFD/53O2qyIbncFqQAG5XxNxwbSxxHJ5GtCB8u5KABCQgAQlIoCgIKCQasAz80Bo+8nX9sFoDmrRoiRIgZqJHjx5BrAWB7iU6jWYZto1KQAISkIAEJFAeBBQS5bHOzlICEpCABCRQEwHzJSABCUwQAYXEBGGzkgQkIAEJSEACEpCABFqLQHH0q5AojnVwFBKQgAQkIAEJSEACEigpAgqJklouB9vaBOxfAhKQgAQkIAEJSOD/CSgk/p+DfyUgAQlIoG0ScFYSkIAEJNBMBBQSzQTWZiUgAQlIQAISkIAEJoSAdUqFgEKiVFbKcUpAAhKQgAQkIAEJSKCICCgkimgxWnso9i8BCUhAAhKQgAQkIIH6ElBI1JeU5SQgAQkUHwFHJAEJSEACEmg1AgqJVkNvxxKQgAQkIAEJlB8BZyyBtkNAIdF21tKZSEACEpCABCQgAQlIoMUIlI2QaDGidiQBCUhAAhKQgAQkIIEyIKCQKINFdooSKFECDlsCEpCABCQggSImoJAo4sVxaBKQgAQkIIHSIuBoJSCBciKgkCin1XauEpCABCQgAQlIQAISKCTQiPcKiUbAs6oEJCABCUhAAhKQgATKlYBColxX3nm3NgH7l4AEJCABCUhAAiVNQCFR0svn4CUgAQlIoOUI2JMEJCABCRQSUEgU0vC9BCQgAQlIQAISkEDbIeBMmpWAQqJZ8dq4BCQgAQlIQAISkIAE2iYBhUTbXNfWnpX9S0ACEpCABCQgAQm0cQIKiTa+wE5PAhKQQP0IWEoCEpCABCTQMAIKiYbxsrQEJCABCUhAAhIoDgKOQgKtTEAh0coLYPcSkIAEJCABCUhAAhIoRQIKiYavmjUkIAEJSEACEpCABCRQ9gQUEmX/FRCABMqBgHOUgAQkIAEJSKCpCSgkmpqo7UlAAhKQgAQk0HgCtiABCRQ9AYVE0S+RA5SABCQgAQlIQAISkEDxEagqJIpvhI6oVgJ///13fPTRR/HKK6/Ezz//XGtZH0pAAhKQgAQkIAEJSKCpCCgkmopkM7Tzyy+/xJ133hkDBw6MU045Ja699tokGj777LP45JNPUo+PPvpoHHzwwbHzzjunZynTP2VGwOlKQAISkIAEJCCBliegkGh55vXqERFx4IEHxrnnnhuzzz57LL744vH999/HbrvtFnvvvXd8/PHHqZ1ZZ501pp122hg7dmz63Jb+DBo0qC1Nx7lIQAIS+B8B30lAAhJoAwQUEkW6iJxEDBkyJK6++urYbLPNYq211op+/frF+eefH19//XXFqLt06RJdu3at+NxW3rz88svx0EMPtZXpOA8JSEACEpCABEqcgMMfn4BCYnwmRZHz+OOPR5ZlQQxEPqAsy2K22WaLAw44IM9qk6/fffddXHbZZTFq1Kg2OT8nJQEJSEACEpCABNoCAYVEHav4559/xvDhw+Oss86Kt956q6L0F198Eeecc0488MADFXlN+QbB8OOPP8Z+++0Xb7zxRkXTE000USy//PIxyyyzVOQVviFmgpiK//73v0EsReGzX3/9NR577LEUc3HyySfHfffdV/g4eP7ggw+m58cdd1xcddVVMWbMmIoyH374Ydxxxx1xzTXXxLvvvpvKXXfddRXPC9989dVX6UThpJNOSicol19+eRIHlMFti3Geeuqpcfzxx8fgwYMr4jv++uuvdOrCacz7778fxx13XFxyySWBuKDuBx98EFdccUUwvksvvTQ+//xzsk0SkIAEJCABCUhAAi1MQCFRB/CffvopjjjiiDjssMMCwzcv/sgjjyRjNv9c+MoNSltvvXWsuuqqtSZiHQrrFb7ffPPNY6WVVkoB1tttt12KlSBGgjLTTz99EBvB+8JEMPYtt9wSTz31VBx77LFx+umnVzzGeCdg+9Zbb41u3bqlG54OOuigZLTnhS644IIgb4455oh55503zjjjjNhhhx0CBhjwxCzsuuuucdppp8Xtt98elOd0pNDViraI17jpppuSCOrfv3/wHuOftgkSZ4yMpUuXLrHwwgsHIuPEE0+ML7/8Mp3CIJTIn3rqqWPllVeO7t27x2STTRYjRoxI4yF/vvnmS8Jk2223rQg8p2+TBCRQBAQcggQkIAEJlAUBhUQdy/zHH3/EQgstFM8991w8/PDDqfRvv/0W9957b8w///yx+uqrp7zCPxjB7Nqzu19b4kSjsF7hewKsr7/++ujdu3fqe5999om11147iYTCcvl7DP0FFlggGf+4Ba233npB/fz5XXfdlQxxxNA666wThx56aEw33XTpxqfXXnstPv3002CHHxGx6aabBkKoR48eMWzYsOBkgPyjjjoqGe0TTzxxbLDBBunZPffcEwibvB9eO3ToELvssktMNdVUfIzFFlssbrzxxnSyM9NMMwWnJZy4MB/mt+GGGwacOP3Isiy6jxMOnTt3jmmmmSZ69eoVSy21VBIziBhOMDbeeOPYaqutYs8990ynHgSkp478IwEJSEACEpDABBGwkgQmhIBCog5qk08+eWy55ZZp55wTBFyZuDGJnfVDDjmk2trffvttPPPMM8GpRW1p5MiR1dbPMzt06BC4+AwdOjQJlldffTXt0O++++6Bm1FejlcMfQzwLMtiyimnjA7j6vL7Ejzj9yUQPhjxjGf48OFJnMwwwwyBUMLNiDo9e/YMjPTff/89Ro8ePZ7bUJZlNBczzjhjzD333OlUZNFFF015Nf1p165dLLPMMtGxY8dYZJFFgs9rrLFGyuM9AigfZ01tkP/EE08EAi53NWMOuGIRQ8L4c9cnypokIAEJSEACEpCABJqfgEKiDsbt27eP1VZbLZVabrnl4umnnw6MXwzjZZddNuVX/YOQwPDlBKO2NGLEiKpVx/tMTMRGG22U4gKIa8AYx8UIdyAEwngVqsmgHIY2BnvheGjryCOPTG5M7P6fd9556fTlwgsvTAIGMVFNc43KQjzwuxecUtAPbk6FMSA1NY7bEy5WheNnfLhw4fqFoKiprvkSkIAEJCABCUhAAk1PQCHRAKbsvuMG9N5778XSSy8d7OhXV53Tgf333z/69+9fa9ppp52qq57yOH1AkKQP4/7MPPPM6fpXBAQi5oUXXki/KzHuUb3/I+6hf5UxYYgTy0EMxQknnJDiQTht2GabbWKJJZaod9v1LUgwNa5XxFksuOCCQSwIJyFRj38IImIyqs4Blyd+S6MeTVhEAo0nYAsSkIAEJCABCSQCComEoX5/pphiiuTyw41NK6ywQhArUL+aDS/F7UrvvPNOpYr0RxwEcRm4JNV3F55TFeIVBg8eHPw+Q2Gj3HpEMPTIkSPT7UjENhBfQTA3/RWWbYr3jPuYY44JxAP9cPvUJJNMUmfTxGEgQgh8LywMgxtuuCG++eabwmzfS0ACEpCABCoI+EYCEmgeAgqJBnLlxqNJJ500xUw0sGqDiw8YMCC4ASq/rYmYAIQAac0110xBzhjSPCeugUQnGOvEE/CeG5UQQBjteRzEmWeema5+5XpXbmVacsklUxA1rkPDhw9PwdUIC1yhMN7ffvvt4CrZ/ISEtjkdoP2aEm0xjnx8eTlOc2iHeXGd7ksvvRSjRo1Kt0jhMsb7LMvS7U24Y1Eed7K55poriAHhpihOS3CJ4kf7uE2LeBUCx/M+fJWABCQgAQlIQAISaH4CzSgkmn/wrdFD165d0y1EiInm7B/3JeIIuO2IW464rejwww9PV7pyqxLGNDv5uPlgVGPoc90rgccY2wgCxocrEO5Ya621Vhx99NHB+GmHoGpuacIFq0uXLin4GXHyn//8JwgiJ6ic26oIbsZgJzaBerT5+uuvpytgiVvgc9WEYU88B7c9ISa47QnhQDncq7ipiXnRz913351+ZI/fzOB3LShPgDuigaB2xo+44LpXGHCbFOU22WSTIPidGJLtt9+epk0SkIAEJCABCUhAAi1IQCHRQNj8fgJXkzawWoOL476E4Y+xjAjAFYiga2Ia+JE6hAaNcgMSwoHrXTldmHPOOWPFFVdMP5bH1a3EYXBbE8Z5v3790o/JcdXq/fffHwRt0zbtcB0rV8PyjF3+HXfcMQ488MB0vSoCYpVVVgl+s4E2udqWK2Q56aBu1cTvPHA9LGLhoYceSrdederUKRXD8D/77LPj5ptvDgK999hjjyDGgWtkmdc888wTiDTmwk1TlGHeiCZOTrgyF/HBOLjNCjcpTyMS2qb5YysSkIAEJCABCUigngTa1bOcxcYRwICtzw1D44o2+r8OHTokgxrBwO9S8MNsGPO4VhUa8OQVJgK9MbjzPOrlQeHEPLCzzw/dEeOB6CgcKPEK1KM+MQkEMPNbErgV8QyBwnMSweaF4yhsByHBla+UI9EXV8bmZRAVtEUb3BZFojxjz8sgDrglizLEeOT58CCPdgl+L3yWl/FVAhKQQDkRcK4SkIAEWouAQqIB5K+44orku8/ueAOqWVQCEpCABCQgAQlIQAI5gTbzqpBowFKOGTMmcL3hKtYGVLOoBCQgAQlIQAISkIAE2hwBhUQDlpQfbONUgtuPGlDNosVCwHFIQAISkIAEJCABCTQZAYVEA1DyGw6kBlSxqAQkIAEJNIKAVSUgAQlIoHgJKCSKd20cmQQkIAEJSEACEig1Ao63jAgoJMposZ2qBCQgAQlIQAISkIAEmoqAQqKpSLZ2O/YvAQlIQAISkIAEJCCBFiSgkGhB2HYlAQlIoJCA7yUgAQlIQAKlTEAhUcqr59glIAEJSEACEmhJAvYlAQkUEFBIFMDwrQQkIAEJSEACEpCABCRQPwKlISTqNxdLSUACEpCABCQgAQlIQAItREAh0UKg7UYC5UbA+UpAAhKQgAQk0LYJKCTa9vo6OwlIQAISkEB9CVhOAhKQQIMIKCQahMvCEpCABCQgAQlIQAISKBYCrTsOhUTr8rd3CUhAAhKQgAQkIAEJlCQBhURJLpuDbm0C9i8BCUhAAhKQgATKnYBCoty/Ac5fAhKQQHkQcJYSkIAEJNDEBBQSTQzU5iQgAQlIQAISkIAEmoKAbRQ7AYVEsa+Q45OABCQgAQlIQAISkEARElBIFOGitPaQ7F8CEpCABCQgAQlIQAJ1EVBI1EXI5xKQgASKn4AjlIAEJCABCbQ4AYVEiyO3QwlIQAISkIAEJCABCZQ+AYVE6a+hM5CABCQgAQlIQAISkECLEyg7IdHihO1QAhKQgAQkIAEJSEACbZCAQqINLqpTkkAbI+B0JCABCUhAAhIoQgIKiSJcFIckAQlIQAISKG0Cjl4CEigHAgqJclhl5ygBCUhAAhKQgAQkIIHaCEzAM4XEBECzigQkIAEJSEACEpCABMqdgEKi3L8Bzr+1Cdi/BCQgAQlIQAISKEkCColz2qBOAAAQAElEQVSSXDYHLQEJSEACrUfAniUgAQlIAAIKCSiYJCABCUhAAhKQgATaLgFn1iwEFBLNgrU4Gv3rr7/it99+i99//704BlQEo/jzzz/jl19+CV6LYDjNOgTWn7k2xfr//fff8ccffyR2zTpoG5eABCQgAQlIoGQIKCTqWCoMzhdffDHuuuuuSun1119Pxujbb79dKf/ee++Nzz//vI5WJ+zxq6++WqmvqmPi83333ZcaHzt2bFx55ZWxwgorxAknnJDyWuFP0XV5zz33xMILLxxDhw4turE19YCef/756NKlSwwYMKBRTSNEaOvggw+OmWeeuVFtWVkCEpCABCQggbZDQCFRx1qyEzt69Oi45JJLYtNNN00JY/3TTz8NdnwRDYMHD4711lsvDj/88Hj88cfju+++q6PVCXtMnw8++GBstdVW0adPnxg+fHi88sorKT3zzDNx1llnxWabbRb8++mnn+Lbb7+NkSNH8tH0D4EFFlggDjnkkFhkkUX+yan7hfWEM7v7dZcunhKzzTZbHH300bHWWms1alCI6e+//z6GDRsWCNRGNWblEiDgECUgAQlIQAL1I6CQqIPTxBNPHBtssEEyPmeYYYaYcsop48QTT4xVV101Jplkklh22WUD43T77bePq6++Oo488siYd95562h1wh6vvvrqccwxx8SKK64Y008/ffTr1y+NC8P4qKOOissvvzzmn3/+1Pgss8wSPXr0SO/98z8Cc801V+yyyy4x33zz/S+zjnfvvfde3HjjjfHrr7/WUbK4HnN6sOeee8bKK6/cqIG1b98+evXqFQiTRjVkZQlIQAISaB4CtiqBViKgkKgH+CzLYsYZZ4xJJ500sixLYoJq7NReeuml8dprr8WZZ56ZXGYmm2wyHjVbmnzyyaNTp07jtY/gmXXWWWPvvfce75kZE06AU6dBgwbFBx98MOGNWFMCEpCABCQgAQm0QQLt2uCcWmRKX375ZVx00UXJlem8886Ljh07tki/NXXy1VdfpR1zTkZqKpPnE4D95ptvBi5axFWMGDEifvjhh/QYw/mxxx6LO++8M6XnnnsuubM8/PDD6TP5TzzxRAq6xeWHz6R333031a/uDz727Orff//9KcbjqaeeSm0Wlv36668D9yzaeuCBB+L9999Pwb2Uwb1s7NixQazKs88+m/oeOXJkEO/wwgsvVJSjLHPDn59nuevXJ598wqOUeE8/H3/8cfrMn59//jlgQJ1hw4bFyy+/HLiR0efFF18c//3vf+PDDz9MvKhLHRLMcWVjzDDDlYx8GH755Zfx9NNPB1zgRD14w50yhYm5w+Tuu+9OfVAHkcoYWB/aJ7300ksBC1ysaI88UmFbhe8ZH7wQuuQTLE2b1GU+X3zxRXKPY134zLgpl6fPPvssPWcM1Pnxxx/zRxWvrC3xQrRBfNA777yTnjHGJ598suI7wzhxA2RdeU/icyrsHwlIQAISkIAESpKAQmIClg1j9Nxzz00GLYbmTDPNVK9WMF5xQ6orXXDBBQ2Ks8B4vuGGG+Kjjz6qcxwYk8R7IIIwxjG2CaLlROWbb75JcR/EYey4445BwsDEKCQ4mdiMnXbaKTBoMSDJp9/99tsvGczVdY7hi4HMnDD6Ma732GOPGDhwYIWYeOutt+KUU05JRidG7xVXXJFOVjBMaXPsOBFB4Hjfvn3j2GOPjSFDhgQnQZy+bLvttsnYpRyJeV122WVpjAiDXXfdNR555BEepXgR5kk7w8YJhpQ57s/NN98cgwYNSgICgxj3NLggMJgnLm2IhDfeeKPiZIL3tIWhzpwY14ABA5Igw1g///zzUyzLVVddleJrECOMZYcddggC9Md1m/6jLMHwiAziMP7973/HbrvtlgTqmDFj4uSTT471118/jjvuuBg1alSqwxrCcp999knCI2VW+cMJCuu89dZbJyHEY9iedNJJKb6G8cGZMtttt13iinCgHIkxMicY8j2BKevEszzh6sX/Dq699tpgPLS18847B6KTMcKof//+afywQTCRKMP/bhA6eVst92pPEpCABCQgAQk0FQGFRANJsiu71157JeN1k002CfzH69vEdNNNF5tvvnmdifgLXJhqa5cAcPpfZpll0s1MGJrsfNdWh2cYyBjlG220URx44IGBoceYLrzwwrj++usDFykM/dVWWy3NbeONN47OnTvHYYcdFgsttFCKC0FQTD311IGAWmeddWL//fePnj170vx4iXFiDK+yyipxwAEHpBgS3mOwI1zYeedEB0OdfhE1xx9/fOobwxvRRlzKYostFgT8YgzPM888ceihhwbGLbvhgwcPruiXGBLmdtBBB6Xgd/jkD+eYY47gc6HBjNBhd5z5woOA+V69eqVTDmIMWGteuekJw51gdsb9n//8J4i32H333VNAc+/eveOaa65JDIlfIVYFgYeIoW04kxAet956az6kwKhm3fbdd99gzIgKjHJOVeiTuJsuXbrE3HPPnWJjsiyLqaaaKhZddNFkoMOqorGCN5yQLbnkkukkJc+ec845Y/HFF0+ijzmwdqeeemridMstt0QuFDDwEXYdOnRInGGNuGG987Z4ZR6IPZ6xtrSFeCDAmzlxOoa46Nq1a/peTTTRROkCgOWXXz6d5nXr1o1mTBKQgARqJ+BTCUigaAkoJCZgaTB+cWXB8MNYrG8T7du3jwUXXLDOhOGIQV9buxiF7KTjcoKxihFel/jAaMZYnmaaaVKQOIYddbhxCkMdI5YyxIOsscYa6VTk0UcfTcPAmEZUsEue5+EKw440t1kRP5IKVvlDYPoUU0wRBIozJ8pxGoF7FAYmRj1CAOMyj/1gbltssUX6DQzGRB3yaAcxw5W2BP7mQcSIjbxbbqvCoEeYYHBj/NMvzzHwmQfvCxMnObhTYVzTB8H1nEIUlil8j5sWLjwY6uy88x1gXRE87PTDFNFCW3CELeOlfJZlyW2K9h566KHAEEdMTDvttCn+Bg64RSHOWB/aQdxw6kG/1OOUhLrLLbdcIOjIq5oYC3PNsqziEX1gvDM+LglgzJShT059YEdhTj44iUBoICYYR/fu3YMYHJ6TOK3hVAgxS7ucTiA0EGq4ZnGKQT3mjkjltAWxy8kElwKw1u3a+X9+YGmSgAQkIAEJlCqBmv4/eanOp9nHjYGGAcVONb7j22yzTfD7DhjVdXWOgcZufl0JlxJcQOpqL3+OwbrhhhtWG4Sdl+EVo504AwxzRA15JHavMVhxvcp9+DlhmH322ZMLEsYyxjqGH6cBuMRgdGLMrrTSSrX2O2zYsHSKgfFKX1VT/rxwPJRhR58TnDvuuIOP9U64EOF6BQ+uw8XI57SgpgayLAvWEncchBJCAKOYU4Sa6hBPgv8/oixfS/rCMC80tmuqn+fzvUG4sX55XnWviBGMdAQXIpbYAvgvscQSSXxUV6cxecOHD08XC9Q2Lr6fcMBFKWfAK/PhtjBERD4GBB9CF5c0yiA88me+SkACEpCABCRQugQUEhOwduxWs0uMSwcuIbyyY1yXmMAV5vbbb4+6En7yGPcNGRquLhjetdXBLYvnxDZgBPKehMHIzjPvOa3gldOCddddNwVHE3zMKQQuSVwnSmAyJwqciDB3yteU2OWnL9xlqiuDCwzj4bXwOePhBqx8PIXPantPDALxDowV96M+ffok3rXVYVcddy8MYIQEbRDXgdFeUz121M8+++zUdtX1rKlO1Xy4kDd69GheakyIE8ZIPArsEUpZlgXir3KlpvmE4K1vS8SoVJ0/LnKFv10BR3ghDnF/IoC/vu1bTgISkIAEJCCB4iWgkJjAtcEw4uQAY5VdfnzE8Q+fwOZapBqGJ0YpRj3BuHmnGPLEH/Ajbfz+RJ6PEY4bDcIG1xfci3A5YjecoGba6tKlS1682ldcaOgLn//CAsQBcGsQz2mP4F7cZfIyGNn0jdtUnlefV9xoEFUETF933XXJJx9xwA56TfWJkSB2gDoYxbgLEZsAp+rqIHBw8cItiR34wjL0X/i5tvf8lkWWZYEgYw3ysgg+XINYE/I4QSLgmlgVvme4YRHHUdMpD3Uakzh1Iggc962q88vbpW/GxXxZ3zyfV4RRoVjg5A43M06LWGNOcjj9oqxJAmVPQAASkIAESpiAQqKei4dxh4sPxdlh5RUXDW424iYcDL8111wzCKbFAK7JAKNeYxJjwEWJ9vNx1NRe/jw/KcHwI4iWAGWMV1x4qItRTJwAfvl8zhP+9AgHbhTCpQvxxKnHlltuGZzEIAKijn8E6mKUEkvB1Z8YkoiG2267Ld1whA89rkcE7iLEmBeJ97hSEYRMF8yB/Pw1z+M1z+c9gb4IFFylEAe4niFU4MZz6he+Uhf3KYxm6mBEE6yNYZ/zozz14YXbD/EOzJ3AcGI4mA/8CHxmfpSnH9rmlc+k/D2vPOP7QowCpx/s4nMqw1h5T+wLgoZ6JH70EE6cBBFvQFwD+bUl+uF5/sp75sFrYV7+Pn8l2J915qYtYk3gwHeaV+ryfeHECPcv1hSRg5sbp0+MG6HAqR3t8b8HYng4uWDM3GLFHBCnzJX2TBKQgAQkIIFSIuBY/0dAIfE/FtW+w3jCGOLkgSBYdsoRDvi38wzDDMOeXWp2WYkZwPjFsMI/v9pGJzATFyNuKsJYY9eXMWGEVtccu8S4+GDMYbgRHI4hjAjYb7/9gutczzjjjOSaw+47/v3EFVRti/iB7t27B37uPMP3HbcVXG2oQ15tacUVVwyuu+XUBuObnXTaxL+fExLaO+qoowIDnleuEr3pppuCeQ0YMCAIkIY788ZYhwEnCLj3EDMQ4/5hzFKPHXwEACcpzJ2ga26HQuwRYwAHAn1ZQ664RTyMq55uMYIJHGiHcsyPOjxnnogpTiwYC0KCNWZuGMS47