OR I G I N A L AR T I C L E Canola meal valorization via acid hydrolysis to generate free amino acids Sumudu N. Warnakulasuriya1,2 | Takuji Tanaka2 | Janitha P. D. Wanasundara1,2 1Agriculture and Agri-Food Canada, Saskatoon Research and Development Centre, Saskatoon, Saskatchewan, Canada 2Department of Food and Bioproduct Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada Correspondence Janitha P. D. Wanasundara, Department of Food and Bioproduct Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, Saskatchewan S7N 5A8, Canada. Email: janitha.wanasunadara@agr.gc.ca Funding information Global Institute of Food Security, University of Saskatchewan, Grant/Award Numbers: J-001661, J-001589 Abstract This study investigated an alternative approach to valorizing canola proteins by hydrolyzing them to generate amino acids (AAs). Pre-treatment of cold-pressed (CP) cake and desolventized-toasted (DT) meal with ethanol (99%, vol/vol) followed by protein separation was studied as process optimizations to maxi- mize protein recovery with higher purity. The optimum ethanol pre-treatment conditions to achieve a meal containing less than 1% oil was reached at a meal-to-ethanol ratio of 1:4 (wt:wt) and 50�C for 30 min extraction. The protein recovery reached the maximum at pH 12 and a meal-to-solvent ratio of 1:10 (wt:vol), yielding 73% and 33% recovery from ethanol pre-treated CP and DT meals, respectively, in a single extraction. Untreated and ethanol pre-treated meals were hydrolyzed with 6 N HCl (protein-to-acid ratio of 5 mg:2 mL) for 24 h at 110�C. The ethanol pre-treatment improved AA recovery and released 373 mg AA/g dry CP meal biomass (dbm) compared to 279 mg AA/g untreated CP cake dbm. However, no improvement in AA recovery upon ethanol pre- treatment of DT meal. Sulfuric acid was examined as an alternative acid. More than 700 mg AA/g CP protein were released with 6 N H2SO4, while for DT meal proteins, a 10 N concentration was needed to achieve a closer value. Commer- cial canola meals can be utilized for generating free AAs; however, the meal processing history may affect the yield. KEYWORDS alkali extraction, canola meal protein, ethanol pre-treatment, free amino acids, H2SO4 hydrolysis, response surface methodology INTRODUCTION Industrial-scale processing fractionates canola seed into oil and meal with an average weight basis recovery of �43% and �57%, respectively (COPA, 2020). The pri- mary canola meal market is for animal feeds that is based on the nutritional quality of protein. The pre-press solvent extraction (PSE) process that yields desolventized- toasted (DT) meal is the most practiced by the canola oil industry and is highly efficient in recovering seed oil. Expeller-pressing (EP) involves only mechanical forces to rupture seeds and express oil. Cold-pressing is a form of EP that only partially recovers seed oil and is practiced on a small scale primarily to obtain organic, non-GMO or virgin canola oil (Ghazani et al., 2014). Bio-diesel industry uses repeated EP to ensure maximum seed oil recovery and produces a full-pressed (FP) meal (CCC, 2019; Gaber et al., 2018; Ghazani et al., 2014). During PSE, seed and meal are exposed to high heat, that is, 80–100�C for 30–35 min in the seed cook- ing step, 66–71�C in the solvent extraction step, and 95–115�C for �30 min in the meal desolventizing and toasting step. In the EP, the frictional forces of the mechanical press can have the material tempera- ture rising to 160�C for a short time that enhancing oil recovery and producing a low residual oil (usually 8%–11%) containing FP meal; however, heat damage to protein is unavoidable (Fetzer et al., 2018; Salazar- Villanea et al., 2017). In contrast, cold-pressing main- tains temperatures of the pressing material below 60�C Received: 6 June 2023 Revised: 22 July 2023 Accepted: 11 August 2023 DOI: 10.1002/aocs.12739 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2023 His Majesty the King in Right of Canada and The Authors. Journal of the American Oil Chemists’ Society published by Wiley Periodicals LLC on behalf of AOCS. Reproduced with the permission of the Minister of Agriculture and Agr-Food Canada. J Am Oil Chem Soc. 2024;101:41–57. wileyonlinelibrary.com/journal/aocs 41 https://orcid.org/0000-0001-7491-8867 mailto:janitha.wanasunadara@agr.gc.ca http://creativecommons.org/licenses/by-nc-nd/4.0/ http://wileyonlinelibrary.com/journal/aocs http://crossmark.crossref.org/dialog/?doi=10.1002%2Faocs.12739&domain=pdf&date_stamp=2023-09-22 and cold-pressed (CP) canola cake contains 13%–16% residual oil (Ghazani et al., 2014). There is no remarkable difference between CP and DT canola meals in their crude protein levels and total amino acid (AA) profiles. Compared to CP meal, components of DT meal pass through high heat and hot apolar solvents that cause excessive structure alterations involving protein–protein and protein–non-protein component inter- actions in addition to the consequences of protein surface modifications. Furthermore, the Maillard reaction occurs due to high heat and leads to crosslinking between pro- tein and non-protein components, such as reducing sugars in the fiber fraction (cellulose, hemicellulose), low- ering the solubility of meal protein (Gaber et al., 2018; Mosenthin et al., 2016). Poor solubility results in less extractable proteins, that is, low protein recovery yields in wet protein extraction processes. In addition, the less competent functional attributes of the recov- ered proteins makes DT canola meal an unsuitable starting material to generate protein products with the competent quality required to be a competitor in the plant protein market. The well-balanced amino acid profile of canola meal protein is highly valued in the feed use and the amino acids remain intact within pro- teins during industrial oil extraction process (Fetzer et al., 2018; Mosenthin et al., 2016). Globally, AAs are produced for food and feed addi- tives, pharmaceuticals (active ingredients and for cul- ture mediums), medical foods, cosmetics and personal care, dietary supplements, and health and wellness products with a rapidly increasing demand for individual AAs. In addition to providing nutritional requirements, AAs can be feed molecules for several industrial prod- ucts, that is, surfactants (Clapés & Infante, 2002), phar- maceuticals (Martnez-Rodrguez et al., 2010), and plant bio-stimulants (Maini, 2006). Amino acids can be functio- nalized to attain desirable attributes with promising appli- cations. For instance, N-acyl AAs are particularly known to have good skin compatibility, antimicrobial activity, and calcium tolerability, which are required attributes as a surfactant in the personal care and detergent industry (Clapés & Infante, 2002). Production of AAs can be through microbial fermentation or chemical synthesis. Hydrolysis of protein can be achieved via chemical or enzymatic means. Chemical hydrolysis using alkali or acid is a comparatively economical, easy-to-control pro- cess that can be scaled up to obtain AAs with a lesser amount of peptides (Fountoulakis & Lahm, 1998). Extraction of AA from protein-rich plant or animal sources is possible through hydrolysis; however, the production of plant-based AA is limited due to the lack of production technologies and the requirement of high capital costs (Leuchtenberger et al., 2005). In 2020, the global AA market was �9.8 million tons and is expected to reach 13.1 million tons by 2026, exhibiting a com- pound annual growth rate (CAGR) of �5% during 2021–2026 (Imarcgroup, 2021). The inadequate supply of AAs to satisfy increasing demand in diverse markets and applications creates opportunities for non-food non- conventional sources for protein-derived AA production. Studies on AA generation from agricultural material pro- cessing co-products include; hydrolysis of cotton seed by acid hydrolysis (Xia et al., 1996), combining microbial fermentation followed by hydrolysis with hydrochloric acid (Zhang et al., 2016), and enzyme-catalyzed