The nutritional quality of plant-based proteins (PBPs) is increasingly scrutinized as global dietary trends shift toward sustainable alternatives to animal-based proteins. This review critically evaluates the impact of food processing on essential amino acids (EAAs), with a particular focus on sulfur amino acids (SAAs), including cysteine and methionine. While processing techniques such as extrusion, autoclaving, fermentation, and high-pressure treatment can enhance the digestibility, functionality, and sensory properties of PBPs, they also induce undesirable chemical modifications. These include oxidation, racemization, and crosslinking, which reduce the biological availability of SAAs and compromise protein quality. Evidence from recent studies highlights significant losses of cysteine during processing, often attributed to the Maillard reaction and oxidative stress. Conventional quantification methods, such as performic acid oxidation, may overestimate SAA content due to the presence of pre-existing oxidized forms in processed foods. Alternative approaches, including Ellman's reagent-based assays, offer more accurate assessments of reactive cysteine and are better suited for evaluating true bioavailability. This review highlights the need for enhanced analytical methods to differentiate between total and biologically available SAAs, particularly in processed PBPs. Accurate quantification is crucial for refining protein quality metrics, informing food formulation, and developing correction factors to compensate for nutrient losses resulting from processing. Addressing these gaps will support the development of high-quality plant protein products and inform strategies to optimize processing conditions, ultimately enhancing the nutritional value of plant-based diets.

Incorporating protein-rich food industry by-products, such as sunflower meal (SFM), into foods aligns with the United Nations Sustainable Development Goals by promoting environmentally less resource-intensive food alternatives for a more secure food future. In this study, SFM and pea protein isolate (PPI) were selected for high-moisture meat analogue (HMMA) production due to their complementary amino acid profiles, low-cost, and relatively low allergenicity compared to common plant proteins like soy and wheat. HMMAs made from two blends of expeller-pressed SFM and PPI (40:60 and 50:50, w/w) were extrusion cooked at two different feed moisture contents (FMC) (48 % and 58 %, w.b.) and three different extruder barrel temperature profiles (60-80-115-125 degrees C, 80-100-125-135 degrees C, and 100-120-135-145 degrees C). The physical (texture and color) and nutritional (protein quality and anti-nutritional factors) quality of the resulting HMMAs were examined. While all HMMAs studied were harder and darker than cooked chicken, they became softer and lighter in color as FMC increased, and extrusion temperature decreased. Overall, all HMMAs studied had comparable protein quality to cooked chicken and beef, with 70-74 % (d.b.) protein content and 86-89 % in-vitro protein digestibility, with tryptophan being the first limiting amino acid. Moreover, extrusion processing significantly (p < 0.05) reduced the levels of anti-nutritional compounds including phytic acid, trypsin inhibitors, and chlorogenic acid. These findings highlight SFM's potential as a novel protein source in meat analogue formulations, demonstrating a sustainable way to add value to an underutilized by-product in the food industry.

The diversification of protein sources used in food formulation has increased the need to assess protein quality beyond traditional food forms. Protein digestibility is a key component of the Protein Digestibility Corrected Amino Acid Score (PDCAAS), the regulatory metric derived from rodent bioassays. Ethical concerns and the high cost of animal testing limit the ability of food formulators to screen protein ingredients and evaluate processing effects on digestibility. Findings from an international collaborative study are presented to position two in vitro methods, the pH-drop and pH-stat assays, as accredited approaches for determining protein digestibility. Nine laboratories participated in the study and analyzed 12 protein ingredients. Relative standard deviations for repeatability ranged from 0.8 to 2.1 % and 0.5-4.8 %, while reproducibility ranged from 1.2 to 3.6 % and 1.1-4.9 % for the pH-drop and pH-stat methods, respectively. Comparison between the two assays demonstrated strong correlation and moderate agreement, with the pH-stat assay yielding slightly lower digestibility values. In vitro protein digestibility coefficients aligned well with literature reported true faecal protein digestibility values for comparable, non-identical protein ingredients. Both methods received official AOCS Uniform Methods Committee approval, offering simple, affordable, reliable, and ethical tools to support informed decisions on protein digestibility during food formulation.