DAfBBrX4xJsjKsUAop5ID4Y74033hgICPrmVIEgcPrjZAcRR6AyAc6c1nCTFn0XJtrmJIDrWnFHK3xW9T1zYz58P/m+ENPBd+D0008PDHhiQjglwiWPcVGfsTD3eeedNxBFnMggKpgj3zWCvVkz1gL3J9zcEJqUy0URYyQOokuXLsktjvngnkaf9MF6I0Lgw/oS70G+SQISkIAEJCCB0iOgkKhjzTD8MB65fpNbZ9iBxohj1xsjncTuLTvhGGmIDMpirGLE1dF8gx6zE4wYwKDlNiQMWozW6hoZO3ZsYOCedtppQawD42U8k0wySbqClZMJ3E0wOLkF6Ygjjqj2R/Uw/ukPYzfvB4MXo59d5zyvplcMR9omKJd2uBIURhiYPKMeQoXdbwxkRBnsMKw5GeA5RmiHDh3SuMmHA4l8mGPQwoVE3AJXtOLKRKIv+qQdhAjuW+TxivDIsixdX0ugNUYtxj83PSG48vFxqsL6UiZ3tcJw5mrTc845JxBkjJ+Aa06paBe3Jb4vnCIg+mibZ8QIEIPB+rDLj/DE4Gce/fr1C045MOL5ffmGkwAAEABJREFUTjHmqolTIm6Vqppf9TNz5/YtRACihNMCxCWxI1zHi/Dhe4345VSE/skjqB5RzEkOa8aaMBYCvvnOkYdYIN6HfC4ewGWJ39Rg/gTiw5/x8H0jlghRkZ/UwIarhPn+INTyfMqbJCABCUhAAhIoLQIKiTrWi11jjF7cNQpTz549K24jKszP32MEIzjqaL5Bj3ElydvPX9ndr64R4gR4lpfDCMY9ibIICK5jxWDGMMe4xMjlWdXE/OmX1/wZgeC0j6tUnlfbK/1yisEuPrf3cA1qYXu4MGFsspNNDAAnClzziuihXXbB83nwygkD40dA8JlEHsIGI7dv377pNzL4wTpODfJxsntO2Tzh+kT7GObUy/Mx7nFz4hkJ452+EGR8zhNCjX5hiPGMYcwzhFHeFq8IGQxx3ucJ456desrTPu3wDDchduzJr5qI+UCE1PS8sDxij/byxNgRB/lnXlkTTl54nyfmQTt8d+HA3PitDMaIiEJAIVAoQ4IT1wfzXSLwPmfKM0QU7fJ7INwARh6nXuTliXUn39RGCTgtCUhAAhJo0wQUEm16eZ1cqRPABYp4FuIMSBjlpT4nxy8BCUhAAsVLwJFJoCEEFBINoWVZCbQwAU5wcLPiR+s4qclPaVp4GHYnAQlIQAISkIAExiOgkBgPSWtk2KcEqifANbsDBw4MApxxwcqyrPqC5kpAAhKQgAQkIIEWJqCQaGHgdieBhhDo1atX+uVt4hUIgm5IXcs2MwGbl4AEJCABCZQ5AYVEmX8BnL4EJCABCUigXAg4TwlIoGkJKCSalqetSUACEpCABCQgAQlIoCwItICQKAuOTlICEpCABCQgAQlIQAJlRUAhUVbL7WQlUE8CFpOABCQgAQlIQAJ1EFBI1AHIxxKQgAQkIIFSIOAYJSABCbQ0AYVESxO3PwlIQAISkIAEJCABCUSUPAOFRMkvoROQgAQkIAEJSEACEpBAyxNQSLQ8c3tsbQL2LwEJSEACEpCABCTQaAIKiUYjtAEJSEACEmhuArYvAQlIQALFR0AhUXxr4ogkIAEJSEACEpBAqRNw/GVAQCFRBovsFCUgAQlIQAISkIAEJNDUBBQSTU20tduzfwlIQAISkIAEJCABCbQAAYVEC0C2CwlIQAK1EfCZBCQgAQlIoBQJKCRKcdUcswQkIAEJSEACrUnAviUggXEEFBLjIPifBCQgAQlIQAISkIAEJNAwAqUlJBo2N0tLQAISkIAEJCABCUhAAs1EQCHRTGBtVgIS+H8C/pWABCQgAQlIoG0SUEi0zXV1VhKQgAQkIIEJJWA9CUhAAvUioJCoFyYLSUACEpCABCQgAQlIoFgJtM64FBKtw91eJSABCUhAAhKQgAQkUNIEFBIlvXwOvrUJ2L8EJCABCUhAAhIoVwIKiXJdeectAQlIoDwJOGsJSEACEmgiAgqJJgJpMxKQgAQkIAEJSEACzUHANouVgEKiWFfGcUlAAhKQgAQkIAEJSKCICSgkinhxWnto9i8BCUhAAhKQgAQkIIGaCCgkaiJjvgQkIIHSI+CIJSABCUhAAi1GQCHRYqibt6PffvstXn311Tj++ONj5MiRzdtZLa2//PLLcf7558fzzz9fS6naH/3555/xzjvvxFlnnRUPPvhg7YWb6OmYMWPijDPOiMsuu6yJWmxcM/A75phj4u23325cQ+Nq//LLL/H444/HxRdfHH/88ce4HP+TgAQkIIHiIeBIJFC6BBQSRbx2m222WSy00EK1pn333Td+//33ePLJJ2OvvfaKU089Nd59991WmdXtt98eBx98cJxyyilJCEzIIP7+++9AjCCIMKRfeOGFCWmmwXW+/fbbuPnmm+O+++5rcN3mqICAuPLKK+Pjjz9udPMnn3xy9OvXL4mkv/76q9Ht2YAEJCABCUhAAhKAQNkKCSZf7OnGG2+Mc889N958882Ye+6546GHHkqnDpw8sFO/zDLLxIcffhiTTDJJrLLKKtG3b98kKlprXhtssEEccsghwQ74hI4hy7Lo3r17bLfddjHttNNOaDMNrjfvvPOmXfvrrruuwXWbo8JWW20V77//fvTs2bPRzR900EHp+4HgbHRjNiABCUhAAhKQgAT+IaCQ+AdEsb4gFjByq46vc+fOyYVolllmqfqoTXyebLLJYuKJJ24Tc3ESTUbAhiQgAQlIQAISKCICCokiWozqhpJlWUw++eTVPYopppgiuTNV+9BMCUhAAhKQQKsTcAASkEBbJqCQqMfqfvnll3HppZfG8ssvn4JVr7322lhppZViww03bJJg2HoMYbwiTz/9dHz11VfRrVu38Z4VZhBzQJzBjjvumFyGGPfll18e33//fSpGbMWCCy4YeRoxYkTypc8/L7zwwnHOOeeksvjak7/eeuvFSy+9lPJq+oN70zXXXBPLLbdc9OjRI8UfEBBeWJ6+iQOhTVx5Xn/99cLHFe+JmfjXv/6V2srHkj/85ptv4uijj07PaAc3Hty9eH7nnXdWzItnAwYMIDs+/fTT2GmnndIzYlDIJCbhiCOOSLEEfC5Mp512WnIxoo0DDzww3nrrrfSYOtQnH76ffPJJyt9yyy1T2+QTOE6A8yuvvBJHHnlkap9g+HXWWSeIAakuZuHXX3+NBx54IHr37h3PPPNMavO7776Le++9N9Zdd9245ZZb4tFHH42NN944zfv6669PZfI/9El9XM0YA2MbNWpU/rjile/1SSedFEsvvXQsu+yyad0ZzxdffBG77bZbxRw4Fcvd7BZZZJHg8w033FDRjm8kIAEJSEACEmhDBBowFYVEHbDwK7/jjjvizDPPjKeeeiowjrkBB2OK1y222CIw3KprBn/7zp07R12pT58+1VWvlIdxiYH3+eefxxtvvJEMc4zoSoWq+YBBiSG50UYbJaOUvjBoMWIRExiQu+66axJEu+yySyyxxBLBnNZee+0UtI3Bvc8++6SWDz/88Jh66qkDA3W++eZLedX9QbwMHjw4Bg0aFHPOOWe8+OKLgRAgmJnyGKtDhgyJE088MXFljMyvd+/e4wUXf/bZZ3H22WengHPqHnDAAXH//ffzNnHfZJNN4rnnngsCvXNjl8DiH3/8MRA8hx56aLz33ntpXgSjU3HmmWdOQeHM4a677kp9XnTRRXHJJZekmBPKkLjJafHFF08cGO9NN90Uw4cPj/XXXz+xnGeeeVLMymyzzRYffPBBEpnUoy245sLo66+/DlhceOGFqa2rr746YAAPXqlTmB577LHktsacmAdCBOF2wgknxD333BMPP/xwYMjzHWSMRx11VKVbsuBz7LHHJjFAPM2KK66YgvEL+0Bs9enTJzp16hT017dv3xTfwhhnmGGGoK/VV189fS8ow9ognFmP/fffPzEobM/3EpCABCQgAQmUHwGFRB1rTiAzRjA7+RTlBOC8885LBvCqq66aAmKr2+2lLLvsGF51JYxMyteW2NHGYOSWJgx6DMssy2qrEuw477HHHsHuN0Ji0kknTQHZGNScSiCE2rVrl4zCFVZYoeKmpammmiq23377mH322QPRkndCezPNNFPssMMO0b59+zx7vNeff/45evXqFbfeemsgpkgY7wMHDkxlCSLGKEfQzDHHHEGcxzbbbBNjx44Ndv9ToX/+jB49OgWcM+fTTz89GeCcmvAYY5hTDdZmxhlnTDvoPXv2DAQXpzUx7h/zIFGWXf1xWamN/JQDoTPrrLMGYglBwHMS4pDTDwQCJzHMe4EFFgjGzQ1PZ511ViAQKEsgPK956tChQ8w111z5x6Au60Y52uPkA2GCwV8QB1JRfrXVVgtONaabbrqURxnmiNBjvWAPJ5gwRsaRixbGdthhh6XTirXWWiuyLIttt9021lhjjdQWfxDHV111VSCSWEviURCPnK4gvGCFmOD7hhhDqFGedWTeW2+9dY3udrRvkoAEJCABCUigPAgoJOqxzhhyGOEUZReYV1LHjh2D3feqLjs8I2HkYsjXldjBpnxtackllwzKYczhWoUBiEFZWx1udsJwXWyxxSqKTTTRRMlNB7EwdOjQlI8BzQ46n3MDnHK4xeA2gwsPBemX25RqitmgDInYDXb7KZdlWRIVa4874cBNh+tMMVSfffbZtOues2GXHgHCTjtt5Ilx0Q6fp5lmGl6S4OAN4oEd9E033TTYmcc4zw1qnpOyLEtGNacS7OSTx0kM7k246fC5usTJD65ElMkNesohPJjLE088kU4hyGtIQkzQHjdSIa4aUheDP8uyWHTRRYP31J1++umTMMq/g4wZwUcZvreU4XuCMOA9CcHG71RwErH33nsHa4BoGDVqVHACgisc5RBA3BrGmPv3759ichDHPDNJQAIQMElAAhIobwIKiWZcfwxxdrrrSrjKNGQYGIa4meByUls9DHee5wYl70kY4F26dInXXnuNj8koxTjOsiyuuOKKdIUsxiQuQj/88EPgpsRON+1xspEqNeAP40VYIDAQNhi9jCF3m4IPvz1B+1xxW9+mp5xyyujTp0/cfffdyVWJEwdOOKrWZ+edOAB21XnGj90xBkQBn6tLjJM5M/aqz7t37x4fffRR8Lzqs9b+jLsUIjAXGtWNB1epn376KcX4cFICfxIiBCHWc9ypTl6PUyncyeBBbEh1rlh5WV8lIAEJSEACRU/AATYpAYVEk+Ks3Bi7zrhC1ZVwralcs+5PGOVVBULVWrgMYfgVuidRJsuywEVmqaWWivwfgeS47vCDdhiM5PPbFAT34oKDEb7mmmsm0cGzhiZ2walDP1mWJUOcU5GqbBrCgp13RAK767g97bzzzoG4oJ/CRIwKrj2cghBjQMwGpzTViYS8HmuHMU5cDG5OeT6vGOrs0nMawOdiSogE1hz3pdrGxfpzisM8CteAuI9CLpzecFKFe9hll10WvOcUrra2fSYBCUhAAhKQQHkQUEgU+TpjtHED0oQME+MZoxAXFvzo8zYw6nFhQSjkebgP4S6FcY7fPW5buBIRWMtuNDcFFQqPvF59Xr/99tsUS0IcBOUxwDHS8fNH5DA/Tgg4IcnjKChXV+JWJmIdiAHghAPjmXaoh/GPQc17Eicu888/fxA/QB1iQsivKXGywYkSpw4EgxeWw00KAYNAIR/RAc+8b+ZTyJsyLZWIdcAtC8HEqUNhv/Dh+4TYYn6cPhHzwZpTjjFzOxnxOHwm1gSXJ2JqEJjcEsUpErE1PDdJQAISkIAEJFDeBBQS9Vh/bkdi95ai+RWfGI3EE2Cw4vPPs+ZIuOEQcEwfed/V9cN4MHAxnnPDED95DGeuG73gggsCNyUMY4Kgcc/hNp/CtnCx4kSAeeXPMLi7dOkSGJ718elHuGCwEh9CkDnjwd2GvnfffffUHW5O3LZ02223peBvbo0iMVYCjWHKXImZYN6p0rg/jGvcS7pZCSOZ3XcMY1yvmBMxJOygU44+MYQpT8JFh/kRpE6cC8Y0+Xlijc5rglEAAAXPSURBVOmPOggBnnPVK+5PBFsT4M24uHaVExBiRfK4A266Io5k0KBBcd999wWnN7RH2xjmBJfTLgyIQ2ANeFZToh/KMw5iOSgHU37hHJ4IBfJIPOfGK3jxHSCwHtFEvAiXAnC6hOGPeOJ7xE1QCMnevXun0yViJHBhY214RdgRF8LaDRgwIHgPL+IliKVgHPx6OW5ojJMxmNoqAeclAQlIQAISqJ2AQqJ2Psn4xmhk95ui+I9jSOHqgW8+Bm2fPn2S8cjzpkzsAHPVKn2wS0x8AoZfdX2cccYZ6ZpUjGt+V+H4448P6nGiQPwBBi4nCvxuAwbwcccdF5w6FLbFzvrBBx+crnfl5iGecWsVO9LcwsRz8mpL7NQTAP3uu+9Gz549AyGCUcrvFeBORF2ukGWnmzyCr7lSl/J77rln+o2CK6+8MvDdx5jGOGfMiAzWAeEwdOjQdHUsNwph/CJIuIWKW4cYK2KCcXOiQn8k3MC49QhBBFfy8oRRTMwJY8Dg5sYkDHR4IVBw9+HmLuIsEChcW8tpD+5BtMGNSowFscbcmSenH5TBdYg5YrjTPom+uAmJutUlrl5lfVg/4hNgxWeuIEZIIGz4DnL6wHwRHDzLA6K5LhdBwekOgo0+u3XrFpxAIYyIrWE94cz6wJOrcxGXXBuLWCDImutwEROIC8aJKEK0wIv5PPLII2SbJCABCUigWAg4Dgm0MAGFRB3A8ePndyTYTcaIZYcXNw9uTyIPw47dYAzNOppq8GPcidiRp1+MO1yMMPCqa4irQNkFZzycniAmiKMg4bKE0YdBSLwDRjonD1mWjdcUxjzl8wcYy7gOYcRn2fjl83L5Ky5SXA/Kzjf9EWPA2DiFILYgL4dQwUhmTsyN8XFdKQY/wgzxwY43u/cYuYghApxhAXeMeVyLOIXA4MYYxjimLicSBA8zlrw/XokZ4USAUxM+54kdd3br2dEnIR44fWHuiAIMatyuOJXAOOcH9vJbvGiDdjkVwTWINUNwMGfEGycsCA3mSdsIPX6LApFC3eoSxjwiirWEA8ICYcj6Mn9OnPgOcuqA4KEcfSPMYNy1a9d0wxfPEKCsKXNgXowNtzKEFqdO5NEm/fGdYV04jYEvAoXvHyKEcSIk6Is58J2HN/kmCUhAAhKQgATKk4BCovHrbgsSkIAEJCABCUhAAhIoOwIKibJbcicsAQlEyEACEpCABCQggcYSUEg0lqD1JSABCUhAAhJofgL2IAEJFB0BhUTRLYkDkoAEJCABCUhAAhKQQPETqEtIFP8MHKEEJCABCUhAAhKQgAQk0OIEFBItjtwOJdDcBGxfAhKQgAQkIAEJND8BhUTzM7YHCUhAAhKQQO0EfCoBCUigBAkoJEpw0RyyBCQgAQlIQAISkEDrErD3CIWE3wIJSEACEpCABCQgAQlIoMEEFBINRmaF1iVg7xKQgAQkIAEJSEACxUBAIVEMq+AYJCABCbRlAs5NAhKQgATaJAGFRJtcViclAQlIQAISkIAEJpyANSVQHwIKifpQsowEJCABCUhAAhKQgAQkUImAQqISjtb+YP8SkIAEJCABCUhAAhIoDQIKidJYJ0cpAQkUKwHHJQEJSEACEihTAgqJMl14py0BCUhAAhIoVwLOWwISaBoCComm4WgrEpCABCQgAQlIQAISKCsCLSgkyoqrk5WABCQgAQlIQAISkECbJqCQaNPL6+Qk0EgCVpeABCQgAQlIQAI1EFBI1ADGbAlIQAISkEApEnDMEpCABFqKgEKipUjbjwQkIAEJSEACEpCABMYnULI5ComSXToHLgEJSEACEpCABCQggdYjoJBoPfb23NoE7F8CEpCABCQgAQlIYIIJKCQmGJ0VJSABCUigpQnYnwQkIAEJFA8BhUTxrIUjkYAEJCABCUhAAm2NgPNpwwQUEm14cZ2aBCQgAQlIQAISkIAEmouAQqK5yLZ2u/YvAQlIQAISkIAEJCCBZiTwfwAAAP//OfJeBAAAAAZJREFUAwCYLUB3wBvcDAAAAABJRU5ErkJggg==\"\u003e\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eFoaming capacity and stability\u003c/h2\u003e\n \u003cp\u003eThe foaming capacity and stability of protein hydrolysates were determined using an automated liquid hander (Hamilton Microlab\u0026reg; STAR, Reno, NV, USA). The hydrolysates were diluted in Milli-Q water at a 1:1 ratio, and 1.8 mL of each diluted sample was added into the wells of a micronic tube plate. Foam generation was facilitated at room temperature (23 \u0026deg;C) by the conductive tips of the automated liquid handler, which pipetted 300 \u0026micro;L of sample from each well and dispersed it into the same well at high speed from the height of 60 mm. This process was repeated 15 times to generate the foam. The sample height was automatically measured before and after foaming using the conductive tips. The foam height was calculated by subtracting the sample height before foaming from the sample height after foaming. The foam height immediately after the foam creation was considered as foam capacity. To determine the foam stability, the drop in foam height was measured at each 5 min for total 55 min. The measurements were taken in technical duplicates for each biological replicate.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eSuspension stability\u003c/h2\u003e\n \u003cp\u003eThe accelerated physical stability of the samples was determined using Lumisizer\u0026reg; (LUM GmbH, Germany). The instrument measures the transmittance (%) across the sample during centrifugation to determine physical stability. Each sample was diluted in deionized water at a 1:1 ratio and loaded (400 \u0026micro;L) into rectangular sample cells. The sample cells were then placed horizontally within the in-built centrifuge of the Lumisizer\u0026reg;. The analysis was conducted under the following parameters: centrifugation speed of 2300 g, temperature set at 22 \u0026deg;C, wavelength of 865 nm, total analysis duration of 60 min, transmission profile recording time of 30 s, and a light factor of 1.0. The instability index, ranging from 0 to 1, was calculated using SEPView software (LUM GmbH, Germany). A value of 0 indicates very stable suspension, while a value 1 indicates complete instability (which is characterized by complete phase separation and 100% transmittance).