hydro- lysis of pea flour (Rondel et al., 2011) to converting AAs into surfactant molecules. In 2021, �78% of Canadian canola production was domestically processed for oil, generating 5.7 million tonnes of canola meal primarily through PSE and expects a continuing increase in domestic proces- sing and meal production (COPA, 2020). Consider- ing the challenges to penetrate as a source for expanding plant protein market opportunities due to the distressed nature of consisting proteins (Östbring et al., 2020), canola meal requires alternative approaches for valorizing. Modification of canola meal proteins as value-added propositions beyond feed applications have been reported, for example, plastics (Manamperi et al., 2010), adhesives (Wang et al., 2014), bio-composites (Li et al., 2018), films with barrier properties (Chang & Nickerson, 2014), surfactants (S�anchez-Vioque et al., 2001), control delivery of bioactives (Akbari & Wu, 2016), and gen- eration of enzymatic hydrolysates to generate pep- tides that can exert antihypertensive (Wu & Muir, 2008), antioxidative (Zhang et al., 2008), anti- fungal (Nioi et al., 2012), and antiviral (Yust, 2004) properties. However, an approach to using proteins of extensively processed canola meal to convert into AAs or uses for AAs from canola meal has not been reported in the literature. The present study reports on the investigation of industrially processed CP cake and DT meal to gener- ate free AAs, including suitable pre-treatments to reduce non-protein components and to obtain protein- enriched fractions prior to protein hydrolysis involving an alternative acid to HCl. MATERIALS AND METHODS Materials Ethanol (99%, denatured by 1% ethyl acetate) was obtained from Commercial Alcohols (Toronto, ON, Canada). All chemicals were of analytical grade and obtained from Sigma-Aldrich (Oakville, ON, Canada) and Fisher Scientific (Toronto, ON, Canada). Canola meals processed at a commercial scale and by two methods were used in the study. CP cake (Virtex Farm Foods, Saskatoon, SK, Canada and Pleasant Valley Oil Mills, Clive, AB, Canada) and DT meal (Bunge Canada, Nipawin, SK, Canada and ADM Pro- cessing, Lloydminster, SK, Canada) were obtained from three random production lots from the same 42 JOURNAL OF THE AMERICAN OIL CHEMISTS’ SOCIETY 15589331, 2024, 1, D ow nloaded from https://aocs.onlinelibrary.w iley.com /doi/10.1002/aocs.12739 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense processing plant. For the experiments, meal samples were milled with Restch Ultra Centrifugal Mill ZM 200 (Restch GmbH, Haan, Germany) and passed through a 250 μm mesh screen. Chemical analysis All meals were analyzed for the contents of moisture, ash and crude protein according to the AOAC method 934.01, 2005(a), 942.05, 2005(b), and 990.03, 2005(c) (con- version factor 6.25), respectively (AOAC International, 2005). Residual oil content was determined by the Swedish tube method (AOCS, 1997). The total phenolic con- tent of methanolic extracts was determined using the Folin–Ciocalteu method and expressed as milligram of sinapic acid equivalent (SAE; Velioglu et al., 1998). Free sugars and their contents Free sugars were extracted into water (meal to solvent ratio 1:10, wt:vol) by stirring at 1400 rpm at 60�C for 1 h using a ThermoMixer C (Eppendorf Canada, Missis- sauga, ON, Canada). The supernatant was cleaned using a mixed ion exchanger Strata ABW cartridge (Phenomenex, Inc., Canada) and analyzed using an ultra-performance liquid chromatography (UPLC) system. The separation was achieved by Acquity UPLC BEH Amide column (1.7 μm, 2.1 � 100 mm) at 85�C using a gradient solvent system (vol/vol): solvent A [95% acetoni- trile, 5% water, 0.1% triethylamine (TEA)], and solvent B (30% acetonitrile, 70% water, 0.1% TEA) at a flow rate of 0.3 mL/min and injection volume of 2–5 μL, and detected by an evaporative light scattering detector. The contents of sucrose, fructose, and glucose were determined using an external calibration curve (200–800 mg/mL). Amino acid analysis The complete amino acid profile (18 amino acids) of untreated meals as received, meals after treatments and their respective protein fractions were determined according to the standard AOAC 994.12, 2005d method (AOAC International, 2005) using three sepa- rate hydrolyses: (a) 6 N HCl hydrolysis; (b) 6 N HCl acid hydrolysis following performic acid pre-treatment; and (c) base hydrolysis as described below. a. Acid hydrolysis (for 15 AAs except for cysteine, methionine, and tryptophan) was carried out by react- ing the sample (2.5 mg protein/mL) with 6 N HCl con- taining 2% (vol/vol) phenol at 110�C for 24 h. Meal hydrolysate was then transferred, neutralized using NaOH, added to a known amount of 20 mM α- aminobutyric acid (internal standard), and brought to the desired volume with water. Hydrolysates were passed through a 0.45-μm filter (Phenex RC syringe filter, Phenomenex, Torrance, CA, USA) and further cleaned using a Waters Oasis HLB cartridge (Waters Oasis, Waters Corp., Mississauga, ON, Canada) before derivatization for HPLC analysis. b. Meal samples (10 mg protein/mL) maintained in an ice bath were added to the cold, freshly prepared performic acid solution, mixed vigorously for 15 min, and incubated at 4�C overnight to ensure conver- sion of thiol and disulfide groups of cysteine and cysteine to cystic acid and methionine to methionine sulfone. Then, the tubes were removed from the ice, and solid sodium metabisulfite was added to each tube to decompose excess performic acid. Samples were stirred for 15–20 min until the bubbling stopped, and 6 N HCl hydrolysis was followed essentially the same as described above in (a), including neutralization, internal standard, and cleaning before derivatizing for HPLC analysis. c. Tryptophan content determination was carried out on a base hydrolyzed sample. The sample (�10 mg protein/mL) was mixed with 4.2 N NaOH and hydro- lyzed at 205�C for 20 min in a microwave digester (CEM Discover SPD). The hydrolysate was quanti- tatively transferred into a volumetric flask containing 6 N HCl and 20 mM 5-methyl tryptophan (internal standard) and brought to the desired volume with water. The filtered hydrolysate (passing through a 0.45-μm Phenex RC syringe) was eluted through Water Oasis HLB cartridge, first with 1.0 mL of 5% (vol/vol) methanol: 5% (vol/vol) acetonitrile: 90% (vol/vol) water, and then with 4.0 mL of 5% (vol/vol) acetonitrile in water and collected into the same flask and brought up to the volume with water. All protein hydrolysates prepared according to hydrolysis procedures (a) and (b) were derivatized according to the Waters AccQtag amino acid analysis method, following the Waters AccQFlour Reagent kit manual (Waters Corp., Mississauga, ON, Canada). Then, samples were run on HPLC (Waters Aliance 2695) with C18 AccQ-Tag Column (3.9 mm � 150 mm) and a fluorescence detector (Waters 2475, excitation at 250 nm and emission at 395 nm), using Eluent A:Waters AccQTag eluent A buffer, Eluent B:HPLC grade acetonitrile, and Eluent C:water, at a flow rate of 0.75 mL/min for cysteine and methionine and 1.0 mL/min for all other amino acids. Hydrolysates prepared by base hydrolysis (procedure c) were analyzed with HPLC directly after cleaning without derivatization. Ethanol pre-treatment Both CP cake and DT meal were pre-treated with etha- nol, and the effect of extraction time, temperature, and meal-to-solvent ratio (wt:vol; three factors or indepen- dent variables) in removing residual oil, proteins, total JOURNAL OF THE AMERICAN OIL CHEMISTS’ SOCIETY 43 15589331, 2024, 1, D ow nloaded from https://aocs.onlinelibrary.w iley.com /doi/10.1002/aocs.12739 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense phenolics, and free sugars (response variables) of meals was examined. Twenty experimental combina- tions designed according to a central composite design (CCD; Table S1) were used, and the response surface methodology (RSM) was employed to find the optimum levels of