The effects of deep eutectic solvent (DES)-based protein extraction on structural and functional properties of fava bean protein isolate (FBPI) were determined in comparison to conventional salt and alkaline extraction methods, and the commercial soybean protein isolate (CS-PI) as the standard protein. The protein content of fava bean isolates extracted from DES (DESE-FBPI), alkaline (ALKE-FBPI), and salt (SSE-FBPI) was observed to be similar (∼92 %). FTIR data indicated that all protein isolates had intermolecular β-sheets as protein aggregates, except in DESE-FBPI. Differences in the extraction methods reflected better solubility, foaming and emulsification properties, and gelling capacity by DESE-FBPI than the conventionally extracted counterparts. In terms of protein quality, significantly higher (p < 0.05) in-vitro-protein digestibility corrected amino acid score (IV-PDCAAS) was exhibited by DESE-FBPI (87.24 ± 0.70%) and SSE-FBPI (86.52 ± 0.83%) than the ALKE-FBPI (80.45 ± 0.08%). These results suggest that different extraction methods have a profound impact on protein functionality and quality.

Lupin is a promising alternative for the protein industry due to its species diversity, protein content and composition, and tolerance to climate stress. Therefore, this study aims to extract protein with high purity and functionality from lupin using a green and sustainable deep eutectic solvent (DES) system, comprising choline chloride and glycerol. Protein extraction conditions were optimized using response surface methodology, and the extracted protein was characterized for physicochemical, functional, and protein quality parameters compared to alkaline-extracted protein isolate. The observed optimum conditions for DES-based protein extraction were a solid-to-liquid ratio of 1:17.5 (w/w), a reaction time of 60 min, and a water content of 54% (w/w). Under optimal conditions, protein content, protein yield, and protein recovery rates of 88.42 +/- 0.09%, 64.47 +/- 0.56%, and 23.76 +/- 0.21%, respectively, were achieved. Alkaline extraction showed a higher protein content but lower yield and recovery (P < 0.05). According to secondary structure analysis, alpha-helix (24.16%) and beta-sheet (66.08%) contents in DES-extracted protein were higher than those of alkaline-extracted protein. Intermolecular beta-sheets (12.51%) as protein aggregates were observed only in alkaline-extracted protein, indicating higher protein aggregation and denaturation. DES-extracted protein had similar or increased functional properties compared to alkaline-extracted protein (P < 0.05). Moreover, remarkable gelation properties were observed with DES-extracted protein. In vitro protein digestibility (IVPD) was not significantly different (P < 0.05) between protein isolates (90.8-91.8%). The DES-extracted protein contained a higher content of sulfur amino acids than the alkaline-extracted protein. DES protein extraction is a promising technique that could serve as an alternative to conventional alkaline extraction to produce sustainable and high-purity protein ingredients from white lupin.

In this study, yellow peas, green lentils, and red lentils were germinated for up to 72 h. The germinated seeds were milled into flour and analyzed for compositional and functional changes, including α-amylase activity, water and oil holding capacity, foaming and emulsifying properties, protein solubility, in vitro protein and starch digestibility, and pasting properties. Overall, germination of pulses had significant impacts on the composition and several functional properties of the flours. The protein content and α-amylase activity increased and the water holding capacity was improved. Germination decreased protein solubility in pea but not in lentils. Germination did not affect the foaming properties or oil holding capacity of the three pulses. The lentil flours from germinated seeds were much lower in pasting viscosity, temperature, and time, while for pea, germination did not have as large of an impact on pasting properties. Germination had minor effects on the nutritional properties of the flours with reduced in-vitro protein digestibility corrected amino acid score (IV-PDCAAS) in lentils and changes in starch digesting properties, mainly in red lentils. The in-vitro protein digestibility (IVPD) was not modified through germination.