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eEmulsion stability\u003c/h2\u003e\n \u003cp\u003eEmulsions (6.8 mL) were prepared by homogenizing the hydrolysates with rapeseed oil using an Ultraturrax homogenizer (T25, IKA, Germany) at 13,500 rpm for 1 min in 15 mL falcon tubes. The resulting emulsions had protein and oil contents of 7% and 12.5%, respectively. The stability of the emulsion was determined using the Lumisizer\u0026reg; following the same procedure as described in the section above.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eGelation\u003c/h2\u003e\n \u003cp\u003eFor gelation, protein hydrolysates were prepared according to the procedure described in the section \u0026ldquo;Partial protein hydrolysis\u0026rdquo; above, but in higher volumes (500 mL) in two biological replicates (n\u0026thinsp;=\u0026thinsp;2). The gelation experiments included two steps, including emulsion preparation and fermentation of emulsions, which is described below.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eEmulsion preparation\u003c/strong\u003e The emulsions were prepared by mixing hydrolysates (6.4% protein w/w), rapeseed oil (10.5% w/w), sucrose (1% w/w), and glucose (1% w/w) using T 25 Digital Ultra-turrax\u0026reg; (IKA, Germany) at 13500 rpm for 2 min. The total weight of the final emulsion was 571 g. The emulsion was further homogenized using a high-pressure homogenizer (PandaPLUS 2000, GEA, Italy) at two stages (150 and 50 bars) in one pass. The homogenized emulsion was pasteurized at 90 \u0026deg;C for 10 min in a water bath and then cooled until the sample reached a temperature of 43 \u0026deg;C by placing it on a cold water for subsequent gelation step.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eFermentation of emulsions\u003c/h2\u003e\n \u003cp\u003eTG (3% v/w of protein) was added to the pasteurized emulsions and the emulsions were then inoculated with Vega\u0026trade; Harmony (0.02% inoculum). The inoculated emulsions were transferred into the sterile sealed cap containers and fermented at 43 \u0026deg;C for 20 h in a dry oven. Another set of gels was prepared using the same method but without adding TG. Initial pH (immediately after the inoculation) and final pH (after 20 h of incubation) of the gels were recorded using a pH meter (Testo 205, Buhl \u0026amp; B\u0026oslash;ns\u0026oslash;e, Sm\u0026oslash;rum, Denmark). Gels were further characterised as detailed below.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eGel texture analysis\u003c/h2\u003e\n \u003cp\u003eProtein gels were subjected to a penetration test using a Texture Analyser (TA.XTplusC, Stable Micro Systems, UK) to measure gel hardness as described in Masi\u0026aacute; et al. (2022) [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]. The hardness was measured at 20\u0026deg;C in the same containers where samples were fermented. The height and diameter of the container was 70 and 55 mm, respectively, and the height of the samples used for gelation was ~\u0026thinsp;30 mm. Once gelation was completed, the gels were penetrated to 6 mm depth with a delrin cylindrical probe (P/10, 10 mm diameter, 45 mm long) at a 1 mm/s speed, return speed of 10 mm/s, and trigger force of 1 g using a 5 kg load cell. The software Exponent 32 (Stable Micro Systems, UK) was used to process the results and the hardness (g) was obtained as the maximum value detected during sample penetration. The measurements were taken in technical triplicates for each biological replicate.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eGel rheological measurements\u003c/h2\u003e\n \u003cp\u003eStorage modulus (G\u0026rsquo;) was measured based on the method described in Masi\u0026aacute; et al. without any modifications [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]. The gels were sliced to 1 mm thickness. The linear viscoelastic region of the gel slices was determined by performing a preliminary amplitude sweep test from 0.01% to 100% strain at 1 Hz for each sample at 20 \u0026deg;C using a rotational rheometer (Kinexus pro+, Malvern, UK). A strain of 0.1%, which was within the linear viscoelastic region of the materials, was then used to perform frequency sweep tests from 0.01 to 10 Hz for all samples at 20 \u0026deg;C. The measurements were taken with a parallel plate-to-plate geometry with flat surfaces (65 mm diameter lower plate and 20 mm diameter top plate) and a 1 mm gap. For the technical duplicates, two different slices of each biological replicate were used.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eMoisture content of gels\u003c/h2\u003e\n \u003cp\u003eGels (1 g each) were dried on an aluminum sample plate in an oven at 105 \u0026deg;C for 4 h, and their weights were recorded after cooling them to room temperature in a desiccator. The drying process was repeated (1 h for each cycle) until constant weights were obtained. The measurements were taken in technical duplicates for each biological replicate. The moisture content was calculated using the following Eq.\u0026nbsp;3:\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eThe results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;one standard deviation (SD). The data were processed using GraphPad (Prism 10, version 10.1.2) and OriginPro 2020 (version 9.7) software. One-way analysis of variance (ANOVA) was performed with Duncan test using IBM SPSS software (version 29) to determine the statistically significant differences between the means. Differences were considered significant when p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cp\u003eIn the present study, partial hydrolysis of pea protein isolate was carried out using Alcalase, Neutrase, and the less studied Novozym, to modify its functional properties. As proteins are known to change their structure and properties as a function of pH and temperature, all hydrolyses were carried out at identical pH (7) and temperature (50\u0026deg;C) conditions, to eliminate protein modifications due to factors other than the enzymatic hydrolysis. The selected conditions fall within the optimal working range for all investigated enzymes. In all experiments, the DH was determined and used as a measure of the level of hydrolysis in the discussions. The least explored enzyme in the literature, Novozym, was studied at four different concentrations (0.01, 0.05, 0.15, and 0.5% based on the supplied enzyme preparation), while two concentrations (0.05 and 0.15%) were selected for the other two enzymes. In the following sections, DH is presented first, followed by characterisation of the hydrolysates (particle size distribution, molecular weight of soluble proteins, ζ-potential, surface tension), their functionality (foamability, viscosity, storage stability) and an application-relevant property (gelation of an emulsion, relevant to cheese analogues).\u003c/p\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eDegree of hydrolysis\u003c/h2\u003e \u003cp\u003eAll tested proteases resulted in partial hydrolysis of the pea proteins, with DH values ranging from 0.4\u0026ndash;5.4% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The DH increased significantly with higher enzyme concentrations for Novozym (DH of 0.4\u0026ndash;5.4% at dosing from 0.01\u0026ndash;0.5%) and Neutrase (DH of 1.8 and 3.4% at dosing of 0.05 and 0.15%, respectively). For Alcalase, under investigated conditions, the DH was low (DH\u0026thinsp;\u0026asymp;\u0026thinsp;0.5%), and similar to that of Novozym-treated PPI at 0.01% enzyme concentration. When the three enzymes were compared at the same dosage (0.05% and 0.15%), the DH order was: Neutrase\u0026thinsp;\u0026gt;\u0026thinsp;Novozym\u0026thinsp;\u0026gt;\u0026thinsp;Alcalase. This could be attributed to variations in enzyme activity and specificity of the different enzymes towards the PPI and its aggregates as a substrate at the tested conditions. Interestingly, higher DH has been previously reported for PPI treated with Alcalase, compared to Neutrase, at enzyme/substrate concentration of 0.5% (both enzymes sourced from the same supplier as in the present work). DH is key in modulating protein functionality of hydrolysates. High DH can lead to generation of bioactive peptides (\u0026lt;\u0026thinsp;10 KDa). However, partially hydrolyzed proteins with low to moderate DH (\u0026lt;\u0026thinsp;10%) is generally regarded beneficial for food applications requiring emulsification, foaming, or gelation, largely due to the potential to control the size and behaviour (e.g., surface properties) of the resulting peptides [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. This is therefore discussed in the present work.