the independent variables that affect these multiple responses (Design Expert software, version 11). At each experiment point, a meal sample (�30 g) was stirred with 99% ethanol at the pre-determined meal-to-solvent ratio for the respective time while maintaining the required temperature, as indicated in Table S1. The resulting meal slurry was filtered under a vacuum (Whatman #1 filter paper), and meal residue was collected, air-dried overnight and stored in air-tight containers at 4�C for further analysis. Moisture content, residual oil content, crude protein, total phenolics, and free sugars of the ethanol pre-treated meal were deter- mined as mentioned previously. Six different samples of CP cake and DT meal obtained on different proces- sing lots were used to verify the applicability of treat- ment conditions found as optimum from data analysis (meal-to-solvent ratio of 1:4 wt/vol, 30 min, 50�C). All samples used in the verification study were ground and characterized for chemical composition before and after ethanol treatment. Protein separation Ethanol pre-treated CP and DT meals with the mini- mum level of non-protein components were used for protein separation. It was expected that separating pro- teins from other meal components may further minimize the effect of non-protein components on hydrolytic reactions and resulting products. A two-factor model using CCD that includes 13 experiment points (Table S2) was used to determine suitable protein extraction conditions and pH and meal-to-solvent ratio (wt:vol) as independent variables. Meal samples (5.0 g) suspended in water were mixed well, and pre- determined levels of 0.1–10 M NaOH was added to reach the required pH. After adding water to achieve the desired meal-to-solvent ratio, the meal slurry was continuously mixed while maintaining the required pH. After 1 h of mixing, the suspension was centrifuged (20 min at 12,429g), and the recovered supernatant was freeze-dried. The contents of total dry matter and crude protein of the freeze-dried supernatant were determined and used in calculating the recovery of dry biomass (dbm) and protein compared to the meal dry matter and meal protein which were the response variables for RSM analysis (Design expert software, version 11). CP and DT meal samples (untreated and ethanol pre-treated) were separately studied for the pH levels and meal-to-solvent ratio that resulted in the maximum level of protein recovery. Those conditions were considered to be suitable to continue in further experiments. Under the optimum extraction conditions determined from data analysis, repeated extractions (second and third times) were carried out to determine the number of multiple extractions that can result in maximum protein recovery. Six different DT and CP meal samples were tested to verify the applicability of conditions determined for ethanol pre-treatment and protein extraction. Suitable H2SO4 acid concentration for meal protein hydrolysis A concentration series of H2SO4 (3–16 N) was tested to understand the effect of acid concentration on releasing free amino acids from both ethanol-treated CP and DT meals. Conditions used in the acid hydroly- sis were the same as the 6 N HCl hydrolysis described above; only the acid and the concentrations were differ- ent. Meals and extracted protein samples prepared to contain 2.5 mg protein/mL with H2SO4 were incubated at 110�C for 24 h, neutralized using NaOH, and added α-aminobutyric acid as the internal standard. The neu- tralized hydrolysate was cleaned following the same procedure as in 6 N HCl hydrolysis (a) and quantified for liberated amino acids. Statistical analysis All experiments were carried out using a minimum of three biological replicates and the analyses were car- ried out with three technical replicates at least, and the means values are reported with standard deviation. In optimizing ethanol pre-treatment and protein extraction conditions in alkali pH ranges, experimental points were determined according to CCD as described in each experiment. In both cases, the analysis of the results was carried out by RSM (Design expert, version 11). To verify the applicability of optimization experiment results, six separate meal samples were subjected to the selected conditions and data analysis was done by one-way ANOVA for each response sepa- rately, and multiple mean comparison was done by Tukey’s, p ≤ 0.05. RESULTS AND DISCUSSION Initial composition analysis showed that CP cake and DT meal primarily differ in oil and protein contents of their dry biomass; CP cake has �3 times more oil and 0.88 times proteins of DT meal (Figure 1). The free sugar fraction of CP meal was composed of glucose, fructose, and sucrose. Only sucrose was detected in DT meal inferring comparatively very low levels of reducing sugars most likely a direct result of PSE pro- cess conditions as pointed out by Adewole et al. (2016). The values for ash, total phenolics, and fiber 44 JOURNAL OF THE AMERICAN OIL CHEMISTS’ SOCIETY 15589331, 2024, 1, D ow nloaded from https://aocs.onlinelibrary.w iley.com /doi/10.1002/aocs.12739 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense and other components of both meals were similar between samples (Figure 1). Extraction of meal with ethanol was tested as a pre-treatment step to reduce the levels of oil, phenolic compounds, and sugars in order to reduce interferences at the protein hydrolysis stage. Ethanol pre-treated meal material with and with- out protein separation was proceeded to acid hydrolysis. Ethanol pre-treatment and condition optimization The residual oil, total phenolics, total sugars, and pro- tein of the resulting CP and DT meals were affected by combinations of time, temperature, and the meal- to-solvent ratio in the ethanol pre-treatment. RSM anal- ysis provided information on the effect of treatment combinations that can maximize the removal of oil, phe- nolics, and sugars (Figures 2a,c,d and 3a,c,d and Table S3) while enriching the protein content (Figures 2b and 3b). In reducing meal oil content, for CP cake (70%–80% reduction), the extraction time, meal-to-solvent ratio, and temperature of ethanol treat- ment were significant (p ≤ 0.05), and for DT meal (78%–93% reduction) meal-to-solvent ratio was the only significant (p ≤ 0.05) factor. In this experimental region, the protein level increase in CP cake by 13.7%– 22.8% and DT meal by 4.0%–8.7% was observed. Both the time and temperature of ethanol treatment showed a significant (p ≤ 0.05) effect on enriching meal protein content of CP cake and DT meal; in addition, the meal- to-solvent ratio also had a significant (p ≤ 0.05) effect on CP cake. Ethanol pre-treatment was able to reduce the level of phenolic compounds in both meals; 7%–35% in CP cake and 2%–27% in DT meal with tem- perature having a significant (p ≤ 0.05) effect. Free sugar level reduction caused by ethanol pre-treatment was by 4.0% in CP meal and by 1%–40% in DT meal with both time and temperature having a significant (p ≤ 0.05) effect. The increase in protein content of meals was due to the removal of ethanol-soluble components during the pre-treatment. Although ethanol without added water was less effective in removing phenolic compounds and free sugars, a significant amount of oil was removed from both meals enriching their meal protein content. Slawski et al. (2012) reported a similar obser- vation for a canola cake (31% protein and 13% oil) that resulted in a final meal containing 40% protein and 1% residual oil after applying a four-step treatment with 75% (vol/vol) ethanol. The reduction of CP and DT meal phenolics by 99% ethanol gave a limited benefit (�30% reduction). Since the solubility of phenolics depends on the chemistry of their structure as well as the interactions with proteins and other seed compo- nents via ionic, hydrogen, covalent bonds, and hydro- phobic interactions (Xu & Diosady, 2000), the solvent polarity plays a vital role in the solubility. Extractable phenolics of defatted canola meal