Acrylamide, a class 2A carcinogen, raises significant food safety concerns. The free amino acid asparagine (ASN) is a precursor of acrylamide formation in foods after high-temperature processing; therefore, accurate measurement of free ASN is essential for risk assessment. This study aimed to compare free ASN determination methods, using a novel rapid enzymatic assay with an established ultra-high-performance liquid chromatography (UHPLC) method, in seeds of field pea (Pisum sativum L.) and lentil (Lens culinaris), important crops and sources of plant-based protein. Quality parameters, such as sensitivity, percentage error, and precision were evaluated. The rapid enzymatic assay demonstrated consistent low percentage error (< 1.7%) and high precision (CV% < 1.20) compared to the UHPLC technique (error from 4.9 to 14.8% and CV% < 0.94). A high Pearson correlation (r > 0.996) between techniques confirmed the enzymatic assay’s reliability for routine asparagine quantification in simple laboratory and industrial settings. These findings support the potential of using a reliable, rapid, and user-friendly technique that eliminates the requirements of sophisticated instrumentation and complex sample preparation for free ASN analysis, and underscore the need for further research on pulse flour to manage acrylamide risks effectively in processed foods.

Twelve GRAS (generally recognized as safe) microorganisms, including seven bacteria (Lactobacillus delbrueckii, Lactobacillus rhamnosus, Weissella confusa, Lactococcus lactis, Leuconostoc pseudomesenteroides, Pediococcus pentosaceus, and Tetragenococcus halophilus), one fungus (Aspergillus oryzae), and four yeasts (Zygosaccharomyces rouxii, Saccharomyces cerevisiae, Kluyveromyces marxianus, and Wickerhamomyces anomalus), were used as starter cultures for solid-state fermentation (SSF) of pea protein isolate (PPI). Improved PPI solubility was found to be the main positive effect of SSF. The greatest improvement in protein solubility, nearly a fourfold increase compared to the solubility of unfermented PPI, was achieved in samples fermented with Z. rouxii. Uninoculated “control” PPI samples, which were fermented solely by indigenous microflora, were found to have nearly threefold higher protein solubility than in unfermented PPI, which was greater than in most samples fermented with starter cultures. Two of the tested strains, W. anomalus and T. halophilus, significantly inhibited the beneficial activity of indigenous microflora, leading to unchanged or diminished protein solubilities after fermentation. Changes in protein solubility following SSF correlated with degrees of protein hydrolysis (DH): most samples, which had protein solubility of > 30

Background and ObjectivesMilling practices, otherwise refined for specific uses in cereal-based foods, have not been thoroughly developed for pulses. This study investigates whether scouring and moisture conditioning pretreatments on yellow peas and green lentils can enhance hull removal, and in turn, whether changes in hull removal alter in vitro protein digestibility and quality.FindingsTotal by-product losses were significant in green lentils when subject to scouring, which was altered by high moisture addition in yellow peas. The scouring pretreatment altered both the protein digestibility and amino acid scores of green lentils, which translated to improved protein quality in all streams, but significantly in the break flour stream. Yellow peas similarly demonstrated significant improvements in protein quality from scouring, as a result of altered amino acid scores.ConclusionThe addition of a scouring procedure can improve the protein quality of yellow peas and green lentils.Significance and NoveltyPulse milling procedures are rarely evaluated for optimization of protein quality. This research establishes milling protocols that may be used to enhance the protein quality of yellow peas and green lentils.