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of hydrolysates: molecular and particle sizes\u003c/h2\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eParticle size distribution\u003c/h2\u003e \u003cp\u003eParticles with sizes up to \u0026asymp;\u0026thinsp;250\u0026micro;m in diameter were determined in all investigated systems, supporting the existence of protein aggregates in the commercial PPI samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), as previously reported [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Excluding the sample treated with Novozym at the highest enzyme concentration (i.e., at 0.5%), all others showed generally bimodal size distributions, with one peak maximum at \u0026asymp;\u0026thinsp;0.1 \u0026micro;m, and one at \u0026asymp;\u0026thinsp;10\u0026micro;m. The surface moment mean diameters (D\u003csub\u003e32\u003c/sub\u003e) is a measure of the volume/surface area of the particles. For the samples with bimodal distribution, D\u003csub\u003e32\u003c/sub\u003e values were unchanged at 80 nm (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This may support a hydrolysis mechanism where the enzymes react largely in the outer layer of the particles, without causing substantial alternations in their sizes (e.g., without diffusing into the particle and breaking it into smaller fragments). It should be noted that treatment with Alcalase at 0.15% enzyme concentration reduced D\u003csub\u003e32\u003c/sub\u003e to 70 nm, which was a significant reduction if statistical analyses excluded the mono-dispersed system (data not shown). This may imply formation of small, fragmented protein particles or aggregated peptides due to increased inter-peptide interactions, as previously hidden hydrophobic regions become exposed (also previously reported) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This will be discussed further in the following sections. The volume moment mean diameter (D\u003csub\u003e43\u003c/sub\u003e) is often influenced by the large aggregates in the sample. The somehow decreasing D\u003csub\u003e43\u003c/sub\u003e of particles after hydrolysis with increasing enzyme concentration may therefore indicate erosion of the large aggregates, which become somehow smaller as the enzymes remove peptides from the surface. This is further supported by the D10, D50, and D90 values, where the enzymatic treatment caused no changes to the \u0026ldquo;fine\u0026rdquo; end of the size distribution (D10), minimal changes to the \u0026ldquo;median\u0026rdquo; (D50), and small but more pronounced changes in the \u0026ldquo;coarse\u0026rdquo; end of the size distribution curve (D90), where hydrolysis reduced the size of the large particles. It is interesting to note that despite its low DH (\u0026lt;\u0026thinsp;1%), Alcalase caused a more prominent reduction in D43 and D90, compared to the other two enzymes in all investigated conditions. Reduction in mean particle size of soy protein isolate has been previously reported on hydrolysis with Alcalase at DH of 2.5%, albeit for substantially smaller particles (of the order of 0.2 \u0026micro;m), and of peanut protein on hydrolysis with papain [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The PPI sample treated with Novozym at 0.5% enzyme/substrate ratio showed a unimodal distribution with maximum at \u0026asymp;\u0026thinsp;10\u0026micro;m and no particles \u0026lt;\u0026thinsp;1\u0026micro;m. This may suggest that the increased Novozym concentration extensively hydrolyzed the small particles to sizes below the detection limit of the used method (\u0026thinsp;\u0026lt;\u0026thinsp;\u0026asymp;\u0026thinsp;0.01\u0026micro;m).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean diameters (D [3;2] and D [4;3]) and particle size distributions (D10, D50, D90) for control and protease treated samples. Here, D [3;2] refers to the area mean diameter, D [4;3] refers to the volume mean diameter, and D10, D50, D90 indicate the particle diameters corresponding to 10%, 50%, and 90% of the cumulative volume, respectively. Significant differences between values within the same column are denoted by different letters (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003eParticle size (\u0026micro;m)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD [3;2]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD [4;3]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eD (10)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eD (50)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eD (90)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.68\u0026thinsp;\u0026plusmn;\u0026thinsp;2.82\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e18.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNovo_0.01%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.18\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNovo_0.05%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNovo_0.15%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.28\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.28\u0026thinsp;\u0026plusmn;\u0026thinsp;2.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNovo_0.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.07\u0026thinsp;\u0026plusmn;\u0026thinsp;3.88\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e46.05\u0026thinsp;\u0026plusmn;\u0026thinsp;13.81\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlca_0.05%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlca_0.15%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeut_0.05%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16.63\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeut_0.15%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.56\u0026thinsp;\u0026plusmn;\u0026thinsp;1.59\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eMolecular weight distribution\u003c/h2\u003e \u003cp\u003eSDS-PAGE was carried out to visualise peptide distribution upon partial protein hydrolysis. The undigested control PPI exhibited complex protein profiles with poor resolution and had several low and high molecular weight (MW) proteins in the range of 14\u0026ndash;200 kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Among them, distinct protein bands at 70, 68, 62, 49, 38, and 20 kDa were observed, which may correspond to subunits of convicilin, albumin, legumin, vicilin, legumin acidic subunit, and legumin basic subunit, respectively, as per previous literature [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. It was evident in the SDS-PAGE gels that even at the lowest DH (0.4%), protease treatment led to the loss of the high MW bands and the formation of new peptide bands, particularly in the low MW range (3\u0026minus;30 kDa). Increasing enzyme concentration increased the number and intensity of the new peptide bands for a single enzyme. Interestingly, treatment with Alcalase at 0.05% and 0.15% showed insignificant differences in DH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), but changes in MW size distributions were (qualitatively) evident, with the higher enzyme concentration resulting in increased hydrolysis of large peptides, suggesting the potency of this non-specific enzyme to reduce MW of proteins. At the most extensively hydrolyzed sample (i.e., the sample treated with Novozym at 0.5%, DH 5.4%), the high molecular weight proteins almost completely disappeared, and the band intensity of peptides at molecular weights 3\u0026ndash;30 kDa also reduced, suggesting the possible formation of smaller peptides and possibly amino acids that cannot be retained on the gels (\u0026lt;\u0026thinsp;3 kDa).\u003c/p\u003e \u003cp\u003eSDS-PAGE further confirmed the different specificities of the three proteases. For example, the band at \u0026asymp;\u0026thinsp;72 kDa (likely convicilin fraction), persisted at all investigated Neutrease treatments despite the relatively high DH. Besides Neutrase, this band has previously shown resistance to other well-known proteases, such as Papain and Flavorozyme [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. It was found susceptible to Alcalase at 0.15% concentration, as also previously reported, and it was further observed to hydrolyze with the less studied Novozym, albeit only at concentrations\u0026thinsp;\u0026ge;\u0026thinsp;0.15% (DH\u0026thinsp;\u0026ge;\u0026thinsp;1.2) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The band at \u0026asymp;\u0026thinsp;62 kDa persisted in all hydrolysates, suggesting the resistance under investigated conditions, albeit band intensity weakened when PPI was hydrolyzed with Novozym at 5.4% DH. This band could be a dissociated subunit of 11S hexameric legumin, and it has previously shown resistance to Flavourzyme and Nautrase, and susceptibility to Papain and Alcalase (at higher DH) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTwo other interesting bands are the one at \u0026asymp;\u0026thinsp;50 kDa and the one at \u0026asymp;\u0026thinsp;26 kDa. The former has been previously associated with vicilin, certain fractions of which have shown allergenic potential [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Alcalase and Neutrase were unable to hydrolyze this band, which has been previously reported [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Interestingly, the less studied Novozym appears more potent in breaking down this fraction, indicating potential for reducing pea allergenicity, but further studies are required. Similarly, the band at 26 kDa (likely albumin fraction) showed resistance to Alcalase and Neutrase, and besides these two enzymes, it has previously shown resistance to other 8 enzymes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Interestingly, its intensity decreased in the Novozym-treated PPI, although it did not disappear completely.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eSurface properties\u003c/h2\u003e \u003cdiv id=\"Sec26\" class=\"Section4\"\u003e \u003ch2\u003eParticle surface properties: ζ-potential and isoelectric point\u003c/h2\u003e \u003cp\u003eZeta potential is an indicator of the average surface charge, and it is therefore key in determining particle-particle and particle-solvent interactions, which subsequently affect food material properties such as rheology, foaming, and stability [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea confirms that all investigated samples carried a net-negative charge, which was expected as the pH of the measurement (\u0026asymp;\u0026thinsp;7) was above the isoelectric point of PPI (\u0026asymp;\u0026thinsp;4.5). The control sample showed the highest absolute ζ-potential value (-30 mV), and protease treatment caused a small but statistically significant reduction (down to -24 mV), with the level of reduction overall following the order of the DH. Similar ζ-potential values have been previously reported for protein isolates from chickpea, faba bean, lupin, pea, soy, quinoa [\u003cspan additionalcitationids=\"CR48 CR49\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In the literature, absolute values of ζ-potential have been reported to moderately reduce upon partial hydrolysis (DH\u0026thinsp;\u0026le;\u0026thinsp;5%) of quinoa protein isolate with Alcalase, to remain constant for PPI treated with Alcalase (DH 5%), and to slightly increase for lentil protein isolate treated with trypsin (DH 5%) and PPI treated with trypsin (DH 2%) and papain (DH 9\u0026ndash;12%) [\u003cspan additionalcitationids=\"CR51 CR52\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. It should be noted that in all works, the changes were moderate, in agreement with the results of the present study. The less studied enzyme