Canola protein processing modifies postprandial plasma amino acid profiles in vivo in healthy, young females

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Hermans, Ines Warnke, Alex Overman, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6733821/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Oct, 2025 Read the published version in Amino Acids → Version 1 posted 9 You are reading this latest preprint version Abstract Canola protein is a rapeseed-derived protein with a complete amino acid profile, making it an interesting protein for human food applications. It is currently unknown whether canola protein processing modulates postprandial plasma amino acid bioavailability in vivo in humans. This study compared postprandial plasma amino acid profiles following the ingestion of unprocessed (native) canola, processed canola, and whey protein isolate in vivo in healthy, young, females. In a randomized, clinical, cross-over design, 15 healthy young females (25 ± 3 y) participated in four test days on which they consumed 20 g protein as either native canola, enzyme processed or heat processed canola protein, or 20 g whey protein. Blood samples were collected for 5 h following protein ingestion to assess plasma amino acid concentrations. Ingestion of native canola protein resulted in lower increases in plasma total amino acid TAA concentrations compared to whey protein (3191 ± 794 vs 4429 ± 84, P < 0.001 ). Canola protein processing resulted in greater peak plasma total amino acids concentrations, reaching statistical significance for enzyme (3599 ± 687 µmol∙L − 1 , P = 0.045 ) but not heat (3565 ± 722 µmol∙L − 1 , P = 0.166 ) treated compared to native canola protein. Plasma total amino acid availability, expressed as incremental area under the curve over a 5 h postprandial period, did not differ between treatments and averaged 163 ± 81, 171 ± 76, 194 ± 82, and 207 ± 85 mmol∙300 min∙L − 1 following ingestion of native, enzyme- and heat processed canola, and whey protein, respectively ( P > 0.05 ). Ingestion of whey protein allows for a more rapid postprandial rise in circulating essential and non-essential amino acids and greater postprandial plasma total amino acid availability when compared to the ingestion of native canola protein. Ingestion of enzyme or heat-processed canola protein accelerates the postprandial rise in circulating amino acids but does not further augment overall plasma amino acid availability throughout a 5 h postprandial period when compared to the ingestion of native canola protein. Plant-derived protein bioavailability protein processing Figures Figure 1 Figure 2 Figure 3 Figure 4 BACKGROUND Ingestion of dietary protein stimulates muscle protein synthesis ( 1 – 4 ). The anabolic response to protein ingestion is regulated at various levels starting from protein digestion, the absorption of free amino acids in the gastrointestinal tract, their (partial) release into the systemic circulation, and their uptake and subsequent incorporation into muscle protein ( 1 , 2 , 5 – 8 ). Both the amount and type of protein that is ingested determine the postprandial rise in circulating amino acid concentrations (i.e. bioavailability) and, as such, modulate the anabolic response to protein ingestion ( 9 , 10 ). Therefore, the postprandial plasma amino acid profile is often used as a proxy for the anabolic properties of a protein source. With the transition towards more sustainable diets, there is an increased interest in the consumption of more plant-based products due to their lower environmental impact and lower risk of developing chronic metabolic diseases ( 11 – 13 ). However, plant-derived proteins are considered to have lesser anabolic properties than animal-derived proteins such as whey or milk ( 14 – 16 ). The lesser anabolic properties of plant-derived proteins have been attributed to lower digestibility, lower essential amino acid content, and/or deficiencies in one or more specific amino acids such as leucine, lysine, or methionine ( 17 – 19 ). Despite their lesser anabolic properties, plant-derived protein isolates and concentrates are increasingly being used in protein supplements and food products, such as meat and dairy analogues ( 20 , 21 ). Due to the increased interest in plant-derived proteins, a number of recent studies have compared the postprandial plasma amino acid profiles following ingestion of various plant- versus animal-derived proteins ( 6 , 14 , 15 , 22 – 24 ). Collectively, these data show an attenuated amino acid response following the ingestion of soy, wheat, potato, corn, and pea protein when compared to the ingestion of equivalent doses of dairy or milk protein. The attenuated amino acid response following ingestion of plant- versus animal-derived proteins can be attributed to differences in protein structure and function that impact protein digestion and amino acid absorption ( 2 , 25 – 27 ). The production of protein isolates requires several industrial processing techniques (i.e. physical and chemical) that may change protein structure and function. For example, the heating of milk protein induces protein glycation due to the Maillard reaction ( 28 – 30 ). High levels of protein glycation result in the inability to absorb lysine ( 31 ), resulting in severely compromised plasma lysine availability ( 32 ). Similar to animal-derived proteins, plant-derived proteins may be susceptible to Maillard reactions during processing, and often contain anti-nutritional factors that may negatively affect protein bioavailability ( 33 , 34 ). In contrast, other techniques may positively impact bioavailability. For example, boiling eggs denatures the available protein, resulting in more rapid and greater postprandial amino acid availability when compared to the ingestion of raw eggs ( 35 , 36 ). In line, partial hydrolysis of micellar casein through enzyme treatment has been shown to accelerate protein digestion and augment the release of protein-derived amino acids in the circulation in vivo in older men ( 37 ). Clearly, more work is required to establish the positive or negative impact of (industrial) processing of both animal and plant-derived proteins on postprandial bioavailability as well as bio-functionality. Rapeseed is the world’s second most produced oilseed after soybean ( 38 , 39 ). Cultivation of the crop is primarily done to produce canola oil. Canola press-cake, which is a by-product from the extraction of rapeseed oil, contains ~ 35–40% protein on a dry weight basis ( 38 , 40 ). Besides its high protein content, canola is a high-quality plant-based protein source due to its amino acid composition which meets human requirements ( 17 , 41 ). Recently it has been shown that ingestion of heat processed canola protein results in greater protein digestibility in pigs, when compared to the ingestion of native canola protein ( 42 ). So far, no data are available on the postprandial plasma amino acid response following native canola protein ingestion in vivo in humans. Furthermore, it remains to be assessed whether processing of native canola protein, via heat or enzyme treatment, modifies the postprandial plasma amino acid response. We hypothesized that heat and enzyme processing facilitates and accelerates protein digestion and amino acid absorption, resulting in a more rapid postprandial rise in circulating amino acid concentrations. To test our hypothesis, fifteen healthy, young females were recruited to participate in a clinical cross-over study in which we assessed the plasma amino acid responses following the ingestion of 20 g native canola protein isolate, 20 g heat-treated canola protein isolate, 20 g enzyme-treated canola protein isolate, and 20 g of a reference whey protein isolate throughout a 5 h postprandial period. METHODS Participants Fifteen healthy, young female subjects volunteered to participate in this randomized double-blind, clinical cross-over study (Table 1 for participants’ characteristics). All procedures involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The study was approved by the Medical Ethical Committee of the Maastricht University Medical Centre+ (azM/UM), Maastricht, The Netherlands (Ethics approval number: METC23-025), and was registered at www.clinicaltrials.gov (NCT06058403). All experimental procedures were conducted between October 2023 and May 2024 at Maastricht University, The Netherlands. Participants were informed about the experimental procedures and possible risks of participation prior to signing informed consent. All participants provided written informed consent to participate in the study. The study was independently monitored by Clinical Trial Centre Maastricht. Table 1 Participant’s characteristics. Parameter Total ( n = 15 ) Age (y) 25 ± 3 Body mass (kg) 63.1 ± 6.7 Height (m) 1.68 ± 0.1 BMI (kg/m 2 ) 22.3 ± 1.6 Body fat (%) 27.8 ± 4.9 Lean body mass (kg) 43.5 ± 4.6 Appendicular lean mass (kg) 19.4 ± 2.3 Systolic blood pressure (mmHg) 106 ± 9 Diastolic blood pressure (mmHg) 66 ± 8 Resting heart rate (bpm) 66 ± 12 Preliminary testing Participants aged 18–35 y, with a BMI between 18–30 kg/m 2 underwent an initial screening to assess eligibility, whereby body height (m), mass (kg) and blood pressure (mmHg) were determined. Participants were deemed healthy based on their response on a routine medical questionnaire. Potential subjects were included if they were non-smoking, recreationally active (exercise ≤ 3 times per week), and had no history of intolerance for dairy. Participants were excluded from participation if suffering from hypertension (> 140/90 mmHg), or gastrointestinal disorders, were smoking, participating in a progressive resistance-type exercise training program, using third generation oral contraceptives, or indicated intolerance to the investigational food products. Body composition was assessed by dual energy X-ray absorptiometry (DEXA). The screening session and the first trial day were separated by at least 3 d. Study design In this randomized, cross-over design, participants ( n = 15) ingested either 20 g canola protein isolate in its unprocessed (native) or processed (heat- or enzyme treated) form, or 20 g whey protein isolate. Subjects performed four test days, separated by at least three days. Arterialized blood samples were collected frequently to assess post-absorptive and postprandial plasma amino acid concentrations. Interventional drink allocation and all analyses were performed in double blinded manner. Standardization of diet and physical activity Three days prior to the first experimental trial day, participants refrained from any sort of heavy physical activity and alcohol consumption. In the two days prior to the first trial day, participants recorded their dietary intake and physical activity. Prior to subsequent trial days, participants received copies of these records and adhered to their own dietary intake and physical activity level. On the evening prior to each of the 4 test days, all participants consumed the same standardized dinner providing 2.5 MJ, with 50 Energy (En) % carbohydrate, 29En% fat, and 16En% protein. Experimental procedures The experimental trial day was scheduled in the first 10 d of the menstrual cycle for participants not taking hormonal contraceptives, which was assessed by self-report to control for hormonal fluctuations. At 07:45 AM, participants reported to the laboratory in an overnight fasted state (~ 10 h). A catheter was inserted into a dorsal hand vein for arterialized blood sampling. To obtain the arterialized blood, the hand was placed into a hotbox (60 ℃) for 10 min prior to every blood sample collection. After taking a baseline blood sample (t = 0 min), 20 g protein (based on the sum of total amino acids, Table 2 ) was dissolved in 300 mL water. All test drinks were flavored with 3 mL vanilla flavor (Dr. Oetker, Amersfoort, The Netherlands) and provided in a non-transparent shaker and consumed within 5 minutes. Immediately after finishing the drink, a 5-hour postprandial period was initiated during which arterialized blood samples were collected at t = 15, 30, 45, 60, 90, 120, 180, 240, and 300 min. Blood samples were collected into EDTA-containing tubes and centrifuged at 1000 g for 10 min at 4 ℃. Aliquots of plasma were frozen in liquid nitrogen and stored at -80 ℃ until later processing. After completion of the experimental protocol, the cannula was removed, and participants received a small meal before leaving the laboratory. Table 2 Amino acid composition of protein isolates Whey Native Canola Canola heat Canola enzyme Alanine 0.9 0.8 0.8 0.8 Arginine 0.4 1.4 1.3 0.8 Aspartic acid 2.2 1.2 1.1 1.2 Cystine 0.4 0.7 0.7 0.3 Glutamic acid 3.6 4.9 5.0 5.0 Glycine 0.3 0.9 0.9 0.9 Histidine 0.3 0.7 0.7 0.7 Hydroxyproline 0.0 0.0 0.1 0.1 Isoleucine 1.3 0.7 0.7 0.7 Leucine 2.1 1.4 1.4 1.5 Lysine 1.9 1.3 1.3 1.3 Methionine 0.4 0.4 0.5 0.2 Phenylalanine 0.6 0.8 0.8 0.8 Proline 1.2 1.6 1.5 1.7 Serine 0.9 0.8 0.8 0.8 Threonine 1.4 0.7 0.7 0.7 Tyrosine 0.6 0.4 0.4 0.4 Valine 1.1 0.9 0.9 1.0 Tryptophan 0.4 0.3 0.3 0.3 Citrulline-Ornithine 0.0 0.0 0.0 0.6 Total NEAA 10.5 12.5 12.4 12.5 Total EAA 9.6 7.3 7.3 7.2 Total AA 20.1 19.8 19.8 19.8 EAA, essential amino acid; NEAA, non-essential amino acid; AA, amino acid. Values for amino acid contents are in grams per 20 g as the provided dose. Whey: 20 g Nutri Whey TM isolate, Canola native: 20 g CanolaPRO® isolate, Canola enzyme: 20 g canola protein isolate processed with peptidyl arginase deiminase enzyme, Canola heat: 20 g canola protein isolate processed at 90°C. Gastrointestinal (dis)comfort Subjects were asked to fill out visual analog scales (VAS) to assess gastrointestinal (GI) comfort. The VAS consisted of 19 questions. Each question started with ‘’to what extent do you experience … right now?’’ and was answered by ticking a 100 mm line (0 mm = not at all, 100 mm = very much). The questions consisted of 8 items related to upper GI discomfort (nausea, general stomach pain, belching, urge to vomit, heartburn, stomach cramps, feeling of fulness, feeling of hunger), 5 items related to lower GI discomfort (flatulence, urge to defecate, intestinal cramps, diarrhea, constipation) and 6 items related to other GI symptoms (dizziness, headache, urge to urinate, bloated feeling, dry mouth, thirst). Proteins and processing Canola protein isolates were supplied by dsm-firmenich AG (Delft, The Netherlands) and whey protein isolate was supplied by FrieslandCampina (Nutri Whey TM Isolate, FrieslandCampina, Amersfoort, The Netherlands). Production of the processed canola proteins was performed from the same batch as the native canola protein. Enzymatic treatment of canola protein was based on protein citrullination. In short, enzyme processed canola protein was prepared by dissolving 5% (w/v) native canola protein in osmosed water at 50°C. The pH was adjusted to 6.0 and 0.17 U/mL peptidylarginine deiminase enzymewas added, and the solution was incubated for 30 mins at 50°C in an double-jacked tank agitated with three axial impellers at 160 rpm. The enzyme was inactivated for 30 min at 65°C. The resulting suspension was spray dried (Extractis Spray Dryer, Dury, France) using an inlet temperature of 150°C and an outlet temperature of 50°C. Heat processed canola protein was produced by dissolving the native canola protein in osmosed water at 55°C in a double-jacked tank with three axial impellers at 160 rpm. The product was left to hydrate overnight at 7°C, mildly stirred at 80 rpm. Thereafter, temperature was increased to 90°C and maintained for 10 min, then decreased to 60°C and the mixture was treated in an in-line high shear mixer (ULTRA-TURRAX UTL 2000 Disperser; IKA, Staufen, Germany) to break up protein aggregates. The resulting suspension was spray dried (Extractis Spray Dryer, Dury, France) using an inlet temperature of 150°C and an outlet temperature of 50°C. Protein analyses Amino acid contents of the protein powders were analyzed by Eurofins in compliance with requirements in DS EN ISO/IEC 17025 DANAK 581. In short, acid hydrolysis (ISO 13903:2005; EU 152/2009), oxidation-hydrolysis to measure cysteine and methionine (ISO 13903:2005; EU 152/2009), and alkaline hydrolysis to measure tryptophan (EU 152/2009) were applied in triplicate. The amino acid compositions of all four protein drinks are presented in Table 2 . Plasma analyses Plasma glucose and insulin concentrations were analyzed using commercially available kits (ref. no. A11A01667, Glucose HK CP, ABX Diagnostics, Montpellier, France; and ref. no. K151BZC-3, Human Insulin Kit, Meso Scale Discovery, Rockville, MD, United States, respectively). Plasma amino acid concentrations were determined by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS; ACQUITY UPLC H-Class with QDa; Waters, Saint-Quentin, France). Specifically, 50 µL blood plasma was deproteinized using 100 µL of 10% SSA with 50 µM of MSK-A2 internal standard (Cambridge Isotope Laboratories, Massachusetts, USA). Subsequently, 50 µL of ultra-pure demineralized water was added and samples were centrifuged (15 min at 21,000 g ). After centrifugation, 10 µL of supernatant was added to 70 µL of Borate reaction buffer (Waters, Saint-Quentin, France). In addition, 20 µL of AccQ/Tag derivatizing reagent solution (Waters, Saint/Quentin, France) was added after which the solution was heated to 55°C for 10 min. An aliquot of 1 µL was injected and measured using ultraperformance liquid chromatograph mass spectrometry. Statistical analyses All data are expressed as mean ± SD (standard deviation). Time-dependent variables were analyzed by two-factor repeated-measures ANOVA with both time and treatment as within-subjects factor. Analyses were carried out for the period right before protein ingestion (t = 0 min) until the end of the experimental trial (t = 300 min). In case of significant Time x Treatment interaction, individual timepoints were analyzed using a one-way ANOVA with the time points as the dependent variable and treatment as the independent variable. Trapezoidal rule adjusted to baseline concentrations (t = 0 min) was applied to calculate the incremental area under the curve (iAUC) of the amino acid concentrations. Non-time-dependent variables (i.e., iAUC, peak concentrations, time to peak) were compared between treatments using a one-way repeated measures ANOVA. All reported P-values were adjusted using the Bonferroni-Holm method to correct for multiple comparisons ( 43 ). Statistical significance was set at P < 0.05 ( 44 ). All calculations were performed using SPSS (version 29.0, IBM Corporation). RESULTS Gastrointestinal (dis)comfort Subjects reported upper GI (nausea, general stomach pain, belching, urge to vomit, heartburn, stomach cramps, feeling of fulness, feeling of hunger) and other GI issues (dizziness, headache, urge to urinate, bloated feeling, dry mouth, thirst) following ingestion of the protein drinks. These symptoms all displayed significant differences over time (Upper: P = 0.005 , other: P < 0.001 , respectively). For all assessed GI complaints (upper, lower, other) no differences between treatments ( P = 0.492 , P = 0.487 , P = 0.444 ) or Time x Treatment interactions ( P = 0.565 , P = 0.369 , and P = 0.514 ) were reported. Plasma glucose and insulin concentrations Plasma glucose concentrations were not different between treatments directly prior to protein ingestion (t = 0 min, Fig. 1 A; P = 0.413 ). Following protein ingestion, plasma glucose concentrations decreased over time (Time, P < 0.001 ) with no differences between treatments (Treatment, P = 0.616 ). Plasma insulin concentrations were not different between treatments prior to protein ingestion (Fig. 1 B; P = 0.604 ). Plasma insulin concentrations were significantly greater following whey compared to native canola protein ingestion (up to t = 45 min, P < 0.001 ). Ingestion of enzyme- and heat processed canola protein resulted in greater plasma insulin concentrations when compared to native canola protein (t = 15 to 45 min, both P < 0.001 ). Plasma amino acid concentrations Concentrations for all measured amino acids over the 5 h postprandial period are visualized in a heat map displaying the fold changes in plasma amino acid concentrations following protein ingestion when compared to baseline values (t = 0 min values set to 1, Fig. 2 ). Plasma essential amino acid (EAA), non-essential amino acid (NEAA), and total amino acid (TAA) concentrations and their 5 h postprandial amino acid availability (iAUC) are presented in Fig. 3 . Plasma EAA concentrations strongly increased following protein ingestion (Fig. 3 A, Time; P < 0.001 ), with less of an increase following native canola protein when compared to whey protein ingestion (Time x Treatment, P < 0.001 ). Ingestion of native canola protein resulted in significantly lower peak plasma EAA concentrations compared to whey protein 1492 ± 388 vs 2367 ± 410 µmol∙L − 1 , P < 0.001) which were also reached later (70 ± 34 vs 47 ± 23 min, P < 0.001). Ingestion of both enzyme- and heat processed canola protein did not result in higher peak plasma EAA concentrations when compared to native canola protein (1687 ± 345, 1674 ± 266, 1492 ± 388 µmol∙L − 1 , P = 0.060 and P = 0.116 , respectively ). The time to reach peak plasma EAA concentrations was also not different following the ingestion of enzyme- and heat processed canola protein compared to native canola protein (44 ± 26, 60 ± 14, 70 ± 23 min, P = 0.064 and P = 0.252 , respectively ). The overall increase in EAAs over the entire 5 h postprandial period, expressed as iAUC, was 46% less following native canola compared to whey protein ingestion (91 ± 35 vs 167 ± 47 mmol∙300 min∙L − 1 , P < 0.001 , Fig. 3 B). Enzyme and heat processing of canola protein resulted in 1.5% and 22% greater EAA iAUC when compared to native canola protein (92 ± 42 and 111 ± 41 vs 91 ± 35 mmol∙300 min∙L − 1 ) but these differences did not reach statistical significance ( P = 0.912 and P = 0.282 , respectively, Fig. 3 B). Plasma NEAA concentrations increased following protein ingestion (Fig. 3 C, Time; P < 0.001 ), with less of an increase following native canola when compared to whey protein ingestion (Time x treatment, P < 0.001 ). Peak plasma NEAA concentrations were not different following native canola versus whey protein ingestion (1712 ± 444 vs 2084 ± 488 µmol∙L − 1 respectively, P = 0.068 ). The time to reach peak plasma NEAA concentrations was not different following native canola versus whey protein ingestion (47 ± 19 vs 60 ± 36 min, P = 0.526 ). The ingestion of both enzyme- and heat processed canola protein resulted in augmented plasma NEAA concentration increases within the first hour following protein ingestion when compared to native canola protein. Despite this, peak plasma NEAA concentrations following the ingestion of enzyme and heat processed canola protein did not significantly differ from native canola protein (1918 ± 413 and 1909 ± 503 vs 1212 ± 444 µmol∙L − 1 , P = 0.072 and P = 0.288 , respectively, Fig. 3 C). In line, the time to reach peak plasma NEAA concentrations was not different between enzyme- and heat processed canola protein compared to native canola protein (64 ± 17 and 54 ± 19 vs 47 ± 19 min, P = 0.052 and P = 0.290 , respectively). The overall increase in NEAAs over the entire 5 h postprandial period, expressed as iAUC (Fig. 3 D), was lowest following whey protein ingestion (40 ± 57 mmol∙300 min∙L − 1 ) and increased following the ingestion of native, enzyme- and heat processed canola protein (73 ± 52 vs 79 ± 43 vs 84 ± 44 mmol∙300 min∙L − 1 ) but these differences were not statistically significant (Treatment, P = 0.107 , Fig. 3 D). Overall, TAA concentrations strongly increased following protein ingestion (Time, P < 0.001 , Fig. 3 E), with less of an increase following native canola protein when compared to whey protein ingestion (Time x treatment, P < 0.001 ). The ingestion of native canola protein resulted in significantly lower peak plasma TAA concentrations compared to whey protein (3191 ± 794 vs 4429 ± 843 µmol∙L − 1 , P < 0.001 ). The time to reach peak plasma TAA concentrations was not different following native canola compared to whey protein ingestion (44 ± 14 vs 60 ± 37 min, P = 0.157 ). The ingestion of both enzyme- and heat processed canola protein augmented plasma TAA kinetics resulting in greater circulating levels of TAA within the first hour following protein ingestion compared to native canola. Peak plasma TAA concentrations were greater for enzyme processed canola vs native canola protein (3599 ± 687 vs 3191 ± 794 µmol∙L − 1 , P = 0.045 ). However, peak TAA concentrations following ingestion of heat processed canola protein were not different from native canola protein (3565 ± 722 vs 3191 ± 794 µmol∙L − 1 , P = 0.166 ). The overall increase in TAAs over the entire 5 h postprandial period, expressed as iAUC, was lower following native canola compared with whey protein ingestion (163 ± 81 vs 207 ± 85 mmol∙300 min∙L − 1 ), but this difference was not statistically significant (Treatment, P = 0.433 ). Additionally, ingestion of enzyme- and heat processed canola protein did not result in different TAA iAUC (Fig. 3 F) when compared to native canola protein (171 ± 76 and 194 ± 82 vs 163 ± 81 mmol∙300 min∙L − 1 respectively). Plasma leucine concentrations increased over time following protein ingestion (Fig. 4 A, Time; P < 0.001 ). Ingestion of native canola protein resulted in lower peak plasma leucine concentrations compared with whey protein (229 ± 67 vs 448 ± 87 µmol∙L − 1 respectively; P < 0.001 ). The time to reach peak leucine concentrations was also longer following native canola compared to whey protein ingestion (71 ± 16 vs 51 ± 14 min, P < 0.001 ). The ingestion of both enzyme- and heat processed canola protein resulted in greater peak plasma leucine concentrations when compared to native canola protein (262 ± 51 and 279 ± 47 µmol∙L − 1 vs 229 ± 67 µmol∙L − 1 , P = 0.012 and P = 0.020 respectively). However, peak plasma leucine concentrations were reached earlier following ingestion of heat processed canola when compared to enzyme processed canola protein (53 ± 20 vs 57 ± 6 min respectively, P = 0.004). Leucine availability over the complete 5 h postprandial period, expressed as iAUC (Fig. 4 B), was 51% less following native canola compared to whey protein ingestion (18 ± 6 vs 37 ± 6 mmol∙300 min∙L − 1 , P < 0.001 ). Leucine iAUC was not different for enzyme processed canola protein compared to native canola protein (18 ± 8 vs 18 ± 6 mmol∙300 min∙L − 1 , P = 0.846 ). Heat processing of canola protein resulted in a 22% greater leucine availability compared to native canola protein, but this difference was not statistically significant (22 ± 7 vs 18 ± 6 mmol∙300 min∙L − 1 , P = 0.177) . Plasma lysine concentrations increased over time following protein ingestion (Time; P < 0.001; Fig. 4 C). Peak plasma lysine concentrations were lower following native canola compared to whey protein ingestion (280 ± 67 vs 446 ± 110 µmol∙L − 1 ; P < 0.001 ). The time to reach peak lysine concentrations was significantly greater following native canola when compared to whey protein ingestion (78 ± 34 vs 43 ± 10 min, respectively; P = 0.004 ). Ingestion of both enzyme- and heat processed canola protein tended to result in higher peak lysine concentrations when compared to native canola protein (325 ± 60 and 326 ± 56 vs 280 ± 67 µmol∙L − 1 ; P = 0.054 and P = 0.098 respectively). Lysine availability over the complete 5-h postprandial period, expressed as iAUC (Fig. 4 D), was 43% less following native canola compared to whey protein ingestion (16 ± 9 vs 29 ± 7 mmol∙300 min∙L − 1 , P < 0.001 ). Compared to native canola protein, the ingestion of enzyme-processed canola protein resulted in 8% lower plasma lysine availability, but this difference was not statistically significant (16 ± 9 vs 15 ± 9, mmol∙300 min∙L − 1 , P = 0.664 ). In contrast, the ingestion of heat processed canola protein resulted in 11% greater plasma lysine availability compared to native canola, however this difference also did not reach statistical significance (16 ± 9 vs 18 ± 7 mmol∙300 min∙L − 1 , P = 0.828 ). Plasma methionine concentrations increased