BACKGROUND:Including fish oil (FO) in laying hen diets is an attractive strategy to increase egg and tissue omega (ω)-3 PUFA, while simultaneously protecting hens from potential immune challenges. OBJECTIVES:This study compared effects of a standard hen diet with FO containing EPA and DHA on plasma oxylipins and splenic cytokine expression in LPS-challenged layers. METHODS:Twenty-four 40-wk-old Dekalb White layers were randomly assigned to either control (no EPA or DHA) or FO (0.4 g/100 g EPA and DHA) diets. After 6 wk, hens were challenged with LPS (8 mg/kg body weight; i.v.) or saline 2 h before termination. PUFA were analyzed by GC, free oxylipins by HPLC/MS/MS, and splenic cytokines by quantitative reverse transcriptase-polymerase chain reaction. Data were analyzed by principal component analysis and analysis of variance. RESULTS:Yolk, liver, and plasma were enriched in ω-3 PUFA and reduced in ω-6 PUFA in FO group. Interestingly, LPS increased liver α-linolenic acid (ALA) and plasma ALA and DHA in FO-fed hens (P < 0.05). FO-fed hens exhibited higher plasma EPA and DHA oxylipins, and lower ω-6 oxylipins. Because of an interaction with FO, LPS increased ∼14% of oxylipins in control hens, particularly octadecanoids from ALA and linoleic acid, whereas in FO-fed hens, LPS decreased ∼31% of oxylipins derived from ω-6 and also from ω-3 PUFA (P < 0.05). Consistent with these putatively anti-inflammatory effects, FO decreased oxylipins associated with higher soluble epoxide hydrolase activity in saline-treated hens. However, FO increased these oxylipins in LPS-treated hens (P < 0.05). LPS induced the expression of splenic cytokines, and this was not altered by diet. CONCLUSIONS:Dietary EPA and DHA enrich ω-3 PUFA in yolk, liver, and plasma of layers, and modulate plasma oxylipins. Whether oxylipin changes mediate or are a consequence of FO effects on LPS-induced inflammation remains to be elucidated.

Background and ObjectivesRoller-milling practices for pulses to establish potential end-use applications are required. Scouring and moisture conditioning may improve milling efficiency and hull removal, in addition to influencing nutritional quality. In this study, the extent of these conditions on the protein digestibility and quality are evaluated in vitro on navy beans and chickpeas.FindingsScouring with no additional moisture conditioning increased protein quality in both navy beans and chickpeas, whereas the addition of 1% moisture conditioning in combination with scouring was detrimental to protein quality. Moisture conditioning at 0.5% without scouring also decreased protein quality in navy beans. Changes in protein quality were primarily due to compositional changes in both protein and amino acid content, reflected in amino acid scoring. Sulfur amino acids were the sole limiting amino acids in navy beans, while tryptophan was the sole limiting amino acid in chickpeas, irrespective of the roller-milled flour stream or Pretreatment.ConclusionScouring with no added moisture conditioning effectively improves the protein quality in both navy beans and Kabuli chickpeas due to changes in their amino acid scoring pattern.Significance and NoveltyBalancing milling practices and nutritional quality is important in developing navy bean and Kabuli chickpea products.

Background and Objectives: This study evaluated the effect of seed protein content on the composition, physicochemical, functional, and quality properties of pea protein isolates recovered through alkaline extraction/isoelectric precipitation. Samples were divided into three high-protein lines (HPL), three low-protein lines (LPL), and one control (medium protein). Findings: Isolates derived from HPLs had higher protein (92.4%-94.5%) compared to LPLs (79.9%-88.7%) and the control (similar to 92%). The protein content was significantly correlated to the legumin/vicilin ratio (1.0-1.8), surface charge (-38 to -23 mV), and surface tension (49-52.7 mN/m). No significant correlation was observed between protein content and functionality. Minor variation was observed for emulsifying (17.7-22.1 m2/g) and foaming (160%-215%) properties. LPLs presented higher amino acid score (0.83), and In Vitro protein digestibility corrected amino acid score (similar to 0.72) than HPLs (similar to 0.72 and similar to 0.64, respectively). Protein digestibility was in the range of similar to 87% for all lines. Conclusions: Seed protein concentration impacted the pea protein isolates' proximate composition, quality, and physicochemical/surface properties. HPLs presented higher total protein content and LPLs presenting overall higher protein quality, whereas functionality did not differ significantly between HPLs versus LPLs. Significance and Novelty: Our findings highlight the importance of sourcing and selecting pea lines tailored to specific applications, with a trade-off between protein quantity versus quality.