have been reported as 1.59–1.84 g/100 g of the meal (Naczk et al., 1998). Aqueous methanol (70%–100%, vol/vol; Cai & Arntfield, 2001) or aqueous ethanol (65%–75%, vol/vol; Kalaydzhiev et al., 2019) are effective solvents for F I GURE 1 Composition of canola meal obtained from (a) cold-pressing (CP), and (b) pre-press solvent extraction and desolventizer-toasting (DT) and used in the study. All values are on moisture free basis as mg/g dry biomass (dbm). CP and DT meals had 65.4 and 92.3 mg moisture/g meal, respectively. CP, cold- pressed; DT, desolventized-toasted. JOURNAL OF THE AMERICAN OIL CHEMISTS’ SOCIETY 45 15589331, 2024, 1, D ow nloaded from https://aocs.onlinelibrary.w iley.com /doi/10.1002/aocs.12739 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense canola seed phenolics. Soluble sugars of oil-free canola were up to 5.7% of the dry matter and consist of glucose, fructose, and sucrose (Pedroche et al., 2004; Wanasundara et al., 2016). Aqueous organic solvents are effective in lowering soluble sugars of the meal; Berot and Briffaud (1983) reported washing of defatted rapeseed meal with 80% (vol/vol) ethanol or methanol that resulted in a meal with soluble sugars (as glucose equivalent) of 0.25% and 0.65%, respectively. Response surface analysis of data gave extraction of either CP cake or DT meal for 30 min at 50�C with a meal-to-solvent ratio of 1:4 (wt:wt) as the optimum treat- ment conditions to remove the maximum amount of oil and to lower the levels of free sugars (Figures 2 and 3). Verification experiments carried out with both meal materials resulted in close values for residual oil, total protein, total phenolic contents, and free sugar levels of resulting meals as the predicted values from the developed model equation (Table 1). Several samples of CP cakes and DT meals treated under optimum conditions gave values that were expected (Table 2) confirming the conditions found optimum were suitable as pre-treatment for canola meal materials. Effect of pre-treatment on meal protein extractability It was assumed that proteins free of non-protein meal components may provide efficient hydrolysis as well as minimize hydrolysis products such as from cell wall car- bohydrates (i.e., cellulose, hemicellulose, and pectin). For both ethanol-treated CP and DT meal, aqueous extraction carried out above pH 8 was studied for the amount of recovered total dbm and protein content in the dbm as the pH and meal-to-solvent ratio changed. Response surfaces (Figure 4a,b) show that the meal- to-solvent ratio had a significant effect on the amount of F I GURE 2 Response surface plots showing the effect of temperature and time of extraction on the contents of: (a) residual oil, (b) protein, (c) total phenolics, and (d) free sugars of CP meal upon ethanol pre-treatment at a meal-to-solvent ratio of 1:4 (wt:wt). CP, cold-pressed, DT, desolventized-toasted. 46 JOURNAL OF THE AMERICAN OIL CHEMISTS’ SOCIETY 15589331, 2024, 1, D ow nloaded from https://aocs.onlinelibrary.w iley.com /doi/10.1002/aocs.12739 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense dbm in the extracts of both meals; however, pH was the most effective factor for protein recovery by solubilizing. Since the extracted dbm includes both protein and non- protein substances, the protein content of dry matter was used to deduce the minimum level of non-protein components (e.g., fiber, phytates, sugars, phenolics, etc.) co-extracted. Within this experimental range, at pH 13.8, a maximum amount of 744 and 539 mg dbm was recovered from 1 g of CP meal and DT meal, respectively. Values of extracted dbm and protein levels of dbm with percentage recovery of meal protein are in the Table S4. According to statistical optimization using RSM, pH 12 and a meal-to-solvent ratio of 1:10 (wt:vol) were the most suitable conditions for protein extraction from both meal types in a single extraction. A comparison of the contents of dry solids recovered from the meal, protein content of those dry solids, and the percentage of meal protein recovery of untreated and ethanol pre-treated CP meal (Figure 5a) and DT meal (Figure 5b) shows that the pre-treatment improved the protein content and their recovery in CP meal but not the DT meal. In a single extraction, under optimum F I GURE 3 Response surface plots showing the effect of temperature and time of extraction on the contents of: (a) residual oil, (b) protein, (c) total phenolics, and (d) free sugars of DT meal as a result of ethanol pre-treatment at a meal-to-solvent ratio of 1:4 (wt:wt). CP, cold-pressed, DT, desolventized-toasted. TAB LE 1 Validation of ethanol extraction conditions for CP and DT meals. Expected values are calculated from the polynomial equation developed by RSM and observed values are actual values resulted in from ethanol pretreatment of meals under optimum conditions.a Parameter CP meal DT meal Expected valueb Observed value Expected valueb Observed value Residual oil, mg/g dbmc 15.0 16.1 ± 0.2 9.50 8.20 ± 0.3 Protein, mg/g dbm 430.7 422.4 ± 5.6 454.0 456.3 ± 1.7 Total phenolics (mg SAEc eq/g dbm) 14.7 14.1 ± 0.4 9.8 8.4 ± 0.4 Free sugars (mg/g dbm) 52.7 53.3 ± 0.8 58.8 46.1 ± 4.4 Abbreviations: CP, cold-pressed, DT, desolventized-toasted; RSM, response surface methodology. aOptimum conditions for ethanol pretreatment are meal-t-solvent ratio 1:4 wt:vol, temperature 50�C, time 30 min. Oil, protein, total phenolics, and free sugars of starting CP meal was 133.9 mg, 363.8 mg, 17.6 mg SAE and 69.9 mg/g dbm, respectively and for DT meal was 43.3 mg, 425.3 mg, 10.9 mg SAE and 75.5 mg/g dbm, respectively. Data are presented as mean ± standard deviation. bCalculated using Equation (S1). cdbm, dry biomass; SAE, Sinapic acid equivalents. JOURNAL OF THE AMERICAN OIL CHEMISTS’ SOCIETY 47 15589331, 2024, 1, D ow nloaded from https://aocs.onlinelibrary.w iley.com /doi/10.1002/aocs.12739 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense TAB LE 2 Values of residual oil, protein, total phenolics, and free sugars after ethanol pre-treatment of randomly obtained CP and DT meal samples. All meals received ethanol pre-treatment under conditions determined as optimum.a Sample Residual oil (mg/g dbmb) Protein (mg/g dbm) Total phenolics (mg SAEb eq/g dbm) Free sugars (mg/g dbm) Untreated After treatment Untreated After treatment Untreated After treatment Untreated After treatment CP meal CP 1 137.2 ± 4.0 23.0 ± 5.0a 368.7 ± 5.6 420.0 ± 4.7bc 16.8 ± 0.3 14.4 ± 0.7a 71.2 ± 2.2 48.5 ± 0.57ab CP 2 140.8 ± 3.1 20.7 ± 2.5a 367.8 ± 7.6 423.6 ± 2.0abc 17.0 ± 0.4 14.1 ± 0.5a 69.6 ± 3.4 51.2 ± 0.56a CP 3 138.8 ± 2.3 22.0 ± 7.2a 370.2 ± 2.9 431.4 ± 9.4ab 16.7 ± 0.3 14.4 ± 0.7a 68.5 ± 4.1 49.4 ± 0.44ab CP 4 141.2 ± 4.3 25.6 ± 5.0a 364.7 ± 3.9 436.8 ± 8.8a 16.9 ± 0.2 14.7 ± 0.5a 70.7 ± 3.0 47.8 ± 1.54b CP 5 140.9 ± 2.3 21.0 ± 4.2a 363.0 ± 3.8 413.6 ± 8.4c 16.6 ± 0.4 14.6 ± 0.5a 72.5 ± 2.3 51.4 ± 0.84a CP 6 141.4 ± 5.2 22.1 ± 3.6a 361.7 ± 4.2 427.2 ± 4.7abc 17.0 ± 0.3 14.7 ± 0.7a 68.4 ± 1.8 49.8 ± 1.69ab DT meal DT1 48.0 ± 5.7 11.1 ± 0.4p 426.5 ± 1.0 443.2 ± 7.4p 11.7 ± 0.2 9.7 ± 1.1pq 78.3 ± 2.7 43.2 ± 1.41qr DT2 52.6 ± 1.0 11.8 ± 0.7p 424.9 ± 1.4 441.6 ± 4.1p 11.5 ± 0.6 10.0 ± 0.2p 80.2 ± 2.1 45.6 ± 0.91pq DT3 59.2 ± 1.0 7.7 ± 0.7qr 409.9 ± 2.6 443.7 ± 6.8p 12.1 ± 0.1 9.4 ± 0.4pq 77.2 ± 1.9 43.5 ± 1.23qr DT4 41.7 ± 5.1 6.6 ± 0.4r 419.7 ± 1.8 443.3 ± 3.2p 11.7 ± 0.3 9.5 ± 0.5pq 78.6 ± 3.1 45.1 ± 0.63pqr DT5 51.9 ± 1.0 9.7 ± 1.1pq 424.3 ± 1.0 447.3 ± 6.6p 10.9 ± 0.2 9.5 ± 0.9pq 80.3 ± 2.5 46.6 ± 1.42p DT6 48.9 ± 3.1 10.2 ± 1.0p 422.8 ± 4.9 448.1 ± 3.5p 10.3 ± 0.3 8.3 ± 0.4q 79.8 ± 2.1 42.5 ± 0.66r Note: Data were analyzed with one-way ANOVA for each response and meal separately. Values sharing same letter are not significantly different. Multiple means comparison by Tukey’s (p ≤ 0.05). Abbreviations: CP, cold-pressed, DT, desolventized-toasted. aOptimum conditions for ethanol pretreatment were meal-to-solvent ratio 1:4 wt:wt, temperature 50�C, time 30 min, untreated n = 3 and after treatment n = 9. bdbm, dry biomass; SAE, Sinapic acid equivalent. F I GURE 4 Effect of pH and meal-to-solvent ratio on extracted dry matter (mg/g meal dbm), protein content in extracted dry matter (mg protein/g dbm), and meal protein recovery (%) of ethanol pre-treated CP meal (a) and DT meal (b). CP, cold-pressed, DT, desolventized-toasted. 