Novozym (at DH 4.6%), and also Alcalase (at DH 6.1%), have been used in one publication to hydrolyze lentil protein, and showed insignificant changes in ζ-potential, but concentrate rather than isolate was used [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In the same study, hydrolysis shifted the pI to lower values, from \u0026asymp;\u0026thinsp;4.7 in the control to \u0026asymp;\u0026thinsp;4 in the hydrolysates. Similar, but more moderate, trend was seen in the present study, where the control sample exhibited a pI of 4.7, and hydrolysis led to a slight decrease in pI values, ranging from 4.7 to 4.4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). More substantial decrease in pI (from \u0026asymp;\u0026thinsp;4.3 to \u0026asymp;\u0026thinsp;2) has been reported upon hydrolysis of rice protein isolate with Flavourzyme (DH 2%), but the authors did not comment on possible reasons [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHigh absolute values of ζ-potential indicate increased electrostatic repulsion between particles, which can enhance stability or lower the tendency for aggregation/flocculation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The absolute value of 30 or 40mV has been suggested as a threshold for (moderate) particle stability, with values\u0026thinsp;\u0026lt;\u0026thinsp;30 mV related to systems with more compromised stability [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In the present work, ζ-potential values are close to the threshold, indicating surfaces that are overall repulsive and can support some level of electrostatic stability, particularly for the control sample, but electrostatic particle-particle interactions cannot be ruled out. It appears that hydrolysis reduced the surface charge, likely due to the overall reduction in ionizable groups.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eSurface tension\u003c/h2\u003e \u003cp\u003eIn food systems, where proteins exist in air-water interfaces, surface tension significantly influences adsorption, aggregation, and interaction properties of proteins, which collectively impact the stability and functionality of the food product [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The control sample had a surface tension of 72 mN/m (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). This was at first surprising, as it equals the equilibrium surface tension of pure water, without any proteins suspended [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The discrepancy can be attributed to kinetic limitations, as at such fast measurements as those used in the present study (\u0026le;\u0026thinsp;100 ms), the reported dynamic surface tension of pure water is 90mN/m, and therefore a 20% reduction in the presence of the PPI can be ascribed [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Partial protein hydrolysis had a small but significant influence on the surface tension of samples, which overall followed the same trend as the DH. At DH\u0026thinsp;\u0026lt;\u0026thinsp;1% (i.e., samples treated with Novozym at 0.01%, and Alcalase at 0.05 and 0.15%), minimal reduction was observed, with surface tension values ranging between 67\u0026ndash;71 mN/m. At DH\u0026thinsp;\u0026gt;\u0026thinsp;1%, Novozym reduced surface tension down to 63 mN/m at 0.5% concentration, and Neutrase resulted in surface tension of 67 mN/m for both investigated enzyme concentrations. Similar small yet significant reduction in surface tension on hydrolysis with Neutrease and other proteases has been reported for milk protein isolate [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. An earlier study has also shown that the surface tension of soy protein isolate was notably decreased after hydrolysis with different proteases including Papain, Pancreatin, and Alcalase [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Interestingly, the authors measured dynamic surface tension, indicating that while for some hydrolysates reduction was evident immediately, the effect of Alcalase was limited at the beginning (as reported in the present study), and reached\u0026thinsp;\u0026asymp;\u0026thinsp;7% after 5 minutes, supporting the kinetic role of protein hydrolysis.\u003c/p\u003e \u003cp\u003eDue to the fast nature of the measurements, one possible factor contributing to the observed reduction in surface tension on enzymatic hydrolysis could be the decrease in MW of the peptides, as small peptides can have a quick effect by diffusing faster to the surface [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Hydrolysis may further increase solubility of the PPI, which can enhance surface tension reduction. Another reason may be related to the observed reduction in aggregation size of the large particles (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), which may have increased their mobility and conformational flexibility, as also previously reported [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. In addition, the possible exposure of the inner hydrophobic regions induced by the enzymatic hydrolysis may further increase affinity to the air/water interface and result in associated reduction in surface tension, as previously reported [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. In one study, surface hydrophobicity of PPI increased after Alcalase treatment with DH of 2%, followed by a modest reduction at DH\u0026thinsp;\u0026gt;\u0026thinsp;6% [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In the same study, Neutrase appeared to moderately reduce hydrophobicity at DH of 2%. However, in another study, Alcalase treatment appeared to reduce surface hydrophobicity by 10-fold even at DH\u0026thinsp;\u0026lt;\u0026thinsp;1% [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. This indicates an important variation of how hydrolysis with the same enzyme can impact protein properties, which could be at least partially attributed to variability in the substrate.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eSuspension viscosity and foamability\u003c/h2\u003e \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e \u003ch2\u003eSuspension Viscosity\u003c/h2\u003e \u003cp\u003eViscosity is key in determining the texture, consistency, and mouth feel of foods. The apparent viscosity curves of the control and hydrolyzed PPI are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea. As the shape of the flow curves confirmed a power-law relationship between viscosity and shear rate for the investigated shear regime, the power-law model was used to analyse the obtained data, and the relevant power-law indices and consistencies are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The control sample exhibited a constant shear viscosity of 19 cP (i.e., about 20 times the viscosity of water), with Newtonian behaviour and a power law index close to 1 (n\u0026thinsp;=\u0026thinsp;0.9) [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Hydrolysis overall increased the apparent viscosity, but interestingly, the effect did not overall follow the order of DH. For example, Alcalase caused\u0026thinsp;\u0026gt;\u0026thinsp;10-fold increase in power-law consistency, and induced shear-thinning (n\u0026thinsp;=\u0026thinsp;0.7) at DH as low as 0.5%, while Neutrase had limited effect on viscosity at DH as high as 3.4%. The effect of Novozym was in-between that of Alcalase and Neutrase, being moderate at DH\u0026thinsp;\u0026le;\u0026thinsp;1.3% and stronger at increasing DH. The highest consistency (\u0026asymp;\u0026thinsp;1300 cP s\u003csup\u003en\u0026minus;1\u003c/sup\u003e) and lowest viscosity index (0.4) were observed for the PPI treated with 0.5% of Novozym. An interesting observation relates to the two Alcalase treatments, which resulted in similar and low DH (both at \u0026asymp;\u0026thinsp;0.5%), but viscosity increased by more than 10-fold when dose increased from 0.05 to 0.15%. Similar differences were observed for the MW of the hydrolysates (section \u0026ldquo;Molecular weight distribution\u0026rdquo;) for the two Alcalase-treated samples, indicating high sensitivity to Alcalase hydrolysis, with minimal increase in DH causing substantial changes in the hydrolysates\u0026rsquo; properties.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eProtein hydrolysis can increase or decrease apparent viscosity, depending on the substrate properties, enzyme, and conditions. For example, the observations made in the present study align with previous work where an increase in the viscosity of lentil proteins was observed after Alcalase and Flavorzyme treatment with DH of 6% and 5%, respectively [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. However, in another study, apparent viscosity of soy protein isolate was decreased by bromelain at DH 2%, but the initial substrate had different properties compared to that used in the present work (e.g., consistency of 7500 cP s\u003csup\u003en\u0026minus;1\u003c/sup\u003e, viscosity index of 0.35). At higher DH (\u0026asymp;\u0026thinsp;10%), hydrolysis with Alcalase and Neutrase have been shown to reduce viscosity of PPI suspensions, ascribed to the extensive hydrolysis [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. For dairy proteins, Doucet et al. observed that whey protein hydrolysates produced using Alcalase treatment exhibited higher viscosity at DH greater than 15% [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe viscosity of protein suspensions vary depending on factors such as the total number of molecules/particles, their size and shape, and their interactions [\u003cspan additionalcitationids=\"CR70\" citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Schematically, hydrolysis of protein-based particles can remove small peptides from the outer layer (e.g., by a mechanism of erosion) or larger entities (e.g., in the form of small fragments). The obtained viscosity data may result from the different patterns of hydrolyses from the three enzymes. Due to its relatively aggressive, non-specific nature, Alcalase may have penetrated the surface of the parent large PPI particles to partially fragment their outer layer to produce small daughter particles even at low DH [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. It