The rising demand for plant-based proteins has intensified interest in pulse crops due to their high protein concentration. Few studies have evaluated protein and amino acid composition or variability in cultivated lentil (Lens culinaris Medik.). We evaluated protein and amino acid composition using near-infrared reflectance spectroscopy in a diversity panel grown in four site-years in Saskatchewan, Canada, followed by genome-wide association analyses with phenology-related traits as covariates. We found little correlation between protein concentration and days from sowing to flowering, region of origin, cotyledon color, or seed size. Reproductive period was correlated with protein concentration, however. We also observed variability among environments and more variability within market classes than among them. We demonstrate the potential for breeders to identify adapted germplasm and select for increased protein and amino acid concentration and quality. We were able to identify several molecular markers for use in marker-assisted breeding to select for protein concentration or quality.

Soybean meals (SBM) SBM ) from different locations differ in their protein content, subsequently influencing their amino acid (AA) AA ) profile. In this study, standardized ileal digestibility (SID) SID ) of AA and growth or production performance were evaluated in pullets and hens fed SBM derived from soybean grown in Western Canada, primarily Manitoba (MB) MB ) labelled as A-, B- and C-SBM compared with that from Eastern Canada (Ontario, ON-SBM) and contained 38.3 f 0.44, 38.6 f 0.61, 39.4 f 0.49, or 44.0 f 0.87% CP, respectively. A Nfree diet was used to determine basal ileal endogenous losses of AA. The study included the grower, developer, and layer phases (9-12, 13-16, and 44/59-64-wk old birds, respectively). Although a lower (P P = 0.029) SID for cysteine was noted in the grower phase for the C-SBM compared with other SBM, the developer phase had higher (P < 0.05) SID for methionine, phenylalanine, cysteine (more by 4.4, 2.4 and 7.2% units, respectively) on average for SBM samples from MB compared with the ON-SBM. Regardless the source of the SBM, no difference in SID of AA was noted in the layer phase. Overall, in all phases the SID values of most AA in the SBM from MB were comparable with the ON-SBM, which may be linked to higher values of these AA per unit of protein content in the former source. In addition, the growth performance including feed intake, BW gain and feed conversion ratio in pullets, and egg production/quality in layers were similar between treatments. These findings show that the MB-SBM have a comparable feeding value with the ON-SBM, hence represent a suitable alternative protein source for poultry.

This study aimed to extract fava bean proteins using an eco-friendly deep eutectic solvent (DES) containing choline chloride and a glycerol-based system. Protein extraction conditions were optimized using response surface methodology, and the extracted protein was characterized in comparison to the protein extracted using a conventional alkaline solution. The optimum conditions obtained using the RSM model were: 28:1 w/w liquid: solid ratio, 1:2 w/w choline chloride: glycerol molar ratio, 50 degree celsius temperature, 1 h extraction time, and 40% w/w water content. Under these optimum conditions, protein content, protein yield, and protein recovery rates of 92.33 +/- 2.28%, 65.42 +/- 6.53%, and 23.15 +/- 2.31% were achieved. Alkaline extraction showed similar protein content (92.50 +/- 1.36%) but lower protein yield (60.76 +/- 1.16%) and recovery rate (21.74 +/- 0.19%) when compared to DES-extracted proteins (P < 0.05). The secondary structure analysis revealed an increased alpha-helix (21.37%) content in DES-extracted proteins compared to alkaline-extracted proteins (10.68%). Moreover, the alkaline-extracted proteins showed intermolecular beta sheets as protein aggregates (7.61%) and an increased percentage of beta-turns (19.71%). The molecular weight distribution pattern of proteins extracted from both methods showed more similarities. Overall, DES protein extraction could be an alternative to the conventional alkaline extraction method, promoting sustainable plant protein production from fava beans for use in new food formulations.