48 JOURNAL OF THE AMERICAN OIL CHEMISTS’ SOCIETY 15589331, 2024, 1, D ow nloaded from https://aocs.onlinelibrary.w iley.com /doi/10.1002/aocs.12739 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense conditions, 67% and 29% of meal protein could be recovered from CP and DT meals, respectively (Table S5 provides expected and observed values). Multiple (three) extractions conducted on the meals showed that two extractions under optimized conditions can reach protein recovery of 79% for CP (Figure S1A) and 38% for DT (Figure S1B) meal. Six random meal samples from each meal type were employed to vali- date the pH and meal-to-solvent ratio of protein extrac- tion (Table 3) and resulted in a wide range of recovery values; 76.5%–85.4% of CP meal protein and 26.2%– 36.4% of DT meal protein in two extractions. Further- more, this estimation showed that DT meal produces substantially lower amount of dry biomass and protein biomass compared to alkali extraction of CP meal. This difference in protein recovery from industrially pro- cessed canola meal directly relates with the way the meal was processed; proteins of highly processed (or distressed) DT meal are sparingly soluble even at highly alkaline pHs with excess solvent, while proteins of lesser processed meals retain the native solubility. Seed storage proteins (SSP) which are 11S cruci- ferin and 2S napin comprise the majority (�80%) of canola meal proteins. In addition, structural proteins associated with oil bodies (2%–8%) and cell walls, and minor proteins, such as enzyme inhibitors and lipid transfer proteins, are also included in the meal protein (Wanasundara, 2011). The solubility behavior differ- ences of SSPs with pH have been reported (Wanasundara et al., 2016; Wu & Muir, 2008); how- ever, at an alkali pH medium higher than pH 11, a considerable fraction of canola meal proteins become soluble (Kalaydzhiev et al., 2019). Results of the pre- sent study, further confirm the alteration of pH-related solubility behavior of canola protein which may be related to the processing history of the meal involving heat and non-polar solvents. Fetzer et al. (2018) also showed that the protein solubility of rapeseed cake from FP or DT processed (exposed to more than 100�C during oil extraction) was lower at alkali pH than that of CP cake. Mosenthin et al. (2016) reported that the high temperatures of the de-solventization and toasting step F I GURE 5 Comparison of untreated and ethanol pre-treated CP (a) and DT (b) canola meal for extracted dry solids, extracted protein, and percentage recovery of meal protein in a single extraction for the extractions carried out at pH 12 and the meal-to-solvent ratio of 1:10 (wt:vol) for 30 min. CP, cold-pressed, DT, desolventized-toasted. JOURNAL OF THE AMERICAN OIL CHEMISTS’ SOCIETY 49 15589331, 2024, 1, D ow nloaded from https://aocs.onlinelibrary.w iley.com /doi/10.1002/aocs.12739 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense of meal lead to denaturation, resulting in protein structure unfolding, exposing hydrophobic regions, and facilitating cross-linkages by intermolecular disulphide bonding and formation of Maillard reaction products, consequently decreasing protein solubility. According to Pedroche et al. (2004), the low solubility of meal proteins results from the change in the net charge of protein molecules as a consequence of SSP interactions with other com- ponents of the meal matrix. An increase in dry bio- mass extractability of commercial canola meal with increasing pH from 10 to 12 has been reported (Gerzhova et al., 2016), most likely from the alkali pH soluble non-protein compounds such as from seed coat and cotyledon (embryo and endosperm) cell walls. Comparison of the extracted dbm and their protein content resulting from a single extraction under the opti- mum conditions showed that ethanol pre-treatment may have contributed to an increase in meal protein extractability; however, the increase depended on the processing history of the meal (Figure 5). The present study showed that ethanol was not efficient in removing a significant amount of non-protein small molecules besides the residual oil of these industrially processed canola meals (Table 2). The levels of total phenolics in DT meal remained fairly unchanged (8%–10% reduc- tions) upon pre-treatment, therefore as described by Xu and Diosady (2000), co-extraction with the protein under alkali pH is possible, leading to the observed poor improvement of DT meal protein level with the subsequent treatments provided. Acid hydrolysability of canola meal protein It is clear that the processing stress on canola seed pro- teins renders them less soluble even at high alkaline condi- tions making lesser or no option to valorize DT meal in the direction of intact protein. The hypothesis tested was that chemical hydrolysis of peptide bonds of meal proteins allows to recover free AAs (FAA) at same levels from both CP and DT meal. Sulfuric acid, having double the normality of that of hydrochloric acid was investigated as an alterna- tive to HCl to generate FAA from oil-free CP and DT meals. Standard 6 N HCl acid hydrolysis and 18 amino acid profile Complete hydrolysis of peptide bonds of protein of meals and separated proteins was expected from standard acid hydrolysis conditions provided with 6 N HCl therefore AA release. The AA profiles of CP and DT meals obtained from standard amino acid analysis (Table 4) were within the range that was reported for canola meal (CCC, 2019). The proteins of untreated CP cake released the lowest amount of amino acids (766 ± 0.6 mg/g protein) however, the oil removal enabled releasing the same amount of AAs as the ethanol-treated DT meals (884 ± 0.5 mg AAs/g protein; Table 4). The separated protein fraction of the CP meal released somewhat closer levels of AAs to that of the ethanol-treated meal; however, a lower amount of free AAs (833 ± 0.9 mg AAs/g protein) was released from the DT meal protein fraction (Table 4). The presence TAB LE 3 Verification of applicability of optimum protein extraction conditions for random samples of CP and DT meals (pre-treated with ethanol).1 Type of meal Extractable meal dry matter (mg/g meal dbm) Protein in extracted dry matter (mg/g dbm) Protein recovery as % (wt/wt) of meal protein CP meal CP 1 506.3 ± 23.4abc 582.9 ± 0.25c 85.4 ± 2.0a CP 2 513.1 ± 13.9ab 592.5 ± 8.2bc 84.4 ± 2.0a CP 3 515.4 ± 4.7a 595.0 ± 0.8b 85.0 ± 1.9a CP 4 462.8 ± 12.7d 617.1 ± 2.8a 76.5 ± 1.3b CP 5 468.0 ± 4.7cd 608.4 ± 3.9a 80.9 ± 0.5ab CP 6 474.6 ± 16.1bcd 612.5 ± 4.6a 79.3 ± 2.6b DT meal DT 1 269.5 ± 16.0pq 496.2 ± 8.7p 30.2 ± 2.0pq DT 2 251.4 ± 23.5q 463.7 ± 10.6q 26.4 ± 3.1q DT3 312.3 ± 19.9p 517.8 ± 6.9p 36.4 ± 2.5p DT 4 304.6 ± 4.8p 510.7 ± 12.8p 35.3 ± 1.6pq DT 5 252.6 ± 10.7q 466.9 ± 4.7q 26.4 ± 1.4q DT 6 252.8 ± 16.1q 464.9 ± 6.9q 26.2 ± 1.7q Note: Data were analyzed with one-way ANOVA for each response and meal separately. Values sharing same letter are not significantly different. Multiple means comparison by Tukey’s (p ≤ 0.05). Abbreviations: CP, cold-pressed, DT, desolventized-toasted. 1Optimum conditions were meal-to-solvent 1:10 wt:vol, pH 12, and two repeated extractions for each sample. 