over time following protein ingestion (Time; P < 0.001 , Fig. 4 E). Peak plasma methionine concentrations were lower following native canola when compared to whey protein ingestion (52 ± 15 vs 68 ± 17 µmol∙L − 1 ; P = 0.048 ). The time to reach peak methionine concentrations was significantly greater following native canola when compared to whey protein ingestion (70 ± 24 vs 41 ± 17 min, respectively; P = 0.032 ). Ingestion of enzyme- and heat processed canola protein did not result in different peak methionine concentrations when compared to native canola protein (62 ± 13 and 63 ± 15 vs 52 ± 15 µmol∙L − 1 , P = 0.123 and P = 0.154 , respectively ) . Methionine availability over the complete 5 h postprandial period, expressed as iAUC (Fig. 4 F), was 20% less following native canola protein when compared to whey protein ingestion (2 ± 1 vs 3 ± 3 mmol∙300 min∙L − 1 ). The ingestion of enzyme- and heat processed canola protein resulted in 22% and 66% greater plasma methionine availability compared to native canola protein (3 ± 2 and 4 ± 1 vs 2 ± 1 mmol∙300 min∙L − 1 , respectively) but none of these differences reached statistical significance (Treatment, P = 0.150 ). In general, increases in plasma concentrations of other measured amino acids revealed significant differences over time following ingestion of whey and all three canola proteins (Time x Treatment, all P < 0.001 ) for all measured amino acids. The increases in plasma amino acid concentrations over the 5 h postprandial period (iAUC) were different following whey vs native canola protein in case of arginine, glycine, isoleucine, ornithine, threonine, tryptophan, tyrosine and valine (all P < 0.001 ). For all other measured amino acids, the iAUC did not differ between treatments (Supplemental Fig. 1). DISCUSSION The present study compared postprandial plasma amino acid profiles following the ingestion of native, heat-processed, and enzyme-processed canola protein as well as whey protein in healthy, young females. The ingestion of whey protein resulted in a more rapid and pronounced increase in circulating amino acids when compared to native canola protein. Ingestion of both enzyme- and heat processed canola protein accelerated the postprandial increase in circulating amino acids, but did not result in greater overall plasma amino acid availability during a 5 h postprandial period when compared to the ingestion of native canola protein. Plant-derived proteins are generally considered of low(er) quality when compared to animal-derived proteins due to differences in protein digestion and amino acid absorption kinetics and a less balanced amino acid profile ( 17 – 19 ). In contrast to many plant-derived proteins ( 17 , 19 , 42 ), canola-derived protein isolate has a well-balanced amino acid profile with no apparent deficiencies in lysine and methionine. In Table 2 , we compared the amino acid composition of native canola protein isolate with whey as a high-quality, animal-derived, reference protein. Despite the marginal differences in amino acid composition between canola and whey protein, we observed marked differences in postprandial plasma amino acid availability following the ingestion of native canola versus whey protein (Fig. 3 ). The ingestion of native canola protein resulted in an attenuated rise in circulating plasma essential amino acids. This was accompanied by a delay in the response, with peak leucine concentrations being reached 20 min later following ingestion of canola compared with whey protein (51 ± 14 vs 71 ± 16 min, P < 0.001 , Fig. 4 A). In line with lower and more delayed postprandial increases in plasma essential amino acids, the overall postprandial plasma essential amino acid availability was also significantly less following ingestion of native canola when compared to whey protein (91 ± 35 vs 167 ± 47 mmol∙300 min∙L − 1 , P < 0.001 , Fig. 3 B). The attenuated postprandial rise in circulating amino acids following ingestion of plant- versus animal-derived proteins has been reported previously ( 6 , 14 , 15 , 22 , 23 , 45 ) and may be attributed to differences in protein structure and function that stimulate amino acid retention in splanchnic tissues ( 2 , 25 – 27 ). Lower postprandial protein-derived amino acid availability lowers protein bioavailability and may even compromise the bio-functionality of a protein or protein source ( 3 ). Therefore, many strategies are being applied to accelerate protein digestion and subsequent amino acid absorption. Protein processing can be used to modulate protein digestion and amino acid absorption, thereby increasing postprandial plasma amino acid availability ( 46 ). Previously, enzymatic hydrolysis of casein has been shown to accelerate protein digestion and increase protein-derived amino acid release in the circulation, resulting in greater overall postprandial plasma amino acid availability in vivo in humans ( 47 ). In the present study, protein processing, either through enzymatic modification or heating, was applied to the native canola protein to study the effects on protein digestion and amino acid absorption. Here, we observed a more rapid postprandial rise in the concentration of some, but certainly not all, amino acids following ingestion of enzyme processed versus native canola, but this did not result in greater overall postprandial plasma amino acid availability (171 ± 76 vs 163 ± 81 mmol∙300 min∙L − 1 respectively, Treatment, P = 0.433 ; Fig. 3 F). So far, only one study has compared postprandial plasma amino acid responses following ingestion of a hydrolyzed versus intact canola protein ( 48 ). Fleddermann et al . reported more rapid increases in circulating amino acid concentrations following ingestion of hydrolyzed versus intact canola protein, without differences in overall plasma amino acid availability. Besides enzymatic hydrolysis, heating can also be applied to proteins with relative low denaturation temperatures. The open protein structure obtained through heating can enhance digestibility and accelerate amino acid absorption kinetics ( 35 , 36 , 49 – 52 ). Previous work in pigs has shown that heat processing can improve canola protein digestibility by 22% ( 42 ). In the present study, ingestion of heat processed canola protein resulted in a more rapid postprandial rise in some of the circulating amino acid concentrations. Even though we also observed a 22% greater increase in plasma essential amino acid availability following the ingestion of heat processed versus intact canola protein (111 ± 41 vs 91 ± 35 mmol∙300 min∙L − 1 , respectively, Fig. 3 B), no significant differences were observed between treatments ( P = 0.282 ). Overall, the present data show a modest impact of canola protein processing on the postprandial increase in circulating plasma amino acid concentrations, without any substantial changes in the overall postprandial increase in plasma amino acid availability. Despite the absence of any substantial impact of the currently tested enzyme or heat processing conditions of canola protein isolate on the postprandial rise in circulating plasma amino acid concentrations, it remains important to evaluate the effects of various processing techniques on protein digestion and subsequent amino acid absorption. Here we assessed the postprandial rise in circulating amino acids, expressed as the incremental area under the curve (iAUC), as a proxy for postprandial protein-derived amino acid availability. However, a more quantitative assessment of protein digestion and amino acid absorption requires a dual isotope tracer approach ( 53 ). The use of intrinsically labeled proteins would be required to provide evidence for the proposed impact of protein processing on protein digestion and amino acid absorption in vivo in humans ( 2 , 32 , 37 , 54 ). Here, we show no significant differences in postprandial plasma amino acid availability following ingestion of both enzyme and heat processed canola protein when compared to the ingestion of native canola protein. However, it should be noted that protein processing techniques may affect specific amino acids. For example, previous work in our group has shown that heating of milk protein can cause glycation, which strongly reduces protein-derived lysine (bio)availability ( 31 , 32 ). Therefore, we also evaluated individual plasma amino acid concentrations and their incremental areas under the curve (Supplemental File 1). In contrast to milk protein, heat processing of canola protein did not negatively impact postprandial plasma lysine availability, and no specific differences in individual amino acid responses were observed following the ingestion of processed versus native canola protein. The present data show a minor role of enzymatic modification and mild heat treatment on postprandial plasma amino acid bioavailability following canola protein ingestion. However, as commercial protein processing techniques vary considerably, the impact of commonly applied processing techniques on postprandial amino acid bioavailability and bio-functionality will need to be addressed ( 55 ). This may be of increasing relevance with regards to the protein transition towards the consumption of more plant-based proteins ( 56 ). The use of plant-derived protein isolates and concentrates in meat and dairy alternatives requires extensive processing and may strongly impact the bioavailability of the available protein(s) in these products. Therefore, more work is needed to assess the impact of the various protein- and food processing procedures on the bioavailability of these proteins or protein sources in vivo in humans. In conclusion, ingestion of native canola protein allows for a more delayed postprandial rise in circulating essential and non-essential amino acids and a lower postprandial plasma amino acid availability when compared to the ingestion of whey protein. Ingestion of enzyme-modified or heat-processed canola protein accelerates the postprandial rise in circulating amino acids but does not further augment overall plasma amino acid availability throughout a 5 hour postprandial period when compared to the ingestion of native canola protein. Declarations Sources of support: The presented research was co-funded by dsm-firmenich AG (Delft, The Netherlands). Conflicts of interest and funding disclosure: The authors have the following interests: This study was co-funded by dsm-firmenich AG (Delft, The Netherlands). The researchers were responsible for the study design, data collection, and analysis, decision to publish, and preparation of the manuscript. The choice of interventional products, and the production thereof was performed by the funder. The funder had no role in data collection, analysis of the main outcome measures, or decision to publish. In preparation of the manuscript, the funder was involved to evaluate aspects of intellectual property rights and confidentiality. LJCvL and LBV have received research grants, consulting fees, speaking honoraria, or a combination of these for research on the impact of exercise and nutrition on muscle metabolism. A full overview on research funding is provided at: https://www.maastrichtuniversity.nl/l.vanloon . Corresponding author: Prof. Luc J.C. van Loon, Department of Human Biology, NUTRIM Institute of Nutrition and Translational Research in Metabolism, Maastricht University Medical Centre+, PO box 616, 6200 MD Maastricht, The Netherlands, Tel: +31 43 388 1397, Email: [email protected] Author Contribution The author contributions were as follows: LJCvL, LBV, WH, NB and IW designed research; NB and WH conducted research; NB, WH, AO, JMXvK, JMS, LBV and LJCvL analyzed data; NB and LJCvL wrote paper; NB and LJCvL had primary responsibility for final content. All authors read and approved the final manuscript. Acknowledgement We thank Lisa M.E. Kuin for medical assistance, Joan M. Senden and Antoine Zorenc for practical assistance and Wendy E. Sluijsmans and Hasibe Aydeniz for their analytical work. We also extend our gratitude to all study participants for their time and commitment. References Boirie Y, Dangin M, Gachon P, Vasson M-P, Maubois J-L, Beaufrère B (1997) Slow and fast dietary proteins differently modulate postprandial protein accretion. Proceedings of the national academy of sciences. ;94(26):14930-5 Groen BB, Horstman AM, Hamer HM, de Haan M, van Kranenburg J, Bierau J et al (2015) Post-Prandial Protein Handling: You Are What You Just Ate. PLoS ONE 10(11):e0141582 Trommelen J, van Lieshout GAA, Nyakayiru J, Holwerda AM, Smeets JSJ, Hendriks FK et al (2023) The anabolic response to protein ingestion during recovery from exercise has no upper limit in magnitude and duration in vivo in humans. 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Lancet 393(10170):447–492 Additional Declarations No competing interests reported. Supplementary Files SupplementalFigure1.docx Cite Share Download PDF Status: Published Journal Publication published 17 Oct, 2025 Read the published version in Amino Acids → Version 1 posted Editorial decision: Revision requested 18 Aug, 2025 Reviews received at journal 08 Aug, 2025 Reviews received at journal 19 Jul, 2025 Reviewers agreed at journal 18 Jul, 2025 Reviewers agreed at journal 17 Jul, 2025 Reviewers invited by journal 15 Jul, 2025 Editor assigned by journal 29 May, 2025 Submission checks completed at journal 28 May, 2025 First submitted to journal 23 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-6733821","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":486136546,"identity":"3da7d94f-8067-4121-8399-ada39fc0e140","order_by":0,"name":"Noortje Boot","email":"","orcid":"","institution":"Maastricht University Medical Centre+","correspondingAuthor":false,"prefix":"","firstName":"Noortje","middleName":"","lastName":"Boot","suffix":""},{"id":486136547,"identity":"4b14c09a-d2a0-4433-ad78-b1552cd51db2","order_by":1,"name":"Wesley J.H. 