The overarching goal of this research was to investigate the effect of trypsin hydrolysis on the functional properties, protein quality, and flavour of chickpea and lentil protein isolates (CPI and LPI, respectively) at varying levels of hydrolysis (5

OBJECTIVE To investigate inflammatory responses to lipopolysaccharide (LPS) injection in layers. ANIMALS 33 40-week-old laying hens were used. METHODS 30 laying hens were divided into 2 groups: the first group was injected with 8 mg/kg LPS, while the second group was injected with sterile saline. At the start of the study, 3 birds served as a baseline and were used as the time 0 controls for both the saline and LPS-treated groups. Blood and spleen tissues were collected at 0 (before) and 1, 2, 3, 4, and 6 hours after injection. RESULTS LPS administration increased splenic mRNA levels of IL-1β, IL-2, IL-6, IL-8, IL-10, interferon-γ, and tumor necrosis factor-α ( P < .001) and serum IL-6 levels ( P < .01) compared to saline injection. The mRNA expression of most cytokine genes increased rapidly toward peak values within 2 hours after the LPS injection, and then the difference between the saline and LPS treatments got smaller as time went on; serum IL-6 reached its highest concentration 2 hours after LPS administration. The magnitude of LPS-induced upregulation of gene expression was the highest for IL-6, followed by IL-1β and IL-8, and tumor necrosis factor-α was the least affected. CLINICAL RELEVANCE The temporal and quantitative profile of these inflammatory mediators generated from this study provides valuable information in identifying the optimal time window and appropriate biomarkers for LPS-induced inflammation, which has significant implications in evaluating the effects of interventions on the immune system of chickens.

The 11S globulin legumin typically accounts for approximately 3% of the total protein in common beans (Phaseolus vulgaris). It was previously reported that a legumin peptide of approximately 20 kDa is resistant to pepsin digestion. Sequence prediction suggested that the pepsin-resistant peptide is located at the C-terminal end of the alpha-subunit, within a glutamic acid-rich domain, overlapping with a chymotrypsin-resistant peptide. Using purified legumin, the peptide of approximately 20 kDa was found to be resistant to pepsin digestion in a pH-dependent manner, and its location was determined by two-dimensional gel electrophoresis and LC-MS-MS. The location of the chymotrypsin-resistant peptide was confirmed by immunoblotting with peptide-specific polyclonal antibodies. The presence of a consensus site for proline hydroxylation and arabinosylation, the detection of hydroxyproline residues, purification by lectin affinity chromatography, and a difference in electrophoretic migration between the chymotrypsin- and pepsin-resistant peptides suggest the presence of a large O-glycan within these peptides.

Pea (Pisum sativum L.) is a nutritious pulse crop and a significant plant-based protein source. Its amino acid composition and protein digestibility influence its nutritional quality. Although different guidelines for protein content claims exist in North America, recommended patterns by FAO/WHO can estimate protein quality for dietary assessment. This study’s primary objective is to examine how adopting the new FAO/WHO recommendations affects the protein quality and protein content claims of field peas, using in vitro methods, and to assess the potential impact on consumer food choices. This work evaluated the amino acid score (AAS) and in vitro protein digestibility (IVPD) to assess pea protein quality. The effect of different amino acid scoring patterns recommended by FAO/WHO (1991, 2013) on AAS was estimated. The in vitro–protein digestibility corrected amino acid score (IV–PDCAAS) and in vitro–digestible indispensable amino acid score (IV–DIAAS) were calculated. Overall, pea protein quality varies with different amino acid scoring patterns. Sulfur amino acids (SAA) were the first-limiting amino acid. The IVPD was 80.6 to 88.6%, the mean IV–PDCAAS was 73.0%, and IV–DIAAS was up to 87.6%. Using the PDCAAS interpretation, all IV–PDCAAS samples qualified for a “good source of protein” claim. For DIAAS interpretation, some IV–DIAAS samples did not meet this claim due to the arbitrary cut-off of 75%. Therefore, the criteria for making protein content claims on food proteins should be reevaluated. This study highlighted significant variations in amino acid nutritional requirements across different life stages, impacting the protein quality assessment of field peas and, consequently, protein content claims, with implications for dietary guidelines and food labeling regulations.