may also have resulted in the formation of small particulate aggregates through increased hydrophobic interactions, as previously discussed. Increased inter-particle interactions due to the increased surface area, potentially supported by exposure of hydrophobic regions previously hidden within the particle, as Alcalase has previously shown to increase surface hydrophobicity at DH\u0026thinsp;\u0026lt;\u0026thinsp;6%, may have caused the observed shear viscosity increase [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. It is known that for the same volume, particle size reduction increases viscosity [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Shear may further weaken these interactions and orient the particles to the flow, further causing the observed shear-thinning [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. On the other hand, the milder Neutrase may have largely hydrolyzed peptides from the outer surface of the large particles at DH up to 3.4%, in an erosion-type mechanism and without fragmenting particles or causing extensive aggregation [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. The resulting small peptides and amino acids will have a reduced effect on friction during shearing, and hence viscosity, further possibly supported by a reduction in surface hydrophobicity, as previously reported for this enzyme [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Novozym appears to have an in-between result that possibly produces both small particles and peptides, depending on the DH. Changes in molecular size and surface charge distribution also affect the hydration layers surrounding these molecules, substantially changing their effective hydrodynamic size, which can further influence viscosity [\u003cspan additionalcitationids=\"CR70\" citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt should be noted that the K values measured for the samples treated with Novozym (0.15 and 0.5%) and Alcalase (0.15%) had a large standard deviation. One possible explanation could be related to the poor suspension stability of these samples, indicating fast particle sedimentation, which may affect homogeneity of the sampling (discussed in section \u0026ldquo;Suspension stability\u0026rdquo;). Despite the large variations, the samples showed significantly different K values.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003epower-law viscosity indices and consistencies of the PPI control and hydrolyzed samples.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK (cP s\u003csup\u003en\u0026minus;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNovo_0.01%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNovo_0.05%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNovo_0.15%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e706\u0026thinsp;\u0026plusmn;\u0026thinsp;360\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNovo_0.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1296\u0026thinsp;\u0026plusmn;\u0026thinsp;579\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esAlca_0.05%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlca_0.15%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e246\u0026thinsp;\u0026plusmn;\u0026thinsp;63\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeut_0.05%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeut_0.15%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eFoaming capacity and foam stability of PPI and hydrolysates\u003c/h3\u003e\n\u003cp\u003eThe foaming of protein solutions is primarily governed by the adsorption kinetics and interfacial properties of proteins at the air-water interface [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan additionalcitationids=\"CR74 CR75\" citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. When protein solutions are agitated, proteins migrate to the air-water interface due to their amphiphilic nature, wherein, their hydrophobic and hydrophilic regions interact with air and water, respectively. At the air-water interface, protein rearrangements and protein-protein interactions facilitate the formation of a viscoelastic film, preventing air bubble coalescence, and ultimately resulting in the formation of stable foam [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. In this study, the control sample showed the lowest foaming capacity with foam height of 8.0 mm when it was measured immediately after the foam generation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Protease treatment increased the foaming capacity with increasing enzyme concentrations, irrespective of type of enzyme. At low DH (\u0026asymp;\u0026thinsp;0.5%, i.e., samples treated with Alcalase at 0.05 and 0.15%, and with Novozym at 0.01%) small improvement in foaming properties was observed. As DH increased to \u0026asymp;\u0026thinsp;1.5% with Novozym or Neutrase (at 0.05% dose), the effect also increased and higher foam heights were recorded (11 mm), which were similar for both enzyme treatments. However, as DH increased further, differentiation between enzymes was observed, with Novozym having lower DH (2.8%) but more pronounced effect on foam height (13.6 mm), compared to Neutrase (DH 3.4%, foam height 12.6 mm). This will be discussed below. It should be noted that further hydrolysis with Novozym (to DH 5.4%) had no apparent effect on foaming properties. A similar observation has been previously reported, where PPI treated with Alcalase, Esperase, and Papain showed foaming capacity that was enhanced at DH in the range of 4\u0026minus;7% and reduced at DH 5\u0026ndash;10% [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. PPI treated with Trypsin has also shown increased foaming capacity, which the authors attributed to the presence of small, mobile peptides [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFoaming capacity of the samples partially correlated with the decrease in surface tension (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), as also previously reported [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. At low DH, surface tension reduction was limited, and improvement in foaming properties was also limited. At increasing DH, the small additional reduction in surface tension correlated with increasing foaming capacity. However, it appears that surface tension alone cannot explain the differences between Novozym and Neutrase at the highest investigated DH. The hydrolysis patterns due to different specificities may be linked with the observed results. In the previous section, it was schematically hypothesised that Alcalase may remove small particles from the protein aggregates, Neutrase may produce peptides without greatly affecting the mother aggregates, and Novozym may result in a mixture of small particles and peptides as DH increases. It appears that the presence of small peptides (evident in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) that can rapidly cover the air/water surface, and small, possibly more flexible compared to large, particles that can mechanically enhance the viscoelastic film surrounding the bubbles may be beneficial for foam development. This is further supported by the somehow enhanced foam stability in the Novozym-treated, compared to the Neutrase-treated samples (15% vs 20% reduced foam height after 55 minutes storage), as it is known that small, flexible particles can sterically enhance foam stability through a Pickering-type mechanism [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eLong-term stability\u003c/h2\u003e \u003cdiv id=\"Sec32\" class=\"Section3\"\u003e \u003ch2\u003eSuspension stability\u003c/h2\u003e \u003cp\u003eAccelerated stability was further measured to evaluate the behaviour of the samples during long-term storage. This was performed by subjecting the samples to centrifugal force of 2300g for 1h and evaluating phase separation in the tube. In a scale from 0 (very stable) to 1 (completely phase separated), all samples had instability indices\u0026thinsp;\u0026gt;\u0026thinsp;0.6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), indicating high tendency to phase separate, with clarification at the top and sedimentation at the bottom of the tube (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Interestingly, ranking of suspension stability did not follow the same order as the DH, but was rather enzyme specific. No effect was seen on treating the PPI with Novozym at DH\u0026thinsp;\u0026le;\u0026thinsp;1.4% (instability index 0.68), but the system became increasingly less stable at DH 2.8 and 5.4% (instability index up to 0.83). For Alcalase, treatment with 0.05% dose did not affect suspension stability, but increasing enzyme concentration to 0.15% increased phase separation (instability index 0.8), even though no significant differences in DH were recorded (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). By contrast, treatment with Neutrase benefited suspension stability, and a small but significantly different reduction in the instability index (down to 0.6) was observed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eObtained results could be correlated with the hydrolysis patterns discussed in the section \u0026ldquo;Degree of hydrolysis\u0026rdquo;. Hypothesizing formation of particles by Alcalase and considering the previously reported possible increase in surface hydrophobicity of the produced hydrolysates, increased instability may be due to the increasing insoluble material and inter-particle interactions [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Novozym shows similar effect at higher DH levels, possibly after hydrolysing the \u0026ldquo;easily accessible\u0026rdquo; targets to peptides, further producing also small particles. On the other hand, Neutrase may have largely produced peptides, without forming insoluble aggregates, thus enhancing stability by a mechanism that may include increased solubility. It should be noted that in the present study, reduction in ζ-potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) showed limited correlation with suspension stability, suggesting that the acquired absolute values (all \u0026gt;\u0026thinsp;25) were sufficient to ensure sufficient repulsions.