50 JOURNAL OF THE AMERICAN OIL CHEMISTS’ SOCIETY 15589331, 2024, 1, D ow nloaded from https://aocs.onlinelibrary.w iley.com /doi/10.1002/aocs.12739 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense of non-amino acid N or the conditions leading to incomplete protein hydrolysis has occurred in CP cake with oil. In the N-based protein values, non-protein N in canola primarily, the glucosinolates (0.4%–2.0%) and tri- amine compounds (e.g., betaine, choline, and sinapine; 2%) that are commonly found in Brassica oilseeds (Wanasundara et al., 2012) are included. Ethanol treatment may have removed these compounds to a certain extent in addition to oil, improving protein hydrolysis. Considering both meals, on average, glu- tamic acid comprised 20%–22% of total AAs followed by Asp (8.7%), Arg (7.5%), Leu (6.7%), Lys (6.2%–7.0%), Pro (5.6%–6.3%), Val (5.2%), Gly (4.7%), and Ser (4.2%) that together making up �72% of the total (Table 4). Hydrolysis with H2SO4 acid and effect of acid concentration on amino acid release In order to find the concentration of H2SO4 that provide protein hydrolysis to the same extent as 6 N HCl, various acid concentrations were tested on separated proteins and the percentage AA release was calculated based on the total AAs obtained during standard acid hydrolysis with 6 N HCl. The total amount of AAs released from the extracted proteins (protein fraction) of ethanol pre-treated CP, increased from 68.7% (3 N) to 82% (8 N), with a small percentage of increase from 6 to 8 N change (Table 5). Hydrolysis of DT meal pro- tein was lower (64.5%) than CP meal protein at 3 N, which was increased up to 78% with 8 N concentration and required 16 N to reach an equivalence of 83% hydrolysis (Table 6). In conventional acid hydrolysis, Asn and Gln are completely hydrolyzed to their respec- tive acid form Asp and Glu. Trp is destroyed completely, while Cys, Met, Ser, Thr, and Tyr are partially lost. Direct H2SO4 hydrolysis, without any protection (no phenol addition compared to HCl hydrolysis), completely or par- tially destroys some of these AAs; therefore, lesser amounts of FAA were recovered; particularly Cys, Met, and Trp (Tables 5 and 6), which were lesser than the amounts recorded from 6 N HCl hydrolysis in Table 4. TAB LE 4 Complete amino acid (AA) profile of untreated and ethanol pre-treated meals and protein fraction obtained from ethanol pre- treated meals. Amino acid Concentration of AA mg/g proteina CP meal DT meal Untreated Ethanol pre-treated Protein fraction Untreated Ethanol pre-treated Protein fraction Ala 35.8 ± 0.7 42.7 ± 0.9 42.7 ± 1.2 45.7 ± 2.4 41.7 ± 1.4 38.3 ± 0.2 Asp + Asn 65.6 ± 0.7 78.3 ± 2.7 79.4 ± 4.0 83.8 ± 1.3 75.8 ± 2.4 73.0 ± 1.0 Arg 58.0 ± 1.7 63.5 ± 8.3 69.2 ± 2.3 68.7 ± 1.1 68.1 ± 1.7 61.7 ± 3.0 Cysteic acidb 32.7 ± 3.2 35.8 ± 1.1 28.1 ± 1.8 35.2 ± 1.8 30.1 ± 0.6 27.0 ± 1.0 Gly 36.5 ± 0.8 40.8 ± 0.4 40.9 ± 0.8 44.1 ± 1.8 42.5 ± 3.3 39.7 ± 0.7 Glu + Gln 157.2 ± 10.1 184.4 ± 3.2 194.3 ± 8.8 194.6 ± 19.8 177.5 ± 10.8 214.3 ± 13.6 His 18.5 ± 0.5 20.3 ± 3.4 21.2 ± 0.7 21.0 ± 1.4 21.3 ± 0.8 19.8 ± 0.8 Ile 29.1 ± 0.6 33.0 ± 1.7 33.1 ± 0.8 35.5 ± 1.4 34.2 ± 1.5 28.8 ± 0.5 Leu 51.3 ± 1.4 58.7 ± 3.1 60.0 ± 1.3 64.2 ± 1.1 60.7 ± 1.5 53.9 ± 0.8 Lys 51.6 ± 1.2 64.4 ± 6.6 61.7 ± 4.4 61.4 ± 2.5 54.4 ± 1.0 47.3 ± 1.2 Metb 20.5 ± 1.7 27.6 ± 3.3 22.5 ± 0.6 25.2 ± 1.5 22.3 ± 1.0 20.2 ± 0.8 Phe 28.9 ± 0.2 31.3 ± 0.3 31.7 ± 0.2 34.5 ± 2.5 35.5 ± 1.4 28.9 ± 0.6 Pro 42.8 ± 0.9 49.4 ± 3.6 49.3 ± 1.1 60.3 ± 3.1 56.1 ± 1.2 50.6 ± 6.7 Ser 32.7 ± 0.9 36.7 ± 3.0 36.7 ± 1.2 40.1 ± 1.6 41.1 ± 1.3 32.2 ± 1.1 Thr 33.1 ± 0.6 37.0 ± 4.6 35.4 ± 1.1 39.2 ± 2.5 38.2 ± 1.9 27.2 ± 1.2 Trpc 7.9 ± 0.2 8.9 ± 0.3 10.9 ± 0.4 10.0 ± 0.5 9.7 ± 0.6 9.3 ± 0.1 Tyr 22.8 ± 0.9 25.0 ± 5.3 25.5 ± 0.9 27.0 ± 4.1 27.1 ± 1.9 19.8 ± 0.4 Val 41.0 ± 0.4 46.1 ± 2.5 46.6 ± 1.5 50.7 ± 2.2 47.9 ± 1.6 40.7 ± 0.8 Total AAs mg/g protein 765.9 ± 43.6 884.0 ± 45.3 889.1 ± 33.1 941.1 ± 52.6 884.2 ± 35.9 832.7 ± 33.2 Total AAs, mg/g dbmd 278.6 ± 8.6 373.4 ± 9.9 543.7 ± 8.9 400.2 ± 10.9 403.5 ± 11.02 382.2 ± 8.8 Note: For total AA, mg/g protein, Mean ± SD is presented. Abbreviations: CP, cold-pressed, DT, desolventized-toasted. aN-based protein content (% N � 6.25). bCysteic acid (from cysteine) and methionine (as methionine sulphone) were determined from acid hydrolysis performed after performic acid oxidation. cTryptophan was determined from base hydrolysis. dBased on percentage protein content of dbm of untreated meal, ethanol treated meal, and protein fraction of ethanol-treated meal: 36.4%, 42.2%, 61.2% for CP meal and 42.5%, 45.6%, 45.9% for DT meal, respectively. Only mean values are presented; n = 3. JOURNAL OF THE AMERICAN OIL CHEMISTS’ SOCIETY 51 15589331, 2024, 1, D ow nloaded from https://aocs.onlinelibrary.w iley.com /doi/10.1002/aocs.12739 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense A higher acid requirement of above 6 N required to reach >80% amino release from DT meal protein (Table 6) could partly be explained by the competing acid-induced reactions by non-protein constituents in DT protein. In the present study, considering the extent of the FAA release and the yield from both CP and DT meal protein fractions at different H2SO4 concentrations, the 8 N level was identified as the concentration that can liberate a substantial amount of AAs. Hydrolysis using 8 N H2SO4 was verified using extracted proteins of six different random meal samples from each meal type (Table 7) and showed 81%–85% liberation of CP meal AAs and 80%–87% from DT meal protein. When the abundance of FAAs in the 8 N H2SO4 hydrolysates is considered, proteins of these industrial meal samples gave a similar pattern of AA release as with 6 N HCl hydrolysis (Table 4), with the same nine amino acids (Glu, Asp, Arg, Leu, Lys, Pro, Val, Gly, Ser) making 73%–76% of the total released AAs (Table 7). As a diprotic acid, H2SO4 provides double the con- centration of protons in the hydrolysis medium compared to monoprotic HCl. The conventional acid hydrolysis uses 6 N HCl, and theoretically, a similar extent of pep- tide bond breakage was expected when 6 N H2SO4 was used. The FAA yields or percentage of total liberated AAs in the present study showed that higher H2SO4 con- centration was required to reach high AA release/ recovery than expected. It has been reported that the H+ ion generated from the second dissociation of H2SO4 is not used in protein hydrolysis reaction, instead involves the degradation of AAs, causing their low yield in the hydrolysate (Flork, 1989). Furthermore, Flork (1989) has reported that a minimum of 12 N H2SO4, giving the same molarity as HCl, is needed, and temper- ature should be maintained at least at 100�C to have a satisfactory AA release in hydrolysis with H2SO4. Several studies have reported the high propensity of H2SO4-assisted hydrolysis to generate small peptides. Alvarez et al. (2012) compared hydrolysis of porcine blood hemoglobin with both HCl and H2SO4 and detected four different fractions as unbroken protein, sol- uble peptides, non-soluble peptides, and free AAs in the hydrolysate. Since protein soon aggregates with the TAB LE 5 Amino acids released from hydrolysis of protein fraction of ethanol-treated cold-pressed (CP) meal under different concentrations of H2SO4 concentrations. Amino acid Released AAs mg/g proteina 3 N 4 N 5 N 6 N 8 N Ala 34.8 ± 1.9 36.7 ± 1.1 39.2 ± 2.5 40.5 ± 2.4 40.7 ± 0.9 Asp + Asn 63.9 ± 2.7 63.5 ± 1.0 67.1 ± 1.6 66.7 ± 1.6 66.5 ± 1.5 Arg 42.5 ± 3.1 46.3 ± 2.1 55.0 ± 4.9 58.9 ± 3.0 61.4 ± 3.0 Cysteic acidb 11.2 ± 1.9 9.1 ± 2.9 8.9 ± 1.9 6.1 ± 1.4 3.3 ± 0.9 Gly 41.8 ± 1.0 42.9 ± 0.3 44.9 ± 2.9 46.5 ± 3.0 47.0 ± 1.2 Glu + Gln 145.2 ± 5.9 149.1 ± 1.4 159.3 ± 11.7 163.3 ± 9.9 162.0 ± 3.4 His 12.3 ± 0.4 13.6 ± 0.9 15.9 ± 2.3 18.1 ± 2.4 19.3 ± 1.4 Ile 14.9 ± 0.4 16.3 ± 0.4 19.1 ± 1.4 22.0 ± 2.7 24.1 ± 1.2 Leu 36.9 ± 1.8 41.3 ± 0.4 46.5 ± 4.2 49.7 ± 5.5 51.1 ± 2.1 Lys 48.1 ± 2.5 44.3 ± 3.0 46.1 ± 3.9 47.0 ± 5.8 46.6 ± 2.6 Metb 1.4 ± 0.4 2.6 ± 0.5 0.9 ± 0.2 1.5 ± 0.4 1.1 ± 0.4 Phe 18.6 ± 0.6 20.6 ± 0.6 22.7 ± 2.8 23.7 ± 3.3 23.5 ± 1.0 Pro 40.7 ± 1.9 44.5 ± 4.7 48.5 ± 2.6 49.7 ± 6.0 48.9 ± 1.3 Ser 30.9 ± 1.6 32.4 ± 1.9 35.2 ± 2.5 37.3 ± 1.9 38.3 ± 0.7 Thr 25.3 ± 1.8 28.4 ± 0.9 32.0 ± 2.9 34.3 ± 2.9 35.6 ± 0.9 Trpc 0 0 0 0 0 Tyr 19.6 ± 0.8 21.0 ± 0.2 23.4 ± 2.5 24.3 ± 3.5 24.3 ± 0.7 Val 23.3 ± 3.2 26.2 ± 1.2 30.3 ± 1.8 33.1 ± 4.2 35.3 ± 0.5 Total AAs released, mg/g protein 611.4 ± 54.1 638.8 ± 23.5 695.0 ± 52.6 722.8 ± 58.9 729.0 ± 47.4 Released amino acids as a % of total amino acids Fraction of released AAs from total, %d 68.7 71.8 78.1 81.3 82.0 aN-based protein content (% N � 6.25). bCysteic acid (from cysteine) and methionine (as methionine sulphone) are reported as it was resulted in from acid hydrolysis. cTryptophan was not detected. dCalculated based on the content of total amino acids of protein fraction obtained from ethanol-treated CP protein values (889.1 mg/g protein) of Table 4. Mean ± SD presented; n = 3. 