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Following collection of a baseline blood sample, a protein drink providing 20 g of protein was ingested. Whey: 20 g Nutri Whey \u003csup\u003eTM\u003c/sup\u003e isolate, Canola native: 20 g CanolaPRO\u003csup\u003e®\u003c/sup\u003e isolate, Canola enzyme: 20 g canola protein isolate processed with peptidyl arginase deiminase enzyme, Canola heat: 20 g canola protein isolate processed at 90°C. Values represent means ± SD\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6733821/v1/b45ea38bf070e560c93cd434.png"},{"id":86965793,"identity":"9b0f49c3-5166-4f7b-8242-257388bc3fa4","added_by":"auto","created_at":"2025-07-17 17:17:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8922,"visible":true,"origin":"","legend":"\u003cp\u003ePostprandial plasma amino acid concentrations following ingestion of 20 g native, enzyme-, or heat-processed canola protein isolate, or 20 g whey protein isolate. The fold changes were compared with baseline (t = 0 min, value set to 1) for all timepoints and all conditions. White: no changes in plasma amino acid concentrations when compared with baseline at the indicated time point. Green: plasma amino acid concentrations are elevated above baseline levels. Red: amino acid concentrations are lower compared to baseline. EAA: essential amino acids, NEAA: non-essential amino acid, TAA: total amino acids.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6733821/v1/84e18a13aa57ec6625661d68.png"},{"id":86965335,"identity":"8fc5745b-2bd3-4bc9-936a-bfb38141b68a","added_by":"auto","created_at":"2025-07-17 17:09:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":19066,"visible":true,"origin":"","legend":"\u003cp\u003ePostprandial plasma EAA (A), NEAA (C), TAA (E) concentrations during the 5-h postprandial period and their incremental area under the curve (iAUC, panel B, D and F) following ingestion of 20 g whey protein isolate, 20 g native canola protein isolate, 20 g enzyme-processed canola protein isolate (Canola enzyme) or 20 g heat-processed canola protein isolate (Canola heat). Values represent means ± SD. Data for plasma amino acid concentrations were analyzed using two-factor repeated-measures analysis of variance. Data for iAUC were analyzed by one-way analysis of variance. Bonferroni-Holm post-hoc testing was applied to locate differences between treatments at each separate time point. ‘’a’’ denotes a significant difference between whey and native canola protein, ‘’b’’ denotes a significant difference between native canola protein and enzyme-processed canola protein, ‘’c’’ denotes a significant difference between native canola protein and heat-processed canola protein, ‘’d’’ denotes a significant difference between the processed canola proteins. Significance was set at \u003cem\u003eP\u0026lt;0.05\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6733821/v1/6bf48e3032cf3c90156c7143.png"},{"id":86964043,"identity":"8647b49f-06ef-4f50-ae5c-a0ccbc0f6ebd","added_by":"auto","created_at":"2025-07-17 17:01:57","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":768940,"visible":true,"origin":"","legend":"\u003cp\u003ePostprandial plasma leucine (A), lysine (C), methionine (E) concentrations during the 5 h postprandial period and their incremental area under the curve (iAUC, panels B, D and F) following ingestion of 20 g whey protein isolate, 20 g native canola protein isolate, 20 g enzyme-processed canola protein isolate (Canola enzyme), or 20 g heat-processed canola protein isolate (Canola heat). Values represent means ± SD. Data for plasma amino acid concentrations were analyzed using two-factor repeated-measures analysis of variance. Data for iAUC were analyzed by one-way analysis of variance. Bonferroni-Holm post-hoc testing was applied to locate differences between treatments at each separate time point. ‘’a’’ denotes a significant difference between whey and native canola protein, ‘’b’’ denotes a significant difference between native canola protein and enzyme-processed canola protein, ‘’c’’ denotes a significant difference between native canola protein and heat-processed canola protein, ‘’d’’ denotes a significant difference between the processed canola proteins. Significance was set at \u003cem\u003eP\u0026lt;0.05\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6733821/v1/eb01509a1f5f0e5816f9ba2e.jpeg"},{"id":93956054,"identity":"aea173a7-508e-44e1-aee1-11793ff80e79","added_by":"auto","created_at":"2025-10-20 16:09:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1729267,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6733821/v1/5880b3c0-01bf-460f-b43b-8760909fef96.pdf"},{"id":86964034,"identity":"f1b1d6e8-af0b-4c88-99f6-9a895e608263","added_by":"auto","created_at":"2025-07-17 17:01:56","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1728972,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFigure1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6733821/v1/04d9c7db4be9ca0d168ac3a6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Canola protein processing modifies postprandial plasma amino acid profiles in vivo in healthy, young females","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eIngestion of dietary protein stimulates muscle protein synthesis (\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). The anabolic response to protein ingestion is regulated at various levels starting from protein digestion, the absorption of free amino acids in the gastrointestinal tract, their (partial) release into the systemic circulation, and their uptake and subsequent incorporation into muscle protein (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Both the amount and type of protein that is ingested determine the postprandial rise in circulating amino acid concentrations (i.e. bioavailability) and, as such, modulate the anabolic response to protein ingestion (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Therefore, the postprandial plasma amino acid profile is often used as a proxy for the anabolic properties of a protein source.\u003c/p\u003e\u003cp\u003eWith the transition towards more sustainable diets, there is an increased interest in the consumption of more plant-based products due to their lower environmental impact and lower risk of developing chronic metabolic diseases (\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). However, plant-derived proteins are considered to have lesser anabolic properties than animal-derived proteins such as whey or milk (\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). The lesser anabolic properties of plant-derived proteins have been attributed to lower digestibility, lower essential amino acid content, and/or deficiencies in one or more specific amino acids such as leucine, lysine, or methionine (\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Despite their lesser anabolic properties, plant-derived protein isolates and concentrates are increasingly being used in protein supplements and food products, such as meat and dairy analogues (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Due to the increased interest in plant-derived proteins, a number of recent studies have compared the postprandial plasma amino acid profiles following ingestion of various plant- versus animal-derived proteins (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Collectively, these data show an attenuated amino acid response following the ingestion of soy, wheat, potato, corn, and pea protein when compared to the ingestion of equivalent doses of dairy or milk protein. The attenuated amino acid response following ingestion of plant- versus animal-derived proteins can be attributed to differences in protein structure and function that impact protein digestion and amino acid absorption (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe production of protein isolates requires several industrial processing techniques (i.e. physical and chemical) that may change protein structure and function. For example, the heating of milk protein induces protein glycation due to the Maillard reaction (\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). High levels of protein glycation result in the inability to absorb lysine (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), resulting in severely compromised plasma lysine availability (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Similar to animal-derived proteins, plant-derived proteins may be susceptible to Maillard reactions during processing, and often contain anti-nutritional factors that may negatively affect protein bioavailability (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). In contrast, other techniques may positively impact bioavailability. For example, boiling eggs denatures the available protein, resulting in more rapid and greater postprandial amino acid availability when compared to the ingestion of raw eggs (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). In line, partial hydrolysis of micellar casein through enzyme treatment has been shown to accelerate protein digestion and augment the release of protein-derived amino acids in the circulation \u003cem\u003ein vivo\u003c/em\u003e in older men (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Clearly, more work is required to establish the positive or negative impact of (industrial) processing of both animal and plant-derived proteins on postprandial bioavailability as well as bio-functionality.\u003c/p\u003e\u003cp\u003eRapeseed is the world\u0026rsquo;s second most produced oilseed after soybean (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Cultivation of the crop is primarily done to produce canola oil. Canola press-cake, which is a by-product from the extraction of rapeseed oil, contains\u0026thinsp;~\u0026thinsp;35\u0026ndash;40% protein on a dry weight basis (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Besides its high protein content, canola is a high-quality plant-based protein source due to its amino acid composition which meets human requirements (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Recently it has been shown that ingestion of heat processed canola protein results in greater protein digestibility in pigs, when compared to the ingestion of native canola protein (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). So far, no data are available on the postprandial plasma amino acid response following native canola protein ingestion \u003cem\u003ein vivo\u003c/em\u003e in humans. Furthermore, it remains to be assessed whether processing of native canola protein, via heat or enzyme treatment, modifies the postprandial plasma amino acid response. We hypothesized that heat and enzyme processing facilitates and accelerates protein digestion and amino acid absorption, resulting in a more rapid postprandial rise in circulating amino acid concentrations. To test our hypothesis, fifteen healthy, young females were recruited to participate in a clinical cross-over study in which we assessed the plasma amino acid responses following the ingestion of 20 g native canola protein isolate, 20 g heat-treated canola protein isolate, 20 g enzyme-treated canola protein isolate, and 20 g of a reference whey protein isolate throughout a 5 h postprandial period.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eParticipants\u003c/h2\u003e\u003cp\u003eFifteen healthy, young female subjects volunteered to participate in this randomized double-blind, clinical cross-over study (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e for participants\u0026rsquo; characteristics). All procedures involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The study was approved by the Medical Ethical Committee of the Maastricht University Medical Centre+ (azM/UM), Maastricht, The Netherlands (Ethics approval number: METC23-025), and was registered at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"https://www.maastrichtuniversity.nl/l.vanloon\" target=\"_blank\"\u003ewww.clinicaltrials.gov\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.clinicaltrials.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (NCT06058403). All experimental procedures were conducted between October 2023 and May 2024 at Maastricht University, The Netherlands. Participants were informed about the experimental procedures and possible risks of participation prior to signing informed consent. All participants provided written informed consent to participate in the study. The study was independently monitored by Clinical Trial Centre Maastricht.\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\u003eParticipant\u0026rsquo;s characteristics.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eTotal (\u003cem\u003en\u0026thinsp;=\u0026thinsp;15\u003c/em\u003e)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge (y)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBody mass (kg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e63.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHeight (m)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBMI (kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e22.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBody fat (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e27.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLean body mass (kg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e43.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAppendicular lean mass (kg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e19.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSystolic blood pressure (mmHg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e106\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDiastolic blood pressure (mmHg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eResting heart rate (bpm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12\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\n\u003ch3\u003ePreliminary testing\u003c/h3\u003e\n\u003cp\u003eParticipants aged 18\u0026ndash;35 y, with a BMI between 18\u0026ndash;30 kg/m\u003csup\u003e2\u003c/sup\u003e underwent an initial screening to assess eligibility, whereby body height (m), mass (kg) and blood pressure (mmHg) were determined. Participants were deemed healthy based on their response on a routine medical questionnaire. Potential subjects were included if they were non-smoking, recreationally active (exercise\u0026thinsp;\u0026le;\u0026thinsp;3 times per week), and had no history of intolerance for dairy. Participants were excluded from participation if suffering from hypertension (\u0026gt;\u0026thinsp;140/90 mmHg), or gastrointestinal disorders, were smoking, participating in a progressive resistance-type exercise training program, using third generation oral contraceptives, or indicated intolerance to the investigational food products. Body composition was assessed by dual energy X-ray absorptiometry (DEXA). The screening session and the first trial day were separated by at least 3 d.