\u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section4\"\u003e \u003ch2\u003eEmulsion stability\u003c/h2\u003e \u003cp\u003eThe emulsifying properties of proteins play a crucial role in determining their suitability for various products containing both oil and water phases, such as plant-based milk, ingredients utilized in the production of meat analogues, and plant-based cheese. In this study, emulsions were prepared using pea protein hydrolysates and rapeseed oil, and emulsion stability was evaluated by Lumisizer\u0026reg; under accelerated centrifugation. Creaming was observed at the top of the control sample with time of centrifugation (Fig. S2), indicating a level of instability, and instability index of 0.22 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Creaming was also observed in all hydrolysates as the sample was centrifuged, with instability indices either similar to that of the control or higher (up to 0.28), except for the sample treated with Alcalase at 0.15% which had an instability index of 0.15. Once again, it was observed that while Alcalase treatment at the two investigated enzyme concentrations resulted in similar DH, the effect on the functional properties was dissimilar. Observed results correlate well with the increased hydrophobicity previously reported for PPI treated with Alcalase at low DH, which can enhance affinity to the oil/water interface by exposing previously buried hydrophobic regions [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. In addition, related the hypothesis discussed in the section \u0026ldquo;suspension viscosity\u0026rdquo;, formation of small, fragmented particles by Alcalase activity may increase flexibility of the produced protein aggregates, further enhancing the emulsifying properties of these hydrolysates. On the other hand, peptides, particularly small peptides, may form less stable emulsions by ways including the limited ability to bind strongly to the oil/water interface due to fewer interaction sites, or potentially increasing aggregation due to exposed hydrophobic regions that are not well attached to the interface [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. These factors may result in larger oil droplets, with higher tendency to cream. It should be noted that at 8% protein content in the aqueous phase, exposed oil surfaces are not expected, and saturated coverage of oil droplets is anticipated due to the excess proteinic substances present.\u003c/p\u003e \u003cp\u003eThe existing literature also demonstrates a varied effects on emulsion stability based on the types of proteins and proteases employed. For example, Liu et al. has shown that hydrolysing a fava bean protein isolate with Alcalase up to DH of 4% did not significantly affect the emulsion stability [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. However, hydrolysing it further up to DH of 9 and 15% resulted in increasing instability when stored for 7 days [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. Shuai et al. have demonstrated improved emulsion stability of pea protein isolate when it hydrolyzed with Flavourzyme, Neutrase, Alcalase, and Trypsin (DH\u0026thinsp;=\u0026thinsp;2\u0026minus;6%) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. On contrary, in another study, a reduction in emulsion stability was observed after hydrolysing the lentil protein isolate with trypsin with DH ranging from 4\u0026minus;20% [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003eGelation\u003c/h2\u003e \u003cp\u003eAs emulsions showed better long-term stability compared to the suspensions (see section above), and fermentation was carried out for 20 h, gels were prepared using emulsified PPI and hydrolysate samples. Gels are formed when a stable three-dimensional protein network with characteristics of a soft solid is produced [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. In this study, proteins and hydrolysates were first fermented, and the resulting pH reduction enhanced protein-protein attractive interactions by decreasing electrostatic repulsions as the pI of the proteins was approached. TG was further used to study the effect of crosslinking on gel mechanical properties. pH monitoring during fermentation (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) confirmed reduction from 6.8\u0026ndash;7.4 to 4.1\u0026ndash;4.3, close to the pI (as determined in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The moisture content in all the gels was measured, and it ranged between 75 and 82% (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eGel texture\u003c/h3\u003e\n\u003cp\u003eWithout addition of TG, all produced gels were soft, with hardness\u0026thinsp;\u0026le;\u0026thinsp;60 g. The hardest one (58 g) was that prepared with the control sample. Partial protein hydrolysis overall decreased the gel hardness, reaching values in the range of 4\u0026ndash;41 g, with increasing enzyme concentrations, irrespective of enzyme type, but the differences were generally considered small due to the low values of hardness (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIncorporating TG during fermentation significantly increased the hardness by 2-4-fold, depending on the treatment. Final gel hardness reached values of 52\u0026minus;171 g in the gels prepared with control and hydrolyzed samples, wherein the control sample yielded the highest hardness (171 g). As also observed in previous sections for other properties, ranking of hardness did not follow the order of DH. It was observed that, among the three investigated enzymes, Alcalase was the most effective, and Neutrase the least effective in reducing gel hardness. For example, at a DH 0.76%, gel hardness was reduced by \u0026asymp;\u0026thinsp;47% when hydrolysis was performed with Alcalase, whereas for significantly similar DH, hydrolysis with Novozym resulted in only a 16% reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Besides, statistically similar gel hardness reductions were observed when the protein was hydrolyzed with Novozym at a DH of 0.44% and with Neutrase at a DH of 1.8% (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and b). Interestingly, increasing Alcalase concentration to 0.15% had marginal effect on DH (which was \u0026asymp;\u0026thinsp;0.5%, see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) but a significant effect on reducing gel hardness.\u003c/p\u003e \u003cp\u003eIn general, protease treatment may enhance or reduce the gelation ability depending on the products and extent of hydrolysis. For example, it can enhance gel formation by increasing the hydrophobic and electrostatic interactions (non-covalent interactions) and disulphide bond formation (covalent interaction) as it exposes buried hydrophobic groups and cysteine residues upon protein unfolding, as reported for partially hydrolyzed soy protein isolate [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Conversely, a reduction in molecular size during hydrolysis and the formation of polypeptides with a poor ability to interact with each other can decrease the ability of proteins to form firm gels [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. Covalent cross-linking can further greatly enhance the stability of the gel structures [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, the large protein particles present in the samples (Section \u0026ldquo;Particle size distribution\u0026rdquo;) imply that the produced gel is of particulate rather than strictly polymer nature. Fragmentation to smaller particles by Alcalase, discussed as a possible mechanism in previous sections, may result in weakening of the particle-based network partially due to steric effects, and the observed reduced gel hardness (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). On the other hand, formation of small peptides through Neutrase action may explain the reduced effect of this enzyme on gel hardness at DH 1.7%, as limited steric effects are expected (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). At similar DH (1.4%), the effect of Novozym on reducing gel hardness was somehow more pronounced, possibly related to the previously hypothesised formation of mixtures of peptides and small fragments (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). As DH increases, disruption of the gel network further increases. In addition, interactions between the possible fragmented particles, the peptides, and the protein aggregate network may further affect gel hardness, for example by a mechanism similar to inactive filler, or repulsive forces.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough the overall gel hardness was lower when hydrolysates were used for gel preparation, it was interesting to note that the extent of increase in gel hardness by TG was notably higher in the gels prepared with hydrolyzed proteins (Table S2). Specifically, the gels prepared with the control sample exhibited a twofold increase in hardness in the presence of TG, while the increase ranged between 2.5 and 4 folds when protein hydrolysates were used. Among the three tested proteases at a concentration of 0.15%, Novozym-treated samples showed the highest increase in hardness (4-fold) compared to Neutrase (3.2-fold) and Alcalase (2.7-fold) treated samples. This observation suggests that Novozym treatment might have resulted in the exposure of more lysine and glutamine residues, thus causing increased protein-protein crosslinking in the presence of TG. However, further increasing the Novozym concentration to 0.5% did not result in an increase in gel hardness in the presence of TG. This indicates that although there were more exposed lysine and glutamine residues, the formation of smaller peptides, due to excessive hydrolysis (as shown in Sections \u0026ldquo;Degree of hydrolysis\u0026rdquo; and Molecular size distribution\u0026rdquo;), resulted in the formation of a weak gel network. These observations further highlight that a balance between the exposure of lysine or glutamine residues and peptide size is crucial to achieve optimal gel hardness.