52 JOURNAL OF THE AMERICAN OIL CHEMISTS’ SOCIETY 15589331, 2024, 1, D ow nloaded from https://aocs.onlinelibrary.w iley.com /doi/10.1002/aocs.12739 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense contact of acid, enough length of time should be given to reduce the fraction of non-soluble peptides. However, Alvarez et al. (2012) showed that the rate of hydrolysis is influenced by the temperature of 50�C without affecting the average peptide size. Increased acid concentrations of H2SO4 from 1 to 3 M produce small sized-peptides; however, at higher acid concentrations, more non-soluble peptides are also produced. Therefore, lower concentra- tions are identified as suitable for soluble peptide produc- tion, with HCl being recognized as a better medium for yielding AAs than H2SO4. Safety issues discourage the use of such high acid concentrations, and consequent neutralization gener- ates high salt levels requiring desalination steps. Cold precipitation (Alvarez et al., 2012) and the addition of water for termination of hydrolysis (Flork, 1989) have been discussed for managing this excess salt produc- tion. The ratio of solid-to-liquid is also a significant fac- tor in determining the hydrolyzing efficiency, and a higher ratio can decrease the yield than lower ratios (Bouhamed & Kechaou, 2017). In the present study, the solid-to-liquid ratio was kept the same as the standard HCl hydrolysis. In large-scale processing, the high corro- sivity of HCl may require glass-lined reactors, whereas H2SO4 hydrolysis can be carried out in stainless steel reactors with a simple protective lining and a closed ves- sel to avoid acid fume emission (Flork, 1989). Further- more, H2SO4-water azeotrope has a boiling point of 338�C at ambient temperature while HCl is 108�C. Protein-H2SO4 solution boiling point is 120�C, and hydro- lysis can be carried out at 110�C without vapor emission (Flork, 1989). Estimation of amino acid yield from CP and DT meal Data from this study was used in estimating the amounts of protein and FAAs that can be obtained from CP and DT meals (Figure S2 provides calculation steps). Figure 6a gives the yield of products (as dbm and protein in dbm) of each processing step when the TAB LE 6 Amino acids released from hydrolysis of protein fraction of ethanol pre-treated desolventized-toasted (DT) meal under different concentrations of H2SO4. Amino acid Released AAs mg/g proteina 3 N 4 N 5 N 6 N 8 N 10 N 12 N 14 N 16 N Ala 29.7 30.4 29.6 32.6 35.4 36.3 37.1 36.7 38.1 Asp + Asn 54.7 53.8 51.2 56.4 60.0 60.6 61.1 59.9 60.8 Arg 33.8 38.8 39.6 46.5 49.3 52.9 52.9 56.5 57.5 Cysteic acidb 10.9 7.7 5.9 4.1 3.6 3.9 1.0 0.0 0.9 Gly 40.2 39.9 38.3 42.0 44.0 44.0 44.7 44.4 45.0 Glu + Gln 149.9 154.4 148.8 164.3 172.6 176.8 176.4 174.9 178.8 His 9.9 12.3 12.4 14.9 15.8 17.5 16.1 17.5 17.8 Ile 11.4 13.3 14.1 17.3 20.4 23.3 24.2 25.8 27.4 Leu 31.7 35.5 35.7 40.7 43.7 48.4 45.9 47.2 48.9 Lys 36.5 34.6 32.2 32.7 33.6 34.4 35 34.1 34.5 Metb 0.8 0.8 0.5 0.0 0.7 0.0 0.3 0.2 0.4 Phe 15.0 17.0 16.0 17.9 17.6 18.2 16.9 17.6 16.8 Pro 35 39.3 39.9 46.9 46.6 49.4 48.8 47.6 49.3 Ser 26.2 28.1 27.4 30.5 31.7 34.7 30.8 30 30.1 Thr 19.6 21.5 21.9 26.3 26.2 28 27.7 28.2 29.1 Trpc 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Tyr 13.0 14.3 14.2 16.4 17.0 18.6 15.1 14.8 13.3 Val 18.5 20.7 21.8 26.6 31.3 34.2 36.4 38.4 41.1 Total AAs released, mg/g protein 536.8 562.4 549.5 616.1 649.5 681.2 670.4 673.81 689.8 Released amino acids as a % of total amino acids Fraction of released AAs from total, %d 64.46 67.54 65.99 73.99 78.00 81.81 80.51 80.92 82.84 aN-based protein content (% N � 6.25). bCysteic acid (from cysteine) and methionine (as methionine sulphone) are reported as it is obtained from acid hydrolysis. cTryptophan was not detected. dCalculated based on the content of total amino acids of protein fraction obtained from ethanol-treated DT protein values (832.7 mg/g protein) of Table 4. Only mean values are presented; n = 3. JOURNAL OF THE AMERICAN OIL CHEMISTS’ SOCIETY 53 15589331, 2024, 1, D ow nloaded from https://aocs.onlinelibrary.w iley.com /doi/10.1002/aocs.12739 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense process starts with 1 kg of meal dbm and the resulting intermediates progress through the process of generat- ing FAAs. For example, the amount of total FAA that can be obtained by processing 1 kg of CP and DT meal dbm after going through ethanol pre-treatment, protein separation, and 4 M H2SO4 hydrolysis. Figure 6b pro- vides the yield of the product when 1 kg of dbm enters into each process step, and this may reflect a process of AA generation from canola meal in a scaling-up set- ting. Although it may be an additional process, the sep- aration of meal protein may be advantageous to reduce dbm of the hydrolysing substrate and the required vol- ume of acid reactant as well as the unwanted products of fiber hydrolysis, particularly sugars. In addition, the remaining residue of protein extraction which is already modified during the alkali conditions of protein extrac- tion can be directed for possible other uses. Glutamic acid was the dominant AA of the canola meal protein and comprised one-fifth of the resulting FAA mixtures of acid hydrolysis. Considering the differences in processing steps involved in generating CP meal and DT meal, proteins of CP meal retained solubility/extractability and the ability to break down into monomeric AAs by chemical hydrolysis. However, the proteins of DT meal may have been structurally modi- fied because of the various reactions that occur under the conditions of oil extraction. This fact explains how DT meal show low extractability/solubility, and even the peptide bond breakdown is difficult under acid hydroly- sis. This study showed that the processing history poses limitations in the chemical hydrolysis of peptide bonds of industrially processed canola meal. However, the FAAs obtained from hydrolysis of either CP or DT meal were the same AA molecule units that can be used directly and to generate compounds for further uses. Reported non-food/feed uses of canola meal protein primarily focus on utilizing SSP. Deliberate structure modifications are required to improve targeted functions such as adhesive (Wang et al., 2014), mechanical (Li et al., 2019; Manamperi et al., 2010), and rheological TAB LE 7 Verification study for AA release due to 8 N H2SO4 hydrolysis of protein fraction of ethanol pre-treated CP and DT canola meal. Amino acid Released AAs mg/g proteina Ethanol pre-treated CP meal protein Ethanol pre-treated DT meal protein CP1 CP2 CP3 CP4 CP5 CP6 DT1 DT2 DT3 DT4 DT5 DT6 Ala 38.2 39.1 36.9 38.4 36.7 37.0 32.8 33.8 32.3 35.8 36.9 33.6 Asp + Asn 66.6 65.4 63.7 69.9 64.9 67.4 62.0 64.8 60.9 64.2 66.4 62.1 Arg 57.1 58.5 53.1 49.6 47.7 48.8 50.6 49.3 49.3 53.3 45.4 49.4 Cysteic acidb 3.4 7.9 6.0 4.8 4.1 3.9 4.5 3.8 3.2 3.7 4.8 3.8 Gly 46.3 46.4 44.0 48.0 45.2 46.6 43.3 45.7 42.6 46.9 48.1 43.9 Glu + Gln 157.9 155.4 147.9 167.2 157.3 160.6 174.7 165.4 172.9 170.4 164.0 175.6 His 20.2 22.0 20.8 22.8 22.9 22.3 17.6 18.5 17.8 19.8 21.7 18.7 Ile 27.8 30.2 36.1 31.3 28.6 29.4 24.2 26.8 23.6 25.9 27.2 24.2 Leu 51.5 52.7 51.7 55.7 52.4 53.3 46.1 48.4 44.9 49.2 49.4 46.1 Lys 41.4 41.2 41.0 43.3 39.5 41.7 29.7 33.7 29.4 35.6 36.8 30.0 Metb 9.2 6.8 8.0 9.1 6.6 7.5 7.4 6.6 6.1 7.8 6.9 6.8 Phe 29.1 27.6 29.4 30.2 29.9 29.7 26.2 24.6 24.5 27.3 28.6 25.5 Pro 53.1 54.7 52.9 56.1 54.3 52.7 54.9 60.8 53.3 57.9 60.4 54.3 Ser 38.3 37.6 36.5 40.0 37.9 38.9 33.0 33.8 33.0 36.9 38.2 33.8 Thr 35.4 36.3 35.5 28.0 35.7 34.6 29.0 29.3 28.0 32.1 33.1 29.1 Trpc 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Tyr 24.8 25.3 24.5 25.5 25.5 25.2 17.4 18.7 17.9 20.2 20.6 18.4 Val 33.1 38.4 34.5 38.1 33.6 34.3 27.9 33.2 27.6 31.0 32.3 27.7 Total AA released, mg/g protein 733.4 745.5 722.5 758.0 722.8 733.9 681.3 697.2 667.3 718.0 720.8 683.0 Released amino acids as a % of total amino acids Fraction of released AA from total, %d 82.5 83.8 81.3 85.3 81.3 82.5 81.8 83.7 80.1 86.2 86.6 82.0 Abbreviations: CP, cold-pressed, DT, desolventized-toasted. aN-based protein content (% N � 6.25). bCysteic acid (from cysteine) and methionine (as methionine sulphone) are reported as it was resulted from acid hydrolysis. cTryptophan was not detected. dCalculated using values of total amino acids of protein fraction obtained for ethanol-treated CP (889.1 mg/g protein) and DT (832.7 mg/g protein) protein as in Table 4. Only mean values are presented; n = 3. 