\u003c/p\u003e\n\u003ch3\u003eStudy design\u003c/h3\u003e\n\u003cp\u003eIn this randomized, cross-over design, participants (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15) ingested either 20 g canola protein isolate in its unprocessed (native) or processed (heat- or enzyme treated) form, or 20 g whey protein isolate. Subjects performed four test days, separated by at least three days. Arterialized blood samples were collected frequently to assess post-absorptive and postprandial plasma amino acid concentrations. Interventional drink allocation and all analyses were performed in double blinded manner.\u003c/p\u003e\n\u003ch3\u003eStandardization of diet and physical activity\u003c/h3\u003e\n\u003cp\u003eThree days prior to the first experimental trial day, participants refrained from any sort of heavy physical activity and alcohol consumption. In the two days prior to the first trial day, participants recorded their dietary intake and physical activity. Prior to subsequent trial days, participants received copies of these records and adhered to their own dietary intake and physical activity level. On the evening prior to each of the 4 test days, all participants consumed the same standardized dinner providing 2.5 MJ, with 50 Energy (En) % carbohydrate, 29En% fat, and 16En% protein.\u003c/p\u003e\n\u003ch3\u003eExperimental procedures\u003c/h3\u003e\n\u003cp\u003eThe experimental trial day was scheduled in the first 10 d of the menstrual cycle for participants not taking hormonal contraceptives, which was assessed by self-report to control for hormonal fluctuations. At 07:45 AM, participants reported to the laboratory in an overnight fasted state (~\u0026thinsp;10 h). A catheter was inserted into a dorsal hand vein for arterialized blood sampling. To obtain the arterialized blood, the hand was placed into a hotbox (60 ℃) for 10 min prior to every blood sample collection.\u003c/p\u003e\u003cp\u003eAfter taking a baseline blood sample (t\u0026thinsp;=\u0026thinsp;0 min), 20 g protein (based on the sum of total amino acids, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) was dissolved in 300 mL water. All test drinks were flavored with 3 mL vanilla flavor (Dr. Oetker, Amersfoort, The Netherlands) and provided in a non-transparent shaker and consumed within 5 minutes. Immediately after finishing the drink, a 5-hour postprandial period was initiated during which arterialized blood samples were collected at t\u0026thinsp;=\u0026thinsp;15, 30, 45, 60, 90, 120, 180, 240, and 300 min. Blood samples were collected into EDTA-containing tubes and centrifuged at 1000\u003cem\u003eg\u003c/em\u003e for 10 min at 4 ℃. Aliquots of plasma were frozen in liquid nitrogen and stored at -80 ℃ until later processing. After completion of the experimental protocol, the cannula was removed, and participants received a small meal before leaving the laboratory.\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\u003eAmino acid composition of protein isolates\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWhey\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNative Canola\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCanola heat\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCanola enzyme\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAlanine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eArginine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAspartic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCystine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlutamic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlycine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHistidine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHydroxyproline\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIsoleucine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLeucine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLysine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMethionine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhenylalanine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProline\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSerine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eThreonine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTyrosine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eValine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTryptophan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCitrulline-Ornithine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal NEAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e12.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e12.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e12.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal EAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e9.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e7.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal AA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e20.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e19.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e19.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eEAA, essential amino acid; NEAA, non-essential amino acid; AA, amino acid.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eValues for amino acid contents are in grams per 20 g as the provided dose. Whey: 20 g Nutri Whey \u003csup\u003eTM\u003c/sup\u003e isolate, Canola native: 20 g CanolaPRO\u0026reg; isolate, Canola enzyme: 20 g canola protein isolate processed with peptidyl arginase deiminase enzyme, Canola heat: 20 g canola protein isolate processed at 90\u0026deg;C.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eGastrointestinal (dis)comfort\u003c/h2\u003e\u003cp\u003eSubjects were asked to fill out visual analog scales (VAS) to assess gastrointestinal (GI) comfort. The VAS consisted of 19 questions. Each question started with \u0026lsquo;\u0026rsquo;to what extent do you experience \u0026hellip; right now?\u0026rsquo;\u0026rsquo; and was answered by ticking a 100 mm line (0 mm\u0026thinsp;=\u0026thinsp;not at all, 100 mm\u0026thinsp;=\u0026thinsp;very much). The questions consisted of 8 items related to upper GI discomfort (nausea, general stomach pain, belching, urge to vomit, heartburn, stomach cramps, feeling of fulness, feeling of hunger), 5 items related to lower GI discomfort (flatulence, urge to defecate, intestinal cramps, diarrhea, constipation) and 6 items related to other GI symptoms (dizziness, headache, urge to urinate, bloated feeling, dry mouth, thirst).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eProteins and processing\u003c/h3\u003e\n\u003cp\u003eCanola protein isolates were supplied by dsm-firmenich AG (Delft, The Netherlands) and whey protein isolate was supplied by FrieslandCampina (Nutri Whey \u003csup\u003eTM\u003c/sup\u003e Isolate, FrieslandCampina, Amersfoort, The Netherlands). Production of the processed canola proteins was performed from the same batch as the native canola protein. Enzymatic treatment of canola protein was based on protein citrullination. In short, enzyme processed canola protein was prepared by dissolving 5% (w/v) native canola protein in osmosed water at 50\u0026deg;C. The pH was adjusted to 6.0 and 0.17 U/mL peptidylarginine deiminase enzymewas added, and the solution was incubated for 30 mins at 50\u0026deg;C in an double-jacked tank agitated with three axial impellers at 160 rpm. The enzyme was inactivated for 30 min at 65\u0026deg;C. The resulting suspension was spray dried (Extractis Spray Dryer, Dury, France) using an inlet temperature of 150\u0026deg;C and an outlet temperature of 50\u0026deg;C. Heat processed canola protein was produced by dissolving the native canola protein in osmosed water at 55\u0026deg;C in a double-jacked tank with three axial impellers at 160 rpm. The product was left to hydrate overnight at 7\u0026deg;C, mildly stirred at 80 rpm. Thereafter, temperature was increased to 90\u0026deg;C and maintained for 10 min, then decreased to 60\u0026deg;C and the mixture was treated in an in-line high shear mixer (ULTRA-TURRAX UTL 2000 Disperser; IKA, Staufen, Germany) to break up protein aggregates. The resulting suspension was spray dried (Extractis Spray Dryer, Dury, France) using an inlet temperature of 150\u0026deg;C and an outlet temperature of 50\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eProtein analyses\u003c/h3\u003e\n\u003cp\u003eAmino acid contents of the protein powders were analyzed by Eurofins in compliance with requirements in DS EN ISO/IEC 17025 DANAK 581. In short, acid hydrolysis (ISO 13903:2005; EU 152/2009), oxidation-hydrolysis to measure cysteine and methionine (ISO 13903:2005; EU 152/2009), and alkaline hydrolysis to measure tryptophan (EU 152/2009) were applied in triplicate. The amino acid compositions of all four protein drinks are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003ePlasma analyses\u003c/h2\u003e\u003cp\u003ePlasma glucose and insulin concentrations were analyzed using commercially available kits (ref. no. A11A01667, Glucose HK CP, ABX Diagnostics, Montpellier, France; and ref. no. K151BZC-3, Human Insulin Kit, Meso Scale Discovery, Rockville, MD, United States, respectively). Plasma amino acid concentrations were determined by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS; ACQUITY UPLC H-Class with QDa; Waters, Saint-Quentin, France). Specifically, 50 \u0026micro;L blood plasma was deproteinized using 100 \u0026micro;L of 10% SSA with 50 \u0026micro;M of MSK-A2 internal standard (Cambridge Isotope Laboratories, Massachusetts, USA). Subsequently, 50 \u0026micro;L of ultra-pure demineralized water was added and samples were centrifuged (15 min at 21,000 \u003cem\u003eg\u003c/em\u003e). After centrifugation, 10 \u0026micro;L of supernatant was added to 70 \u0026micro;L of Borate reaction buffer (Waters, Saint-Quentin, France). In addition, 20 \u0026micro;L of AccQ/Tag derivatizing reagent solution (Waters, Saint/Quentin, France) was added after which the solution was heated to 55\u0026deg;C for 10 min. An aliquot of 1 \u0026micro;L was injected and measured using ultraperformance liquid chromatograph mass spectrometry.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analyses\u003c/h2\u003e\u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (standard deviation). Time-dependent variables were analyzed by two-factor repeated-measures ANOVA with both time and treatment as within-subjects factor. Analyses were carried out for the period right before protein ingestion (t\u0026thinsp;=\u0026thinsp;0 min) until the end of the experimental trial (t\u0026thinsp;=\u0026thinsp;300 min). In case of significant \u003cem\u003eTime x Treatment\u003c/em\u003e interaction, individual timepoints were analyzed using a one-way ANOVA with the time points as the dependent variable and treatment as the independent variable. Trapezoidal rule adjusted to baseline concentrations (t\u0026thinsp;=\u0026thinsp;0 min) was applied to calculate the incremental area under the curve (iAUC) of the amino acid concentrations. Non-time-dependent variables (i.e., iAUC, peak concentrations, time to peak) were compared between treatments using a one-way repeated measures ANOVA. All reported P-values were adjusted using the Bonferroni-Holm method to correct for multiple comparisons (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Statistical significance was set at \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). All calculations were performed using SPSS (version 29.0, IBM Corporation).\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eGastrointestinal (dis)comfort\u003c/h2\u003e\u003cp\u003eSubjects reported upper GI (nausea, general stomach pain, belching, urge to vomit, heartburn, stomach cramps, feeling of fulness, feeling of hunger) and other GI issues (dizziness, headache, urge to urinate, bloated feeling, dry mouth, thirst) following ingestion of the protein drinks. These symptoms all displayed significant differences over time (Upper: \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.005\u003c/em\u003e, other: \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e, respectively). For all assessed GI complaints (upper, lower, other) no differences between treatments (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.492\u003c/em\u003e, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.487\u003c/em\u003e, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.444\u003c/em\u003e) or Time x Treatment interactions (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.565\u003c/em\u003e, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.369\u003c/em\u003e, and \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.514\u003c/em\u003e) were reported.