\u003c/p\u003e\n\u003ch3\u003eGel rheology\u003c/h3\u003e\n\u003cp\u003eThe rheological characterization provides valuable insights into the viscoelastic behaviour of gels. The G\u0026rsquo; of the gels prepared by fermentation of control samples was 7408 Pa, which increased to 8548 Pa when the control samples were treated with TG during fermentation (refer to Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Generally, gels prepared with protein hydrolysates exhibited a lower G\u0026rsquo; (ranging from 36 to 6650 Pa) than the control samples, regardless of the type of protease used. Furthermore, the addition of TG during fermentation increased the G\u0026rsquo; of gels prepared with hydrolysates, although the values did not surpass those of the control samples treated with TG. In the case of gels prepared in the presence of TG using hydrolysates obtained from Novozym treatment, a decrease in G\u0026rsquo; was observed with increasing enzyme concentration. Conversely, enzyme concentration did not have any significant impact on the G\u0026rsquo; of gels prepared with hydrolysates obtained from Neutrase and Alcalase-treatment in the presence of TG. The reduction in molecular sizes and changes in other intermolecular interactions induced by hydrolysis can make the proteins less effective in forming extensive networks within the gel matrix, which may impact the elastic behaviour of the gels. Hydrolysis can also result in different extents of solubility of the proteins, which may also affect the elastic properties of the gels. A previous study showed that higher soluble proteins result in higher G\u0026rsquo; in fermented pea protein gels than insoluble proteins [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. Insoluble proteins can act as inactive fillers and can weaken the emerging gel matrix [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe present study highlights the importance of protease specificity, complementary to the DH, on determining the functionality of pea-protein hydrolysates. For example, at similar DH, Novozym treatment led to an increase in suspension viscosity, whereas Neutrase treatment had no significant effect on viscosity compared to the control. For all investigated systems, absolute value of zeta potential, pI, and surface tension were slightly reduced after enzymatic hydrolysis, while the effect of proteolysis on functional properties, including suspension viscosity, foaming capacity, and gel hardness, was largely enzyme specific. Based on the SDS-PAGE profiling, particle size analysis, and DH characterisation of partial hydrolysates, the study suggests that the size and properties of the released peptides play a key role in controlling functional properties of the material. Therefore, selecting the appropriate protease is essential for achieving desired functional properties. A possible mechanism of hydrolysis is suggested where the three enzymes result in small, fragmented particles or aggregates, peptides, or their mixture. Acquired results support this hypothesis, albeit full verification requires further work. From a product development perspective, hydrolysates with high viscosity (e.g., from Novozym treatment) could be utilized in thickened sauces, soups, or yogurt preparations, while hydrolysates with minimal viscosity change (e.g., from Neutrase treatment) may be ideal for beverages. Further research involving characterization and sequencing of the resulting peptides using mass spectrometry is needed to establish clearer structure\u0026ndash;function relationships. Future research is also needed to design protein hydrolysate-based systems with predictable behavior for food applications. In addition, more studies are required to better understand protein functionality in large-scale production systems.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNovo\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNovozym\u0026reg;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAlca\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAlcalase\u0026reg;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNeut\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNeutrase\u0026reg;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDegree of hydrolysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTransglutaminase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGDL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlucono-\u0026#120575;-lactone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSDS-PAGE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSodium dodecyl-sulphate polyacrylamide gel electrophoresis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT author statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAshwitha Amin: Conceptualization, Methodology, Formal Analysis, Investigation, Validation, Visualization, Writing - Original Draft, Writing - Editing.\u003c/p\u003e\n\u003cp\u003eOurania Gouseti: Conceptualization, Methodology, Validation, Investigation, Writing-review \u0026amp; editing, Supervision.\u003c/p\u003e\n\u003cp\u003eGernot J. Abel: Methodology, Resources\u003c/p\u003e\n\u003cp\u003ePoul Erik Jensen: Conceptualization, Methodology, Validation, Investigation, Resources, Writing-review \u0026amp; editing, Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Milk Levy Fund (PLANTCURD: Funktionelle planteproteiner som ostemasse/ PLANTCURD: Functional Plant Proteins for Cheese Curd.)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to acknowledge Signe Munk Rydtoft, Line Friis Bakmann Christensen, and Søren Lillevang (Arla Foods Innovation Centre, Aarhus, Denmark) and Hanna Maria Lilbæk (Novonesis, Lyngby, Denmark) for scientific discussions and suggestions.The authors would like to thank Natascha Krog Bager (Novonesis, Lyngby, Denmark) for technical help in physical stability analyses.The authors would like to thank Roquette, Lestrem, France and Novonesis, Lyngby, Denmark for donating pea protein isolate and enzymes, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data used in this study are presented in the illustrated figures and Supplementary Information, and the raw data will be made available upon request to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eMasi\u0026aacute; C, Jensen PE, Petersen IL, Buldo P (2022) Design of a Functional Pea Protein Matrix for Fermented Plant-Based Cheese, Foods 2022, 11, Page 178. 11 178. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/FOODS11020178\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eDenkova Z, Filipov E, Goranov B, Dobrev I, Yanakieva V (2015) Yogurts Enriched with Pea Protein, Food Environ. 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Food Hydrocoll 108:106036. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.FOODHYD.2020.106036\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-food-research-and-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [European Food Research and Technology](https://link.springer.com/journal/217)","snPcode":"217","submissionUrl":"https://submission.springernature.com/new-submission/217/3","title":"European Food Research and Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"partial hydrolysis, fermentation, functional properties, protein gelation, protein-crosslinking, Transglutaminase","lastPublishedDoi":"10.21203/rs.3.rs-8923282/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8923282/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlant proteins are promising candidates for the green transition in foods, but they have low functionality. Partial hydrolysis is gaining interest as a way to modify the functionality of plant proteins. The degree of hydrolysis (DH) is the \u0026ldquo;golden standard\u0026rdquo; to characterize partial hydrolysis. In this work, we hydrolyzed pea protein isolate (PPI) with three enzymes (Novozym, Alcalase, and Neutrase) at a range of enzyme concentrations, and we characterized the hydrolysates for DH and a range of food relevant functionalities including fermentation induced gel formation. The results indicate that the DH was insufficient to correlate with functional properties. For example, hydrolysis increased viscosity in all systems, with more extensive hydrolysis resulting in a greater increase in viscosity for individual enzymes. However, at low DH (\u0026asymp;\u0026thinsp;0.5\u0026ndash;1%), Alcalase hydrolysates exhibited\u0026thinsp;~\u0026thinsp;400% higher viscosity than those from Novozym, while Neutrase hydrolysates showed the lowest viscosity despite having the highest DH. Similarly, hydrolysis increased the foaming ability of PPI, but the sample with the highest DH (hydrolyzed with Neutrase) showed a medium effect, and at a similar DH, Alcalase hydrolysis increased foamability to a larger extent. These findings indicate that DH alone was insufficient to explain variations in PPI functionality, and that enzyme specificity, leading to distinct hydrolysate compositions, played a key role. Moreover, hydrolysates formed harder gels during fermentation (4\u0026ndash;171 g) only when transglutaminase was added.\u003c/p\u003e","manuscriptTitle":"Effect of proteases on the functional properties of pea protein isolates","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-12 12:46:09","doi":"10.21203/rs.3.rs-8923282/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-03-09T14:06:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-23T01:07:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-23T01:06:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Food Research and Technology","date":"2026-02-20T07:12:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-food-research-and-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [European Food Research and Technology](https://link.springer.com/journal/217)","snPcode":"217","submissionUrl":"https://submission.springernature.com/new-submission/217/3","title":"European Food Research and Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0b7b913b-be8c-4e0b-8f0d-aa12018da913","owner":[],"postedDate":"March 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-12T12:46:10+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-12 12:46:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8923282","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8923282","identity":"rs-8923282","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}