54 JOURNAL OF THE AMERICAN OIL CHEMISTS’ SOCIETY 15589331, 2024, 1, D ow nloaded from https://aocs.onlinelibrary.w iley.com /doi/10.1002/aocs.12739 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense (Tan et al., 2014) properties of isolated canola proteins. Highly distressed proteins, such as those found in com- mercial DT canola meal, cannot be the competitive start- ing material for the routes mentioned above, if it is not used in the feed industry, it may end up as waste mate- rial or incorporated back into the soil. In the global AA market, glutamic acid is the most demanding AA (�50% of the demand), followed by L-Asp, L-Phe, L-Lys, and L-Thr (Grandviewresearch, 2023). Depending on the raw material(s) used, both animal- and plant-based AAs are available, while plant-origin AAs have a higher demand than animal sources due to the rising preference for vegan and sustainable production systems (Leuchtenberger et al., 2005). Regenerating mono- meric AAs that consist of protein’s primary structure via extensive protein hydrolysis may be a strategic approach to obtaining base chemicals for tangible product development. Recovered FAAs can be used as amino acid mixtures of high potency or converted into new molecules and compounds with various functions such as surface tension reduction (Bordes & Holmberg, 2015), antimicrobial (Pinazo et al., 2016) or plant growth stimu- lating (Ertani et al., 2019). Therefore, the generation of free AAs is another approach for valorizing industrially processed canola meal. CONCLUSIONS A pre-treatment with ethanol (99%) can produce a de- oiled CP canola meal by removing �88% of residual oil and �20% reduction of phenolics, and �25% reduction in free sugars. DT meal oil content can also be lowered to reach less than 1% of dbm with a limited effect (�20% reduction) on phenolic compounds and free sugars. Therefore, pre-treatment of 99% ethanol effec- tively eliminates the influence of the residual oil in meals to further application, with partial removal of phe- nolics and free sugars. The recovery of proteins from untreated CP meal and DT meals at pH 12 may be affected by the conditions of oil extraction than residual oil content, shown as the resulting lesser protein recov- ery from DT meal (38% of meal protein) than CP meal (79% of meal protein). There was no negative effect on the protein recovery from either CP or DT meal due to ethanol pre-treatment. Only CP meal showed a small (�2%) increment. Proteins of CP and DT canola meals can be con- verted to FAA mixtures using H2SO4 acid-assisted hydrolysis. Interestingly, the protein fraction recovered from CP and DT meals had a different propensity to acid hydrolysis, with DT meal protein requiring high acid concentration. On average, �83% of protein- bound amino acids of extracted protein of these meals can be released by using 4 M H2SO4 acid with a lesser recovery of sulfur amino acids and no recovery of tryp- tophan. Proteins obtained from DT meal showed com- paratively low FAA yield (69%) than proteins of CP meal (74%) under the conditions of alternative acid (8 N) hydrolysis. Ethanol pre-treatment of meal and recovering alkali soluble components reduced non- protein compounds and enhanced FAA recovery from the proteins of CP and DT meal upon hydrolysis. This study showed that generation of FAA is possible as F I GURE 6 Summary of ethanol pre-treatment, protein extraction (two extractions) and hydrolysis of CP and DT canola meals showing protein and free amino acid (FAA) yields. (a) Amount dry bio mass (dbm) and protein biomass that can be obtained from each meal based on 1 kg dry weight of starting meal, and (b) amount of product (protein and FAA) that can be obtained from 1 kg dbm of input (meal or protein extract) in the process involving ethanol pre-treatment, protein extraction (two extractions), and hydrolysis. Values obtained from the industrial meal assessment in the study are extrapolated to 1 kg meal material. CP, cold-pressed, DT, desolventized-toasted. JOURNAL OF THE AMERICAN OIL CHEMISTS’ SOCIETY 55 15589331, 2024, 1, D ow nloaded from https://aocs.onlinelibrary.w iley.com /doi/10.1002/aocs.12739 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense another utilization approach for industrially-processed canola meals and release AAs can be useful feed mol- ecules for various compounds and applications. AUTHOR CONTRIBUTIONS Janitha P. D. Wanasundara: Conceptualization, experimental design of the study and reviewing and editing of MS. Sumudu N. Warnakulasuriya: Conduct- ing experiments, data collection and analysis, manu- script writing and editing. Takuji Tanaka: Review of experiment data presentation, and manuscript review- ing editing. ACKNOWLEDGMENTS Funding support for the study was from the Global Insti- tute of Food Security, University of Saskatchewan, Project # J-001661, and Agriculture and Agri-Food Canada Project # J-001589. The authors thank Tara C. 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See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense https://doi.org/10.1007/s11746-010-1616-8 https://doi.org/10.1186/s40104-016-0095-7 https://doi.org/10.1186/s40104-016-0095-7 https://doi.org/10.1016/j.foodchem.2012.04.017 https://doi.org/10.3390/foods9010019 https://doi.org/10.3390/foods9010019 https://doi.org/10.1016/j.foodchem.2004.01.045 https://doi.org/10.1016/j.foodchem.2004.01.045 https://doi.org/10.1016/j.cis.2015.11.007 https://doi.org/10.1016/j.cis.2015.11.007 https://doi.org/10.1007/s11743-011-1283-2 https://doi.org/10.1007/s11743-011-1283-2 https://doi.org/10.1007/s11746-017-2960-8 https://doi.org/10.1007/s11746-017-2960-8 https://doi.org/10.1006/jcis.2001.7932 https://doi.org/10.1006/jcis.2001.7932 https://doi.org/10.1007/s10499-011-9476-2 https://doi.org/10.1007/s10499-011-9476-2 https://doi.org/10.1016/j.foodres.2014.04.055 https://doi.org/10.1016/j.foodres.2014.04.055 https://doi.org/10.1007/s11746-011-1975-9 https://doi.org/10.1007/s11746-011-1975-9 https://doi.org/10.1051/ocl/2016028 https://doi.org/10.1111/j.1750-3841.2008.00675.x https://doi.org/10.1111/j.1750-3841.2008.00675.x https://doi.org/10.1016/j.foodchem.2003.11.020 https://doi.org/10.1016/j.foodchem.2003.11.020 https://doi.org/10.1002/jctb.4602 https://doi.org/10.1007/s11746-008-1217-y https://doi.org/10.1002/aocs.12739 Canola meal valorization via acid hydrolysis to generate free amino acids INTRODUCTION MATERIALS AND METHODS Materials Chemical analysis Free sugars and their contents Amino acid analysis Ethanol pre-treatment Protein separation Suitable H2SO4 acid concentration for meal protein hydrolysis Statistical analysis RESULTS AND DISCUSSION Ethanol pre-treatment and condition optimization Effect of pre-treatment on meal protein extractability Acid hydrolysability of canola meal protein Standard 6N HCl acid hydrolysis and 18 amino acid profile Hydrolysis with H2SO4 acid and effect of acid concentration on amino acid release Estimation of amino acid yield from CP and DT meal CONCLUSIONS AUTHOR CONTRIBUTIONS ACKNOWLEDGMENTS CONFLICT OF INTEREST STATEMENT ETHICS STATEMENT REFERENCES