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003ePlasma glucose and insulin concentrations\u003c/h2\u003e\u003cp\u003ePlasma glucose concentrations were not different between treatments directly prior to protein ingestion (t\u0026thinsp;=\u0026thinsp;0 min, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA; \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.413\u003c/em\u003e). Following protein ingestion, plasma glucose concentrations decreased over time (Time, \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) with no differences between treatments (Treatment, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.616\u003c/em\u003e). Plasma insulin concentrations were not different between treatments prior to protein ingestion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB; \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.604\u003c/em\u003e). Plasma insulin concentrations were significantly greater following whey compared to native canola protein ingestion (up to t\u0026thinsp;=\u0026thinsp;45 min, \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). Ingestion of enzyme- and heat processed canola protein resulted in greater plasma insulin concentrations when compared to native canola protein (t\u0026thinsp;=\u0026thinsp;15 to 45 min, both \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003ePlasma amino acid concentrations\u003c/h2\u003e\u003cp\u003eConcentrations for all measured amino acids over the 5 h postprandial period are visualized in a heat map displaying the fold changes in plasma amino acid concentrations following protein ingestion when compared to baseline values (t\u0026thinsp;=\u0026thinsp;0 min values set to 1, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePlasma essential amino acid (EAA), non-essential amino acid (NEAA), and total amino acid (TAA) concentrations and their 5 h postprandial amino acid availability (iAUC) are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Plasma EAA concentrations strongly increased following protein ingestion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Time; \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), with less of an increase following native canola protein when compared to whey protein ingestion (Time x Treatment, \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). Ingestion of native canola protein resulted in significantly lower peak plasma EAA concentrations compared to whey protein 1492\u0026thinsp;\u0026plusmn;\u0026thinsp;388 \u003cem\u003evs\u003c/em\u003e 2367\u0026thinsp;\u0026plusmn;\u0026thinsp;410 \u0026micro;mol∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) which were also reached later (70\u0026thinsp;\u0026plusmn;\u0026thinsp;34 \u003cem\u003evs\u003c/em\u003e 47\u0026thinsp;\u0026plusmn;\u0026thinsp;23 min, \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/em\u003e Ingestion of both enzyme- and heat processed canola protein did not result in higher peak plasma EAA concentrations when compared to native canola protein (1687\u0026thinsp;\u0026plusmn;\u0026thinsp;345, 1674\u0026thinsp;\u0026plusmn;\u0026thinsp;266, 1492\u0026thinsp;\u0026plusmn;\u0026thinsp;388 \u0026micro;mol∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.060\u003c/em\u003e and \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.116\u003c/em\u003e, respectively\u003cem\u003e).\u003c/em\u003e The time to reach peak plasma EAA concentrations was also not different following the ingestion of enzyme- and heat processed canola protein compared to native canola protein (44\u0026thinsp;\u0026plusmn;\u0026thinsp;26, 60\u0026thinsp;\u0026plusmn;\u0026thinsp;14, 70\u0026thinsp;\u0026plusmn;\u0026thinsp;23 min, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.064\u003c/em\u003e and \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.252\u003c/em\u003e, respectively\u003cem\u003e).\u003c/em\u003e The overall increase in EAAs over the entire 5 h postprandial period, expressed as iAUC, was 46% less following native canola compared to whey protein ingestion (91\u0026thinsp;\u0026plusmn;\u0026thinsp;35 \u003cem\u003evs\u003c/em\u003e 167\u0026thinsp;\u0026plusmn;\u0026thinsp;47 mmol∙300 min∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Enzyme and heat processing of canola protein resulted in 1.5% and 22% greater EAA iAUC when compared to native canola protein (92\u0026thinsp;\u0026plusmn;\u0026thinsp;42 and 111\u0026thinsp;\u0026plusmn;\u0026thinsp;41 \u003cem\u003evs\u003c/em\u003e 91\u0026thinsp;\u0026plusmn;\u0026thinsp;35 mmol∙300 min∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) but these differences did not reach statistical significance (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.912\u003c/em\u003e and \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.282\u003c/em\u003e, respectively, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePlasma NEAA concentrations increased following protein ingestion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, Time; \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), with less of an increase following native canola when compared to whey protein ingestion (Time x treatment, \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). Peak plasma NEAA concentrations were not different following native canola versus whey protein ingestion (1712\u0026thinsp;\u0026plusmn;\u0026thinsp;444 \u003cem\u003evs\u003c/em\u003e 2084\u0026thinsp;\u0026plusmn;\u0026thinsp;488 \u0026micro;mol∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e respectively, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.068\u003c/em\u003e). The time to reach peak plasma NEAA concentrations was not different following native canola versus whey protein ingestion (47\u0026thinsp;\u0026plusmn;\u0026thinsp;19 vs 60\u0026thinsp;\u0026plusmn;\u0026thinsp;36 min, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.526\u003c/em\u003e). The ingestion of both enzyme- and heat processed canola protein resulted in augmented plasma NEAA concentration increases within the first hour following protein ingestion when compared to native canola protein. Despite this, peak plasma NEAA concentrations following the ingestion of enzyme and heat processed canola protein did not significantly differ from native canola protein (1918\u0026thinsp;\u0026plusmn;\u0026thinsp;413 and 1909\u0026thinsp;\u0026plusmn;\u0026thinsp;503 \u003cem\u003evs\u003c/em\u003e 1212\u0026thinsp;\u0026plusmn;\u0026thinsp;444 \u0026micro;mol∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.072\u003c/em\u003e and \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.288\u003c/em\u003e, respectively, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In line, the time to reach peak plasma NEAA concentrations was not different between enzyme- and heat processed canola protein compared to native canola protein (64\u0026thinsp;\u0026plusmn;\u0026thinsp;17 and 54\u0026thinsp;\u0026plusmn;\u0026thinsp;19 \u003cem\u003evs\u003c/em\u003e 47\u0026thinsp;\u0026plusmn;\u0026thinsp;19 min, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.052\u003c/em\u003e and \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.290\u003c/em\u003e, respectively). The overall increase in NEAAs over the entire 5 h postprandial period, expressed as iAUC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), was lowest following whey protein ingestion (40\u0026thinsp;\u0026plusmn;\u0026thinsp;57 mmol∙300 min∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and increased following the ingestion of native, enzyme- and heat processed canola protein (73\u0026thinsp;\u0026plusmn;\u0026thinsp;52 \u003cem\u003evs\u003c/em\u003e 79\u0026thinsp;\u0026plusmn;\u0026thinsp;43 \u003cem\u003evs\u003c/em\u003e 84\u0026thinsp;\u0026plusmn;\u0026thinsp;44 mmol∙300 min∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) but these differences were not statistically significant (Treatment, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.107\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eOverall, TAA concentrations strongly increased following protein ingestion (Time, \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), with less of an increase following native canola protein when compared to whey protein ingestion (Time x treatment, \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). The ingestion of native canola protein resulted in significantly lower peak plasma TAA concentrations compared to whey protein (3191\u0026thinsp;\u0026plusmn;\u0026thinsp;794 \u003cem\u003evs\u003c/em\u003e 4429\u0026thinsp;\u0026plusmn;\u0026thinsp;843 \u0026micro;mol∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). The time to reach peak plasma TAA concentrations was not different following native canola compared to whey protein ingestion (44\u0026thinsp;\u0026plusmn;\u0026thinsp;14 \u003cem\u003evs\u003c/em\u003e 60\u0026thinsp;\u0026plusmn;\u0026thinsp;37 min, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.157\u003c/em\u003e). The ingestion of both enzyme- and heat processed canola protein augmented plasma TAA kinetics resulting in greater circulating levels of TAA within the first hour following protein ingestion compared to native canola. Peak plasma TAA concentrations were greater for enzyme processed canola \u003cem\u003evs\u003c/em\u003e native canola protein (3599\u0026thinsp;\u0026plusmn;\u0026thinsp;687 \u003cem\u003evs\u003c/em\u003e 3191\u0026thinsp;\u0026plusmn;\u0026thinsp;794 \u0026micro;mol∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.045\u003c/em\u003e). However, peak TAA concentrations following ingestion of heat processed canola protein were not different from native canola protein (3565\u0026thinsp;\u0026plusmn;\u0026thinsp;722 \u003cem\u003evs\u003c/em\u003e 3191\u0026thinsp;\u0026plusmn;\u0026thinsp;794 \u0026micro;mol∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.166\u003c/em\u003e). The overall increase in TAAs over the entire 5 h postprandial period, expressed as iAUC, was lower following native canola compared with whey protein ingestion (163\u0026thinsp;\u0026plusmn;\u0026thinsp;81 \u003cem\u003evs\u003c/em\u003e 207\u0026thinsp;\u0026plusmn;\u0026thinsp;85 mmol∙300 min∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), but this difference was not statistically significant (Treatment, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.433\u003c/em\u003e). Additionally, ingestion of enzyme- and heat processed canola protein did not result in different TAA iAUC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) when compared to native canola protein (171\u0026thinsp;\u0026plusmn;\u0026thinsp;76 and 194\u0026thinsp;\u0026plusmn;\u0026thinsp;82 \u003cem\u003evs\u003c/em\u003e 163\u0026thinsp;\u0026plusmn;\u0026thinsp;81 mmol∙300 min∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e respectively).\u003c/p\u003e\u003cp\u003ePlasma leucine concentrations increased over time following protein ingestion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Time; \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). Ingestion of native canola protein resulted in lower peak plasma leucine concentrations compared with whey protein (229\u0026thinsp;\u0026plusmn;\u0026thinsp;67 \u003cem\u003evs\u003c/em\u003e 448\u0026thinsp;\u0026plusmn;\u0026thinsp;87 \u0026micro;mol∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e respectively; \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). The time to reach peak leucine concentrations was also longer following native canola compared to whey protein ingestion (71\u0026thinsp;\u0026plusmn;\u0026thinsp;16 \u003cem\u003evs\u003c/em\u003e 51\u0026thinsp;\u0026plusmn;\u0026thinsp;14 min, \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). The ingestion of both enzyme- and heat processed canola protein resulted in greater peak plasma leucine concentrations when compared to native canola protein (262\u0026thinsp;\u0026plusmn;\u0026thinsp;51 and 279\u0026thinsp;\u0026plusmn;\u0026thinsp;47 \u0026micro;mol∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u003cem\u003evs\u003c/em\u003e 229\u0026thinsp;\u0026plusmn;\u0026thinsp;67 \u0026micro;mol∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.012\u003c/em\u003e and \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.020\u003c/em\u003e respectively). However, peak plasma leucine concentrations were reached earlier following ingestion of heat processed canola when compared to enzyme processed canola protein (53\u0026thinsp;\u0026plusmn;\u0026thinsp;20 \u003cem\u003evs\u003c/em\u003e 57\u0026thinsp;\u0026plusmn;\u0026thinsp;6 min respectively, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.004).\u003c/em\u003e Leucine availability over the complete 5 h postprandial period, expressed as iAUC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), was 51% less following native canola compared to whey protein ingestion (18\u0026thinsp;\u0026plusmn;\u0026thinsp;6 \u003cem\u003evs\u003c/em\u003e 37\u0026thinsp;\u0026plusmn;\u0026thinsp;6 mmol∙300 min∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). Leucine iAUC was not different for enzyme processed canola protein compared to native canola protein (18\u0026thinsp;\u0026plusmn;\u0026thinsp;8 \u003cem\u003evs\u003c/em\u003e 18\u0026thinsp;\u0026plusmn;\u0026thinsp;6 mmol∙300 min∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.846\u003c/em\u003e). Heat processing of canola protein resulted in a 22% greater leucine availability compared to native canola protein, but this difference was not statistically significant (22\u0026thinsp;\u0026plusmn;\u0026thinsp;7 \u003cem\u003evs\u003c/em\u003e 18\u0026thinsp;\u0026plusmn;\u0026thinsp;6 mmol∙300 min∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.177)\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePlasma lysine concentrations increased over time following protein ingestion (Time; \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001;\u003c/em\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Peak plasma lysine concentrations were lower following native canola compared to whey protein ingestion (280\u0026thinsp;\u0026plusmn;\u0026thinsp;67 \u003cem\u003evs\u003c/em\u003e 446\u0026thinsp;\u0026plusmn;\u0026thinsp;110 \u0026micro;mol∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). The time to reach peak lysine concentrations was significantly greater following native canola when compared to whey protein ingestion (78\u0026thinsp;\u0026plusmn;\u0026thinsp;34 \u003cem\u003evs\u003c/em\u003e 43\u0026thinsp;\u0026plusmn;\u0026thinsp;10 min, respectively; \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.004\u003c/em\u003e). Ingestion of both enzyme- and heat processed canola protein tended to result in higher peak lysine concentrations when compared to native canola protein (325\u0026thinsp;\u0026plusmn;\u0026thinsp;60 and 326\u0026thinsp;\u0026plusmn;\u0026thinsp;56 \u003cem\u003evs\u003c/em\u003e 280\u0026thinsp;\u0026plusmn;\u0026thinsp;67 \u0026micro;mol∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.054\u003c/em\u003e and \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.098\u003c/em\u003e respectively). Lysine availability over the complete 5-h postprandial period, expressed as iAUC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), was 43% less following native canola compared to whey protein ingestion (16\u0026thinsp;\u0026plusmn;\u0026thinsp;9 \u003cem\u003evs\u003c/em\u003e 29\u0026thinsp;\u0026plusmn;\u0026thinsp;7 mmol∙300 min∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). Compared to native canola protein, the ingestion of enzyme-processed canola protein resulted in 8% lower plasma lysine availability, but this difference was not statistically significant (16\u0026thinsp;\u0026plusmn;\u0026thinsp;9 \u003cem\u003evs\u003c/em\u003e 15\u0026thinsp;\u0026plusmn;\u0026thinsp;9, mmol∙300 min∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.664\u003c/em\u003e). In contrast, the ingestion of heat processed canola protein resulted in 11% greater plasma lysine availability compared to native canola, however this difference also did not reach statistical significance (16\u0026thinsp;\u0026plusmn;\u0026thinsp;9 \u003cem\u003evs\u003c/em\u003e 18\u0026thinsp;\u0026plusmn;\u0026thinsp;7 mmol∙300 min∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.828\u003c/em\u003e).\u003c/p\u003e\u003cp\u003ePlasma methionine concentrations increased over time following protein ingestion (Time; \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Peak plasma methionine concentrations were lower following native canola when compared to whey protein ingestion (52\u0026thinsp;\u0026plusmn;\u0026thinsp;15 \u003cem\u003evs\u003c/em\u003e 68\u0026thinsp;\u0026plusmn;\u0026thinsp;17 \u0026micro;mol∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.048\u003c/em\u003e). The time to reach peak methionine concentrations was significantly greater following native canola when compared to whey protein ingestion (70\u0026thinsp;\u0026plusmn;\u0026thinsp;24 \u003cem\u003evs\u003c/em\u003e 41\u0026thinsp;\u0026plusmn;\u0026thinsp;17 min, respectively; \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.032\u003c/em\u003e). Ingestion of enzyme- and heat processed canola protein did not result in different peak methionine concentrations when compared to native canola protein (62\u0026thinsp;\u0026plusmn;\u0026thinsp;13 and 63\u0026thinsp;\u0026plusmn;\u0026thinsp;15 \u003cem\u003evs\u003c/em\u003e 52\u0026thinsp;\u0026plusmn;\u0026thinsp;15 \u0026micro;mol∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.123\u003c/em\u003e and \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.154\u003c/em\u003e, respectively\u003cem\u003e)\u003c/em\u003e. Methionine availability over the complete 5 h postprandial period, expressed as iAUC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), was 20% less following native canola protein when compared to whey protein ingestion (2\u0026thinsp;\u0026plusmn;\u0026thinsp;1 \u003cem\u003evs\u003c/em\u003e 3\u0026thinsp;\u0026plusmn;\u0026thinsp;3 mmol∙300 min∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The ingestion of enzyme- and heat processed canola protein resulted in 22% and 66% greater plasma methionine availability compared to native canola protein (3\u0026thinsp;\u0026plusmn;\u0026thinsp;2 and 4\u0026thinsp;\u0026plusmn;\u0026thinsp;1 \u003cem\u003evs\u003c/em\u003e 2\u0026thinsp;\u0026plusmn;\u0026thinsp;1 mmol∙300 min∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively) but none of these differences reached statistical significance (Treatment, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.150\u003c/em\u003e).\u003c/p\u003e\u003cp\u003eIn general, increases in plasma concentrations of other measured amino acids revealed significant differences over time following ingestion of whey and all three canola proteins (Time x Treatment, all \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) for all measured amino acids. The increases in plasma amino acid concentrations over the 5 h postprandial period (iAUC) were different following whey \u003cem\u003evs\u003c/em\u003e native canola protein in case of arginine, glycine, isoleucine, ornithine, threonine, tryptophan, tyrosine and valine (all \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). For all other measured amino acids, the iAUC did not differ between treatments (Supplemental Fig.\u0026nbsp;1).\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe present study compared postprandial plasma amino acid profiles following the ingestion of native, heat-processed, and enzyme-processed canola protein as well as whey protein in healthy, young females. The ingestion of whey protein resulted in a more rapid and pronounced increase in circulating amino acids when compared to native canola protein. Ingestion of both enzyme- and heat processed canola protein accelerated the postprandial increase in circulating amino acids, but did not result in greater overall plasma amino acid availability during a 5 h postprandial period when compared to the ingestion of native canola protein.\u003c/p\u003e\u003cp\u003ePlant-derived proteins are generally considered of low(er) quality when compared to animal-derived proteins due to differences in protein digestion and amino acid absorption kinetics and a less balanced amino acid profile (\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). In contrast to many plant-derived proteins (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e), canola-derived protein isolate has a well-balanced amino acid profile with no apparent deficiencies in lysine and methionine. In Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, we compared the amino acid composition of native canola protein isolate with whey as a high-quality, animal-derived, reference protein. Despite the marginal differences in amino acid composition between canola and whey protein, we observed marked differences in postprandial plasma amino acid availability following the ingestion of native canola versus whey protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The ingestion of native canola protein resulted in an attenuated rise in circulating plasma essential amino acids. This was accompanied by a delay in the response, with peak leucine concentrations being reached 20 min later following ingestion of canola compared with whey protein (51\u0026thinsp;\u0026plusmn;\u0026thinsp;14 \u003cem\u003evs\u003c/em\u003e 71\u0026thinsp;\u0026plusmn;\u0026thinsp;16 min, \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In line with lower and more delayed postprandial increases in plasma essential amino acids, the overall postprandial plasma essential amino acid availability was also significantly less following ingestion of native canola when compared to whey protein (91\u0026thinsp;\u0026plusmn;\u0026thinsp;35 \u003cem\u003evs\u003c/em\u003e 167\u0026thinsp;\u0026plusmn;\u0026thinsp;47 mmol∙300 min∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The attenuated postprandial rise in circulating amino acids following ingestion of plant- versus animal-derived proteins has been reported previously (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e) and may be attributed to differences in protein structure and function that stimulate amino acid retention in splanchnic tissues (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Lower postprandial protein-derived amino acid availability lowers protein bioavailability and may even compromise the bio-functionality of a protein or protein source (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Therefore, many strategies are being applied to accelerate protein digestion and subsequent amino acid absorption.\u003c/p\u003e\u003cp\u003eProtein processing can be used to modulate protein digestion and amino acid absorption, thereby increasing postprandial plasma amino acid availability (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Previously, enzymatic hydrolysis of casein has been shown to accelerate protein digestion and increase protein-derived amino acid release in the circulation, resulting in greater overall postprandial plasma amino acid availability \u003cem\u003ein vivo\u003c/em\u003e in humans (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). In the present study, protein processing, either through enzymatic modification or heating, was applied to the native canola protein to study the effects on protein digestion and amino acid absorption. Here, we observed a more rapid postprandial rise in the concentration of some, but certainly not all, amino acids following ingestion of enzyme processed versus native canola, but this did not result in greater overall postprandial plasma amino acid availability (171\u0026thinsp;\u0026plusmn;\u0026thinsp;76 \u003cem\u003evs\u003c/em\u003e 163\u0026thinsp;\u0026plusmn;\u0026thinsp;81 mmol∙300 min∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e respectively, Treatment, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.433\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). So far, only one study has compared postprandial plasma amino acid responses following ingestion of a hydrolyzed versus intact canola protein (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Fleddermann \u003cem\u003eet al\u003c/em\u003e. reported more rapid increases in circulating amino acid concentrations following ingestion of hydrolyzed versus intact canola protein, without differences in overall plasma amino acid availability. Besides enzymatic hydrolysis, heating can also be applied to proteins with relative low denaturation temperatures. The open protein structure obtained through heating can enhance digestibility and accelerate amino acid absorption kinetics (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan additionalcitationids=\"CR50 CR51\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Previous work in pigs has shown that heat processing can improve canola protein digestibility by 22% (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). In the present study, ingestion of heat processed canola protein resulted in a more rapid postprandial rise in some of the circulating amino acid concentrations. Even though we also observed a 22% greater increase in plasma essential amino acid availability following the ingestion of heat processed versus intact canola protein (111\u0026thinsp;\u0026plusmn;\u0026thinsp;41 \u003cem\u003evs\u003c/em\u003e 91\u0026thinsp;\u0026plusmn;\u0026thinsp;35 mmol∙300 min∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), no significant differences were observed between treatments (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.282\u003c/em\u003e). Overall, the present data show a modest impact of canola protein processing on the postprandial increase in circulating plasma amino acid concentrations, without any substantial changes in the overall postprandial increase in plasma amino acid availability.\u003c/p\u003e\u003cp\u003eDespite the absence of any substantial impact of the currently tested enzyme or heat processing conditions of canola protein isolate on the postprandial rise in circulating plasma amino acid concentrations, it remains important to evaluate the effects of various processing techniques on protein digestion and subsequent amino acid absorption. Here we assessed the postprandial rise in circulating amino acids, expressed as the incremental area under the curve (iAUC), as a proxy for postprandial protein-derived amino acid availability. However, a more quantitative assessment of protein digestion and amino acid absorption requires a dual isotope tracer approach (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). The use of intrinsically labeled proteins would be required to provide evidence for the proposed impact of protein processing on protein digestion and amino acid absorption \u003cem\u003ein vivo\u003c/em\u003e in humans (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Here, we show no significant differences in postprandial plasma amino acid availability following ingestion of both enzyme and heat processed canola protein when compared to the ingestion of native canola protein. However, it should be noted that protein processing techniques may affect specific amino acids. For example, previous work in our group has shown that heating of milk protein can cause glycation, which strongly reduces protein-derived lysine (bio)availability (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Therefore, we also evaluated individual plasma amino acid concentrations and their incremental areas under the curve (Supplemental File 1). In contrast to milk protein, heat processing of canola protein did not negatively impact postprandial plasma lysine availability, and no specific differences in individual amino acid responses were observed following the ingestion of processed versus native canola protein.\u003c/p\u003e\u003cp\u003eThe present data show a minor role of enzymatic modification and mild heat treatment on postprandial plasma amino acid bioavailability following canola protein ingestion. However, as commercial protein processing techniques vary considerably, the impact of commonly applied processing techniques on postprandial amino acid bioavailability and bio-functionality will need to be addressed (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). This may be of increasing relevance with regards to the protein transition towards the consumption of more plant-based proteins (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). The use of plant-derived protein isolates and concentrates in meat and dairy alternatives requires extensive processing and may strongly impact the bioavailability of the available protein(s) in these products. Therefore, more work is needed to assess the impact of the various protein- and food processing procedures on the bioavailability of these proteins or protein sources \u003cem\u003ein vivo\u003c/em\u003e in humans.\u003c/p\u003e\u003cp\u003eIn conclusion, ingestion of native canola protein allows for a more delayed postprandial rise in circulating essential and non-essential amino acids and a lower postprandial plasma amino acid availability when compared to the ingestion of whey protein. Ingestion of enzyme-modified or heat-processed canola protein accelerates the postprandial rise in circulating amino acids but does not further augment overall plasma amino acid availability throughout a 5 hour postprandial period when compared to the ingestion of native canola protein.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cspan lang=\"EN-CA\"\u003eSources of support:\u003c/span\u003e\u003c/strong\u003e\u003cspan lang=\"EN-CA\"\u003e\u0026nbsp;The presented research was co-funded by dsm-firmenich AG (Delft, The Netherlands).\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eConflicts of interest and funding disclosure: The authors have the following interests: This study was co-funded by dsm-firmenich AG (Delft, The Netherlands). The researchers were responsible for the study design, data collection, and analysis, decision to publish, and preparation of the manuscript. The choice of interventional products, and the production thereof was performed by the funder. The funder had no role in data collection, analysis of the main outcome measures, or decision to publish. In preparation of the manuscript, the funder was involved to evaluate aspects of intellectual property rights and confidentiality. LJCvL and LBV have received research grants, consulting fees, speaking honoraria, or a combination of these for research on the impact of exercise and nutrition on muscle metabolism. A full overview on research funding is provided at: \u003cspan lang=\"EN-US\"\u003ehttps://www.maastrichtuniversity.nl/l.vanloon\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003eCorresponding author: Prof. Luc J.C. van Loon, Department of Human Biology, NUTRIM Institute of Nutrition and Translational Research in Metabolism, Maastricht University Medical Centre+, PO box 616, 6200 MD Maastricht, The Netherlands, Tel: +31 43 388 1397, Email: [email protected]\u0026nbsp;\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThe author contributions were as follows: LJCvL, LBV, WH, NB and IW designed research; NB and WH conducted research; NB, WH, AO, JMXvK, JMS, LBV and LJCvL analyzed data; NB and LJCvL wrote paper; NB and LJCvL had primary responsibility for final content. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Lisa M.E. Kuin for medical assistance, Joan M. Senden and Antoine Zorenc for practical assistance and Wendy E. Sluijsmans and Hasibe Aydeniz for their analytical work. We also extend our gratitude to all study participants for their time and commitment.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBoirie Y, Dangin M, Gachon P, Vasson M-P, Maubois J-L, Beaufr\u0026egrave;re B (1997) Slow and fast dietary proteins differently modulate postprandial protein accretion. Proceedings of the national academy of sciences. ;94(26):14930-5\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGroen BB, Horstman AM, Hamer HM, de Haan M, van Kranenburg J, Bierau J et al (2015) Post-Prandial Protein Handling: You Are What You Just Ate. 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J Nutr\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTrommelen J, Tom\u0026eacute; D, van Loon LJ (2021) Gut amino acid absorption in humans: concepts and relevance for postprandial metabolism. Clin Nutr Open Sci 36:43\u0026ndash;55\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKashyap S, Shivakumar N, Varkey A, Duraisamy R, Thomas T, Preston T et al (2018) Ileal digestibility of intrinsically labeled hen's egg and meat protein determined with the dual stable isotope tracer method in Indian adults. Am J Clin Nutr 108(5):980\u0026ndash;987\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKashyap S, Varkey A, Shivakumar N, Devi S, Reddy BHR, Thomas T et al (2019) True ileal digestibility of legumes determined by dual-isotope tracer method in Indian adults. 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Br J Nutr 123(5):545\u0026ndash;552\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003evan Lieshout GA, Trommelen J, Nyakayiru J, van Kranenburg J, Senden JM, Gijsen AP et al (2025) Protein glycation compromises the bioavailability of milk protein-derived lysine in vivo in healthy adult males: a double-blind randomized cross-over trial. Am J Clin Nutr\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHerreman L, Nommensen P, Pennings B, Laus MC (2020) Comprehensive overview of the quality of plant-And animal‐sourced proteins based on the digestible indispensable amino acid score. Food Sci Nutr 8(10):5379\u0026ndash;5391\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoughan PJ (2021) Population protein intakes and food sustainability indices: the metrics matter. Global Food Secur 29:100548\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEvenepoel P, Geypens B, Luypaerts A, Hiele M, Ghoos Y, Rutgeerts P (1998) Digestibility of cooked and raw egg protein in humans as assessed by stable isotope techniques. J Nutr 128(10):1716\u0026ndash;1722\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFuchs CJ, Hermans WJ, Smeets JS, Senden JM, van Kranenburg J, Gorissen SH et al (2022) Raw Eggs To Support Postexercise Recovery in Healthy Young Men: Did Rocky Get It Right or Wrong? J Nutr 152(11):2376\u0026ndash;2386\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKoopman R, Crombach N, Gijsen AP, Walrand S, Fauquant J, Kies AK et al (2009) Ingestion of a protein hydrolysate is accompanied by an accelerated in vivo digestion and absorption rate when compared with its intact protein. Am J Clin Nutr 90(1):106\u0026ndash;115\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCampbell L, Rempel CB, Wanasundara JP (2016) Canola/Rapeseed Protein: Future Opportunities and Directions-Workshop Proceedings of IRC 2015. Plants (Basel). ;5(2)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSarwar M, Ahmad N, Siddiqui Q, Rajput A, Tofique M (2003) Efficiency of different chemicals on Canola strain Rainbow (Brassica napus L.) for aphids control. Asian J Plant Sci 2(11):831\u0026ndash;833\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWickramasuriya SS, Yi YJ, Yoo J, Kang NK, Heo JM (2015) A review of canola meal as an alternative feed ingredient for ducks. J Anim Sci Technol 57:29\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBos C, Airinei G, Mariotti F, Benamouzig R, B\u0026eacute;rot S, Evrard J et al (2007) The poor digestibility of rapeseed protein is balanced by its very high metabolic utilization in humans. 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J Agric Food Chem 60(10):2569\u0026ndash;2576\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBax ML, Buffi\u0026egrave;re C, Hafnaoui N, Gaudichon C, Savary-Auzeloux I, Dardevet D et al (2013) Effects of meat cooking, and of ingested amount, on protein digestion speed and entry of residual proteins into the colon: a study in minipigs. PLoS ONE 8(4):e61252\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMansour E, Dworsch\u0026aacute;k E, Lugasi A, Ga\u0026aacute;l \u0026Ouml;, Barna E, Gergely A (1993) Effect of processing on the antinutritive factors and nutritive value of rapeseed products. Food Chem 47(3):247\u0026ndash;252\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBadshah A, Sattar A, Bibi A (1993) Effect of irradiation and other processing methods on in-vitro digestibility of rapeseed protein. J Sci Food Agric 61(2):273\u0026ndash;275\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTrommelen J, van Loon LJC (2024) Quantification and interpretation of postprandial whole-body protein metabolism using stable isotope methodology: a narrative review. Front Nutr 11:1391750\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003evan Loon LJ, Boirie Y, Gijsen AP, Fauquant J, de Roos AL, Kies AK et al (2009) The production of intrinsically labeled milk protein provides a functional tool for human nutrition research. J Dairy Sci 92(10):4812\u0026ndash;4822\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJuul F, Parekh N, Martinez-Steele E, Monteiro CA, Chang VW (2022) Ultra-processed food consumption among US adults from 2001 to 2018. Am J Clin Nutr 115(1):211\u0026ndash;221\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWillett W, Rockstr\u0026ouml;m J, Loken B, Springmann M, Lang T, Vermeulen S et al (2019) Food in the Anthropocene: the EAT-Lancet Commission on healthy diets from sustainable food systems. Lancet 393(10170):447\u0026ndash;492\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"amino-acids","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"amac","sideBox":"Learn more about [Amino Acids](http://link.springer.com/journal/726)","snPcode":"726","submissionUrl":"https://submission.nature.com/new-submission/726/3","title":"Amino Acids","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Plant-derived protein, bioavailability, protein processing","lastPublishedDoi":"10.21203/rs.3.rs-6733821/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6733821/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCanola protein is a rapeseed-derived protein with a complete amino acid profile, making it an interesting protein for human food applications. It is currently unknown whether canola protein processing modulates postprandial plasma amino acid bioavailability \u003cem\u003ein vivo\u003c/em\u003e in humans. This study compared postprandial plasma amino acid profiles following the ingestion of unprocessed (native) canola, processed canola, and whey protein isolate \u003cem\u003ein vivo\u003c/em\u003e in healthy, young, females. In a randomized, clinical, cross-over design, 15 healthy young females (25\u0026thinsp;\u0026plusmn;\u0026thinsp;3 y) participated in four test days on which they consumed 20 g protein as either native canola, enzyme processed or heat processed canola protein, or 20 g whey protein. Blood samples were collected for 5 h following protein ingestion to assess plasma amino acid concentrations. Ingestion of native canola protein resulted in lower increases in plasma total amino acid TAA concentrations compared to whey protein (3191\u0026thinsp;\u0026plusmn;\u0026thinsp;794 vs 4429\u0026thinsp;\u0026plusmn;\u0026thinsp;84, \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). Canola protein processing resulted in greater peak plasma total amino acids concentrations, reaching statistical significance for enzyme (3599\u0026thinsp;\u0026plusmn;\u0026thinsp;687 \u0026micro;mol∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.045\u003c/em\u003e) but not heat (3565\u0026thinsp;\u0026plusmn;\u0026thinsp;722 \u0026micro;mol∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.166\u003c/em\u003e) treated compared to native canola protein. Plasma total amino acid availability, expressed as incremental area under the curve over a 5 h postprandial period, did not differ between treatments and averaged 163\u0026thinsp;\u0026plusmn;\u0026thinsp;81, 171\u0026thinsp;\u0026plusmn;\u0026thinsp;76, 194\u0026thinsp;\u0026plusmn;\u0026thinsp;82, and 207\u0026thinsp;\u0026plusmn;\u0026thinsp;85 mmol∙300 min∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e following ingestion of native, enzyme- and heat processed canola, and whey protein, respectively (\u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e). Ingestion of whey protein allows for a more rapid postprandial rise in circulating essential and non-essential amino acids and greater postprandial plasma total amino acid availability when compared to the ingestion of native canola protein. Ingestion of enzyme or heat-processed canola protein accelerates the postprandial rise in circulating amino acids but does not further augment overall plasma amino acid availability throughout a 5 h postprandial period when compared to the ingestion of native canola protein.\u003c/p\u003e","manuscriptTitle":"Canola protein processing modifies postprandial plasma amino acid profiles in vivo in healthy, young females","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-17 17:01:52","doi":"10.21203/rs.3.rs-6733821/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-18T13:31:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-08T05:50:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-19T19:32:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54163713601821031829936722030326490234","date":"2025-07-18T12:34:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"236134595648656194434311712268530468065","date":"2025-07-17T23:51:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-15T12:40:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-29T17:19:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-28T15:22:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Amino Acids","date":"2025-05-23T14:23:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"amino-acids","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"amac","sideBox":"Learn more about [Amino Acids](http://link.springer.com/journal/726)","snPcode":"726","submissionUrl":"https://submission.nature.com/new-submission/726/3","title":"Amino Acids","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"310c4b88-c126-43f0-a7a3-751d77f4c7bd","owner":[],"postedDate":"July 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-20T16:03:27+00:00","versionOfRecord":{"articleIdentity":"rs-6733821","link":"https://doi.org/10.1007/s00726-025-03482-1","journal":{"identity":"amino-acids","isVorOnly":false,"title":"Amino Acids"},"publishedOn":"2025-10-17 15:57:11","publishedOnDateReadable":"October 17th, 2025"},"versionCreatedAt":"2025-07-17 17:01:52","video":"","vorDoi":"10.1007/s00726-025-03482-1","vorDoiUrl":"https://doi.org/10.1007/s00726-025-03482-1","workflowStages":[]},"version":"v1","identity":"rs-6733821","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6733821","identity":"rs-6733821","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}